Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum

MISSING IMAGE

Material Information

Title:
Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum
Physical Description:
Mixed Material
Language:
English
Creator:
Al-Khedery, Basima
Lundgren, Anna M.
Stuen, Snorre
Granquist, Erik G.
Munderloh, Ulrike G.
Nelson, Curtis M.
Publisher:
BioMed Central (BMC Genomics)
Publication Date:

Notes

Abstract:
Background: Anaplasma phagocytophilum is an intracellular organism in the Order Rickettsiales that infects diverse animal species and is causing an emerging disease in humans, dogs and horses. Different strains have very different cell tropisms and virulence. For example, in the U.S., strains have been described that infect ruminants but not dogs or rodents. An intriguing question is how the strains of A. phagocytophilum differ and what different genome loci are involved in cell tropisms and/or virulence. Type IV secretion systems (T4SS) are responsible for translocation of substrates across the cell membrane by mechanisms that require contact with the recipient cell. They are especially important in organisms such as the Rickettsiales which require T4SS to aid colonization and survival within both mammalian and tick vector cells. We determined the structure of the T4SS in 7 strains from the U.S. and Europe and revised the sequence of the repetitive virB6 locus of the human HZ strain. Results: Although in all strains the T4SS conforms to the previously described split loci for vir genes, there is great diversity within these loci among strains. This is particularly evident in the virB2 and virB6 which are postulated to encode the secretion channel and proteins exposed on the bacterial surface. VirB6-4 has an unusual highly repetitive structure and can have a molecular weight greater than 500,000. For many of the virs, phylogenetic trees position A. phagocytophilum strains infecting ruminants in the U.S. and Europe distant from strains infecting humans and dogs in the U.S. Conclusions: Our study reveals evidence of gene duplication and considerable diversity of T4SS components in strains infecting different animals. The diversity in virB2 is in both the total number of copies, which varied from 8 to 15 in the herein characterized strains, and in the sequence of each copy. The diversity in virB6 is in the sequence of each of the 4 copies in the single locus and the presence of varying numbers of repetitive units in virB6-3 and virB6-4. These data suggest that the T4SS should be investigated further for a potential role in strain virulence of A. phagocytophilum. Keywords: Anaplasma, phagocytophilum, Rickettsiales, T4SS, Comparative genomics
General Note:
Publication of this article was funded in part by the University of Florida Open-Access publishing Fund. In addition, requestors receiving funding through the UFOAP project are expected to submit a post-review, final draft of the article to UF's institutional repository, IR@UF, (www.uflib.ufl.edu/UFir) at the time of funding. The institutional Repository at the University of FLorida community, with research, news, outreach, and educational materials.
General Note:
Al-Khedery et al. BMC Genomics 2012, 13:678 http://www.biomedcentral.com/1471-2164/13/678; Pages 1-15
General Note:
doi:10.1186/1471-2164-13-678 Cite this article as: Al-Khedery et al.: Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum. BMC Genomics 2012 13:678.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All rights reserved by the source institution.
System ID:
AA00013685:00001


This item is only available as the following downloads:


Full Text


Al-Khedery et al. BMC Genomics 2012, 13:678
http://www.biomedcentral.com/1471-2164/13/678


BM ics
Genomics


Structure of the type IV secretion system in

different strains of Anaplasma phagocytophilum

Basima Al-Khederyl, Anna M Lundgren', Snorre Stuen 2, Erik G Granquist2, Ulrike G Munderloh3, Curtis M Nelson3,
A Rick Alleman4, Suman M Mahan5 and Anthony F Barbet*


Abstract
Background: Anaplasma phagocytophilum is an intracellular organism in the Order Rickettsiales that infects diverse
animal species and is causing an emerging disease in humans, dogs and horses. Different strains have very different
cell tropisms and virulence. For example, in the U.S., strains have been described that infect ruminants but not dogs
or rodents. An intriguing question is how the strains of A. phagocytophilum differ and what different genome loci
are involved in cell tropisms and/or virulence. Type IV secretion systems (T4SS) are responsible for translocation of
substrates across the cell membrane by mechanisms that require contact with the recipient cell. They are especially
important in organisms such as the Rickettsiales which require T4SS to aid colonization and survival within both
mammalian and tick vector cells. We determined the structure of the T4SS in 7 strains from the U.S. and Europe
and revised the sequence of the repetitive virB6 locus of the human HZ strain.
Results: Although in all strains the T4SS conforms to the previously described split loci for vir genes, there is great
diversity within these loci among strains. This is particularly evident in the virB2 and virB6 which are postulated to
encode the secretion channel and proteins exposed on the bacterial surface. VirB6-4 has an unusual highly
repetitive structure and can have a molecular weight greater than 500,000. For many of the virs, phylogenetic trees
position A. phagocytophilum strains infecting ruminants in the U.S. and Europe distant from strains infecting
humans and dogs in the U.S.
Conclusions: Our study reveals evidence of gene duplication and considerable diversity of T4SS components in
strains infecting different animals. The diversity in virB2 is in both the total number of copies, which varied from 8
to 15 in the herein characterized strains, and in the sequence of each copy. The diversity in virB6 is in the sequence
of each of the 4 copies in the single locus and the presence of varying numbers of repetitive units in virB6-3 and
virB6-4. These data suggest that the T4SS should be investigated further for a potential role in strain virulence of A.
phagocytophilum.
Keywords: Anaplasma, phagocytophilum, Rickettsiales, T4SS, Comparative genomics


Background
Anaplasma phagocytophilum is a tick-borne pathogen in
the Order Rickettsiales that is increasingly recognized as
a cause of disease in humans and animals world-wide
[1,2]. It causes the potentially fatal disease of human
granulocytic anaplasmosis, which typically manifests as a
flu-like illness accompanied by leukopenia, thrombo-
cytopenia and anemia. It was initially recognized in the
early 1990's when patients from Wisconsin and Minnesota

* Correspondence barbet@ufl edu
Department of Infectious Diseases and Pathology, College of Veterinary
Medicine, University of Florida, Gainesville, FL, USA
Full list of author information is available at the end of the article


developed febrile illness following a tick bite [3]. Since that
time the number of human cases has increased annually;
between 2000 and 2007 the reported incidence in the U.S.
increased from 1.4 to 3.0 cases/million persons/year [4].
The case fatality rate was 0.6% and the hospitalization
rate was 36%. In Massachusetts during the 2009 trans-
mission season there were 33 confirmed cases with 14
(42%) requiring hospitalization [5]. The human disease is
also present in Europe and Asia [2]. A recent study of 83
A. phagocytophilum-infected patients in China reported
a mortality rate in this cohort of 26.5% [6]. In the U.S.,
there has been a parallel increase in cases of the disease
[7] and seroprevalence [8] in dogs in the eastern and


0 2012 AI-Khedery et al., licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Biollf Med Central Creative Commons Attribution License (http//creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.






Al-Khedery et aL BMC Genomics 2012, 13:678
http://www.biomedcentral.com/1471-2164/13/678


upper Midwestern states. The tick vectors in the U.S.
are Ixodes scapularis and Ixodes pacificus and wild
rodents are the main reservoirs of human infections.
A. phagocytophilum also infects numerous other mam-
malian species including ruminants, horses, cats, and
bears and the symptoms are extremely variable, with
some mammalian species exhibiting acute disease and
others only persistent asymptomatic infections [9,10].
For example, A. phagocytophilum strains isolated from
deer in the U.S. can have a slightly different 16S
rRNA sequence and be uninfective to mice and it is
thought, humans [11-13]. In Europe, this agent has been
known to cause disease of ruminants for >100 years, yet
there have been few human infections [14]. The genome
sequence is available for a single strain of A. phagocyto-
philum derived from an infected human in the U.S. and
it is apparent that, although this strain lacks Type II, III,
V and VI secretion systems, a Type IV secretion system
(T4SS) is present [15]. As in other members of the
Rickettsiales, the T4SS of A. phagocytophilum is organized
differently from most gram-negative bacteria with the
component vir genes distributed between three major
genome locations [16].
The T4SS typically encodes a membrane-spanning
multiprotein complex that forms a transmembrane
channel through which solutes can pass into host cells.
It can mediate transfer of DNA and proteins into
eukaryotic host cells, interfere with host signaling, and is
essential for the survival of intracellular bacteria [17]. In
A. phagocytophilum, which preferentially colonize neu-
trophilic white blood cells, it is thought that the T4SS
secretes virulence factors that are responsible for sub-
verting innate immunity and inhibiting host cell apop-
tosis [16]. Interestingly, there appears to be differential
transcription of the T4SS in ticks and in the mammalian
host with virB6 and virB9 upregulated during infection
of human neutrophils and different virB2 paralogs
expressed in mammalian and tick cells [18]. There is evi-
dence that VirB2, VirB6 and VirB9 are exposed on the
outer membrane surface in the Rickettsiales [18-20],
which has stimulated interest in their potential use as
vaccine candidates. This possibility has been investigated
more extensively in the related organism Anaplasma
marginale [21-25]. In A. marginale, unlike many other
surface-exposed proteins, the T4SS proteins are con-
served between strains [26]. Also, cattle immunized with
outer membranes and protected against challenge infec-
tion respond with IgG and T cells to Vir proteins, not-
ably VirB2, VirB9 and VirB10. To date, only two T4SS
substrates have been identified and partially characterized
in A. phagocytophilum: the ankyrin repeat domain-
containing protein, AnkA, and the Anaplasma translo-
cated substrate 1, Ats-1. AnkA translocates to the host
nucleus and interacts with DNA [27,28], while Ats-1 is


imported into the mitochondria where it is proposed to
interfere with the induction of apoptosis [29].
In this study, we compared the structure and diversity
of the T4SS in different strains of A. phagocytophilum
infecting humans, dogs, rodents and ruminants. Most
diversity was found in the proteins thought to be surface-
exposed, which may be associated with the different
virulence and cell invasion properties of this species.

Results and discussion
The vir loci were sequenced in eight strains of A. phago-
cytophilum; seven of these were strains for which previ-
ous structural information was not available and
included organisms originally isolated from U.S. dogs
(ApDogl, ApDog2), a rodent (ApJM), a horse (ApMRK),
the ruminant Ap variant 1 strain (ApVarl) and two
strains from Norwegian sheep (ApNorVl, ApNorV2).
The human HZ strain was also resequenced, as optical
mapping had suggested a possible error in the previously
sequenced virB6-4 locus. The data indicated consider-
able diversity in the individual vir loci between strains
that will be discussed below. In all strains, however, as
noted previously [20,30], the vir loci were distributed
mainly in three gene clusters comprising: virB8-1, virB9-
1, virBlO, virBll and virD4; virB27s and virB4-2; and
virB3, virB4-1, and the four virB6 paralogs (Figure 1).
These three loci may each be transcribed polycistroni-
cally [31], although it is clear that T4SS structure in the
Rickettsiales is unique and more complex than initially
thought. The number of virB2 paralogs was different be-
tween strains with the human HZ strain having the least
(8 total paralogs) and the ruminant strains having the
most (up tol5 total paralogs). The description of the
T4SS components presented here follows the functional
classification described by Alvarez-Martinez and Christie
[20].

Inner membrane channel/scaffold subunits: VirB3, VirB6,
VirB8 and VirB10O
The most conserved of these subunits are VirB3, VirB8
and VirBlO, with few differences between strains. VirB3
has been linked in Agrobacterium tumefaciens with pilus
assembly and substrate translocation [32,33]. It is abso-
lutely conserved between strains with no amino acid
changes and conforms to the typical VirB3 structure.
Two alpha-helical domains for insertion into the cyto-
plasmic membrane are strongly predicted by TMpred.
VirB8, proposed to function as a nucleation factor dur-
ing the assembly of T4SS [34,35], is also well conserved,
particularly VirB8-1 in the polycistronic transcription
locus (one amino acid change between all strains).
VirBO1, proposed to function as a scaffold across the
entire cell envelope [36], is also generally well-
conserved with the exception of one ruminant strain,


Page 2 of 15








Al-Khedery et al. BMC Genomics 2012, 13:678
http://www.biomedcentral.com/1471-2164/13/678


ApJM -..
ApDogl
ApDog2
ApMRK
ApNorLamb- V2

ApVar 1


ApNorLamb-V1


Page 3 of 15


additional irB4-t in |
ApNorLanb-Vi and -V2

B2-8 B2-7B2-6 B2-5 B24 23 22 21
ArHz .... .......... ....... ....... ....


IC


-a< <-
-a< -
-a3-C<--

-

Figure 1 Distribution and content of vir gene clusters in eight diverse A. phagocytophilum strains. Top panel. Schematic representation of
all vir loci (colored arrows) showing the three conserved gene cluster islands (see text). VirB-7, virB8-2 and virB9-2 are not part of vir gene clusters,
but their location relative to surrounding genes is also highly conserved among strains. A small cluster comprising truncated (t) virB6 and virB4
gene fragments is present in all strains, but the Norwegian lamb strains have one additional virB4-t Bottom panel. .1 of the virB2 gene
cluster. Numbering of paralogs 1-8 is based on the original ApHZ annotated genome (GenBank CP000235). Artificial gaps (stippled lines) were
introduced to allow alignment of the more spatially conserved paralogs B2-1, 2-2 and 2-3 at one end, and B2-7 and 2-8 at the other end of the
cluster. With the exception of virB2-9, lacking in ApHZ, the number and arrangement (but not necessarily sequence) of virB2 genes is highly
conserved in all but the US ruminant ApVar-1 and ApNorLamb-VI, which have several additional virB2 genes. In both strains a sub-cluster of 6
distinct genes was present. Due to the repetitive nature of sequences in this region, combined with the relatively short length of 454 reads
(<550 bp), their placement could not be confidently ascertained (highlighted by arrows and '?'). Maps are drawn to scale. Double lines designate
interruption in sequences. Genes belonging to the same grouping have the same col o. oriC; origin of replication.


ApNorLamb-V1, which has 31 amino acid substitutions
with respect to ApHZ (data not shown). However, all A.
phagocytophilum VirB10's, including ApNorLamb-V1,
have two strongly predicted transmembrane domains,
which supports their function as membrane scaffolding
subunits in these organisms.
Of these inner membrane channel subunits, the data
on VirB6 are the most interesting. All VirB6 subunits


that have been described possess a highly hydrophobic
membrane domain including five or more predicted
transmembrane domains [20]. Some VirB6 proteins also
have an extended C-terminal hydrophilic domain that
has been proposed to protrude through the T4SS into
the target cell, or may be proteolytically released from
the N-terminal domain and then translocated into the
target cell. Evidence has been obtained for surface


ApHz-VirB6-1 ApHz-VirB6-2 ApHz-VirB6-3


ApDogl-VirB6-1 ApMRK-VirB6-2 ApDogl-VirB6-3


ApDog2-VirB6-1 ApNorV2-VirB6-2 ApMRK-VirB6-3


ApMRK-VirB6-1 ApVarl-VirB6-2 ApNorV2-VirB6-3


ApNorV2-VirB6-1 ApNorVl-VirB6-2 ApVarl-VirB6-3


ApVarl-VirB6-1 ApDogl-VirB6-2 ApNorVl-VirB6-3


ApNorVl-VirB6-1 ApDog2-VirB6-2 ApDog2-VirB6-3


ApJM-VirB6-1 Ap]M-VirB6-2 Ap3M-VirB6-3


Figure 2 Phylogenetic tree to show the relationship of syntenic VirB6 proteins from different strains of A. phagocytophilum. A scale bar
is shown underneath representing the number of amino acid substitutions/site.


C







Al-Khedery et al. BMC Genomics 2012, 13:678
http://www.biomedcentral.com/1471-2164/13/678


exposure of extended VirB6 in some Rickettsiales [37].
Of all the membrane channel subunits, the most se-
quence diversity between A. phagocytophilum strains
was in the four VirB6 paralogs (Figure 1). Although
there were no amino acid changes in the VirB6-1, VirB6-
2 and VirB6-3 paralogs between human, dog and rodent
strains, the ruminant and horse strains had numerous
substitutions throughout each molecule, agreeing with
the closer evolutionary relationship between strains
infecting humans and dogs in the U.S. (Figure 2). Fur-
thermore, major differences in repeat number and se-
quence were found in the C-terminal repeat region of
VirB6-3 (yellow boxes in Figure 3A and Additional file 1:
Figure Sl) in ruminant and horse strains, with the horse
strain showing the least variability from ApHZ.
The only amino acid differences detected between the
human, dog and rodent strains were in the VirB6-4 sub-
unit. VirB6-4 in these strains contains four repeat
regions (R1-R4 in Figure 3A) and variability in repeat
number, order and sequence were found mainly in R3


virB6-4
in CP000235


virB6-3


virB6-4


1 kb


and R4 (Additional file 2: Figure S2). Within Ri
(Figure 3A), the only difference detected was in ApDog2
which had 4 and 1 partial of 231 bp repeat units (data
not shown), compared to 3 and 1 partial repeats in the
ApDogl, ApJM and ApHZ virB6-4 R1. Optical mapping
of the Dogi genome and comparison with ApHZ sug-
gested that the sequence obtained previously for the
human HZ strain virB6-4 was incorrect (Figure 3B). This
was confirmed by PCR and sequencing, and mapped
specifically to the 3'-most R4 region (Figure 3C). Be-
cause of its size and unusual composition it was only
possible to resolve this sequence using the long read-
length Pacific Biosciences technology (see Methods).
The corrected virB6-4 R4 of ApHZ, totaling 6.89 kb, dif-
fered from the original by 5.88 kb of additional sequence
composed exclusively of 84 bp [type 1, a and b (Tia,
Tib); light/dark blue boxes, respectively, in Figure 3A]
and 162 bp [type 2, a and b (T2a, T2b); light/dark or-
ange boxes, respectively, in Figure 3A] repeat units, giv-
ing a complex repeat structure containing 53 and 1


2.7 kb
1.15 kb


AB1395


B
CP000235 v6-4

R4:8+1p
CP000235 corrected

R4:53+lp
Dogl optical map

Dogi genome sequence

JM genome sequence

R4:81+lp 1 k


rt rt~~ ri 3 <-
N RO N ^O O SO >
=. =. a, a a a ,


AB1395/1466:R4 AB1393/1466:R3+R4


Figure 3 The 3' end of A. phagocytophilum virB6-4 genes is composed of an unusually large tandem repeat region, which exhibits
dramatic variability among strains. A. Map of the human HZ strain virB6-3 and virB6-4 genes, highlighting the location and structure of several
repeat regions (RIl-R4). The most variability occurred in R4; this region is 5.88 kb larger than previously reported for the ApHZ genome
(CP000235). The original sequence is diagrammed above the map, with the dashed line representing the segment missing in CP000235. Larger
repeated R4 segments of 2.7 kb and 1.15 kb are indicated above. Vertical black bars within each gene designate segments encoding predicted
transmembrane domains. BamHI sites, of which there is one in all R4 type 2 repeats (see Figure S2B), are indicated. Also shown are the positions
of PCR primers used in C. B. BamHI genomic maps depicting the virB6-4 locus (black arrows).The segment encompassing R4 is highlighted below
each respective map. In the regions outside the virB6-4 locus, corresponding BamHI fragments are shown in the same color. Overall, the optical
map sizes were in good agreement with the actual sizes, except within R4. This is attributed to the limitation of optical mapping in resolving
fragments <2 kb. Despite these discrepancies, the cumulative size of the genomic region encompassing virB6-4 in the optical map is in close
agreement with that in the ApDogl genome sequence. C. The variability in size of PCR products spanning virB6-4 repeat regions R4 and R3/R4 in
diverse A. phagocytophilum strains.


Page 4 of 15


BamHI: R1 R3 R4






Al-Khedery et aL BMC Genomics 2012, 13:678
http://www.biomedcentral.com/1471-2164/13/678


partial repeat units compared to 8 and 1 partial in the
original sequence. Further, the 5'- and 3'-most 2.7 kb of
this complex structure are identical in sequence, and the
3'-most 1.15 kb of each of these segments is repeated
again in the center of R4 (Figure 3A and Additional file
2: Figure S2). Although the possibility exists that the
ApHZ population from which we isolated gDNA differs
within the virB6-4 R3/R4 repeat regions from the popu-
lation used to generate CP000235, the fact that all
strains investigated herein presented expansive R3/R4
regions (Figure 3C) would contradict that. Instead, it is
more plausible that the existence of 2.7 kb of identical
repeats at the ends of the ApHZ R4 may have lead to
the excision of most of its sequence during construc-
tion/propagation of those libraries. Interestingly, virB6-4
R3 and R4 were identical both in size and sequence in
the Dogi and rodent strains despite differing markedly
from the HZ and Dog2 strain regions (Additional file 2:
Figure S2A). Within R3, these strains had 2 additional
405 bp repeats compared to ApHZ and one more com-
pared to the Dog2 strain. However, differences between
strains were most dramatic within R4. Not only was this
region in ApDogl/ApJM 2.87 kb larger than in ApHZ
bringing the total number of repeats to 81 and 1 partial,
but intriguingly, the repeat pattern was completely unre-
lated to that in the HZ strain. Also, the Dogi and rodent
strain R4 lacked T1b repeat units, while having a third
type 2 repeat variant, namely T2c, which differed from
T2b by 1 SNP and a 12 bp deletion (Additional file 2:
Figure S2). Partial analysis of the ApDog2 454 reads
spanning R4 (estimated at -8 kb by PCR; Figure 3C)
showed that the order of the 5'- and 3'-most three repeat
units differed from either the HZ or Dogl/rodent strain
R4 repeat patterns (Additional file 2: Figure S2A). Not-
ably, our preliminary analyses of the horse and ruminant
454 reads suggest the absence of distinct R3 and R4
regions in virB6-4 in these strains. Rather, the few repeat
units identified to date appear to be a combination of R3
and R4 repeats (data not shown). It is also unclear if the
~17 kb and -25 kb PCR products generated with pri-
mers AB1393/1466 in ApVar-1 and ApNorLamb-V2, re-
spectively (Figure 3C), are composed mainly of repeats,
or alternatively if a fifth virB-6 gene paralog exists in
these strains. Taken together, the data presented here
clearly demonstrate the extreme variability of the T4SS
VirB6-4 subunit among A. phagocytophilum strains. Al-
though the differences between the more closely related
human, dog and rodent US strains were mainly within
repeat-laden regions, the fact that an extensive, distinct
repeat pattern was maintained in two strains would
speak against the possibility that the variability may be
attributed solely to the highly recombinogenic nature of
such structures. Worth noting, Camp Ripley, where the
infected jumping mouse was captured (2001) is only -20


miles away from the city of Baxter, MN, where Dogi
resides. Although there are no records of where this dog
may have actually acquired the infection, it presented
with severe clinical disease in 2007.
The unusual structure and likely antigenicity of the
C-terminal region of the A. phagocytophilum VirB6-4's is
apparent in hydrophobicity plots (Figure 4). What spe-
cific properties these distinct repeat patterns may confer
onto each strain awaits functional analysis of these pro-
teins in A. phagocytophilum. The corrected VirB6-4
translated protein had a predicted molecular weight of
470,695 Da containing 4,322 amino acid residues com-
pared to molecular weights of 90,742, 103,204 and
158,321 Da for the HZ strain VirB6-1, VirB6-2 and
VirB6-3, respectively. Interestingly, the predicted acidity
of the VirB6's also increased from VirB6-1 to VirB6-4
(pI's of 8.4, 6.8, 5.1 and 4.0 for the ApHZ VirB6-1, VirB6-
2, VirB6-3 and VirB6-4, respectively). The ApDogl/ApJM
VirB6-4 polypeptides had a predicted molecular weight
of 603,529 Da containing 5,550 amino acids, and a pI of
3.96. Despite these dissimilarities, at least eight trans-
membrane segments were predicted for all VirB6
paralogs.

Periplasmic/outer membrane channel subunits: VirB2,
VirB7 and VirB9
Several other T4SS subunits contribute to the secretion
channel across the periplasm and outer membrane.
VirB7 subunits are typically small lipoproteins that may
stabilize VirB9 [38,39]. In A. phagocytophilum strains a
putative VirB7 is absolutely conserved between strains
and may be lipid modified through an N-terminal cyst-
eine on the mature molecule. VirB9 is hydrophilic and
also localizes to the periplasm and outer membrane. In
A. tumefaciens the C-terminal region of VirB9 is part of
the outer membrane protein channel and is surface ac-
cessible [40]. There is also evidence for surface exposure
of VirB9 in Ehrlichia chaffeensis and A. phagocytophilum
[18,19,41]. VirB9-1, which is encoded on the polycistro-
nic virB8-1-virD4 transcript [31], has a strongly pre-
dicted signal peptide and two transmembrane helices. Of
all the potentially exposed components of the T4SS,
VirB9 of A. phagocytophilum appears to be the least di-
verse among strains. There are some amino acid substi-
tutions in ruminant and horse strains (2-6 total
compared to ApHZ) but in the other strains VirB9's are
unchanged (data not shown).
Unlike VirB9's, VirB2's are the most diverse of all
T4SS subunits in A. phagocytophilum, in terms of both
copy number and sequence. VirB2 proteins are typically
constituents of pili and of the secretion channel and
their diversity in Anaplasma suggests the possibility of
exposed, antigenically variable structures. In A. margin-
ale, VirB2 is expressed together with the major outer


Page 5 of 15







AI-Khedery et al. BMC Genomics 2012, 13:678
http://www.biomedcentral.com/1471-2164/13/678


1000


Position
Figure 4 Hydrophobicity plots of VirB6-4 proteins from A. phagocytophilum HZ (top) or Dog 1 (bottom) strains.


membrane protein MSP3 on a sequence-variable poly-
cistronic transcript [25,42]. The mechanism of expres-
sion in A. phagocytophilum is not known. VirB2's of
other genera are typically small hydrophobic proteins
with a long signal peptide sequence and two hydropho-
bic alpha helices for integration into the cytoplasmic
membrane. This also appears to be the case for A.


phagocytophilum. The VirB2 paralogs in the different
strains are predicted to have two hydrophobic alpha-
helices of lengths 22+/-3 and 20+/-0.2 amino acids and
signal peptides of length 27+/-2 amino acids. This is
true despite their sequence diversity (Figure 5). As with
many other T4SS components, the ruminant and horse
strains are more distant taxonomically in VirB2


Page 6 of 15


500 1000 1500 2000 2500 3000 3500 4000
Position


4000


5000







Al-Khedery et al. BMC Genomics 2012, 13:678
http://www.biomedcentral.com/1471-2164/13/678


sequence compared to VirB2's of human and dog
strains. Alignment of all VirB2 paralogs and orthologs
shows that sequence diversity is primarily localized to
two hypervariable regions either preceding an N-
terminal cysteine or close to the C-terminus (Figure 6).
This is similar to the hypervariable regions found among
VirB2 paralogs of A. marginale [25].

Energetic subunits: VirB4 and VirB11
ATPases are typically used in T4SS to energize substrate
transfer and have been found in every T4SS described.
In gram-negative bacteria these are typically integral
membrane proteins encoded by genes residing upstream
of virB2 (encoding pilin). This is true for all strains of A.
phagocytophilum and it has been suggested that this ar-
rangement of multiple virB2 paralogs and virB4-2 may
allow assembly of an antigenically variable surface or-
ganelle [20]. The energetic subunit itself, VirB4-2, is
however, well conserved between strains. The most dis-
tant taxonomic relationship was found between human
and ruminant strains (29 total amino acid substitutions
in ApNorLamb-V1 compared to ApHZ, Figure 7). The
other energetic subunit, VirB11, was also well-conserved


between strains (6 amino acid substitutions between
ApNorLamb-V1 and ApHZ; data not shown).

Type 4 coupling protein: VirD4
Type 4 coupling proteins such as VirD4 are ATPases
that function in substrate recognition and translocation
using the T4SS. They are associated with most effector
translocator systems. They typically possess a minimum
of two N-terminal transmembrane domains. Often most
heterogeneity exists in these N-terminal regions [20].
The A. phagocytophilum VirD4's conform somewhat to
this stereotype with three strongly predicted N-terminal
transmembrane segments. As with the other ATPases of
the A. phagocytophilum T4SS, there is little variation in
VirD4, a total of 17 amino acid substitutions of which 4
are N-terminal but more (12) are C-terminal. Again, the
evolutionary relationships among VirD4 sequences pos-
ition the ruminant and horse strains more distantly to
the U.S. dog, human and rodent strains (Figure 8).

Conclusions
A. phagocytophilum represents a recent reclassification of
intracellular organisms infecting different animal species


Page 7 of 15


ApH-OVlC 2-1 A V- 2- 2 AH.-VI 3 AH,-V, B2- A





A)M-VI2- ApVarl-VirB22 A0M iWrB2 3 ApNo -V2-VB-4








-A- AD2pHz- V62-S
ApDv-rE,2-5 Aovz.lrB2-3 A pM V-24-








ApNOM VS A-g.2 V-6 pO -2 7

ApAg2 5irB2-8
ApiM VirS2S AoDo -VloB2-6 AaDglvaiB-2 7





AAJM-VirB2-f





Figure 5 Phylogenetic trees to show the relationship of syntenic VirB2 proteins from different strains of A. phagocytophilum.







Al-Khedery et aL BMC Genomics 2012, 13:678
http://www.biomedcentral.com/1471-2164/13/678


and humans and causing diverse disease symptomatology
[43]. These bacteria were previously known as Ehrlichia
phagocytophila, Ehrlichia equi, and the agent of human
granulocytic ehrlichiosis. Despite the differences within
this species, the overall genome structure and synteny of
the T4SS is maintained. However, gene structural analysis
reveals evidence of gene duplication and considerable di-
versity of T4SS components in strains infecting different
animals. Taxonomic trees suggest a close evolutionary re-
lationship of A. phagocytophilum strains infecting U.S.
humans, mice and dogs and a more distant relationship
with ruminant and horse strains. This relationship is not
unique to the T4SS but is also supported by similar taxo-
nomic trees of other A. phagocytophilum proteins of con-
served metabolic function (Figure 9). Within the T4SS
multicomponent membrane complex, the energetic and
internal scaffolding protein components are the most con-
served. In contrast, components that form the proposed
exposed structures of the T4SS, such as VirB2 and VirB6,
are more variable. T4SS are important virulence


determinants of bacteria, therefore these differences may
result in the different infectivity and virulence profiles
observed with different strains. It will be of interest to de-
termine the molecular architecture of VirB6 paralogs in
different strains, including interactions with other T4SS
components and effectors. Of the known surface exposed
components of the T4SS, VirB9 is the most conserved.
This protein has been proposed as a vaccine component
against A. marginale and may also be suitable against
A. phagocytophilum.

Methods
A. phagocytophilum strains, cell culture, and experimental
infection
The A. phagocytophilum U.S. strains HZ (human-origin,
NY), MRK (horse-origin, CA), JM (rodent-origin, MN)
and Dogi (dog-origin, MN) were propagated in HL-60
cells in RPMI-1640 medium (Thermo Fisher Scientific,
Inc., Waltham, MA) supplemented with final 10% heat-
inactivated fetal bovine serum (Thermo Scientific) and


Page 8 of 15


AMR V"r2-

ApMVr2 2 V- 2-
Apo2 --r 2-2
A VIr ~r2 2 4
2 or2 -~r2 2-4
Alor" _~r2 2 R
ApJM-V,r22 5


por2 Vr2- 2
2A JM-Vr I


2 12r Vr2n 1
oV V

pr V-r22 no-1el-4-
ApVr- _Vr2nvl ~t
AFVorl, V,r 22no2 -4
ApoV V~r.2 no -p- -

Ap orV' V,r2 n
Ap'orVI V-r22-1
ApDogl V-r2 2 6
ApJM-V,r22 6
po 2 ,~ 2_6
ApJM -2r2 ,
A,--K V-r22- 1

_2M ~r2 7

2p~r v~rv2 7
A ....V" V-r2-7
2J Vri -


Al jr2 -
ApJog2V~rl2-
2 -JM V Vr 2 -3
poV2 Vr2 3

Figure 6 Multiple sequence alignment of VirB2 amino acid sequences from different strains of A. phagocytophilum.







AI-Khedery et al. BMC Genomics 2012, 13:678
http://www.biomedcentral.com/1471-2164/13/678


ApHz-VirD4



ApDogl-VirD4



ApDog2-VirD4



ApJM-VirD4


-VirD4


ApVarl-VirD4
10.01
Figure 8 Phylogenetic tree to show the relationship
VirD4 proteins from different strains of A. phagocy


4 mM L-glutamine (Lonza, Rockland, ME), and in the
absence of antibiotics. ApHZ and ApMRK have been
described previously [15,44]. The ApJM strain (CR01-
1258) originated from a meadow jumping mouse (Zapus
hudsonius) trapped at Camp Ripley, MN [45]. The
ApDogI strain originated from the blood of a dog from
Baxter, MN naturally infected with A. phagocytophilum,
as evidenced by the detection of distinctive morulae in a
diagnostic blood sample, and sequencing of the Expres-
sion Site-linked msp2/p44 gene. Briefly, whole blood was
collected from the animal with EDTA as an anticoagu-
lant. The buffy coat layer was collected after low-speed
centrifugation of the whole-blood, washed in lx phos-
phate buffered saline (PBS, Hyclone, cat. no. SH30256.01),
then added to a culture of uninfected HL-60 cells. The
ApNorV1-VirD4 culture was left undisturbed for 3 days, after which mor-
ulae began to appear. The ApDog2 strain also originated
from a MN dog and was passage to and maintained in
the Ixodes scapularis ISE6 tick cell line as described [46].
The Ap variant 1 CRT35 strain (tick-origin, MN),
maintained in ISE6 cells, has been described [47]. For
DNA isolation, cultures were maintained until 90-100%
of cells were infected with mature morulae. Cells were
pelleted by centrifugation at 2500 x g for 20 min at 4'C.
p of syntenic Pellets were gently resuspended in 1.5 ml cold PBS, trans-
tophilum.o screw-cap microfuge tubes, and centrifuge at
f erred to screw-cap microfuge tubes, and centrifuged at


Page 9 of 15


ApHz-VirB4-1 ApHz-VirB4-2



ApMRK-VirB4-1 --ApMRK-VirB4-2



ApDogl-VirB4-1 ApNorV2-VirB4-2



ApDog2-VirB4-1 ApVarl-VirB4-2



ApJM-VirB4-1 -- ApNorVl-VirB4-2



ApNorV2-VirB4-1 ApDogl-VirB4-2



ApNorV1-VirB4-1 ApJM-VirB4-2



ApVarl-VirB4-1 ApDog2-VirB4-2
,0.01 ,0.01

Figure 7 Phylogenetic trees to show the relationship of syntenic VirB4 proteins from different strains of A. phagocytophilum.







Al-Khedery et al. BMC Genomics 2012, 13:678
http://www.biomedcentral.com/1471-2164/13/678


1500 x g for 20 min at 4C. Supernatants were removed
and the cell pellets stored at -80C until further use.
Two naturally occurring Norwegian lamb A. phagocy-
tophilum strains differing in the 16S rRNA gene and de-
gree of virulence were used to experimentally infect
lambs raised in an indoor environment with barriers
against tick entry and tick infestation. Lamb 00186 was
infected with the more virulent variant 1 (identical to
GenBank M73220) and lamb 0054 with variant 2 (identi-
cal to GenBank AF336220) [48], to be referred to as
ApNorLamb-V1 and -V2 from here on. Infections were
monitored by microscopy and blood was harvested at
maximum parasitemia. To purify buffy coats containing
the infected neutrophils, approximately 2.5 1 of Na-
citrated blood was collected from each animal. The


blood was transferred to 1 1 centrifuge bottles and cen-
trifuged at 2,500-3,000 x g in a swing-out bucket rotor
for 30 min at 4C. After removing most of the plasma
layer, the buffy coat layer was collected with minimal
contamination of red blood cells. The cells were diluted
1:3 with PBS, mixed gently and centrifuged at 1,500x g
for 20 min at 4C. Following three PBS washes, superna-
tants were removed and the cell pellets stored at -80C.
The experimental study in sheep was approved by the
Norwegian Animal Research Authority.


Purification of host cell-free A. phagocytophilum and
genomic DNA (gDNA) isolation
For the HZ, JM, Dogi, MRK and NorLamb-V1 and -V2
strains, intact, host cell-free organisms with minimal


Page 10 of 15


ApHz-PQ4A ApHz-LeuS ApHz-AtpA


ApDg1-PolA ApMRK-LeuS ApDogl-AtpA


ApMRK-PotA ApVarl-euS ApDog2-AtpA


ApNorV2-PolA ApNLOrV-LS APM-At


ApNorV1-PoIA ApNorV2-LeuS ApWRK-AtpA


ApVar -PolA ApDogl-LeuS ApNOrV2-AtpA


ApDogO2-OIA ApDog2-LeuS ApVarl AtpA


Ap)M-PolA ApJM-LeuS ApNoNV-AtpA



ApH-ValIS ApHz-RG ApHz-UgA


ApMRK-Rec ApMRK-bgA


AQMRK-ValS ApDogl-Req c ApNOrV -LgA


ApNo -VaIS ApDog2-Rec ApNV2- LgA


ApNoN2-ValS ApNO&Vl-RecG ApVar I-LgA


ApVar1.ValS Aprn2.RcG ApO1 .LUgA


ApDOgl-VlS ApVar .ReCG ApDOg2-LgA


Ap)M-V.IS ApJN-Re ApJM- gA
,001 l 001

Figure 9 Phylogenetic tree to show the relationship of other conserved proteins from different strains of A. phagocytophilum. These
proteins are: PolA, DNA polymerase I; LeuS, leucyl-tRNA synthetase; AtpA, ATPsynthase F1, alpha subunit; ValS,valyl-tRNA synthetase; RecG,
ATP-dependent DNA helicase; LigA, NAD-dependent DNA ligase.






Al-Khedery et al. BMC Genomics 2012, 13:678
http://www.biomedcentral.com/1471-2164/13/678


host cell gDNA/RNA contamination were purified from
frozen PBS pellets of infected cells prepared as above.
Samples and reagents were maintained on ice through-
out the entire procedure, and all centrifugations per-
formed at 4C. Following a quick thaw, host cells were
disrupted by vigorous vortexing for 5 min. An equal vol-
ume of PBS was added and vortexing continued for
3 min. Cellular debris was removed by centrifugation at
200 x g for 15 min. After removing most of the superna-
tants to fresh tubes, these were passed several times
through a 31 G needle and saved on ice. Pellets were
resuspended well in final 500 tl PBS then passed serially
through 22 G, 25 G, 28 G and, when possible, 31 G nee-
dles attached to a 1 ml syringe. 3-5 volumes PBS were
added and mixed by vortexing. Debris was removed by
centrifugation at 200 x g for 10 min. Supernatants were
pooled to those from the previous centrifugation step.
RNaseA was added to a final 250-300 tg/ml and the
samples incubated 45-60 min at 37C. Samples were
centrifuged at 21,000 x g for 30 min and the superna-
tants removed completely. Pellets were resuspended in
50-100 tl PBS each and transferred to fresh tubes. To
ensure homogeneity of the suspension, initially a drawn-
out 10 tl pipette tip was used to disrupt the pellet by
swirling followed by up/down pipetting and gentle vor-
texing, before switching to a larger tip. The sample was
further homogenized by several passes through a 28-
31 G needle. PBS was added to final 500-700 tl and
DNasel to final 250 tg/ml. Following 45-60 min incuba-
tion at 37'C the samples were centrifuged at 21,000 x g
for 30 min. Pellets were homogenized as above and the
DNasel treatment repeated. EDTA (pH 8.0) was added
to final 25 mM and the samples centrifuged as above.
Tubes were washed twice with PBS without disturbing
the pellets and residual PBS was removed after 3 min
centrifugation at 21,000 x g. Pellets were homogenized
as above in 600-800 tl RPMI culture medium (contain-
ing 10% fetal bovine serum) added incrementally and
transferred to a 50 ml tube. Culture medium was added
to a final volume of 6 ml before passage through a pre-
wet, 2 pm pore-size, 25 mm, GMF-150 glass microfiber
syringe filter (Puradisc 25GD; Whatman Inc., Florham
Park, NJ). The filter was washed 3-4x with culture
medium. Washes were pooled to the filtrate and centri-
fuged at 22,000 x g for 30 min. The pellets, comprised of
free, non-viable organisms and host cell mitochondria,
were resuspended in PBS, transferred to microfuge tubes
and re-pelleted at 21,000 x g for 30 min. Supernatants
were removed completely and the pellets were processed
immediately or stored at -20'C. For every 108 host cells
used at 90-100% infectivity, enough organisms were
recovered to yield on average 1-1.5 tg high-quality
DNA using either the Gentra Puregene Yeast/Bact. kit
(Qiagen Inc., Valencia, CA) or the QIAGEN Blood &


Cell Culture DNA mini kit following the manufacturer's
protocols.
For the Dog2 and Ap variant 1 strains, organisms were
cultured and isolated from ISE6 tick cells as described
[49]. Host cell-free bacteria were prepared from two cul-
tures in 25 cm2 flasks, collected by centrifugation for
10 min at 11,000 xg at 4oC, and lysed in Gentra Pure-
gene lysis buffer (Qiagen) at 80oC for 5 min. Since these
DNA samples also contained a considerable amount of
small (<500 bp) DNA species naturally associated with
the ISE6 host cell line, the A. phagocytophilum gDNA
was further purified by electroelution from agarose gels,
followed by phenol/chloroform extraction and EtOH
precipitation using conventional protocols.

Preparation of host cell-free A. phagocytophilum agarose
plugs for optical mapping
ApDogl was initially selected for complete genome se-
quencing to compare with the published HZ strain.
When a draft genome was assembled for ApDogl it was
largely syntenic with HZ except for the virB6 locus, indi-
cating a possible error in the sequence of one or both of
the strains. Accordingly, the ApDogl draft genome se-
quence was verified by Optical Mapping. In preparation
for Optical Mapping (performed by OpGen Inc.,
Gaithersburg, MD), host cell-free organisms were em-
bedded in 0.5% low-melting point agarose plugs and
subsequently lysed, allowing access to the intact,
~1.48 Mb circular A. phagocytophilum chromosome. A
procedure recommended by OpGen was followed. All
solutions were made fresh using OpGen suggested
reagents. Intact ApDogl organisms were purified as
above, except that the pellet of free organisms obtained
following centrifugation of the filtrate was resupended
and washed in cell suspension buffer [200 mM NaC1,
100 mM EDTA-Na2 (pH 8.0), 10 mM Tris (pH 7.2)].
Plugs were made immediately on completion of the iso-
lation procedure. Briefly, following the final centrifuga-
tion of the purified organisms, the pellet was
resuspended in cell suspension buffer using 40-50 tl for
every 108 host cells used at >95% infectivity. The sample
was passed 2x through a 31 G needle (3/10 ml capacity
Insulin Syringe with fused 8 mm long needle, BD
#328438; Becton, Dickinson & Co., Franklin Lakes, NJ)
to ensure homogeneity of the thick suspension, and an
equal volume of 1% low melting point SeaPlaque GTG
agarose [(Lonza #50111) dissolved in DEPC-treated
water (Invitrogen #750023; Carlsbad, CA) and held at 55oC]
was immediately added. Following mixing, 100 tl aliquots
were dispensed into plug molds (Bio-Rad #170-3713;
Hercules, CA) and allowed to set for 1 hr at 4oC prior to
transfer into a 50 ml tube containing 5-10 ml, 50C NDSK
solution [filter sterile NDS solution (1% N-lauroylsarcosine
(Sigma #L5000; St. Louis, MO) in 0.5 M EDTA-Na2


Page 11 of 15






Al-Khedery et al. BMC Genomics 2012, 13:678
http://www.biomedcentral.com/1471-2164/13/678


(pH 9.5), supplemented with final 2 mg/ml proteinase
K (Pierce #17916; Rockford, IL) immediately prior to
use]. The tube was incubated upright at 50C with
mild shaking (40 rpm) for 8-24 hrs, until the plugs
turned clear and colorless. Plugs were gently washed
3x in 5 ml 0.5 M EDTA-Na2 (pH 9.5), then transferred
to a fresh tube and stored in EDTA at 4C. Optical
Mapping data generated from the BamHI-digested
ApDogl chromosome was analyzed using the OpGen
MapSolver software.

454 Genome sequencing and bioinformatics
Isolated DNA was provided to the Interdisciplinary Cen-
ter for Biotechnology Research (ICBR) core facilities,
University of Florida for library construction and pyrose-
quencing on the Roche/454 Genome Sequencer accord-
ing to standard manufacturer protocols. Regular read
libraries were generated for all strains. Additionally, 3 kb
paired end libraries were made for ApHZ, ApDogl and
ApMRK. Genome coverage range was 31.3x to 72.1x.
For each strain, the SFF format flow files, returned by
ICBR for bioinformatics analysis, were first combined
and converted to .fasta and .qual files (or the two com-
bined in .fastq format) using Roche/454 Genome Se-
quencer FLX System software. Genome drafts were
assembled using the CLC Genomics Workbench soft-
ware suite (version 4.0-4.9) by mapping reads initially
against the fully annotated, Sanger sequenced ApHZ
genome (GenBank CP000235), then against the com-
pleted ApDogl genome. Default parameters were used:
length fraction, 0.5; similarity, 0.8; and for paired end
reads, minimum distance, 1500/maximum distance,
4500. To obtain the vir loci, the resulting consensus se-
quence and underlying aligned reads were inspected for
conflicts and mismatched paired ends suggesting the
presence of insertions and/or deletions not mirrored in
the consensus. These were manually corrected. Gaps
were also manually closed where possible. Briefly, over-
lapping reads covering at least 2 kb of sequence on both
sides of a gap and extending into it were individually
extracted from the alignment. A new consensus for each
side was obtained by assembling the reads against each
other, and 250 N's were added to its ends. These were
individually used as the reference sequence against
which all the 454 reads were re-mapped to pull out
novel reads extending into the unknown region. The
process was repeated multiple times, allowing for the in-
cremental filling of the gap. PCR, followed by sequen-
cing was performed when sequences extrapolated in this
fashion spanned complex tandem repeat regions such as
repeat regions 1 and 3 (Ri and R3 in Figure 3A) of the
virB6-4 gene, or when gap closure could not be com-
pleted due to such structures, as was the case with the
extremely long virB6-4 R4 (Figure 3A) region.


Amino acid sequences were aligned with MAFFT [50]
and displayed with CHROMA [51]. Taxonomic relation-
ships used a neighbor-joining tree and the ITT substitu-
tion model [52] and were displayed using Archaeopteryx
(http://www.phylosoft.org/archaeopteryx). Hydrophobicity
analyses were conducted using the method of Hopp and
Woods [53,54] at web.expasy.org and transmembrane seg-
ments were predicted with TMpred at http://www.ch.
embnet.org/software/TMPRED_form.html.

PCR amplification of virB6-4 gene repeat regions, cloning,
and Pacific Biosciences sequencing
Due to difficulties in amplifying tandem repeat-containing
DNA, all PCR reactions spanning the virB6-4 gene repeat
regions were performed in the presence of 1.5-1.7 M
Betaine (Sigma). The 8.36 kb PCR product spanning R3
and R4 in the ApHZ strain (Figure 3A, 3C, and Additional
file 2: Figure S2A) was amplified using the iProof
High-Fidelity DNA Polymerase system with GC buffer
(Bio-Rad). Reactions totaled 50 tl with 5 ng purified A.
phagocytophilum gDNA, 1.0 U polymerase, 1.5 mM MgC12,
200 tM each dNTP, and 250 nM each primer (AB1393:
5'-CGGGATCTAAGACAGATGATGATTC-3', forward;
AB1466: 5'-CTCATCCTGATGCGTCTCCTTAG-3', re-
verse; Figure 3A). 35 cycles of 30 sec denaturing at 98oC,
20 sec annealing at 67oC, and 5 min extension at 72'C were
performed. PCR products spanning R4 in ApJM and
ApDogl (both -10.3 kb; Figure 3C) were derived using
Takara's PrimeSTAR GXL DNA Polymerase system (Clon-
tech Laboratories, Mountain View, CA). Reactions con-
tained 5 ng gDNA, 1.25 U polymerase, 1.0 mM MgCl2,
200 tM each dNTP, and 200 nM each primer (AB1395: 5'-
CACCAGAGGATGCAGCATTAG-3', forward; AB1466,
reverse; Figure 3A) in total 50 pl. Following the manufac-
turer's recommendations, 2-step PCR was performed with
30 cycles of 10 sec denaturing at 98'C and 10 min anneal-
ing/extension at 68C. PCR products were analyzed on
0.5% agarose gels alongside the 1 kb Plus (Invitrogen) and
the GeneRulerHighRange (Fermentas, Inc., Glen Burnie,
MD) DNA ladders. In order to TA-clone the amplicons, A-
overhangs were added to the ends using 0.5-1.0 units
AmpliTaq DNA polymerase (Applied Biosystems, Foster
City, CA) in a 10-15 min reaction at 72C. Products puri-
fied from agarose gels (before or after A-overhang addition)
were cloned into the pCR-XL-TOPO vector (Invitrogen)
and transformed into E. coli Stbl2 (Invitrogen), which is
more permissive to repeat-laden foreign DNA. Recombi-
nants containing the correct size insert were end sequenced
to verify their identity.
In preparation for sequencing with the long-read
length Pacific Biosciences (PacBio) next-generation se-
quencing RS instrument, constructs were linearized with
restriction enzymes which cut the vector only, but on
opposite sides of the insert within the Multiple Cloning


Page 12 of 15







Al-Khedery et aL BMC Genomics 2012, 13:678
http://www.biomedcentral.com/1471-2164/13/678


Site. For ApHZ, equimolar amounts of the TA clone
were cut with either HindIII or EcoRV. Following pool-
ing and EtOH precipitation, the linearized DNA mix
was submitted to ICBR/UF for SMRTbell library con-
struction and sequencing. Libraries were constructed
using a commercial strobe library preparation kit
(#001-326-530; Pacific Biosciences, Menlo Park, CA)
following standard manufacturer protocols. To further
increase the likelihood of full coverage, the strobe-
sequencing run was performed using two different
conditions: I) 45 min light period (continuous collec-
tion time); and II) (5 min light period, 10 min dark
period), followed by (45 min light period, 10 min dark
period). The ApJM and ApDogl constructs were
double-digested with HindIII/XbaI to excise the -10.3 kb
inserts. Following separation on 0.5% agarose gels, the
inserts were recovered from agarose slices by electroelu-
tion and further purified and concentrated by passage
over QIAquick spin columns following the PCR Purifica-
tion kit protocol (Qiagen). SMRTbell libraries were made
as above then sequenced using a single 75 min movie
time run.
Due to the repetitive nature of the cloned gene frag-
ments, combined with the relatively high error-rate of
the PacBio system, all attempts to assemble the reads de
novo failed to yield a sequence of the expected size.
Therefore, for each construct, reads >3 kb were selected
from the multi-fasta files using the Galaxy suite [55],
and imported into the CLC Genomics Workbench for
assembly and further analysis. These were assembled at
low stringency initially against a consensus sequence
representing an entire (vector and insert sequence) lin-
ear construct to which sufficient N's were added based
on the estimated gap-size. Starting with reads initiating
outside the repeat region, the longest of the assembled
reads were visually inspected for the presence of virB6-4
R4 repeat signature-sequences (Additional file 2: Figure
S2) and their sequence manually corrected where neces-
sary. The extended sequences were used to replace N's
in the consensus and the process repeated several times
until sufficient reads with >2 kb sequence overlap were
recovered spanning the entire insert region. For verifica-
tion, the completed sequence for each strain was used as
the reference to re-map all the respective >3 kb PacBio
reads and the Roche/454 reads at higher stringency.

GenBank Accession Numbers: for each isolate, the vir
genes are listed in order
The sequences of vir loci are complete for strains
ApDogl and ApJM. The sequence of the repetitive
virB6-4 locus was incomplete (ApDog2) or not deter-
mined for the other strains except ApHz. We provide a
revised sequence of virB6-4 for the previously sequenced
[15] ApHZ strain.


ApDogl:JX415845 JX415868
B2-1 B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-9,
B3, B4-1, B4-tl, B4-2, B6-1, B6-2, B6-3, B6-4, B8-1, B8-
2, B9-1, B9-2, B10, Bll, D4

ApJM:JX415869 JX415892
B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-9,
B3, B4-1, B4-tl, B4-2, B6-1, B6-2, B6-3, B6-4, B8-1, B8-
2, B9-1, B9-2, B10, Bll, D4

ApDog2:JX415893 JX415915 (virB6-4 submitted
separately as gapped)
B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-9,
B3, B4-1, B4-tl, B4-2, B6-1, B6-2, B6-3, B8-1, B8-2, B9-
1, B9-2, B10, Bll, D4

ApNorLambV2:JX415916 JX415938
B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-9,
B3, B4-1, B4-tl, B4-2, B6-1, B6-2, B6-3, B8-1, B8-2, B9-
1, B9-2, B10, Bll, D4

ApNorLambVl:JX415939 JX415966
B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-
novell, B2- novel, B2-novel3, B2-novel4, B2-novel5,
B2-novel6, B3, B4-1, B4- tl, B4-2, B6-1, B6-2, B6-3, B8-
1, B8-2, B9-1, B9-2, B10, Bll, D4

ApHZvirB6-4:JX415967

ApVarl:JX415968 JX415996
B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-
novell, B2- novel, B2-novel3, B2-novel4, B2-novel5,
B2-novel6, B2-novel7, B3, B4-1, B4-tl, B4-2, B6-1,
B6-2, B6-3, B8-1, B8-2, B9-1, B9-2, B10, Bll, D4

ApMRK:JX415997 JX416019
B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-9,
B3, B4-1, B4-tl, B4-2, B6-1, B6-2, B6-3, B8-1, B8-2, B9-
1, B9-2, B10, Bll, D4

ApDog2virB6-4Gapped:JX416020.

Additional files

Additional file 1: Figure S1. Multiple sequence alignment of
VirB6-3 amino acid sequences from different strains of A
phagocytophilum Arrows indicate the locations of C-terminal 41 -mer
repeats
Additional file 2: Figure S2. Structure of the virB6-4 repeat regions R3
and R4 in four US A phagocytophilum strains A Comparative maps of
AB1393/AB1466 PCR products detailing the repeat unit content of R3 and
R4 in the human, rodent and dog strains ApJM and ApDogl have
identical virB6-4 genes and are, therefore, represented by one map
Moderate variability in the number and sequence of the R3 405 bp
repeat units (light blue arrows) is apparent The small bar at the end of
R3 corresponds to the 3'-most partial repeat unit present in all strains


Page 13 of 15








Al-Khedery et al. BMC Genomics 2012, 13:678
http://www.biomedcentral.com/1471-2164/13/678


The colored arrows within R4 represent the five repeat types T1a (yellow),
T1 b (green), T2a, (red), T2b (dark blue) and T2c (grey) The repeat pattern
in ApHZ shows no relationship to that of ApJM/ApDogl, which is also
287 kb larger, totaling 976 kb This region was not fully characterized in
ApDog2 as indicated by a broken line, but the repeat pattern of the 5'-
and 3'-most repeats is clearly different from that of the other strains The
small bar downstream of the second repeat unit represents a partially
characterized type 2 repeat unit Lines above and below the ApHZ and
ApJM/ApDogl maps delineate segments of sequence identity within the
respective R4 regions Their sizes are specified B Alignment of the
nucleic acid sequence of all virB6-4 R4 repeat unit types identified to
date Type 1 repeats are shown in black, type 2 in blue Differences
between sub-types are highlighted A single BamHI site present in all
type 2 repeats is underlined With the exception of only a few
nucleotides at each end, type 1 and type 2 repeat units do not share any
sequences C Alignment of the amino acid sequences of the repeat units
shown in B The single nucleotide differences between sub-types do not
lead to changes in amino acid sequence


Competing interests
The authors declare that they have no competing interests


Authors' contributions
BAK and AFB conceived the study, performed bioinformatics analyses and
drafted the manuscript BAK grew infected HL-60 cell cultures, purified
organisms, isolated gDNA, designed and supervised PCR and submitted
sequences to GenBank AML performed PCR analyses and cloning and
supervised data transfer between units 55 and EGG isolated the European
sheep strains, infected and monitored sheep, and prepared organisms at
maximal parasitemia UGM and CMN isolated and cultured in vitro the JM,
MRK, Dog2 and Ap variant 1 strains, and prepared Dog2 and Ap variant 1
strain gDNA ARA and SMM established the Dog1 strain All authors read and
approved the final manuscript


Acknowledgements
The research described here received support from grants R01 GM081714
and GM081714-0351 and from Pfizer Animal Health We thank Dr Roberta
Veluci-Marlow, Susan Benda and Adam Webster for help with culturing cells
infected with A phagocytophilum, and Dr Savita Shanker for high-
throughput DNA sequencing

Author details
'Department of Infectious Diseases and Pathology, College of Veterinary
Medicine, University of Florida, Gainesville, FL, USA Department of
Production Animal Sciences, Section of Small Ruminant Research, Norwegian
School of Veterinary Science, Sandnes, Norway Department of Entomology,
University of Minnesota, St Paul, MN, USA 4Physiological Sciences, College of
Veterinary Medicine, University of Florida, Gainesville, FL, USA Pfizer Animal
Health, Kalamazoo M, M, USA

Received: 10 July 2012 Accepted: 20 November 2012
Published: 29 November 2012


References
1 Dumler JS, Choi KS, Garcia-Garcia JC, Barat NS, Scorpio DG, Garyu JW, et alo
Human granulocytic anaplasmosis and Anaplasma phagocytophilum.
Emerg Infect Dis 2005, 11 1828-1834
2 Jin H, Wei F, Liu Q, Qian J Epidemiology and control of human
granulocytic anaplasmosis: a systematic review. Vector Borne Zoonotic Dis
2012, 12269-274
3 Bakken JS, Dumler JS Clinical diagnosis and treatment of human
granulocytotropic anaplasmosis. Ann N YAcod Sc 2006, 1078'236-247
4 Dahlgren FS, Mandel EJ, Krebs JW, Massung RF, McQuiston JH Increasing
incidence of Ehrlichia chaffeensis and Anaplasma phagocytophilum in the
United States, 2000-2007. Am J Trop Med Hyg 2011, 85'124-131
5 Weil AA, Baron EL, Brown CM, Drapkin MS Clinical findings and diagnosis
in human granulocytic anaplasmosis: a case series from Massachusetts.
Mavo Clin Proc 2012, 87'233-239


6 Li H, Zhou Y, Wang W, Guo D, Huang S, Jie S The clinical characteristics
and outcomes of patients with human granulocytic anaplasmosis in
China. Int J Infect Dis 2011, 15'e859-e866
7 Eberts MD, Beall MJ, Stillman BA, Chandrashekar R, Breitschwerdt EB Typical
and atypical manifestations of Anaplasma phagocytophilum infection in
dogs. J Am Anim Hosp Assoc 2011, 47e86-e94
8 Bowman D, Little SE, Lorentzen L, Shields J, Sullivan MP, Carlin EP
Prevalence and geographic distribution of Dirofilaria immitis, Borrelia
burgdorferi, Ehrlichia canis, and Anaplasma phagocytophilum in dogs in
the United States: results of a national clinic-based serologic survey. Vet
Porsito/ 2009, 160'138-148
9 Foley J, Nieto NC, Madigan J, Sykes J Possible differential host tropism in
Anaplasma phagocytophilum strains in the Western United States. Ann N
YAcod Sc 2008, 1149'94-97
10 Foley JE, Nieto NC, Massung R, Barbet A, Madigan J, Brown RN Distinct
ecologically relevant strains of Anaplasma phagocytophilum. Emerg Infect
Dis 2009, 15'842-843
11 Massung RF, Mather TN, Priestley RA, Levin ML Transmission efficiency of
the AP-variant 1 strain of Anaplasma phagocytophila. Ann NY Acod Sc
2003, 990 75-79
12 Massung RF, Priestley RA, Miller NJ, Mather TN, Levin ML Inability of a
variant strain of Anaplasma phagocytophilum to infect mice. J Infect Dis
2003, 188 1757-1763
13 Massung RF, Courtney JW, Hiratzka SL, Pitzer VE, Smith G, Dryden RL
Anaplasma phagocytophilum in white-tailed deer. Emerg Infect Dis 2005,
11 1604-1606
14 Stuen S Anaplasma phagocytophilum the most widespread tick-borne
infection in animals in Europe. Vet Res Commun 2007, 31 (Suppl 1)79-84
15 Dunning Hotopp JC, Lin M, Madupu R, Crabtree J, Angiuoli SV, Eisen J, et al
Comparative genomics of emerging human ehrlichiosis agents. PLoS
Genet 2006, 2'e21
16 Rikihisa Y, Lin M, Niu H Type IV secretion in the obligatory intracellular
bacterium Anaplasma phagocytophilum. Cell Microbiol 2010, 12'1213-1221
17 Waksman G, Fronzes R Molecular architecture of bacterial type IV
secretion systems. Trends Biochem Sc 2010, 35'691-698
18 Niu H, Rikihisa Y, Yamaguchi M, Ohashi N' Differential expression of VirB9
and VirB6 during the life cycle of Anaplasma phagocytophilum in human
leucocytes is associated with differential binding and avoidance of
lysosome pathway. Cell Microbiol 2006, 8'523-534
19 Ge Y, Rikihisa Y Identification of novel surface proteins of Anaplasma
phagocytophilum by affinity purification and proteomics. J Bocterio/2007,
1897819-7828
20 Alvarez Martinez CE, Christie PJ Biological diversity of prokaryotic type IV
secretion systems. Microbio Mol Bio Rev 2009, 73 775-808
21 Lopez JE, Palmer GH, Brayton KA, Dark MJ, Leach SE, Brown WC
Immunogenicity of Anaplasma marginale type IV secretion system
proteins in a protective outer membrane vaccine. Infect Immun 2007,
75'2333-2342
22 Morse K, Norimine J, Palmer GH, Sutten EL, Baszler TV, Brown WC
Association and evidence for linked recognition of type IV secretion
system proteins VirB9-1, VirB9-2, and VirB10 in Anaplasma marginale.
Infect Immun 2012, 80'215-227
23 Morse K, Norimine J, Hope JC, Brown WC Breadth of the CD4(+) T cell
response to Anaplasma marginale VirB9-1, VirB9-2 and VirB10 and MHC
class II DR and DQ restriction elements. Immunogenetics 2012,
64507-523
24 Araujo FR, Costa CM, Ramos CA, Farias TA, Souza II, Melo ES, et oal
IgG and IgG2 antibodies from cattle naturally infected with Anaplasma
marginale recognize the recombinant vaccine candidate antigens
VirB9, VirB10, and elongation factor-Tu. Mem Inst Oswoldo Cruz 2008,
103186-190
25 Sutten EL, Norimine J, Beare PA, Heinzen RA, Lopez JE, Morse K, et oal
Anaplasma marginale type IV secretion system proteins VirB2, VirB7,
VirB11, and VirD4 are immunogenic components of a protective
bacterial membrane vaccine. Infect Immun 2010, 78'1314-1325
26 Dark MJ, Al-Khedery B, Barbet AF Multistrain genome analysis identifies
candidate vaccine antigens of Anaplasma marginale. Vaccine 2011,
294923-4932
27 Park J, Kim KJ, Choi KS, Grab DJ, Dumler JS Anaplasma phagocytophilum
AnkA binds to granulocyte DNA and nuclear proteins. Cell Microbiol 2004,


Page 14 of 15








Al-Khedery et al BMC Genomics 2012, 13:678
http://www.biomedcentral.com/1471-2164/13/678


28 Garcia-Garcia JC, Rennoll-Bankert KE, Pelly S, Milstone AM, Dumler JS'
Silencing of host cell CYBB gene expression by the nuclear effector
AnkA of the intracellular pathogen Anaplasma phagocytophilum. Infect
Immune 2009, 77 2385-2391
29 Niu H, Kozjak Pavlovic V, Rudel T, Rikihisa Y Anaplasma phagocytophilum
Ats-1 is imported into host cell mitochondria and interferes with
apoptosis induction. PLoS Pathog 2010, 6'e000774
30 Gillespie JJ, Brayton KA, Williams KP, Diaz MA, Brown WC, Azad AF, Sobral
BW Phylogenomics reveals a diverse Rickettsiales type IV secretion
system. Infect immun 2010, 78'1809-1823
31 Ohashi N, Zhi N, Lin Q, Rikihisa Y Characterization and transcriptional
analysis of gene clusters for a type IV secretion machinery in human
granulocytic and monocytic ehrlichiosis agents. Infect Immun 2002,
70'2128-2138
32 Berger BR, Christie PJ Genetic complementation analysis of the
Agrobacterium tumefaciens virB operon: virB2 through virB11 are
essential virulence genes. J Bacterio/ 1994, 176'3646-3660
33 Yuan Q, Carle A, Gao C, Sivanesan D, Aly KA, Hoppner C, et oal Identification
of the VirB4-VirB8-VirB5-VirB2 pilus assembly sequence of type IV
secretion systems. J Bio Chem 2005, 280'26349-26359
34 Judd PK, Kumar RB, Das A Spatial location and requirements for the
assembly of the Agrobacterium tumefaciens type IV secretion apparatus.
Proc Nat/ Acod Sd USA 2005, 102'11498-11503
35 Kumar RB, Xie YH, Das A Subcellular localization of the Agrobacterium
tumefaciens T-DNA transport pore proteins: VirB8 is essential for the
assembly of the transport pore. Mol Microbiol 2000, 36'608-617
36 Cascales E, Christie PJ Agrobacterium VirB10, an ATP energy sensor
required for type IV secretion. Proc Nat/Acod Sc USA 2004,
101 17228-17233
37 Rances E, Voronin D, Tran-Van V, Mavingui P Genetic and functional
characterization of the type IV secretion system in Wolbachia.
J Bocterio/ 2008, 190 5020-5030
38 Anderson LB, Hertzel AV, Das A Agrobacterium tumefaciens VirB7 and
VirB9 form a disulfide-linked protein complex. Proc Nat Acod Sc USA
1996, 93'8889-8894
39 Spudich GM, Fernandez D, Zhou XR, Christie PJ Intermolecular disulfide
bonds stabilize VirB7 homodimers and VirB7/VirB9 heterodimers during
biogenesis of the Agrobacterium tumefaciens T-complex transport
apparatus. Proc Nat/ Acod Sc USA 1996, 937512-7517
40 Bayliss R, Harris R, Coutte L, Monier A, Fronzes R, Christie PJ, et o/ NMR
structure of a complex between the VirB9/VirB7 interaction domains of
the pKM101 type IV secretion system. Proc Nat Acod Sc USA 2007,
104'1673-1678
41 Ge Y, Rikihisa Y Surface-exposed proteins of Ehrlichia chaffeensis. Infect
Immune 2007, 75'3833-3841
42 Meeus PF, Brayton KA, Palmer GH, Barbet AF Conservation of a gene
conversion mechanism in two distantly related paralogues of Anaplasma
marginale. Mol Microbiol 2003, 47'633-643
43 Dumler JS, Barbet AF, Bekker CP, Dasch GA, Palmer GH, Ray SC, et o/
Reorganization of genera in the families Rickettsiaceae and
Anaplasmataceae in the order Rickettsiales: unification of some species
of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with
Neorickettsia, descriptions of six new species combinations and
designation of Ehrlichia equi and 'HGE agent' as subjective
synonyms of Ehrlichia phagocytophila. Int J Syst Evol Microbiol 2001,
51 2145-2165
44 Gribble DH Equine ehrlichiosis. J Am Vet Med Assoc 1969, 155462-469
45 Johnson RC, Kodner C, Jarnefeld J, Eck DK, Xu Y' Agents of human
anaplasmosis and Lyme disease at Camp Ripley, Minnesota. Vector Borne
Zoonotic Dis 2011, 11 1529-1534
46 Munderloh UG, Jauron SD, Fingerle V, Leitritz L, Hayes SF, Hautman JM, et
o/ Invasion and intracellular development of the human granulocytic
ehrlichiosis agent in tick cell culture. J Clin Microbiol 1999, 37'2518-2524
47 Massung RF, Levin ML, Munderloh UG, Silverman DJ, Lynch MJ, Gaywee JK,
Kurtti TJ Isolation and propagation of the Ap-Variant 1 strain of
Anaplasma phagocytophilum in a tick cell line. J Clin Microbio 2007,
45'2138-2143
48 Granquist EG, Bardsen K, Bergstrom K, Stuen S Variant and individual
dependent nature of persistent Anaplasma phagocytophilum infection.
Arto Vet Scond 2010. 5225


49 Felsheim RF, Herron MJ, Nelson CM, Burkhardt NY, Barbet AF, Kurtti TJ,
Munderloh UG Transformation of Anaplasma phagocytophilum. BMC
Biotechnol 2006, 642
50 Katoh K, Toh H Recent developments in the MAFFT multiple sequence
alignment program. Brief Bioinform 2008, 9'286-298
51 Goodstadt L, Ponting CP CHROMA: consensus-based colouring of
multiple alignments for publication. Bioinformatics 2001, 17845-846
52 Jones DT, Taylor WR, Thornton JM The rapid generation of mutation data
matrices from protein sequences. Comput App/ Biosc 1992, 8 275-282
53 Hopp TP, Woods KR A computer program for predicting protein
antigenic determinants. Mol immunol 1983, 20483-489
54 Hopp TP Use of hydrophilicity plotting procedures to identify protein
antigenic segments and other interaction sites. Methods Enzymol 1989,
178571-585
55 Goecks J, Nekrutenko A, Taylor J Galaxy: a comprehensive approach for
supporting accessible, reproducible, and transparent computational
research in the life sciences. Genome Biol 2010, 11 R86

doi:10.1186/1471-2164-13-678
Cite this article as: Al-Khedery et al Structure of the type IV secretion
system in different strains of Anaplasma phagocytophilum. BMC
Genomics 2012 13'678


Page 15 of 15


Submit your next manuscript to BioMed Central
and take full advantage of:

* Convenient online submission
* Thorough peer review
* No space constraints or color figure charges
* Immediate publication on acceptance
* Inclusion in PubMed, CAS, Scopus and Google Scholar
* Research which is freely available for redistribution

Submit your manuscript at nt
www.biomedcentral.com/submit 3illied Central




Full Text

PAGE 1

A. B C .T1a/T1b QEKD--------------------------GNTATELPREVVPEATEYGTKPDD 28 T2a/T2b QDGDKGDLRPERLDPDIGDGSAIEDEVEVRSSRSSESTDSVPSEVTERDAQRDD 54 T2c QDGDKGDLRPERLDPDIGDGSAIEDEVEVRSSRSSESTDSVPSEVTE ---RDD 50 *: .. ::* .* .*.** ** T1a CAGGAGAAGGAT---GGTAATACTGCGACGGAA---CT---TCCT-----AGAGAA--GT 44 T1b CAGGAGAAGGAT---GGTAATACTGCGACGGAA---CT---TCCT-----AGAGAA--GT 44 T2a CAGGATGGTGATAAGGGTGATTTAAGGCCTGAAAGACTGGATCC TGATATAGGGGATGGT 60 T2b CAGGATGGTGATAAGGGTGATTTAAGGCCTGAAAG G CTGGATCC TGATATAGGGGATGGT 60 T2c CAGGATGGTGATAAGGGTGATTTAAGGCCTGAAAG G CTGGATCC TGATATAGGGGATGGT 60 ***** *** *** ** *** ** **** ** ** T1a AGTAC------------------------------------------------------84 T1b AGTAC------------------------------------------------------84 T2a AGTGCTATAGAAGATGAGGTTGAGGTTAGATCTTCTAGGAGTAGTGAATCAACTGATAG 119 T2b AGTGCTATAGAAGATGAGGTTGAGGTTAGATCTTCTAGGAGTAGTGAATCAACTGATAG 119 T2c AGTGCTATAGAAGATGAGGTTGAGGTTAGATCTTCTAGGAGTAGTGAATCAACTGATAG 119 *** T1a --------CTGAAGCTACCGAGTATGGTACTAAACCTGATGAT 84 T1b --------CTGAAGCTAC T GAGTATGGTACTAAACCTGATGAT 84 T2a CGTGCCATCTGAAGTAACTGAACGTGATGCTCAACGTGATGAT 162 T2b CGTGCCATCTGAAGTAACTGAACGTGATGCTCAACGTGATGAT 162 T2c T GTGCCATCTGAAGTAACTGAA -----------CGTGATGAT 150 ****** ** ** ** ** *** ******* R3R4 R3R4Ap Hz Ap JM/ Ap Dog1 1 2 3 4 5 6 7 8 9 10 11 12 kb R3R4Ap Dog21.15 kb 2.7 kb 3.7 kb 1.07 kbAB1393 AB1466


!DOCTYPE art SYSTEM 'http:www.biomedcentral.comxmlarticle.dtd'
ui 1471-2164-13-678
ji 1471-2164
fm
dochead Research article
bibl
title
p Structure of the type IV secretion system in different strains of it Anaplasma phagocytophilum
aug
au id A1 snm Al-Khederyfnm Basimainsr iid I1 email balkhedery@ufl.edu
A2 Lundgrenmi MAnnalundgrena@ufl.edu
A3 StuenSnorreI2 Snorre.Stuen@nvh.no
A4 GranquistGErikErikGeorg.Granquist@nvh.no
A5 MunderlohGUlrikeI3 munde001@umn.edu
A6 NelsonMCurtisnelso015@umn.edu
A7 AllemanA RickI4 allemanr@ufl.edu
A8 MahanMSumanI5 Suman.Mahan@pfizer.com
A9 ca yes BarbetFAnthonybarbet@ufl.edu
insg
ins Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA
Department of Production Animal Sciences, Section of Small Ruminant Research, Norwegian School of Veterinary Science, Sandnes, Norway
Department of Entomology, University of Minnesota, St Paul, MN, USA
Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA
Pfizer Animal Health, Kalamazoo, MI, USA
source BMC Genomics
section Comparative and evolutionary genomicsissn 1471-2164
pubdate 2012
volume 13
issue 1
fpage 678
url http://www.biomedcentral.com/1471-2164/13/678
xrefbib pubidlist pubid idtype doi 10.1186/1471-2164-13-678pmpid 23190684
history rec date day 10month 7year 2012acc 20112012pub 29112012
cpyrt 2012collab Al-Khedery et al.; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
kwdg
kwd
Anaplasma
phagocytophilum
Rickettsiales
T4SS
Comparative genomics
abs
sec
st
Abstract
Background
Anaplasma phagocytophilum is an intracellular organism in the Order Rickettsiales that infects diverse animal species and is causing an emerging disease in humans, dogs and horses. Different strains have very different cell tropisms and virulence. For example, in the U.S., strains have been described that infect ruminants but not dogs or rodents. An intriguing question is how the strains of A. phagocytophilum differ and what different genome loci are involved in cell tropisms and/or virulence. Type IV secretion systems (T4SS) are responsible for translocation of substrates across the cell membrane by mechanisms that require contact with the recipient cell. They are especially important in organisms such as the Rickettsiales which require T4SS to aid colonization and survival within both mammalian and tick vector cells. We determined the structure of the T4SS in 7 strains from the U.S. and Europe and revised the sequence of the repetitive virB6 locus of the human HZ strain.
Results
Although in all strains the T4SS conforms to the previously described split loci for vir genes, there is great diversity within these loci among strains. This is particularly evident in the virB2 and virB6 which are postulated to encode the secretion channel and proteins exposed on the bacterial surface. VirB6-4 has an unusual highly repetitive structure and can have a molecular weight greater than 500,000. For many of the virs, phylogenetic trees position A. phagocytophilum strains infecting ruminants in the U.S. and Europe distant from strains infecting humans and dogs in the U.S.
Conclusions
Our study reveals evidence of gene duplication and considerable diversity of T4SS components in strains infecting different animals. The diversity in virB2 is in both the total number of copies, which varied from 8 to 15 in the herein characterized strains, and in the sequence of each copy. The diversity in virB6 is in the sequence of each of the 4 copies in the single locus and the presence of varying numbers of repetitive units in virB6-3 and virB6-4. These data suggest that the T4SS should be investigated further for a potential role in strain virulence of A. phagocytophilum.
bdy
Background
Anaplasma phagocytophilum is a tick-borne pathogen in the Order Rickettsiales that is increasingly recognized as a cause of disease in humans and animals world-wide abbrgrp
abbr bid B1 1
B2 2
. It causes the potentially fatal disease of human granulocytic anaplasmosis, which typically manifests as a flu-like illness accompanied by leukopenia, thrombocytopenia and anemia. It was initially recognized in the early 1990's when patients from Wisconsin and Minnesota developed febrile illness following a tick bite
B3 3
. Since that time the number of human cases has increased annually; between 2000 and 2007 the reported incidence in the U.S. increased from 1.4 to 3.0 cases/million persons/year
B4 4
. The case fatality rate was 0.6% and the hospitalization rate was 36%. In Massachusetts during the 2009 transmission season there were 33 confirmed cases with 14 (42%) requiring hospitalization
B5 5
. The human disease is also present in Europe and Asia
2
. A recent study of 83 A. phagocytophilum-infected patients in China reported a mortality rate in this cohort of 26.5%
B6 6
. In the U.S., there has been a parallel increase in cases of the disease
B7 7
and seroprevalence
B8 8
in dogs in the eastern and upper Midwestern states. The tick vectors in the U.S. are Ixodes scapularis and Ixodes pacificus and wild rodents are the main reservoirs of human infections. A. phagocytophilum also infects numerous other mammalian species including ruminants, horses, cats, and bears and the symptoms are extremely variable, with some mammalian species exhibiting acute disease and others only persistent asymptomatic infections
B9 9
B10 10
. For example, A. phagocytophilum strains isolated from deer in the U.S. can have a slightly different 16S rRNA sequence and be uninfective to mice and it is thought, humans
B11 11
B12 12
B13 13
. In Europe, this agent has been known to cause disease of ruminants for >100 years, yet there have been few human infections
B14 14
. The genome sequence is available for a single strain of A. phagocytophilum derived from an infected human in the U.S. and it is apparent that, although this strain lacks Type II, III, V and VI secretion systems, a Type IV secretion system (T4SS) is present
B15 15
. As in other members of the Rickettsiales, the T4SS of A. phagocytophilum is organized differently from most gram-negative bacteria with the component vir genes distributed between three major genome locations
B16 16
.The T4SS typically encodes a membrane-spanning multiprotein complex that forms a transmembrane channel through which solutes can pass into host cells. It can mediate transfer of DNA and proteins into eukaryotic host cells, interfere with host signaling, and is essential for the survival of intracellular bacteria
B17 17
. In A. phagocytophilum, which preferentially colonize neutrophilic white blood cells, it is thought that the T4SS secretes virulence factors that are responsible for subverting innate immunity and inhibiting host cell apoptosis
16
. Interestingly, there appears to be differential transcription of the T4SS in ticks and in the mammalian host with virB6 and virB9 upregulated during infection of human neutrophils and different virB2 paralogs expressed in mammalian and tick cells
B18 18
. There is evidence that VirB2, VirB6 and VirB9 are exposed on the outer membrane surface in the Rickettsiales
18
B19 19
B20 20
, which has stimulated interest in their potential use as vaccine candidates. This possibility has been investigated more extensively in the related organism Anaplasma marginale
B21 21
B22 22
B23 23
B24 24
B25 25
. In A. marginale, unlike many other surface-exposed proteins, the T4SS proteins are conserved between strains
B26 26
. Also, cattle immunized with outer membranes and protected against challenge infection respond with IgG and T cells to Vir proteins, notably VirB2, VirB9 and VirB10. To date, only two T4SS substrates have been identified and partially characterized in A. phagocytophilum: the ankyrin repeat domain-containing protein, AnkA, and the Anaplasma translocated substrate 1, Ats-1. AnkA translocates to the host nucleus and interacts with DNA
B27 27
B28 28
, while Ats-1 is imported into the mitochondria where it is proposed to interfere with the induction of apoptosis
B29 29
.In this study, we compared the structure and diversity of the T4SS in different strains of A. phagocytophilum infecting humans, dogs, rodents and ruminants. Most diversity was found in the proteins thought to be surface-exposed, which may be associated with the different virulence and cell invasion properties of this species.
Results and discussion
The vir loci were sequenced in eight strains of A. phagocytophilum; seven of these were strains for which previous structural information was not available and included organisms originally isolated from U.S. dogs (ApDog1, ApDog2), a rodent (ApJM), a horse (ApMRK), the ruminant Ap variant 1 strain (ApVar1) and two strains from Norwegian sheep (ApNorV1, ApNorV2). The human HZ strain was also resequenced, as optical mapping had suggested a possible error in the previously sequenced virB6
4 locus. The data indicated considerable diversity in the individual vir loci between strains that will be discussed below. In all strains, however, as noted previously
20
B30 30
, the vir loci were distributed mainly in three gene clusters comprising: virB8
1, virB9
1, virB10, virB11 and virD4; virB2′s and virB4
2; and virB3, virB4
1, and the four virB6 paralogs (Figure figr fid F1 1). These three loci may each be transcribed polycistronically
B31 31
, although it is clear that T4SS structure in the Rickettsiales is unique and more complex than initially thought. The number of virB2 paralogs was different between strains with the human HZ strain having the least (8 total paralogs) and the ruminant strains having the most (up to15 total paralogs). The description of the T4SS components presented here follows the functional classification described by Alvarez-Martinez and Christie
20
.
fig Figure 1caption Distribution and content of vir gene clusters in eight diverse A. phagocytophilum strainstext
b Distribution and content of vir gene clusters in eight diverse A. phagocytophilum strains. Top panel. Schematic representation of all vir loci (colored arrows) showing the three conserved gene cluster islands (see text). VirB-7, virB8-2 and virB9-2 are not part of vir gene clusters, but their location relative to surrounding genes is also highly conserved among strains. A small cluster comprising truncated (t) virB6 and virB4 gene fragments is present in all strains, but the Norwegian lamb strains have one additional virB4-t. Bottom panel. Magnification of the virB2 gene cluster. Numbering of paralogs 1–8 is based on the original ApHZ annotated genome (GenBank CP000235). Artificial gaps (stippled lines) were introduced to allow alignment of the more spatially conserved paralogs B2-1, 2–2 and 2–3 at one end, and B2-7 and 2–8 at the other end of the cluster. With the exception of virB2-9, lacking in ApHZ, the number and arrangement (but not necessarily sequence) of virB2 genes is highly conserved in all but the US ruminant ApVar-1 and ApNorLamb-V1, which have several additional virB2 genes. In both strains a sub-cluster of 6 distinct genes was present. Due to the repetitive nature of sequences in this region, combined with the relatively short length of 454 reads (≤550 bp), their placement could not be confidently ascertained (highlighted by arrows and ‘?’). Maps are drawn to scale. Double lines designate interruption in sequences. Genes belonging to the same grouping have the same color. oriC; origin of replication.
graphic file 1471-2164-13-678-1
Inner membrane channel/scaffold subunits: VirB3, VirB6, VirB8 and VirB10
The most conserved of these subunits are VirB3, VirB8 and VirB10, with few differences between strains. VirB3 has been linked in Agrobacterium tumefaciens with pilus assembly and substrate translocation
B32 32
B33 33
. It is absolutely conserved between strains with no amino acid changes and conforms to the typical VirB3 structure. Two alpha-helical domains for insertion into the cytoplasmic membrane are strongly predicted by TMpred. VirB8, proposed to function as a nucleation factor during the assembly of T4SS
B34 34
B35 35
, is also well conserved, particularly VirB8-1 in the polycistronic transcription locus (one amino acid change between all strains). VirB10, proposed to function as a scaffold across the entire cell envelope
B36 36
, is also generally well-conserved with the exception of one ruminant strain, ApNorLamb-V1, which has 31 amino acid substitutions with respect to ApHZ (data not shown). However, all A. phagocytophilum VirB10sup ′s, including ApNorLamb-V1, have two strongly predicted transmembrane domains, which supports their function as membrane scaffolding subunits in these organisms.Of these inner membrane channel subunits, the data on VirB6 are the most interesting. All VirB6 subunits that have been described possess a highly hydrophobic membrane domain including five or more predicted transmembrane domains
20
. Some VirB6 proteins also have an extended C-terminal hydrophilic domain that has been proposed to protrude through the T4SS into the target cell, or may be proteolytically released from the N-terminal domain and then translocated into the target cell. Evidence has been obtained for surface exposure of extended VirB6 in some Rickettsiales
B37 37
. Of all the membrane channel subunits, the most sequence diversity between A. phagocytophilum strains was in the four VirB6 paralogs (Figure 1). Although there were no amino acid changes in the VirB6-1, VirB6-2 and VirB6-3 paralogs between human, dog and rodent strains, the ruminant and horse strains had numerous substitutions throughout each molecule, agreeing with the closer evolutionary relationship between strains infecting humans and dogs in the U.S. (Figure F2 2). Furthermore, major differences in repeat number and sequence were found in the C-terminal repeat region of VirB6-3 (yellow boxes in Figure F3 3A and Additional file supplr sid S1 1: Figure S1) in ruminant and horse strains, with the horse strain showing the least variability from ApHZ.
Figure 2Phylogenetic tree to show the relationship of syntenic VirB6 proteins from different strains of A. phagocytophilum
Phylogenetic tree to show the relationship of syntenic VirB6 proteins from different strains of A. phagocytophilum. A scale bar is shown underneath representing the number of amino acid substitutions/site.
1471-2164-13-678-2
Figure 3The 3′ end of A. phagocytophilum virB6-4 genes is composed of an unusually large tandem repeat region, which exhibits dramatic variability among strains
The 3′ end of A. phagocytophilum virB6-4 genes is composed of an unusually large tandem repeat region, which exhibits dramatic variability among strains.A. Map of the human HZ strain virB6-3 and virB6-4 genes, highlighting the location and structure of several repeat regions (R1-R4). The most variability occurred in R4; this region is 5.88 kb larger than previously reported for the ApHZ genome (CP000235). The original sequence is diagrammed above the map, with the dashed line representing the segment missing in CP000235. Larger repeated R4 segments of 2.7 kb and 1.15 kb are indicated above. Vertical black bars within each gene designate segments encoding predicted transmembrane domains. BamHI sites, of which there is one in all R4 type 2 repeats (see Figure S2B), are indicated. Also shown are the positions of PCR primers used in C. B. BamHI genomic maps depicting the virB6-4 locus (black arrows).The segment encompassing R4 is highlighted below each respective map. In the regions outside the virB6-4 locus, corresponding BamHI fragments are shown in the same color. Overall, the optical map sizes were in good agreement with the actual sizes, except within R4. This is attributed to the limitation of optical mapping in resolving fragments <2 kb. Despite these discrepancies, the cumulative size of the genomic region encompassing virB6-4 in the optical map is in close agreement with that in the ApDog1 genome sequence. C. The variability in size of PCR products spanning virB6-4 repeat regions R4 and R3/R4 in diverse A. phagocytophilum strains.
1471-2164-13-678-3
suppl
Additional file 1
Figure S1. Multiple sequence alignment of VirB6-3 amino acid sequences from different strains of A. phagocytophilum. Arrows indicate the locations of C-terminal 41-mer repeats.
name 1471-2164-13-678-S1.pdf
Click here for file
The only amino acid differences detected between the human, dog and rodent strains were in the VirB6-4 subunit. VirB6-4 in these strains contains four repeat regions (R1-R4 in Figure 3A) and variability in repeat number, order and sequence were found mainly in R3 and R4 (Additional file S2 2: Figure S2). Within R1 (Figure 3A), the only difference detected was in ApDog2 which had 4 and 1 partial of 231 bp repeat units (data not shown), compared to 3 and 1 partial repeats in the ApDog1, ApJM and ApHZ virB6-4 R1. Optical mapping of the Dog1 genome and comparison with ApHZ suggested that the sequence obtained previously for the human HZ strain virB6-4 was incorrect (Figure 3B). This was confirmed by PCR and sequencing, and mapped specifically to the 3′-most R4 region (Figure 3C). Because of its size and unusual composition it was only possible to resolve this sequence using the long read-length Pacific Biosciences technology (see Methods). The corrected virB6-4 R4 of ApHZ, totaling 6.89 kb, differed from the original by 5.88 kb of additional sequence composed exclusively of 84 bp [type 1, a and b (T1a, T1b); light/dark blue boxes, respectively, in Figure 3A]and 162 bp [type 2, a and b (T2a, T2b); light/dark orange boxes, respectively, in Figure 3A] repeat units, giving a complex repeat structure containing 53 and 1 partial repeat units compared to 8 and 1 partial in the original sequence. Further, the 5′- and 3′-most 2.7 kb of this complex structure are identical in sequence, and the 3′-most 1.15 kb of each of these segments is repeated again in the center of R4 (Figure 3A and Additional file 2: Figure S2). Although the possibility exists that the ApHZ population from which we isolated gDNA differs within the virB6-4 R3/R4 repeat regions from the population used to generate CP000235, the fact that all strains investigated herein presented expansive R3/R4 regions (Figure 3C) would contradict that. Instead, it is more plausible that the existence of 2.7 kb of identical repeats at the ends of the ApHZ R4 may have lead to the excision of most of its sequence during construction/propagation of those libraries. Interestingly, virB6-4 R3 and R4 were identical both in size and sequence in the Dog1 and rodent strains despite differing markedly from the HZ and Dog2 strain regions (Additional file 2: Figure S2A). Within R3, these strains had 2 additional 405 bp repeats compared to ApHZ and one more compared to the Dog2 strain. However, differences between strains were most dramatic within R4. Not only was this region in ApDog1/ApJM 2.87 kb larger than in ApHZ bringing the total number of repeats to 81 and 1 partial, but intriguingly, the repeat pattern was completely unrelated to that in the HZ strain. Also, the Dog1 and rodent strain R4 lacked T1b repeat units, while having a third type 2 repeat variant, namely T2c, which differed from T2b by 1 SNP and a 12 bp deletion (Additional file 2: Figure S2). Partial analysis of the ApDog2 454 reads spanning R4 (estimated at ~8 kb by PCR; Figure 3C) showed that the order of the 5′- and 3′-most three repeat units differed from either the HZ or Dog1/rodent strain R4 repeat patterns (Additional file 2: Figure S2A). Notably, our preliminary analyses of the horse and ruminant 454 reads suggest the absence of distinct R3 and R4 regions in virB6-4 in these strains. Rather, the few repeat units identified to date appear to be a combination of R3 and R4 repeats (data not shown). It is also unclear if the ~17 kb and ~25 kb PCR products generated with primers AB1393/1466 in ApVar-1 and ApNorLamb-V2, respectively (Figure 3C), are composed mainly of repeats, or alternatively if a fifth virB-6 gene paralog exists in these strains. Taken together, the data presented here clearly demonstrate the extreme variability of the T4SS VirB6-4 subunit among A. phagocytophilum strains. Although the differences between the more closely related human, dog and rodent US strains were mainly within repeat-laden regions, the fact that an extensive, distinct repeat pattern was maintained in two strains would speak against the possibility that the variability may be attributed solely to the highly recombinogenic nature of such structures. Worth noting, Camp Ripley, where the infected jumping mouse was captured (2001) is only ~20 miles away from the city of Baxter, MN, where Dog1 resides. Although there are no records of where this dog may have actually acquired the infection, it presented with severe clinical disease in 2007.
Additional file 2
Figure S2. Structure of the virB6-4 repeat regions R3 and R4 in four US A.phagocytophilum strains.A. Comparative maps of AB1393/AB1466 PCR products detailing the repeat unit content of R3 and R4 in the human, rodent and dog strains. ApJM and ApDog1 have identical virB6-4 genes and are, therefore, represented by one map. Moderate variability in the number and sequence of the R3 405 bp repeat units (light blue arrows) is apparent. The small bar at the end of R3 corresponds to the 3′-most partial repeat unit present in all strains. The colored arrows within R4 represent the five repeat types T1a (yellow), T1b (green), T2a, (red), T2b (dark blue) and T2c (grey). The repeat pattern in ApHZ shows no relationship to that of ApJM/ApDog1, which is also 2.87 kb larger, totaling 9.76 kb. This region was not fully characterized in ApDog2 as indicated by a broken line, but the repeat pattern of the 5′- and 3′-most repeats is clearly different from that of the other strains. The small bar downstream of the second repeat unit represents a partially characterized type 2 repeat unit. Lines above and below the ApHZ and ApJM/ApDog1 maps delineate segments of sequence identity within the respective R4 regions. Their sizes are specified. B. Alignment of the nucleic acid sequence of all virB6-4 R4 repeat unit types identified to date. Type 1 repeats are shown in black, type 2 in blue. Differences between sub-types are highlighted. A single BamHI site present in all type 2 repeats is underlined. With the exception of only a few nucleotides at each end, type 1 and type 2 repeat units do not share any sequences. C. Alignment of the amino acid sequences of the repeat units shown in B. The single nucleotide differences between sub-types do not lead to changes in amino acid sequence.
1471-2164-13-678-S2.pdf
Click here for file
The unusual structure and likely antigenicity of the C-terminal region of the A. phagocytophilum VirB6-4′s is apparent in hydrophobicity plots (Figure F4 4). What specific properties these distinct repeat patterns may confer onto each strain awaits functional analysis of these proteins in A. phagocytophilum. The corrected VirB6-4 translated protein had a predicted molecular weight of 470,695 Da containing 4,322 amino acid residues compared to molecular weights of 90,742, 103,204 and 158,321 Da for the HZ strain VirB6-1, VirB6-2 and VirB6-3, respectively. Interestingly, the predicted acidity of the VirB6′s also increased from VirB6-1 to VirB6-4 (pI’s of 8.4, 6.8, 5.1 and 4.0 for the ApHZ VirB6-1, VirB6-2, VirB6-3 and VirB6-4, respectively). The ApDog1/ApJM VirB6-4 polypeptides had a predicted molecular weight of 603,529 Da containing 5,550 amino acids, and a pI of 3.96. Despite these dissimilarities, at least eight transmembrane segments were predicted for all VirB6 paralogs.
Figure 4Hydrophobicity plots of VirB6-4 proteins from A. phagocytophilum HZ (top) or Dog 1 (bottom) strains
Hydrophobicity plots of VirB6-4 proteins from
A.
phagocytophilum
HZ (top) or Dog 1 (bottom) strains.
1471-2164-13-678-4
Periplasmic/outer membrane channel subunits: VirB2, VirB7 and VirB9
Several other T4SS subunits contribute to the secretion channel across the periplasm and outer membrane. VirB7 subunits are typically small lipoproteins that may stabilize VirB9
B38 38
B39 39
. In A. phagocytophilum strains a putative VirB7 is absolutely conserved between strains and may be lipid modified through an N-terminal cysteine on the mature molecule. VirB9 is hydrophilic and also localizes to the periplasm and outer membrane. In A. tumefaciens the C-terminal region of VirB9 is part of the outer membrane protein channel and is surface accessible
B40 40
. There is also evidence for surface exposure of VirB9 in Ehrlichia chaffeensis and A. phagocytophilum
18
19
B41 41
. VirB9-1, which is encoded on the polycistronic virB8
1
virD4 transcript
31
, has a strongly predicted signal peptide and two transmembrane helices. Of all the potentially exposed components of the T4SS, VirB9 of A. phagocytophilum appears to be the least diverse among strains. There are some amino acid substitutions in ruminant and horse strains (2–6 total compared to ApHZ) but in the other strains VirB9′s are unchanged (data not shown).Unlike VirB9′s, VirB2′s are the most diverse of all T4SS subunits in A. phagocytophilum, in terms of both copy number and sequence. VirB2 proteins are typically constituents of pili and of the secretion channel and their diversity in Anaplasma suggests the possibility of exposed, antigenically variable structures. In A. marginale, VirB2 is expressed together with the major outer membrane protein MSP3 on a sequence-variable polycistronic transcript
25
B42 42
. The mechanism of expression in A. phagocytophilum is not known. VirB2′s of other genera are typically small hydrophobic proteins with a long signal peptide sequence and two hydrophobic alpha helices for integration into the cytoplasmic membrane. This also appears to be the case for A. phagocytophilum. The VirB2 paralogs in the different strains are predicted to have two hydrophobic alpha-helices of lengths 22+/−3 and 20+/−0.2 amino acids and signal peptides of length 27+/−2 amino acids. This is true despite their sequence diversity (Figure F5 5). As with many other T4SS components, the ruminant and horse strains are more distant taxonomically in VirB2 sequence compared to VirB2′s of human and dog strains. Alignment of all VirB2 paralogs and orthologs shows that sequence diversity is primarily localized to two hypervariable regions either preceding an N-terminal cysteine or close to the C-terminus (Figure F6 6). This is similar to the hypervariable regions found among VirB2 paralogs of A. marginale
25
.
Figure 5Phylogenetic trees to show the relationship of syntenic VirB2 proteins from different strains of A. phagocytophilum
Phylogenetic trees to show the relationship of syntenic VirB2 proteins from different strains of
A.
phagocytophilum.
1471-2164-13-678-5
Figure 6Multiple sequence alignment of VirB2 amino acid sequences from different strains of A. phagocytophilum
Multiple sequence alignment of VirB2 amino acid sequences from different strains of
A.
phagocytophilum.
1471-2164-13-678-6
Energetic subunits: VirB4 and VirB11
ATPases are typically used in T4SS to energize substrate transfer and have been found in every T4SS described. In gram-negative bacteria these are typically integral membrane proteins encoded by genes residing upstream of virB2 (encoding pilin). This is true for all strains of A. phagocytophilum and it has been suggested that this arrangement of multiple virB2 paralogs and virB4
2 may allow assembly of an antigenically variable surface organelle
20
. The energetic subunit itself, VirB4-2, is however, well conserved between strains. The most distant taxonomic relationship was found between human and ruminant strains (29 total amino acid substitutions in ApNorLamb-V1 compared to ApHZ, Figure F7 7). The other energetic subunit, VirB11, was also well-conserved between strains (6 amino acid substitutions between ApNorLamb-V1 and ApHZ; data not shown).
Figure 7Phylogenetic trees to show the relationship of syntenic VirB4 proteins from different strains of A. phagocytophilum
Phylogenetic trees to show the relationship of syntenic VirB4 proteins from different strains of
A.
phagocytophilum
.
1471-2164-13-678-7
Type 4 coupling protein: VirD4
Type 4 coupling proteins such as VirD4 are ATPases that function in substrate recognition and translocation using the T4SS. They are associated with most effector translocator systems. They typically possess a minimum of two N-terminal transmembrane domains. Often most heterogeneity exists in these N-terminal regions
20
. The A. phagocytophilum VirD4′s conform somewhat to this stereotype with three strongly predicted N-terminal transmembrane segments. As with the other ATPases of the A. phagocytophilum T4SS, there is little variation in VirD4, a total of 17 amino acid substitutions of which 4 are N-terminal but more (12) are C-terminal. Again, the evolutionary relationships among VirD4 sequences position the ruminant and horse strains more distantly to the U.S. dog, human and rodent strains (Figure F8 8).
Figure 8Phylogenetic tree to show the relationship of syntenic VirD4 proteins from different strains of A. phagocytophilum
Phylogenetic tree to show the relationship of syntenic VirD4 proteins from different strains of
A.
phagocytophilum
.
1471-2164-13-678-8
Conclusions
A. phagocytophilum represents a recent reclassification of intracellular organisms infecting different animal species and humans and causing diverse disease symptomatology
B43 43
. These bacteria were previously known as Ehrlichia phagocytophila, Ehrlichia equi, and the agent of human granulocytic ehrlichiosis. Despite the differences within this species, the overall genome structure and synteny of the T4SS is maintained. However, gene structural analysis reveals evidence of gene duplication and considerable diversity of T4SS components in strains infecting different animals. Taxonomic trees suggest a close evolutionary relationship of A. phagocytophilum strains infecting U.S. humans, mice and dogs and a more distant relationship with ruminant and horse strains. This relationship is not unique to the T4SS but is also supported by similar taxonomic trees of other A. phagocytophilum proteins of conserved metabolic function (Figure F9 9). Within the T4SS multicomponent membrane complex, the energetic and internal scaffolding protein components are the most conserved. In contrast, components that form the proposed exposed structures of the T4SS, such as VirB2 and VirB6, are more variable. T4SS are important virulence determinants of bacteria, therefore these differences may result in the different infectivity and virulence profiles observed with different strains. It will be of interest to determine the molecular architecture of VirB6 paralogs in different strains, including interactions with other T4SS components and effectors. Of the known surface exposed components of the T4SS, VirB9 is the most conserved. This protein has been proposed as a vaccine component against A. marginale and may also be suitable against A. phagocytophilum.
Figure 9Phylogenetic tree to show the relationship of other conserved proteins from different strains of A. phagocytophilum
Phylogenetic tree to show the relationship of other conserved proteins from different strains of A. phagocytophilum. These proteins are: PolA, DNA polymerase I; LeuS, leucyl-tRNA synthetase; AtpA, ATPsynthase F1, alpha subunit; ValS,valyl-tRNA synthetase; RecG, ATP-dependent DNA helicase; LigA, NAD-dependent DNA ligase.
1471-2164-13-678-9
Methods
A. phagocytophilum strains, cell culture, and experimental infection
The A. phagocytophilum U.S. strains HZ (human-origin, NY), MRK (horse-origin, CA), JM (rodent-origin, MN) and Dog1 (dog-origin, MN) were propagated in HL-60 cells in RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA) supplemented with final 10% heat-inactivated fetal bovine serum (Thermo Scientific) and 4 mM L-glutamine (Lonza, Rockland, ME), and in the absence of antibiotics. ApHZ and ApMRK have been described previously
15
B44 44
. The ApJM strain (CR01-1258) originated from a meadow jumping mouse (Zapus hudsonius) trapped at Camp Ripley, MN
B45 45
. The ApDog1 strain originated from the blood of a dog from Baxter, MN naturally infected with A. phagocytophilum, as evidenced by the detection of distinctive morulae in a diagnostic blood sample, and sequencing of the Expression Site-linked msp2/p44 gene. Briefly, whole blood was collected from the animal with EDTA as an anticoagulant. The buffy coat layer was collected after low-speed centrifugation of the whole-blood, washed in 1x phosphate buffered saline (PBS, Hyclone, cat. no. SH30256.01), then added to a culture of uninfected HL-60 cells. The culture was left undisturbed for 3 days, after which morulae began to appear. The ApDog2 strain also originated from a MN dog and was passaged to and maintained in the Ixodes scapularis ISE6 tick cell line as described
B46 46
. The Ap variant 1 CRT35 strain (tick-origin, MN), maintained in ISE6 cells, has been described
B47 47
. For DNA isolation, cultures were maintained until 90-100% of cells were infected with mature morulae. Cells were pelleted by centrifugation at 2500 x g for 20 min at 4°C. Pellets were gently resuspended in 1.5 ml cold PBS, transferred to screw-cap microfuge tubes, and centrifuged at 1500 x g for 20 min at 4°C. Supernatants were removed and the cell pellets stored at −80°C until further use.Two naturally occurring Norwegian lamb A. phagocytophilum strains differing in the 16S rRNA gene and degree of virulence were used to experimentally infect lambs raised in an indoor environment with barriers against tick entry and tick infestation. Lamb 00186 was infected with the more virulent variant 1 (identical to GenBank M73220) and lamb 0054 with variant 2 (identical to GenBank AF336220)
B48 48
, to be referred to as ApNorLamb-V1 and -V2 from here on. Infections were monitored by microscopy and blood was harvested at maximum parasitemia. To purify buffy coats containing the infected neutrophils, approximately 2.5 l of Na-citrated blood was collected from each animal. The blood was transferred to 1 l centrifuge bottles and centrifuged at 2,500-3,000 x g in a swing-out bucket rotor for 30 min at 4°C. After removing most of the plasma layer, the buffy coat layer was collected with minimal contamination of red blood cells. The cells were diluted 1:3 with PBS, mixed gently and centrifuged at 1,500x g for 20 min at 4°C. Following three PBS washes, supernatants were removed and the cell pellets stored at −80°C. The experimental study in sheep was approved by the Norwegian Animal Research Authority.
Purification of host cell-free A. phagocytophilum and genomic DNA (gDNA) isolation
For the HZ, JM, Dog1, MRK and NorLamb-V1 and -V2 strains, intact, host cell-free organisms with minimal host cell gDNA/RNA contamination were purified from frozen PBS pellets of infected cells prepared as above. Samples and reagents were maintained on ice throughout the entire procedure, and all centrifugations performed at 4°C. Following a quick thaw, host cells were disrupted by vigorous vortexing for 5 min. An equal volume of PBS was added and vortexing continued for 3 min. Cellular debris was removed by centrifugation at 200 x g for 15 min. After removing most of the supernatants to fresh tubes, these were passed several times through a 31 G needle and saved on ice. Pellets were resuspended well in final 500 μl PBS then passed serially through 22 G, 25 G, 28 G and, when possible, 31 G needles attached to a 1 ml syringe. 3–5 volumes PBS were added and mixed by vortexing. Debris was removed by centrifugation at 200 x g for 10 min. Supernatants were pooled to those from the previous centrifugation step. RNaseA was added to a final 250–300 μg/ml and the samples incubated 45–60 min at 37°C. Samples were centrifuged at 21,000 x g for 30 min and the supernatants removed completely. Pellets were resuspended in 50–100 μl PBS each and transferred to fresh tubes. To ensure homogeneity of the suspension, initially a drawn-out 10 μl pipette tip was used to disrupt the pellet by swirling followed by up/down pipetting and gentle vortexing, before switching to a larger tip. The sample was further homogenized by several passes through a 28-31 G needle. PBS was added to final 500–700 μl and DNaseI to final 250 μg/ml. Following 45–60 min incubation at 37°C the samples were centrifuged at 21,000 x g for 30 min. Pellets were homogenized as above and the DNaseI treatment repeated. EDTA (pH 8.0) was added to final 25 mM and the samples centrifuged as above. Tubes were washed twice with PBS without disturbing the pellets and residual PBS was removed after 3 min centrifugation at 21,000 x g. Pellets were homogenized as above in 600–800 μl RPMI culture medium (containing 10% fetal bovine serum) added incrementally and transferred to a 50 ml tube. Culture medium was added to a final volume of 6 ml before passage through a pre-wet, 2 μm pore-size, 25 mm, GMF-150 glass microfiber syringe filter (Puradisc 25GD; Whatman Inc., Florham Park, NJ). The filter was washed 3-4x with culture medium. Washes were pooled to the filtrate and centrifuged at 22,000 x g for 30 min. The pellets, comprised of free, non-viable organisms and host cell mitochondria, were resuspended in PBS, transferred to microfuge tubes and re-pelleted at 21,000 x g for 30 min. Supernatants were removed completely and the pellets were processed immediately or stored at −20°C. For every 108 host cells used at 90-100% infectivity, enough organisms were recovered to yield on average 1–1.5 μg high-quality DNA using either the Gentra Puregene Yeast/Bact. kit (Qiagen Inc., Valencia, CA) or the QIAGEN Blood & Cell Culture DNA mini kit following the manufacturer’s protocols.For the Dog2 and Ap variant 1 strains, organisms were cultured and isolated from ISE6 tick cells as described
B49 49
. Host cell-free bacteria were prepared from two cultures in 25 cm2 flasks, collected by centrifugation for 10 min at 11,000 xg at 4°C, and lysed in Gentra Puregene lysis buffer (Qiagen) at 80°C for 5 min. Since these DNA samples also contained a considerable amount of small (<500 bp) DNA species naturally associated with the ISE6 host cell line, the A. phagocytophilum gDNA was further purified by electroelution from agarose gels, followed by phenol/chloroform extraction and EtOH precipitation using conventional protocols.
Preparation of host cell-free A. phagocytophilum agarose plugs for optical mapping
ApDog1 was initially selected for complete genome sequencing to compare with the published HZ strain. When a draft genome was assembled for ApDog1 it was largely syntenic with HZ except for the virB6 locus, indicating a possible error in the sequence of one or both of the strains. Accordingly, the ApDog1 draft genome sequence was verified by Optical Mapping. In preparation for Optical Mapping (performed by OpGen Inc., Gaithersburg, MD), host cell-free organisms were embedded in 0.5% low-melting point agarose plugs and subsequently lysed, allowing access to the intact, ~1.48 Mb circular A. phagocytophilum chromosome. A procedure recommended by OpGen was followed. All solutions were made fresh using OpGen suggested reagents. Intact ApDog1 organisms were purified as above, except that the pellet of free organisms obtained following centrifugation of the filtrate was resupended and washed in cell suspension buffer [200 mM NaCl, 100 mM EDTA-Nasub 2 (pH 8.0), 10 mM Tris (pH 7.2)]. Plugs were made immediately on completion of the isolation procedure. Briefly, following the final centrifugation of the purified organisms, the pellet was resuspended in cell suspension buffer using 40–50 μl for every 108 host cells used at >95% infectivity. The sample was passed 2x through a 31 G needle (3/10 ml capacity Insulin Syringe with fused 8 mm long needle, BD #328438; Becton, Dickinson & Co., Franklin Lakes, NJ) to ensure homogeneity of the thick suspension, and an equal volume of 1% low melting point SeaPlaque GTG agarose [(Lonza #50111) dissolved in DEPC-treated water (Invitrogen #750023; Carlsbad, CA) and held at 55°C] was immediately added. Following mixing, 100 μl aliquots were dispensed into plug molds (Bio-Rad #170-3713; Hercules, CA) and allowed to set for 1 hr at 4°C prior to transfer into a 50 ml tube containing 5–10 ml, 50°C NDSK solution [filter sterile NDS solution (1% N-lauroylsarcosine (Sigma #L5000; St. Louis, MO) in 0.5 M EDTA-Na2 (pH 9.5), supplemented with final 2 mg/ml proteinase K (Pierce #17916; Rockford, IL) immediately prior to use]. The tube was incubated upright at 50°C with mild shaking (40 rpm) for 8–24 hrs, until the plugs turned clear and colorless. Plugs were gently washed 3x in 5 ml 0.5 M EDTA-Na2 (pH 9.5), then transferred to a fresh tube and stored in EDTA at 4°C. Optical Mapping data generated from the BamHI-digested ApDog1 chromosome was analyzed using the OpGen MapSolver software.
454 Genome sequencing and bioinformatics
Isolated DNA was provided to the Interdisciplinary Center for Biotechnology Research (ICBR) core facilities, University of Florida for library construction and pyrosequencing on the Roche/454 Genome Sequencer according to standard manufacturer protocols. Regular read libraries were generated for all strains. Additionally, 3 kb paired end libraries were made for ApHZ, ApDog1 and ApMRK. Genome coverage range was 31.3x to 72.1x. For each strain, the SFF format flow files, returned by ICBR for bioinformatics analysis, were first combined and converted to .fasta and .qual files (or the two combined in .fastq format) using Roche/454 Genome Sequencer FLX System software. Genome drafts were assembled using the CLC Genomics Workbench software suite (version 4.0-4.9) by mapping reads initially against the fully annotated, Sanger sequenced ApHZ genome (GenBank CP000235), then against the completed ApDog1 genome. Default parameters were used: length fraction, 0.5; similarity, 0.8; and for paired end reads, minimum distance, 1500/maximum distance, 4500. To obtain the vir loci, the resulting consensus sequence and underlying aligned reads were inspected for conflicts and mismatched paired ends suggesting the presence of insertions and/or deletions not mirrored in the consensus. These were manually corrected. Gaps were also manually closed where possible. Briefly, overlapping reads covering at least 2 kb of sequence on both sides of a gap and extending into it were individually extracted from the alignment. A new consensus for each side was obtained by assembling the reads against each other, and 250 N’s were added to its ends. These were individually used as the reference sequence against which all the 454 reads were re-mapped to pull out novel reads extending into the unknown region. The process was repeated multiple times, allowing for the incremental filling of the gap. PCR, followed by sequencing was performed when sequences extrapolated in this fashion spanned complex tandem repeat regions such as repeat regions 1 and 3 (R1 and R3 in Figure 3A) of the virB6-4 gene, or when gap closure could not be completed due to such structures, as was the case with the extremely long virB6-4 R4 (Figure 3A) region.Amino acid sequences were aligned with MAFFT
B50 50
and displayed with CHROMA
B51 51
. Taxonomic relationships used a neighbor-joining tree and the ITT substitution model
B52 52
and were displayed using Archaeopteryx (http://www.phylosoft.org/archaeopteryx). Hydrophobicity analyses were conducted using the method of Hopp and Woods
B53 53
B54 54
at web.expasy.org and transmembrane segments were predicted with TMpred at http://www.ch.embnet.org/software/TMPRED_form.html.
PCR amplification of virB6-4 gene repeat regions, cloning, and Pacific Biosciences sequencing
Due to difficulties in amplifying tandem repeat-containing DNA, all PCR reactions spanning the virB6-4 gene repeat regions were performed in the presence of 1.5-1.7 M Betaine (Sigma). The 8.36 kb PCR product spanning R3 and R4 in the ApHZ strain (Figure 3A, 3C, and Additional file 2: Figure S2A) was amplified using the iProof High-Fidelity DNA Polymerase system with GC buffer (Bio-Rad). Reactions totaled 50 μl with 5 ng purified A. phagocytophilum gDNA, 1.0 U polymerase, 1.5 mM MgCl2, 200 μM each dNTP, and 250 nM each primer (AB1393: 5′-CGGGATCTAAGACAGATGATGATTC-3′, forward; AB1466: 5′-CTCATCCTGATGCGTCTCCTTAG-3′, reverse; Figure 3A). 35 cycles of 30 sec denaturing at 98°C, 20 sec annealing at 67°C, and 5 min extension at 72°C were performed. PCR products spanning R4 in ApJM and ApDog1 (both ~10.3 kb; Figure 3C) were derived using Takara’s PrimeSTAR GXL DNA Polymerase system (Clontech Laboratories, Mountain View, CA). Reactions contained 5 ng gDNA, 1.25 U polymerase, 1.0 mM MgCl2, 200 μM each dNTP, and 200 nM each primer (AB1395: 5′-CACCAGAGGATGCAGCATTAG-3′, forward; AB1466, reverse; Figure 3A) in total 50 μl. Following the manufacturer’s recommendations, 2-step PCR was performed with 30 cycles of 10 sec denaturing at 98°C and 10 min annealing/extension at 68°C. PCR products were analyzed on 0.5% agarose gels alongside the 1 kb Plus (Invitrogen) and the GeneRulerHighRange (Fermentas, Inc., Glen Burnie, MD) DNA ladders. In order to TA-clone the amplicons, A-overhangs were added to the ends using 0.5-1.0 units AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA) in a 10–15 min reaction at 72°C. Products purified from agarose gels (before or after A-overhang addition) were cloned into the pCR-XL-TOPO vector (Invitrogen) and transformed into E. coli Stbl2 (Invitrogen), which is more permissive to repeat-laden foreign DNA. Recombinants containing the correct size insert were end sequenced to verify their identity.In preparation for sequencing with the long-read length Pacific Biosciences (PacBio) next-generation sequencing RS instrument, constructs were linearized with restriction enzymes which cut the vector only, but on opposite sides of the insert within the Multiple Cloning Site. For ApHZ, equimolar amounts of the TA clone were cut with either HindIII or EcoRV. Following pooling and EtOH precipitation, the linearized DNA mix was submitted to ICBR/UF for SMRTbell library construction and sequencing. Libraries were constructed using a commercial strobe library preparation kit (#001-326-530; Pacific Biosciences, Menlo Park, CA) following standard manufacturer protocols. To further increase the likelihood of full coverage, the strobe-sequencing run was performed using two different conditions: I) 45 min light period (continuous collection time); and II) (5 min light period, 10 min dark period), followed by (45 min light period, 10 min dark period). The ApJM and ApDog1 constructs were double-digested with HindIII/XbaI to excise the ~10.3 kb inserts. Following separation on 0.5% agarose gels, the inserts were recovered from agarose slices by electroelution and further purified and concentrated by passage over QIAquick spin columns following the PCR Purification kit protocol (Qiagen). SMRTbell libraries were made as above then sequenced using a single 75 min movie time run.Due to the repetitive nature of the cloned gene fragments, combined with the relatively high error-rate of the PacBio system, all attempts to assemble the reads de novo failed to yield a sequence of the expected size. Therefore, for each construct, reads >3 kb were selected from the multi-fasta files using the Galaxy suite
B55 55
, and imported into the CLC Genomics Workbench for assembly and further analysis. These were assembled at low stringency initially against a consensus sequence representing an entire (vector and insert sequence) linear construct to which sufficient N’s were added based on the estimated gap-size. Starting with reads initiating outside the repeat region, the longest of the assembled reads were visually inspected for the presence of virB6
4 R4 repeat signature-sequences (Additional file 2: Figure S2) and their sequence manually corrected where necessary. The extended sequences were used to replace N’s in the consensus and the process repeated several times until sufficient reads with >2 kb sequence overlap were recovered spanning the entire insert region. For verification, the completed sequence for each strain was used as the reference to re-map all the respective >3 kb PacBio reads and the Roche/454 reads at higher stringency.
GenBank Accession Numbers: for each isolate, the vir genes are listed in order
The sequences of vir loci are complete for strains ApDog1 and ApJM. The sequence of the repetitive virB6
4 locus was incomplete (ApDog2) or not determined for the other strains except ApHz. We provide a revised sequence of virB6
4 for the previously sequenced
15
ApHZ strain.indent 1
ApDog1:JX415845 JX415868B2-1 B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-9, B3, B4-1, B4-t1, B4-2, B6-1, B6-2, B6-3, B6-4, B8-1, B8-2, B9-1, B9-2, B10, B11, D4
ApJM:JX415869 JX415892B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-9, B3, B4-1, B4-t1, B4-2, B6-1, B6-2, B6-3, B6-4, B8-1, B8-2, B9-1, B9-2, B10, B11, D4
ApDog2:JX415893 JX415915 (virB6-4 submitted separately as gapped)B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-9, B3, B4-1, B4-t1, B4-2, B6-1, B6-2, B6-3, B8-1, B8-2, B9-1, B9-2, B10, B11, D4
ApNorLambV2:JX415916 JX415938B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-9, B3, B4-1, B4-t1, B4-2, B6-1, B6-2, B6-3, B8-1, B8-2, B9-1, B9-2, B10, B11, D4
ApNorLambV1:JX415939 JX415966B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-novel1, B2- novel2, B2-novel3, B2-novel4, B2-novel5, B2-novel6, B3, B4-1, B4- t1, B4-2, B6-1, B6-2, B6-3, B8-1, B8-2, B9-1, B9-2, B10, B11, D4
ApHZvirB6-4:JX415967
ApVar1:JX415968 JX415996B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-novel1, B2- novel2, B2-novel3, B2-novel4, B2-novel5, B2-novel6, B2-novel7, B3, B4-1, B4-t1, B4-2, B6-1, B6-2, B6-3, B8-1, B8-2, B9-1, B9-2, B10, B11, D4
ApMRK:JX415997 JX416019B2-1, B2-2, B2-3, B2-4, B2-5, B2-6, B2-7, B2-8, B2-9, B3, B4-1, B4-t1, B4-2, B6-1, B6-2, B6-3, B8-1, B8-2, B9-1, B9-2, B10, B11, D4ApDog2virB6-4Gapped:JX416020.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
BAK and AFB conceived the study, performed bioinformatics analyses and drafted the manuscript. BAK grew infected HL-60 cell cultures, purified organisms, isolated gDNA, designed and supervised PCR and submitted sequences to GenBank. AML performed PCR analyses and cloning and supervised data transfer between units. SS and EGG isolated the European sheep strains, infected and monitored sheep, and prepared organisms at maximal parasitemia. UGM and CMN isolated and cultured in vitro the JM, MRK, Dog2 and Ap variant 1 strains, and prepared Dog2 and Ap variant 1 strain gDNA. ARA and SMM established the Dog1 strain. All authors read and approved the final manuscript.
bm
ack
Acknowledgements
The research described here received support from grants RO1 GM081714 and GM081714-03S1 and from Pfizer Animal Health. We thank Dr. Roberta Veluci-Marlow, Susan Benda and Adam Webster for help with culturing cells infected with A. phagocytophilum, and Dr. Savita Shanker for high-throughput DNA sequencing.
refgrp Human granulocytic anaplasmosis and Anaplasma phagocytophilumDumlerJSChoiKSGarcia-GarciaJCBaratNSScorpioDGGaryuJWetal Emerg Infect Dis2005111828lpage 183410.3201/eid1112.050898pmcid 3367650link fulltext 16485466Epidemiology and control of human granulocytic anaplasmosis: a systematic reviewJinHWeiFLiuQQianJVector Borne Zoonotic Dis20121226927410.1089/vbz.2011.075322217177Clinical diagnosis and treatment of human granulocytotropic anaplasmosisBakkenJSDumlerJSAnn N Y Acad Sci2006107823624710.1196/annals.1374.04217114714Increasing incidence of Ehrlichia chaffeensis and Anaplasma phagocytophilum in the United States, 2000–2007DahlgrenFSMandelEJKrebsJWMassungRFMcQuistonJHAm J Trop Med Hyg20118512413110.4269/ajtmh.2011.10-0613312235621734137Clinical findings and diagnosis in human granulocytic anaplasmosis: a case series from MassachusettsWeilAABaronELBrownCMDrapkinMSMayo Clin Proc20128723323910.1016/j.mayocp.2011.09.008349839422386178The clinical characteristics and outcomes of patients with human granulocytic anaplasmosis in ChinaLiHZhouYWangWGuoDHuangSJieSInt J Infect Dis201115e859e86610.1016/j.ijid.2011.09.00822015246Typical and atypical manifestations of Anaplasma phagocytophilum infection in dogsEbertsMDBeallMJStillmanBAChandrashekarRBreitschwerdtEBJ Am Anim Hosp Assoc201147e86e9410.5326/JAAHA-MS-557822058372Prevalence and geographic distribution of Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis, and Anaplasma phagocytophilum in dogs in the United States: results of a national clinic-based serologic surveyBowmanDLittleSELorentzenLShieldsJSullivanMPCarlinEPVet Parasitol200916013814810.1016/j.vetpar.2008.10.09319150176Possible differential host tropism in Anaplasma phagocytophilum strains in the Western United StatesFoleyJNietoNCMadiganJSykesJAnn N Y Acad Sci20081149949710.1196/annals.1428.06619120182Distinct ecologically relevant strains of Anaplasma phagocytophilumFoleyJENietoNCMassungRBarbetAMadiganJBrownRNEmerg Infect Dis20091584284310.3201/eid1505.081502268702319402993Transmission efficiency of the AP-variant 1 strain of Anaplasma phagocytophilaMassungRFMatherTNPriestleyRALevinMLAnn N Y Acad Sci2003990757910.1111/j.1749-6632.2003.tb07340.x12860603Inability of a variant strain of Anaplasma phagocytophilum to infect miceMassungRFPriestleyRAMillerNJMatherTNLevinMLJ Infect Dis20031881757176310.1086/37972514639548Anaplasma phagocytophilum in white-tailed deerMassungRFCourtneyJWHiratzkaSLPitzerVESmithGDrydenRLEmerg Infect Dis2005111604160610.3201/eid1110.041329336673516318705Anaplasma phagocytophilum the most widespread tick-borne infection in animals in EuropeStuenSVet Res Commun200731Suppl 1798417682851Comparative genomics of emerging human ehrlichiosis agentsDunning HotoppJCLinMMadupuRCrabtreeJAngiuoliSVEisenJPLoS Genet20062e2110.1371/journal.pgen.0020021136649316482227Type IV secretion in the obligatory intracellular bacterium Anaplasma phagocytophilumRikihisaYLinMNiuHCell Microbiol2010121213122110.1111/j.1462-5822.2010.01500.x20670295Molecular architecture of bacterial type IV secretion systemsWaksmanGFronzesRTrends Biochem Sci20103569169810.1016/j.tibs.2010.06.00220621482Differential expression of VirB9 and VirB6 during the life cycle of Anaplasma phagocytophilum in human leucocytes is associated with differential binding and avoidance of lysosome pathwayNiuHRikihisaYYamaguchiMOhashiNCell Microbiol2006852353410.1111/j.1462-5822.2005.00643.x16469062Identification of novel surface proteins of Anaplasma phagocytophilum by affinity purification and proteomicsGeYRikihisaYJ Bacteriol20071897819782810.1128/JB.00866-07216872717766422Biological diversity of prokaryotic type IV secretion systemsAlvarez-MartinezCEChristiePJMicrobiol Mol Biol Rev200973775808278658319946141Immunogenicity of Anaplasma marginale type IV secretion system proteins in a protective outer membrane vaccineLopezJEPalmerGHBraytonKADarkMJLeachSEBrownWCInfect Immun2007752333234210.1128/IAI.00061-07186577617339347Association and evidence for linked recognition of type IV secretion system proteins VirB9-1, VirB9-2, and VirB10 in Anaplasma marginaleMorseKNorimineJPalmerGHSuttenELBaszlerTVBrownWCInfect Immun20128021522710.1128/IAI.05798-11325568822038917Breadth of the CD4(+) T cell response to Anaplasma marginale VirB9-1, VirB9-2 and VirB10 and MHC class II DR and DQ restriction elementsMorseKNorimineJHopeJCBrownWCImmunogenetics20126450752310.1007/s00251-012-0606-422361828IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-TuAraujoFRCostaCMRamosCAFariasTASouzaIIMeloESMem Inst Oswaldo Cruz200810318619010.1590/S0074-0276200800020001018425271Anaplasma marginale type IV secretion system proteins VirB2, VirB7, VirB11, and VirD4 are immunogenic components of a protective bacterial membrane vaccineSuttenELNorimineJBearePAHeinzenRALopezJEMorseKInfect Immun2010781314132510.1128/IAI.01207-09282595120065028Multistrain genome analysis identifies candidate vaccine antigens of Anaplasma marginaleDarkMJAl-KhederyBBarbetAFVaccine2011294923493210.1016/j.vaccine.2011.04.131313368521596083Anaplasma phagocytophilum AnkA binds to granulocyte DNA and nuclear proteinsParkJKimKJChoiKSGrabDJDumlerJSCell Microbiol2004674375110.1111/j.1462-5822.2004.00400.x15236641Silencing of host cell CYBB gene expression by the nuclear effector AnkA of the intracellular pathogen Anaplasma phagocytophilumGarcia-GarciaJCRennoll-BankertKEPellySMilstoneAMDumlerJSInfect Immun2009772385239110.1128/IAI.00023-09268735719307214Anaplasma phagocytophilum Ats-1 is imported into host cell mitochondria and interferes with apoptosis inductionNiuHKozjak-PavlovicVRudelTRikihisaYPLoS Pathog20106e100077410.1371/journal.ppat.1000774282475220174550Phylogenomics reveals a diverse Rickettsiales type IV secretion systemGillespieJJBraytonKAWilliamsKPDiazMABrownWCAzadAFSobralBWInfect Immun2010781809182310.1128/IAI.01384-09286351220176788Characterization and transcriptional analysis of gene clusters for a type IV secretion machinery in human granulocytic and monocytic ehrlichiosis agentsOhashiNZhiNLinQRikihisaYInfect Immun2002702128213810.1128/IAI.70.4.2128-2138.200212784811895979Genetic complementation analysis of the Agrobacterium tumefaciens virB operon: virB2 through virB11 are essential virulence genesBergerBRChristiePJJ Bacteriol1994176364636602055548206843Identification of the VirB4-VirB8-VirB5-VirB2 pilus assembly sequence of type IV secretion systemsYuanQCarleAGaoCSivanesanDAlyKAHoppnerCJ Biol Chem2005280263492635910.1074/jbc.M50234720015901731Spatial location and requirements for the assembly of the Agrobacterium tumefaciens type IV secretion apparatusJuddPKKumarRBDasAProc Natl Acad Sci USA2005102114981150310.1073/pnas.0505290102118360216076948Subcellular localization of the Agrobacterium tumefaciens T-DNA transport pore proteins: VirB8 is essential for the assembly of the transport poreKumarRBXieYHDasAMol Microbiol20003660861710844650Agrobacterium VirB10, an ATP energy sensor required for type IV secretionCascalesEChristiePJProc Natl Acad Sci USA2004101172281723310.1073/pnas.040584310153537715569944Genetic and functional characterization of the type IV secretion system in WolbachiaRancesEVoroninDTran-VanVMavinguiPJ Bacteriol20081905020503010.1128/JB.00377-08244701718502862Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complexAndersonLBHertzelAVDasAProc Natl Acad Sci USA1996938889889410.1073/pnas.93.17.8889385648799123Intermolecular disulfide bonds stabilize VirB7 homodimers and VirB7/VirB9 heterodimers during biogenesis of the Agrobacterium tumefaciens T-complex transport apparatusSpudichGMFernandezDZhouXRChristiePJProc Natl Acad Sci USA1996937512751710.1073/pnas.93.15.7512387768755505NMR structure of a complex between the VirB9/VirB7 interaction domains of the pKM101 type IV secretion systemBaylissRHarrisRCoutteLMonierAFronzesRChristiePJProc Natl Acad Sci USA20071041673167810.1073/pnas.0609535104178526417244707Surface-exposed proteins of Ehrlichia chaffeensisGeYRikihisaYInfect Immun2007753833384110.1128/IAI.00188-07195197517517859Conservation of a gene conversion mechanism in two distantly related paralogues of Anaplasma marginaleMeeusPFBraytonKAPalmerGHBarbetAFMol Microbiol20034763364310.1046/j.1365-2958.2003.03331.x12535066Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocytophilaDumlerJSBarbetAFBekkerCPDaschGAPalmerGHRaySCInt J Syst Evol Microbiol2001512145216510.1099/00207713-51-6-214511760958Equine ehrlichiosisGribbleDHJ Am Vet Med Assoc19691554624695819585Agents of human anaplasmosis and Lyme disease at Camp Ripley, MinnesotaJohnsonRCKodnerCJarnefeldJEckDKXuYVector Borne Zoonotic Dis2011111529153410.1089/vbz.2011.0633323178921867420Invasion and intracellular development of the human granulocytic ehrlichiosis agent in tick cell cultureMunderlohUGJauronSDFingerleVLeitritzLHayesSFHautmanJMJ Clin Microbiol199937251825248527110405394Isolation and propagation of the Ap-Variant 1 strain of Anaplasma phagocytophilum in a tick cell lineMassungRFLevinMLMunderlohUGSilvermanDJLynchMJGayweeJKKurttiTJJ Clin Microbiol2007452138214310.1128/JCM.00478-07193299917475757Variant and individual dependent nature of persistent Anaplasma phagocytophilum infectionGranquistEGBardsenKBergstromKStuenSActa Vet Scand2010522510.1186/1751-0147-52-25285976920398321Transformation of Anaplasma phagocytophilumFelsheimRFHerronMJNelsonCMBurkhardtNYBarbetAFKurttiTJMunderlohUGBMC Biotechnol200664210.1186/1472-6750-6-42163503517076894Recent developments in the MAFFT multiple sequence alignment programKatohKTohHBrief Bioinform2008928629810.1093/bib/bbn01318372315CHROMA: consensus-based colouring of multiple alignments for publicationGoodstadtLPontingCPBioinformatics20011784584610.1093/bioinformatics/17.9.84511590103The rapid generation of mutation data matrices from protein sequencesJonesDTTaylorWRThorntonJMComput Appl Biosci199282752821633570A computer program for predicting protein antigenic determinantsHoppTPWoodsKRMol Immunol19832048348910.1016/0161-5890(83)90029-96191210Use of hydrophilicity plotting procedures to identify protein antigenic segments and other interaction sitesHoppTPMethods Enzymol19891785715852481215Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciencesGoecksJNekrutenkoATaylorJGenome Biol201011R8610.1186/gb-2010-11-8-r86294578820738864


xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID EHE4OOA6P_5U7WGU INGEST_TIME 2013-03-05T20:21:09Z PACKAGE AA00013685_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES



PAGE 1

Fig. S1 Ap HZ-VirB6-3 ML -S R S LLFI A IVVL SS C GG T CI E P GAG V SSSS Q E V Q V P VY P D GA D H KR P V T YWV H S G YR V G E K DE L K I T V D R T I D LC P L D T K A K P VA I K MY P E H F ST -Ap Dog1-VirB6-3 ML -S R S LLFI A IVVL SS C GG T CI E P GAG V SSSS Q E V Q V P VY P D GA D H KR P V T YWV H S G YR V G E K DE L K I T V D R T I D LC P L D T K A K P VA I K MY P E H F ST -Ap Dog2-VirB6-3 ML -S R S LLFI A IVVL SS C GG T CI E P GAG V SSSS Q E V Q V P VY P D GA D H KR P V T YWV H S G YR V G E K DE L K I T V D R T I D LC P L D T K A K P VA I K MY P E H F ST -Ap JM-VirB6-3 ML -S R S LLFI A IVVL SS C GG T CI E P GAG V SSSS Q E V Q V P VY P D GA D H KR P V T YWV H S G YR V G E K DE L K I T V D R T I D LC P L D T K A K P VA I K MY P E H F ST -Ap MRK-VirB6-3 ML -S R S LLFI A IVVL SS C GG T CI E P GAG V SSSS Q E V Q V P VY P D GA D H KR P V T YWV H S G YR V G E K DE L K I T V D R T I D LC P L D T K A K P VA I K MY P E H F ST -Ap NorV2-VirB6-3 ML LR R L LLFI A IVVL SS C GG T CI E P GAG V SSSS Q E V Q V P VY P D GA D H N R P V T YWV H S G YR V G E K DE L K I T V D R T I D LC P L D T K A K P VA I K MY P E H F ST -Ap Var1-VirB6-3 ML LR LF LFI A IVVL SS C GG T CI E P GAG V SSSS Q E V Q V P VY P D GA D Q KR P V T YWV N S G HR V G E K DE L K I T V D R T I D LC P L D T K A K P AV I R MY P E H F ST -Ap NorV1-VirB6-3 M MLR L LL V I A IV A L SS C GG T CI E P GAG V SSSS Q E V Q V P VY P D GA D Q KR P V T YWV H S G HR V G E K DE I K I T V D R T I D LC P L D T K A K P AV I R MY P E H L ST SI Consensus/80% ML..R.LLFIAIVVLSSCGGTCIEPGAGV SSSSQEVQVPVYPDGADpKRPVTYWVHSGaRVGEKDELKITVDRTIDLCPL DTKAKPssI+MYPEHFST.. Ap HZ-VirB6-3 P ST E FY D T YI D V Q E G D Q L R F SS VLY D L S F P D C K E I S R E H S P VI FH NN E VIF R D E A C KK T V A I EE LCL S GA TS E VL G D G S KK G LL H V K D A K G E C K E V P AG Ap Dog1-VirB6-3 P ST E FY D T YI D V Q E G D Q L R F SS VLY D L S F P D C K E I S R E H S P VI FH NN E VIF R D E A C KK T V A I EE LCL S GA TS E VL G D G S KK G LL H V K D A K G E C K E V P AG Ap Dog2-VirB6-3 P ST E FY D T YI D V Q E G D Q L R F SS VLY D L S F P D C K E I S R E H S P VI FH NN E VIF R D E A C KK T V A I EE LCL S GA TS E VL G D G S KK G LL H V K D A K G E C K E V P AG Ap JM-VirB6-3 P ST E FY D T YI D V Q E G D Q L R F SS VLY D L S F P D C K E I S R E H S P VI FH NN E VIF R D E A C KK T V A I EE LCL S GA TS E VL G D G S KK G LL H V K D A K G E C K E V P AG Ap MRK-VirB6-3 P ST E FY D T YI D V Q E G D Q L R F SS VLY D L S F P D C K E I S S E H S P VI FH NN E VIF R D E A C KK T V A I EE LCL S GA TS E VL G D G S KK G LL H V K D A K G E C K E L P AG Ap NorV2-VirB6-3 P ST E FY D T YI D V Q E G D Q L R F SS VLY D L S F P D C K E I S S E H S P VI FH NN E VIF Q D S S C KK A V E I EE LCL S GA TS E VL G D G S KK G LL H V K D A K G N C K E L P AG Ap Var1-VirB6-3 P ST E FY D T YI D V Q E G D Q L R F GA VLY D L N F P N C K E I S S E H S P VI YH NN E VIF Q D S S C KK A V E A EE LCL S GA TS E VL G D G S KK G LL Y V K D A G G E C K E L P AG Ap NorV1-VirB6-3 E P ST E FY D T H I D V Q E G D Q L R F GA VLY D L N F P N C K E I S S E H S P VI YH NN E VIF R D G A C KK A V E A EE LCL S GA L S E KV G E-KK G LL Y V K D S K G E C QA L P ES Consensus/80% .PSTEFYDTYIDVQEGDQLRFttVLYDLs FPsCKEISpEHSPVIaHNNEVIFpDptCKKsV.hEELCLSGATSEVLGDGS KKGLLaVKDAKGECKElPAG Ap HZ-VirB6-3 V Q I P R V GG S II Q T P R M Q F P L G YI G VV D SS V S G E L S F S Y NN VI T N G R I H D A R V P K I N M S P K L D Q E S C E LL K G S N I T L R L S N I D S I D Q L K D R D A H N KH E R S Y Ap Dog1-VirB6-3 V Q I P R V GG S II Q T P R M Q F P L G YI G VV D SS V S G E L S F S Y NN VI T N G R I H D A R V P K I N M S P K L D Q E S C E LL K G S N I T L R L S N I D S I D Q L K D R D A H N KH E R S Y Ap Dog2-VirB6-3 V Q I P R V GG S II Q T P R M Q F P L G YI G VV D SS V S G E L S F S Y NN VI T N G R I H D A R V P K I N M S P K L D Q E S C E LL K G S N I T L R L S N I D S I D Q L K D R D A H N KH E R S Y Ap JM-VirB6-3 V Q I P R V GG S II Q T P R M Q F P L G YI G VV D SS V S G E L S F S Y NN VI T N G R I H D A R V P K I N M S P K L D Q E S C E LL K G S N I T L R L S N I D S I D Q L K D R D A H N KH E R S Y Ap MRK-VirB6-3 V Q I P R V GG S II Q T P R M Q F P L G YI G VV D SS V S G E L S F S Y NN VI T N G R I H D A R V P K I N M S P K L D H E S C E LL K G S N I T L R L S N I D S V D Q L K D R D A H N KH E R S Y Ap NorV2-VirB6-3 V Q I P R V GG S II Q T P R M Q F P L G YI G VV D SS V S G E L S F S Y NN VI T N G R I H D A R V P K I N M S P K L D E E S C A LL K E S N I T L R L S N I D S V D Q L K D R D A H N KH E R S Y Ap Var1-VirB6-3 V Q I P R V GG S II Q T P R M Q F P L G YI G VV D SS V S G E L S F S Y NN VI T N G R I H D A R V P K M N M S P K L D Q E S C E LL K G S N L T L R V S N I D S V D Q L K D R D A H N KH E R S Y Ap NorV1-VirB6-3 V Q I P R V GG S II Q T S R M Q Y P L G YI G VV D SS V S G E L S F S Y NN VI T N G R I H D A R V P K M N A S P K L D Q E S C E LL K G S N L T L R V S N I D S V D Q L K D R D A H N KH K R S Y Consensus/80% VQIPRVGGSIIQTPRMQFPLGYIGVVDSS VSGELSFSYNNVITNGRIHDARVPKbNMSPKLDpESCELLKGSNlTLRlSN IDSlDQLKDRDAHNKHERSY Ap HZ-VirB6-3 T Y G S N K A F TT G KR F G K P S E T EEE KK Q R E K EE K I AA LL S E Y T E Y D L N C H C G YVC K P S N Q V DDD CI R S IV T VM D G N VVC P T Q T K F N G K D H EED A G N QQ H N I P Ap Dog1-VirB6-3 T Y G S N K A F TT G KR F G K P S E T EEE KK Q R E K EE K I AA LL S E Y T E Y D L N C H C G YVC K P S N Q V DDD CI R S IV T VM D G N VVC P T Q T K F N G K D H EED A G N QQ H N I P Ap Dog2-VirB6-3 T Y G S N K A F TT G KR F G K P S E T EEE KK Q R E K EE K I AA LL S E Y T E Y D L N C H C G YVC K P S N Q V DDD CI R S IV T VM D G N VVC P T Q T K F N G K D H EED A G N QQ H N I P Ap JM-VirB6-3 T Y G S N K A F TT G KR F G K P S E T EEE KK Q R E K EE K I AA LL S E Y T E Y D L N C H C G YVC K P S N Q V DDD CI R S IV T VM D G N VVC P T Q T K F N G K D H EED A G N QQ H N I P Ap MRK-VirB6-3 T Y G S N K A F TT G KR F S K P S E T EEE KK Q R E K EE K I AA LL S E Y T E Y D L N C H C G YVC K P S N Q V DDD CI R S IV T VM D G N VVC P T Q T K F N G K D H EED A G N QQ H N I P Ap NorV2-VirB6-3 T Y G S N K A F TT G KR F S K P L E T EEE KK Q R E K EE K I AA LL S E Y T E Y D L N C H C G YVC K P S N Q V DDD CI R S IV T VM D G N VVC P T Q T K F N G K D H EED A G N QQ H N I P Ap Var1-VirB6-3 T Y G S N K P F TT G KR F S K P S E T E Q E KK Q R E K EE K I AA LL S E Y T E Y D L N C H C G YVC K P S N H V DDD CI R S IV T VM D G N VVC P T Q T K F N G K D D E K D A S QQ H N I P Ap NorV1-VirB6-3 T D G DS K P F TT G KR F K K QL E T K EE K E Q R E K EE K I AA LL S E Y T E Y D L N C H C G Y I C K P S D K I D GS C V R S IV T VM D G N VVC P T Q T K F N S K D D EE G A I K QQ R N I P Consensus/80% TYGSNKsFTTGKRFtKP.ETEEEKKQREK EEKIAALLSEYTEYDLNCHCGYVCKPSNpVDDDCIRSIVTVMDGNVVCPTQ TKFNGKDcEEDAhsQQHNIP

PAGE 2

cont. Fig. S1 Ap HZ-VirB6-3 D I R G D L S P E A F A R S V S S L T GQHF G QN V A Q K D S V I S V E K A Y E L A E G VI A VIV S I E N G KK I LP S D I A Q YC N D H Q G K C K YL P E G I K S L K S S Q S G L T F E K G S L Ap Dog1-VirB6-3 D I R G D L S P E A F A R S V S S L T GQHF G QN V A Q K D S V I S V E K A Y E L A E G VI A VIV S I E N G KK I LP S D I A Q YC N D H Q G K C K YL P E G I K S L K S S Q S G L T F E K G S L Ap Dog2-VirB6-3 D I R G D L S P E A F A R S V S S L T GQHF G QN V A Q K D S V I S V E K A Y E L A E G VI A VIV S I E N G KK I LP S D I A Q YC N D H Q G K C K YL P E G I K S L K S S Q S G L T F E K G S L Ap JM-VirB6-3 D I R G D L S P E A F A R S V S S L T GQHF G QN V A Q K D S V I S V E K A Y E L A E G VI A VIV S I E N G KK I LP S D I A Q YC N D H Q G K C K YL P E G I K S L K S S Q S G L T F E K G S L Ap MRK-VirB6-3 D I R G D L S P E A F A R S V S S L T GQHF G QN V A Q K D S V I S V E K A Y E L A E G VI A VIV S I E N G KK I LP S D I A Q YC N D H Q G K C K YL P E G I K S L K S S Q S G L T F E K G S L Ap NorV2-VirB6-3 D I R G D L S P E A F A R S V S S L T GQRF G QN D V A QN D S V I S V E K A Y E L A E G VI A VIV S I E N G KK I PE G E V A K YC N D H Q E K C K YL P E G I K S L K S S Q S G L T F E K G S L Ap Var1-VirB6-3 D I R K M S QK D F A S S V A S L A---G QN V L P K D S T I S V K T A Y E L A E G VI A VIV S V E N G KK I PE G G V A K YC N D H Q G K C K YL P E G I K S L K V G Q S G L T F E K G S L Ap NorV1-VirB6-3 D VY-N M S P E D MK K GL G A L T ---G QN V L P K D S T I G V K T A Y E L A E G VI A VIV S V E N G KK I PE G G V A E YC N D H Q E K C K YL P D G I K S L K D G Q S G L T F GD G S L Consensus/80% DIR.cbSPEsFA+SVtSLT....GQN.Vh .KDSsISVcpAYELAEGVIAVIVSlENGKKI..tslApYCNDHQ.KCKYLP EGIKSLKstQSGLTFEKGSL Ap HZ-VirB6-3 K L D S D YI A P G S G R LYL A YW P Y F G E L G KK V K E RK G S E A A VV AA S G S SS Q R G L T V S R A K N Y S E N S LL S A L A Q RR FFW K Q G M S S G S GG FL S S N D AG N T YV P L S Ap Dog1-VirB6-3 K L D S D YI A P G S G R LYL A YW P Y F G E L G KK V K E RK G S E A A VV AA S G S SS Q R G L T V S R A K N Y S E N S LL S A L A Q RR FFW K Q G M S S G S GG FL S S N D AG N T YV P L S Ap Dog2-VirB6-3 K L D S D YI A P G S G R LYL A YW P Y F G E L G KK V K E RK G S E A A VV AA S G S SS Q R G L T V S R A K N Y S E N S LL S A L A Q RR FFW K Q G M S S G S GG FL S S N D AG N T YV P L S Ap JM-VirB6-3 K L D S D YI A P G S G R LYL A YW P Y F G E L G KK V K E RK G S E A A VV AA S G S SS Q R G L T V S R A K N Y S E N S LL S A L A Q RR FFW K Q G M S S G S GG FL S S N D AG N T YV P L S Ap MRK-VirB6-3 K L D S D YI A P G S G R LYL A YW P Y F G E L G KK V K E RK G S E A A VV AA S G G SS Q R G L T V S R A K N Y S E N S LL S A L A Q RR FFW K Q D M S S G S GG FL S S N D AG N T YV P L S Ap NorV2-VirB6-3 K L D S D YI A P G S G R LYL A YW P Y F G E L G KK V K E RK GR E A T V A AA S G S SS Q S G L T V S R A K N Y S E N S LL S A L A Q RR FFW K Q D M S S G S GG FL S S N D AG N T YV P L S Ap Var1-VirB6-3 K L D S D YI A P G S G R LYL A YW P S L G E L G KK V K E R RE N N A -V AA S G S SS Q R G L T V S R A K N Y S E N S LL S A L A Q RR FFW K Q Y M S F A S GG FL P S N D AG N A YV S L S Ap NorV1-VirB6-3 K L D S D YI A P G S G R LYL A YW P S L G E L G KK V K E RK E S K A A VV V A S G G SS Q R G S T V S ST K K Y Y E D S LL S A L A Q RR FFW K Q D M S F A S GG FL P S N D AG N A YV S L S Consensus/80% KLDSDYIAPGSGRLYLAYWP.bGELGKKV KERK.scAsVVAASGtSSQRGLTVSRAKNYSENSLLSALAQRRFFWKQsMS .tSGGFLsSNDAGNsYVsLS Ap HZ-VirB6-3 R DE G I TT I S Y S V A S AA T P V S AG K P A S ER R I S V A R T G E D I A V Q G FY S L N V HR T CY A TS G Q K LYMYI G D T PP T A L P G K Q Q GA I P L D F E K I N SS K S D K EE Ap Dog1-VirB6-3 R DE G I TT I S Y S V A S AA T P V S AG K P A S ER R I S V A R T G E D I A V Q G FY S L N V HR T CY A TS G Q K LYMYI G D T PP T A L P G K Q Q GA I P L D F E K I N SS K S D K EE Ap Dog2-VirB6-3 R DE G I TT I S Y S V A S AA T P V S AG K P A S ER R I S V A R T G E D I A V Q G FY S L N V HR T CY A TS G Q K LYMYI G D T PP T A L P G K Q Q GA I P L D F E K I N SS K S D K EE Ap JM-VirB6-3 R DE G I TT I S Y S V A S AA T P V S AG K P A S ER R I S V A R T G E D I A V Q G FY S L N V HR T CY A TS G Q K LYMYI G D T PP T A L P G K Q Q GA I P L D F E K I N SS K S D K EE Ap MRK-VirB6-3 R DE G I TT I S Y S V A S AA T P V S AG K P A S ER R I S V A R T G E D I A V Q G FY S L N V HR T CY A TS G Q K LYMYI G D T PP T A L P G K Q Q GA I P L D F E K I N SS K S D K E A Ap NorV2-VirB6-3 R DE G I TT I S Y S -I A S AA M P V S AG N T A S ER R I S V A R T G E D I A V Q G FY S L N V HR T CY A TS G Q K LYMYI G D T PP T A L P G K Q L GA I P L D F E K I N SS K G D K EE Ap Var1-VirB6-3 H DE G A TT I S Y S T V A A AA A P V S AG K T A S Q A R M S V A H T S E N T T K V Q G FY A L N V HR T CY A TS G Q K LYMYI G D T PP T A L P G K Q Q GA I P L D F E K I N SS K S D D K EE Ap NorV1-VirB6-3 H DE G A TT I S Y S T V A A AA A P V S AG K T V S QS H I S V A R N S E N T T V Q G FY S L S V R R T CY A TS G Q R LYMYI G D T PP T E L P G K Q T GA I S L D F E EM N K S ---K K E Consensus/80% +DEGhTTISYS.VAtAAsPVSAGKsASpp RISVARTtEshs.VQGFYSLNVHRTCYATSGQKLYMYIGDTPPTALPGKQp GAIPLDFEKINSSKt.DKEE Ap HZ-VirB6-3 K E W S Y K I N S GA E KR Q G YIYF G V D V D P G Y E A K L K Q A NN S D N YY A V H LWV P K W T P IF SS FF N FL Q G VLL H VLY G T D L P T M G Q D T K A V E A S K VI G R A M S P E Y Ap Dog1-VirB6-3 K E W S Y K I N S GA E KR Q G YIYF G V D V D P G Y E A K L K Q A NN S D N YY A V H LWV P K W T P IF SS FF N FL Q G VLL H VLY G T D L P T M G Q D T K A V E A S K VI G R A M S P E Y Ap Dog2-VirB6-3 K E W S Y K I N S GA E KR Q G YIYF G V D V D P G Y E A K L K Q A NN S D N YY A V H LWV P K W T P IF SS FF N FL Q G VLL H VLY G T D L P T M G Q D T K A V E A S K VI G R A M S P E Y Ap JM-VirB6-3 K E W S Y K I N S GA E KR Q G YIYF G V D V D P G Y E A K L K Q A NN S D N YY A V H LWV P K W T P IF SS FF N FL Q G VLL H VLY G T D L P T M G Q D T K A V E A S K VI G R A M S P E Y Ap MRK-VirB6-3 K E W S Y K I N S GA E KR Q G YIYF G V D V D P G Y E A K L K Q A NN S D N YY A V H LWV P K W T P IF SS FF N FL Q G VLL H VLY G T D L P T M G Q D T K A V E A S K VI G R A M S P E Y Ap NorV2-VirB6-3 K E W S Y K I N S GA K K Q Q G YIYF G V D V D P G Y E A K L K Q A NN S D N YY A V H LWV P K W T P IF SS FF N FL Q G VLL H VLY G T D L P T M G Q D T K A V E A S K VI G R A M S P E Y Ap Var1-VirB6-3 K E W S Y K I N S GA E KR Q G YIYF G V D V D P G Y E A K L K Q A NN S D N YY A V H LWV P K W T P IF SS FF N FL Q G VLL H VLY G T D L P T M G Q D T K A V E A S K VI G R A M S P E Y Ap NorV1-VirB6-3 K E W S Y K I N ADK D H K Q K G YIYF G V A V D P A Y E A N L K K A S N TE N YY S V H LWV P K W T P IF SS FF N L L Q G M LL H VLY G T D L P T M G Q D T R A V E A A K A I G KV M S P E Y Consensus/80% KEWSYKINSGA-K+Q.GYIYFGVDVDPGY EAKLKQANNSDNYYAVHLWVPKWTPIFSSFFNFLQGVLLHVLYGTDLPTMG QDTKAVEASKVIGRAMSPEY

PAGE 3

cont. Fig. S1 Ap HZ-VirB6-3 I G I Q GGG QQ KK AG VV QQ IY NNQ V ST K P FWF A V R A LLVLYLMF S VL G YII G II Q V T KH D IFV R I A K I A LII T LV S P G S W K FF T E H CF S IFIL G I P D II S A F Ap Dog1-VirB6-3 I G I Q GGG QQ KK AG VV QQ IY NNQ V ST K P FWF A V R A LLVLYLMF S VL G YII G II Q V T KH D IFV R I A K I A LII T LV S P G S W K FF T E H CF S IFIL G I P D II S A F Ap Dog2-VirB6-3 I G I Q GGG QQ KK AG VV QQ IY NNQ V ST K P FWF A V R A LLVLYLMF S VL G YII G II Q V T KH D IFV R I A K I A LII T LV S P G S W K FF T E H CF S IFIL G I P D II S A F Ap JM-VirB6-3 I G I Q GGG QQ KK AG VV QQ IY NNQ V ST K P FWF A V R A LLVLYLMF S VL G YII G II Q V T KH D IFV R I A K I A LII T LV S P G S W K FF T E H CF S IFIL G I P D II S A F Ap MRK-VirB6-3 I G I Q GGG QQ KK AG VV QQ IY NNQ V ST K P FWF A V R A LLVLYLMF S VL G YII G II Q V T KH D IFV R I A K I A LII T LV S P G S W K FF T E H CF S IFIL G I P D II S A F Ap NorV2-VirB6-3 I G I Q GGG QQ KK AG VV QQ IY NNQ V ST K P FWF A V R A LLVLYLMF S VL G YII G II Q V T KH D IFV R I A K I A LII T LV S P G S W K FF T E H CF S IFIL G I P D II S A F Ap Var1-VirB6-3 I G I Q GG E QQ KK AG VV QQ IY NNQ V ST K P FWF A V R A LLVLYLMF S VL G YII G II Q V T KH D IFV R I A K I A LII T LV S P G S W K FF T E H CF S IFIL G I P D II S A F Ap NorV1-VirB6-3 I G V Q G S G -K KK T G A V QQ IY NNQ V L T K P FW A A V R A LLVLYLMF S VL G YII G V I Q V T K Y D V FV R V A K I A LI V T LV S P G S W Q FF T E H CF S IFIL G I P D II S A F Consensus/80% IGIQGGGQQKKAGVVQQIYNNQVSTKPFW FAVRALLVLYLMFSVLGYIIGIIQVTKHDIFVRIAKIALIITLVSPGSWKF FTEHCFSIFILGIPDIISAF Ap HZ-VirB6-3 N G YL GG D SS F A FL D ST L G IML TS E FWL R ML S LFM AG P V G WL A FI G I IW A LF S FFL A MM R A IILYLFIMV G L A FLL T L A P IFI T FLLF Q V T K G LF D G WL K M Ap Dog1-VirB6-3 N G YL GG D SS F A FL D ST L G IML TS E FWL R ML S LFM AG P V G WL A FI G I IW A LF S FFL A MM R A IILYLFIMV G L A FLL T L A P IFI T FLLF Q V T K G LF D G WL K M Ap Dog2-VirB6-3 N G YL GG D SS F A FL D ST L G IML TS E FWL R ML S LFM AG P V G WL A FI G I IW A LF S FFL A MM R A IILYLFIMV G L A FLL T L A P IFI T FLLF Q V T K G LF D G WL K M Ap JM-VirB6-3 N G YL GG D SS F A FL D ST L G IML TS E FWL R ML S LFM AG P V G WL A FI G I IW A LF S FFL A MM R A IILYLFIMV G L A FLL T L A P IFI T FLLF Q V T K G LF D G WL K M Ap MRK-VirB6-3 N G YL GG D SS F A FL D ST L G IML TS E FWL R ML S LFM AG P V G WL A FI G V IW A LF S FFL A MM R A IILYLFIMV G L A FLL T L A P IFI T FLLF Q V T K G LF D G WL K M Ap NorV2-VirB6-3 N G YL GG D SS F A FL D ST L G IML TS E FWL R ML S LFM AG P V G WL A FI G I IW A LF S FFL A MM R A IILYLFIMV G L A FLL T L A P IFI T FLLF Q V T K G LF D G WL K M Ap Var1-VirB6-3 N G YL GG D SS F A FL D ST L G IML TS E FWL R ML S LFM AG P V G WL A FI G I IW A LF S FFL A MM R A IILYLFIMV G L A FLL T L A P IFI T FLLF Q V T K G LF D G WL K M Ap NorV1-VirB6-3 N G YL GG D T S F A FL D ST L G IML TS E FWL R ML S LFM AG P V G WL A FI G V IW A LF S FFL A MM R A IILYLF V MV G L A FLL T L A P IFI T FLLF Q I T K G LF D G WL K M Consensus/80% NGYLGGDSSFAFLDSTLGIMLTSEFWLRM LSLFMAGPVGWLAFIGlIWALFSFFLAMMRAIILYLFIMVGLAFLLTLAPI FITFLLFQVTKGLFDGWLKM Ap HZ-VirB6-3 LV N FML Q P IILF AA L A FL NQ VII TS L H A V T D F AA C E S C A V G F N I SS K D S K AA P G Q S D ICII P A LL P M G Y A F E L P V S D R I R E G L A R G D I G FM G L P F S M A ML Ap Dog1-VirB6-3 LV N FML Q P IILF AA L A FL NQ VII TS L H A V T D F AA C E S C A V G F N I SS K D S K AA P G Q S D ICII P A LL P M G Y A F E L P V S D R I R E G L A R G D I G FM G L P F S M A ML Ap Dog2-VirB6-3 LV N FML Q P IILF AA L A FL NQ VII TS L H A V T D F AA C E S C A V G F N I SS K D S K AA P G Q S D ICII P A LL P M G Y A F E L P V S D R I R E G L A R G D I G FM G L P F S M A ML Ap JM-VirB6-3 LV N FML Q P IILF AA L A FL NQ VII TS L H A V T D F AA C E S C A V G F N I SS K D S K AA P G Q S D ICII P A LL P M G Y A F E L P V S D R I R E G L A R G D I G FM G L P F S M A ML Ap MRK-VirB6-3 LV N FML Q P IILF AA L A FL NQ VII TS L H A V T D F AA C E S C A V G F N I SS K D S K AA P G Q S D ICII P A LL P M G Y A F E L P V S D R I R E G L A R G D I G FM G L P F S M A ML Ap NorV2-VirB6-3 LV N FML Q P IILF AA L A FL NQ VII TS L H A V T D F AA C E S C A V G F N I SS K D S K AA P G Q S D ICII P A LL P M G Y A F E L P V S D R I R E G L A R G D I G FM G L P F S M A ML Ap Var1-VirB6-3 LV N FML Q P V ILF AA L A FL NQ VII TS L H A V T D F AA C E S C A V G F N I SS K D S K AA P G Q S D ICII P A LL P M G Y A F E L P V S D R I R E G L A R G D I G FM G L P F S M A ML Ap NorV1-VirB6-3 LV N FML Q P IILF AA L A FL NQ VII TS L H A V T D F AA C E A C A V G F N I P S K D S K A G P G Q S D ICII P A LL P T G F A LD L P V S D R I R E G L A R G D I G FM G L P F S M A ML Consensus/80% LVNFMLQPIILFAALAFLNQVIITSLHAV TDFAACESCAVGFNISSKDSKAAPGQSDICIIPALLPMGYAFELPVSDRIR EGLARGDIGFMGLPFSMAML Ap HZ-VirB6-3 MVLIL A C K A T R E F G D I A E VM A H S I S G S M S G M T AAA V GA T Q S ML S VV G L DD A T Q H LI R S A V A M D P V A S D K V R F D A ED S V Q P RH D G V K ---D N S GA D S P G K Ap Dog1-VirB6-3 MVLIL A C K A T R E F G D I A E VM A H S I S G S M S G M T AAA V GA T Q S ML S VV G L DD A T Q H LI R S A V A M D P V A S D K V R F D A ED S V Q P RH D G V K ---D N S GA D S P G K Ap Dog2-VirB6-3 MVLIL A C K A T R E F G D I A E VM A H S I S G S M S G M T AAA V GA T Q S ML S VV G L DD A T Q H LI R S A V A M D P V A S D K V R F D A ED S V Q P RH D G V K ---D N S GA D S P G K Ap JM-VirB6-3 MVLIL A C K A T R E F G D I A E VM A H S I S G S M S G M T AAA V GA T Q S ML S VV G L DD A T Q H LI R S A V A M D P V A S D K V R F D A ED S V Q P RH D G V K ---D N S GA D S P G K Ap MRK-VirB6-3 MVLIL A C K A T R E F G D I A E VM A H S I S G S V S G M T AAA V GA T Q S ML S VV G L DD A T Q H LI R S A V A M D P V A S D K V R F D A ED S V Q P RH D G V K ---D N S GA D S P G K Ap NorV2-VirB6-3 MVLIL A C K A T R E F G D I A E VM A H S I S G S V S G M T AAA V GA T Q S ML S VV G L DD A T Q H LI R S A V A M D P V A S D K V R F D A ED S V Q P RH D G V K ---D N S GA D S P G K Ap Var1-VirB6-3 MV F IL A C K A T R E F G D I A E VM A H S I S G S V S G M T AAA V GA T Q S ML S VV G L DD A T Q H LI R S A V A M D P V A S D K V R F D A ED S V Q P RH D G V K ---D N S GA D S P G K Ap NorV1-VirB6-3 MVLIL A C K A T R E F G D I A E VM A H S I S G S V S G V T S AA V GA T Q S ML S VV G L DD A T Q H LI K S A I A M D P V GT D K V R F D A ED S V Q P RH D G I K GPVDGS S GA D S L G K Consensus/80% MVLILACKATREFGDIAEVMAHSISGShS GMTAAAVGATQSMLSVVGLDDATQHLIRSAVAMDPVASDKVRFDAEDSVQP RHDGVK....DNSGADSPGK

PAGE 4

cont. Fig. S1 Ap HZ-VirB6-3 E G S D S V Q GA S N A R GAGAG E ---G P GA P V D N D G S N V G R S G ED N V S GG D S GG D I RR P GGG SS G S M Q AG R P Q F ED LL S DD P G I N D R V N A F R E Y R P G D GG V G E Ap Dog1-VirB6-3 E G S D S V Q GA S N A R GAGAG E ---G P GA P V D N D G S N V G R S G ED N V S GG D S GG D I RR P GGG SS G S M Q AG R P Q F ED LL S DD P G I N D R V N A F R E Y R P G D GG V G E Ap Dog2-VirB6-3 E G S D S V Q GA S N A R GAGAG E ---G P GA P V D N D G S N V G R S G ED N V S GG D S GG D I RR P GGG SS G S M Q AG R P Q F ED LL S DD P G I N D R V N A F R E Y R P G D GG V G E Ap JM-VirB6-3 E G S D S V Q GA S N A R GAGAG E ---G P GA P V D N D G S N V G R S G ED N V S GG D S GG D I RR P GGG SS G S M Q AG R P Q F ED LL S DD P G I N D R V N A F R E Y R P G D GG V G E Ap MRK-VirB6-3 E G S D S V Q GA S N A R GAGAG E ---G P GA P V D N D G S N V G R S G ED N V S GG D S GG D I RR P GGG SS G S M Q AG R P Q F ED LL S DD P G I N D R V N A F R E Y R P G D GG V G E Ap NorV2-VirB6-3 E G S D S V Q GA S N T R GAGAG E ---G P GA P V D N D G S N V G R S G ED N V S GG D R GG D I RR P GGG SS G S M Q AG R P Q F ED LL S DD P G I N N R V N A F R E Y R P G D GG V G E Ap Var1-VirB6-3 E G S D S V Q GA S N A R GAGAG E ---G P GA P V D N D G S N V G R S G ED N V S GG Y S GG D I RR P GGG SS G S M Q AG R P Q F ED LL S DD P G I N D R V N A F R E Y R P G D GG V G E Ap NorV1-VirB6-3 E G S D S V Q GA S D A R R AG V G AHADV G P GA S V D N D S S N V G R S G ED N I S GG D S GG D I RR P GG R SS G GVR A EGS Q L ED LL G DD L G I Q D R V S A F K E Y H P G D G V G E Consensus/80% EGSDSVQGASNARGAGAGE....GPGAPV DNDGSNVGRSGEDNVSGGDSGGDIRRPGGGSSGSMQAGRPQFEDLLSDDPG INDRVNAFREYRPGDGGVGE Ap HZ-VirB6-3 D D R I S G STT AG S G V S GG E A D V D A R V S H S ---------------------------------------R E Y R P G D G G V G E G D D R V Y G S T T AG S G I S G D A Ap Dog1-VirB6-3 D D R I S G STT AG S G V S GG E A D V D A R V S H S ---------------------------------------R E Y R P G D G G V G E G D D R V Y G S T T AG S G I S G D A Ap Dog2-VirB6-3 D D R I S G STT AG S G V S GG E A D V D A R V S H S ---------------------------------------R E Y R P G D G G V G E G D D R V Y G S T T AG S G I S G D A Ap JM-VirB6-3 D D R I S G STT AG S G V S GG E A D V D A R V S H S ---------------------------------------R E Y R P G D G G V G E G D D R V Y G S T T AG S G I S G D A Ap MRK-VirB6-3 D D R I S G STT AG S G V S GG E A D V D A R V S H S ---------------------------------------R E Y R P G D G G V G E G D D R V Y G S T T AG S G I S G D A Ap NorV2-VirB6-3 D D R I S G STT AG S G I S G DA A G V D A R V S H S REYRPGDGGVGEDDRISGSTTAGSGISGDAAGVDARVSHS R E Y R P G D G G I G E G E D R V Y G S T T AG S G I S G D A Ap Var1-VirB6-3 D D R I S G STT AG S G V S GG E A D V D A R V S H S REYRPGDGGV-----------------------------------------------------------Ap NorV1-VirB6-3 D V D R VH G PA T AG --S GG E A D V D A R V S H F---------------------------------------R E Y S P G G E G V V E V D D R V S G S N T AG G G I S G D E Consensus/80% D.DRISGSTTAGSGlSGGEADVDARVSHS ........................................REYpPGs.Gls Es-DRV.GSsTAGtGISGD. Ap HZ-VirB6-3 AG I D A R G D H A R E H Q P A D V G V G E G A D R V S G S A T AG S G I S G D A V G V D A R G D H A R E H Q P A D V G V G E GA D R V S G S A T AG S G I S G DE AG V D A R G D H A R E HR L ED Ap Dog1-VirB6-3 AG I D A R G D H A R E H Q P A D V G V G E G A D R V S G S A T AG S G I S G D A V G V D A R G D H A R E H Q P A D V G V G E GA D R V S G S A T AG S G I S G DE AG V D A R G D H A R E HR L ED Ap Dog2-VirB6-3 AG I D A R G D H A R E H Q P A D V G V G E G A D R V S G S A T AG S G I S G D A V G V D A R G D H A R E H Q P A D V G V G E GA D R V S G S A T AG S G I S G DE AG V D A R G D H A R E HR L ED Ap JM-VirB6-3 AG I D A R G D H A R E H Q P A D V G V G E G A D R V S G S A T AG S G I S G D A V G V D A R G D H A R E H Q P A D V G V G E GA D R V S G S A T AG S G I S G DE AG V D A R G D H A R E HR L ED Ap MRK-VirB6-3 AG V D A R G D H A R E H Q P A D V G V G E G A D R V S G S A T AG S G I S G D A A G V D A R G D H A R E H Q P A D V G V G E GA D R V S G S A T AG S G I S G DE AG V D A R G D H A R E HR L ED Ap NorV2-VirB6-3 AG V D A R G G H A R E H Q P A D V G V G E D A D R V S G S A T AG S G I S G D E A G V D A R G D H A R E H R P A D V G V G G E D A D R V S G S A T AG S G I S G DE AG V D A R G D H S R E HR L ED Ap Var1-VirB6-3 ------------------------------------------------------------G E GA D R V S G S A T AG S G I S G D A AG V D A R G D H A R E HR L ED Ap NorV1-VirB6-3 AG V D A R G D Y V R E H Q P A D V G V G E G A D P G S G P A T AG S G I S S D A A G V D A R G D Y V R E H Q P A D V G V G E GA D PG S G S A T AG G G I S G DE AG V D A R G D H S R E HR L ED Consensus/80% AGlDARGsasREHQPADVGVGEsAD.sSG sATAGSGIStD.sGVDARGDasREHpPADVGV.GEGADRVSGSATAGSGIS GDEAGVDARGDHtREHRLED Ap HZ-VirB6-3 G D T GAG V D N S D A S A D V N G D K V R GA V R DD L K D G K DD K Ap Dog1-VirB6-3 G D T GAG V D N S D A S A D V N G D K V R GA V R DD L K D G K DD K Ap Dog2-VirB6-3 G D T GAG V D N S D A S A D V N G D K V R GA V R DD L K D G K DD K Ap JM-VirB6-3 G D T GAG V D N S D A S A D V N G D K V R GA V R DD L K D G K DD K Ap MRK-VirB6-3 G D T GAG V D N S D A S A D V N G D K V R GA V R DD L K D G K DD K Ap NorV2-VirB6-3 G D T GAG V D N S D A S A D V N G D K V R GA V R DD L K D G K DD K Ap Var1-VirB6-3 G D T GAG V D N S D A Y A D V N G D K V R GA V R DD L K D G K DD K Ap NorV1-VirB6-3 G D T GAG V D N S D A S A D V SD D K V R GA V R DD L K D G K DD K Consensus/80% GDTGAGVDNSDASADVNGDKVRGAVRDDL KDGKDDK



PAGE 1

RESEARCHARTICLEOpenAccessStructureofthetypeIVsecretionsystemin differentstrainsof AnaplasmaphagocytophilumBasimaAl-Khedery1,AnnaMLundgren1,SnorreStuen2,ErikGGranquist2,UlrikeGMunderloh3,CurtisMNelson3, ARickAlleman4,SumanMMahan5andAnthonyFBarbet1*AbstractBackground: Anaplasmaphagocytophilum isanintracellularorganismintheOrder Rickettsiales thatinfectsdiverse animalspeciesandiscausinganemergingdiseaseinhumans,dogsandhorses.Differentstrainshaveverydifferent celltropismsandvirulence.Forexample,intheU.S.,strainshavebeendescribedthatinfectruminantsbutnotdogs orrodents.Anintriguingquestionishowthestrainsof A phagocytophilum differandwhatdifferentgenomeloci areinvolvedincelltropismsand/orvirulence.TypeIVsecretionsystems(T4SS)areresponsiblefortranslocationof substratesacrossthecellmembranebymechanismsthatrequirecontactwiththerecipientcell.Theyareespecially importantinorganismssuchasthe Rickettsiales whichrequireT4SStoaidcolonizationandsurvivalwithinboth mammalianandtickvectorcells.WedeterminedthestructureoftheT4SSin7strainsfromtheU.S.andEurope andrevisedthesequenceoftherepetitive virB6 locusofthehumanHZstrain. Results: AlthoughinallstrainstheT4SSconformstothepreviouslydescribedsplitlocifor vir genes,thereisgreat diversitywithintheselociamongstrains.Thisisparticularlyevidentinthe virB2 and virB6 whicharepostulatedto encodethesecretionchannelandproteinsexposedonthebacterialsurface. VirB6 4 hasanunusualhighly repetitivestructureandcanhaveamolecularweightgreaterthan500,000.Formanyofthe virs ,phylogenetictrees position A phagocytophilum strainsinfectingruminantsintheU.S.andEuropedistantfromstrainsinfecting humansanddogsintheU.S. Conclusions: OurstudyrevealsevidenceofgeneduplicationandconsiderablediversityofT4SScomponentsin strainsinfectingdifferentanimals.Thediversityin virB2 isinboththetotalnumberofcopies,whichvariedfrom8 to15inthehereincharacterizedstrains,andinthesequenceofeachcopy.Thediversityin virB6 isinthesequence ofeachofthe4copiesinthesinglelocusandthepresenceofvaryingnumbersofrepetitiveunitsin virB6 3 and virB6 4 .ThesedatasuggestthattheT4SSshouldbeinvestigatedfurtherforapotentialroleinstrainvirulenceof A phagocytophilum Keywords: Anaplasma phagocytophilum Rickettsiales ,T4SS,ComparativegenomicsBackgroundAnaplasmaphagocytophilum isatick-bornepathogenin theOrder Rickettsiales thatisincreasinglyrecognizedas acauseofdiseaseinhumansandanimalsworld-wide [1,2].Itcausesthepotentiallyfataldiseaseofhuman granulocyticanaplasmosis,whichtypicallymanifestsasa flu-likeillnessaccompaniedbyleukopenia,thrombocytopeniaandanemia.Itwasinitiallyrecognizedinthe early1990'swhenpatientsfromWisconsinandMinnesota developedfebrileillnessfollowingatickbite[3].Sincethat timethenumberofhumancaseshasincreasedannually; between2000and2007thereportedincidenceintheU.S. increasedfrom1.4to3.0cases/millionpersons/year[4]. Thecasefatalityratewas0.6%andthehospitalization ratewas36%.InMassachusettsduringthe2009transmissionseasontherewere33confirmedcaseswith14 (42%)requiringhospitalization[5].Thehumandiseaseis alsopresentinEuropeandAsia[2].Arecentstudyof83 A phagocytophilum -infectedpatientsinChinareported amortalityrateinthiscohortof26.5%[6].IntheU.S., therehasbeenaparallelincreaseincasesofthedisease [7]andseroprevalence[8]indogsintheeasternand *Correspondence: barbet@ufl.edu1DepartmentofInfectiousDiseasesandPathology,CollegeofVeterinary Medicine,UniversityofFlorida,Gainesville,FL,USA Fulllistofauthorinformationisavailableattheendofthearticle 2012Al-Khederyetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Al-Khedery etal.BMCGenomics 2012, 13 :678 http://www.biomedcentral.com/1471-2164/13/678

PAGE 2

upperMidwesternstates.ThetickvectorsintheU.S. are Ixodesscapularis and Ixodespacificus andwild rodentsarethemainreservoirsofhumaninfections. A phagocytophilum alsoinfectsnumerousothermammalianspeciesincludingruminants,horses,cats,and bearsandthesymptomsareextremelyvariable,with somemammalianspeciesexhibitingacutediseaseand othersonlypersistentasymptomaticinfections[9,10]. Forexample, A phagocytophilum strainsisolatedfrom deerintheU.S.canhaveaslightlydifferent16S rRNAsequenceandbeuninfectivetomiceanditis thought,humans[11-13].InEurope,thisagenthasbeen knowntocausediseaseofruminantsfor>100years,yet therehavebeenfewhumaninfections[14].Thegenome sequenceisavailableforasinglestrainof A phagocytophilum derivedfromaninfectedhumanintheU.S.and itisapparentthat,althoughthisstrainlacksTypeII,III, VandVIsecretionsystems,aTypeIVsecretionsystem (T4SS)ispresent[15].Asinothermembersofthe Rickettsiales ,theT4SSof A phagocytophilum isorganized differentlyfrommostgram-negativebacteriawiththe component vir genesdistributedbetweenthreemajor genomelocations[16]. TheT4SStypicallyencodesamembrane-spanning multiproteincomplexthatformsatransmembrane channelthroughwhichsolutescanpassintohostcells. ItcanmediatetransferofDNAandproteinsinto eukaryotichostcells,interferewithhostsignaling,andis essentialforthesurvivalofintracellularbacteria[17].In A phagocytophilum ,whichpreferentiallycolonizeneutrophilicwhitebloodcells,itisthoughtthattheT4SS secretesvirulencefactorsthatareresponsibleforsubvertinginnateimmunityandinhibitinghostcellapoptosis[16].Interestingly,thereappearstobedifferential transcriptionoftheT4SSinticksandinthemammalian hostwith virB6 and virB9 upregulatedduringinfection ofhumanneutrophilsanddifferent virB2 paralogs expressedinmammalianandtickcells[18].ThereisevidencethatVirB2,VirB6andVirB9areexposedonthe outermembranesurfaceinthe Rickettsiales [18-20], whichhasstimulatedinterestintheirpotentialuseas vaccinecandidates.Thispossibilityhasbeeninvestigated moreextensivelyintherelatedorganism Anaplasma marginale [21-25].In A marginale ,unlikemanyother surface-exposedproteins,theT4SSproteinsareconservedbetweenstrains[26].Also,cattleimmunizedwith outermembranesandprotectedagainstchallengeinfectionrespondwithIgGandTcellstoVirproteins,notablyVirB2,VirB9andVirB10.Todate,onlytwoT4SS substrateshavebeenidentifiedandpartiallycharacterized in A phagocytophilum :theankyrinrepeatdomaincontainingprotein,AnkA,andthe Anaplasma translocatedsubstrate1,Ats-1.AnkAtranslocatestothehost nucleusandinteractswithDNA[27,28],whileAts-1is importedintothemitochondriawhereitisproposedto interferewiththeinductionofapoptosis[29]. Inthisstudy,wecomparedthestructureanddiversity oftheT4SSindifferentstrainsof A phagocytophilum infectinghumans,dogs,rodentsandruminants.Most diversitywasfoundintheproteinsthoughttobesurfaceexposed,whichmaybeassociatedwiththedifferent virulenceandcellinvasionpropertiesofthisspecies.ResultsanddiscussionThe vir lociweresequencedineightstrainsof A phagocytophilum ;sevenofthesewerestrainsforwhichpreviousstructuralinformationwasnotavailableand includedorganismsoriginallyisolatedfromU.S.dogs ( Ap Dog1, Ap Dog2),arodent( Ap JM),ahorse( Ap MRK), theruminant Ap variant1strain( Ap Var1)andtwo strainsfromNorwegiansheep( Ap NorV1, Ap NorV2). ThehumanHZstrainwasalsoresequenced,asoptical mappinghadsuggestedapossibleerrorinthepreviously sequenced virB6 4 locus.Thedataindicatedconsiderablediversityintheindividual vir locibetweenstrains thatwillbediscussedbelow.Inallstrains,however,as notedpreviously[20,30],the vir lociweredistributed mainlyinthreegeneclusterscomprising: virB8 1 virB9 1 virB10 virB11 and virD4 ; virB2 0 sand virB4 2 ;and virB3 virB4 1 ,andthefour virB6 paralogs(Figure1). Thesethreelocimayeachbetranscribedpolycistronically[31],althoughitisclearthatT4SSstructureinthe Rickettsiales isuniqueandmorecomplexthaninitially thought.Thenumberof virB2 paralogswasdifferentbetweenstrainswiththehumanHZstrainhavingtheleast (8totalparalogs)andtheruminantstrainshavingthe most(upto15totalparalogs).Thedescriptionofthe T4SScomponentspresentedherefollowsthefunctional classificationdescribedbyAlvarez-MartinezandChristie [20].Innermembranechannel/scaffoldsubunits:VirB3,VirB6, VirB8andVirB10ThemostconservedofthesesubunitsareVirB3,VirB8 andVirB10,withfewdifferencesbetweenstrains.VirB3 hasbeenlinkedin Agrobacteriumtumefaciens withpilus assemblyandsubstratetranslocation[32,33].Itisabsolutelyconservedbetweenstrainswithnoaminoacid changesandconformstothetypicalVirB3structure. Twoalpha-helicaldomainsforinsertionintothecytoplasmicmembranearestronglypredictedbyTMpred. VirB8,proposedtofunctionasanucleationfactorduringtheassemblyofT4SS[34,35],isalsowellconserved, particularlyVirB8-1inthepolycistronictranscription locus(oneaminoacidchangebetweenallstrains). VirB10,proposedtofunctionasascaffoldacrossthe entirecellenvelope[36],isalsogenerallywellconservedwiththeexceptionofoneruminantstrain,Al-Khedery etal.BMCGenomics 2012, 13 :678 Page2of15 http://www.biomedcentral.com/1471-2164/13/678

PAGE 3

Ap NorLamb-V1,whichhas31aminoacidsubstitutions withrespectto Ap HZ(datanotshown).However,all A phagocytophilum VirB100s,including Ap NorLamb-V1, havetwostronglypredictedtransmembranedomains, whichsupportstheirfunctionasmembranescaffolding subunitsintheseorganisms. Oftheseinnermembranechannelsubunits,thedata onVirB6arethemostinteresting.AllVirB6subunits thathavebeendescribedpossessahighlyhydrophobic membranedomainincludingfiveormorepredicted transmembranedomains[20].SomeVirB6proteinsalso haveanextendedC-terminalhydrophilicdomainthat hasbeenproposedtoprotrudethroughtheT4SSinto thetargetcell,ormaybeproteolyticallyreleasedfrom theN-terminaldomainandthentranslocatedintothe targetcell.Evidencehasbeenobtainedforsurface virB9 -2 v i rB 3 v i rB 4 1 virB6 -1 v i rB 6 -2 virB6 -3 v i rB 6 4 virB4 t v i rB8 -2 virB 7 virB4 -2 virB2 -8 virD4 v i rB9 -1 v i rB 8 1 v i rB 6-t virB4 t additionalvirB4-t in ApNorLamb-V1 and -V2: v i rB2 -7 v i rB 2 6 v i rB 2 5 v i rB 2 -4 v i rB2 -3 v i rB 2 2 v i rB 2 1 oriC v i rB11 v i rB10 B2-8B2-7B2-6B2-5 B2-9 B2-4B2-3B2-2B2-1ApHzApJMApDog1ApDog2ApMRKApNorLamb-V2ApVar-1ApNorLamb-V1 ? ? 1kb 1kb Figure1 Distributionandcontentof vir geneclustersineightdiverse A.phagocytophilum strains. Toppanel.Schematicrepresentationof all vir loci(coloredarrows)showingthethreeconservedgeneclusterislands(seetext). VirB 7 virB8 2 and virB9 2 arenotpartof vir geneclusters, buttheirlocationrelativetosurroundinggenesisalsohighlyconservedamongstrains.Asmallclustercomprisingtruncated(t) virB6 and virB4 genefragmentsispresentinallstrains,buttheNorwegianlambstrainshaveoneadditional virB4 t .Bottompanel.Magnificationofthe virB2 gene cluster.Numberingofparalogs1 – 8isbasedontheoriginal Ap HZannotatedgenome(GenBankCP000235).Artificialgaps(stippledlines)were introducedtoallowalignmentofthemorespatiallyconservedparalogs B2 1 2 – 2 and 2 – 3 atoneend,and B2 7 and 2 – 8 attheotherendofthe cluster.Withtheexceptionof virB2 9 ,lackingin Ap HZ,thenumberandarrangement(butnotnecessarilysequence)of virB2 genesishighly conservedinallbuttheUSruminant Ap Var-1and Ap NorLamb-V1,whichhaveseveraladditional virB2 genes.Inbothstrainsasub-clusterof6 distinctgeneswaspresent.Duetotherepetitivenatureofsequencesinthisregion,combinedwiththerelativelyshortlengthof454reads ( 550bp),theirplacementcouldnotbeconfidentlyascertained(highlightedbyarrowsand ‘ ? ’ ).Mapsaredrawntoscale.Doublelinesdesignate interruptioninsequences.Genesbelongingtothesamegroupinghavethesamecolor. oriC ;originofreplication. Figure2 PhylogenetictreetoshowtherelationshipofsyntenicVirB6proteinsfromdifferentstrainsof A phagocytophilum Ascalebar isshownunderneathrepresentingthenumberofaminoacidsubstitutions/site. Al-Khedery etal.BMCGenomics 2012, 13 :678 Page3of15 http://www.biomedcentral.com/1471-2164/13/678

PAGE 4

exposureofextendedVirB6insome Rickettsiales [37]. Ofallthemembranechannelsubunits,themostsequencediversitybetween A phagocytophilum strains wasinthefourVirB6paralogs(Figure1).Although therewerenoaminoacidchangesintheVirB6-1,VirB62andVirB6-3paralogsbetweenhuman,dogandrodent strains,theruminantandhorsestrainshadnumerous substitutionsthroughouteachmolecule,agreeingwith thecloserevolutionaryrelationshipbetweenstrains infectinghumansanddogsintheU.S.(Figure2).Furthermore,majordifferencesinrepeatnumberandsequencewerefoundintheC-terminalrepeatregionof VirB6-3(yellowboxesinFigure3AandAdditionalfile1: FigureS1)inruminantandhorsestrains,withthehorse strainshowingtheleastvariabilityfrom Ap HZ. Theonlyaminoaciddifferencesdetectedbetweenthe human,dogandrodentstrainswereintheVirB6-4subunit. VirB6 4 inthesestrainscontainsfourrepeat regions(R1-R4inFigure3A)andvariabilityinrepeat number,orderandsequencewerefoundmainlyinR3 andR4(Additionalfile2:FigureS2).WithinR1 (Figure3A),theonlydifferencedetectedwasin Ap Dog2 whichhad4and1partialof231bprepeatunits(data notshown),comparedto3and1partialrepeatsinthe Ap Dog1, Ap JMand Ap HZ virB6 4 R1.Opticalmapping oftheDog1genomeandcomparisonwith Ap HZsuggestedthatthesequenceobtainedpreviouslyforthe humanHZstrain virB6 4 wasincorrect(Figure3B).This wasconfirmedbyPCRandsequencing,andmapped specificallytothe30-mostR4region(Figure3C).Becauseofitssizeandunusualcompositionitwasonly possibletoresolvethissequenceusingthelongreadlengthPacificBiosciencestechnology(seeMethods). Thecorrected virB6 4 R4of Ap HZ,totaling6.89kb,differedfromtheoriginalby5.88kbofadditionalsequence composedexclusivelyof84bp[type1,aandb(T1a, T1b);light/darkblueboxes,respectively,inFigure3A] and162bp[type2,aandb(T2a,T2b);light/darkorangeboxes,respectively,inFigure3A]repeatunits,givingacomplexrepeatstructurecontaining53and1 4 6 B r i v 3 6 B r i v 2.7kb 1.15kb virB6-4 inCP000235 R1 R2 4 R 3 R AB1393 AB1395 AB1466 AB1466Bam HI: 1kbCP000235 CP000235 corrected Dog1 optical map Dog1genome sequence 1kb kb 12 10 9 8 7 6 5 kb 48.5 24.5 17.0 13.8 12.1 10.2Ap Hz Ap Dog1 Ap Dog2 Ap JM Ap MRK Ap Var-1 Ap NorLamb -V2virB6-4 2.32 4.81 4.30 3.61 3.19 2.23 2.11 3.40 4.22 4.36 2.46 4.90 16.637.2516.687.38 4 11 Ap Hz Ap Dog1 Ap JM AB1395/1466:R4 AB1393/1466:R3 R4JMgenome sequence B A C R4:811p R4:531p R4:81p + + + + Figure3 The30endof A.phagocytophilumvirB6 4 genesiscomposedofanunusuallylargetandemrepeatregion,whichexhibits dramaticvariabilityamongstrains.A .MapofthehumanHZstrain virB6 3 and virB6 4 genes,highlightingthelocationandstructureofseveral repeatregions(R1-R4).ThemostvariabilityoccurredinR4;thisregionis5.88kblargerthanpreviouslyreportedforthe Ap HZgenome (CP000235).Theoriginalsequenceisdiagrammedabovethemap,withthedashedlinerepresentingthesegmentmissinginCP000235.Larger repeatedR4segmentsof2.7kband1.15kbareindicatedabove.Verticalblackbarswithineachgenedesignatesegmentsencodingpredicted transmembranedomains.BamHIsites,ofwhichthereisoneinallR4type2repeats(seeFigureS2B),areindicated.Alsoshownarethepositions ofPCRprimersusedinC. B .BamHIgenomicmapsdepictingthe virB6 4 locus(blackarrows).ThesegmentencompassingR4ishighlightedbelow eachrespectivemap.Intheregionsoutsidethe virB6 4 locus,correspondingBamHIfragmentsareshowninthesamecolor.Overall,theoptical mapsizeswereingoodagreementwiththeactualsizes,exceptwithinR4.Thisisattributedtothelimitationofopticalmappinginresolving fragments<2kb.Despitethesediscrepancies,thecumulativesizeofthegenomicregionencompassing virB6 4 intheopticalmapisinclose agreementwiththatinthe Ap Dog1genomesequence. C .ThevariabilityinsizeofPCRproductsspanning virB6 4 repeatregionsR4andR3/R4in diverse A phagocytophilum strains. Al-Khedery etal.BMCGenomics 2012, 13 :678 Page4of15 http://www.biomedcentral.com/1471-2164/13/678

PAGE 5

partialrepeatunitscomparedto8and1partialinthe originalsequence.Further,the50-and30-most2.7kbof thiscomplexstructureareidenticalinsequence,andthe 30-most1.15kbofeachofthesesegmentsisrepeated againinthecenterofR4(Figure3AandAdditionalfile 2:FigureS2).Althoughthepossibilityexiststhatthe Ap HZpopulationfromwhichweisolatedgDNAdiffers withinthe virB6 4 R3/R4repeatregionsfromthepopulationusedtogenerateCP000235,thefactthatall strainsinvestigatedhereinpresentedexpansiveR3/R4 regions(Figure3C)wouldcontradictthat.Instead,itis moreplausiblethattheexistenceof2.7kbofidentical repeatsattheendsofthe Ap HZR4mayhaveleadto theexcisionofmostofitssequenceduringconstruction/propagationofthoselibraries.Interestingly, virB6 4 R3andR4wereidenticalbothinsizeandsequencein theDog1androdentstrainsdespitedifferingmarkedly fromtheHZandDog2strainregions(Additionalfile2: FigureS2A).WithinR3,thesestrainshad2additional 405bprepeatscomparedto Ap HZandonemorecomparedtotheDog2strain.However,differencesbetween strainsweremostdramaticwithinR4.Notonlywasthis regionin Ap Dog1/ Ap JM2.87kblargerthanin Ap HZ bringingthetotalnumberofrepeatsto81and1partial, butintriguingly,therepeatpatternwascompletelyunrelatedtothatintheHZstrain.Also,theDog1androdent strainR4lackedT1brepeatunits,whilehavingathird type2repeatvariant,namelyT2c,whichdifferedfrom T2bby1SNPanda12bpdeletion(Additionalfile2: FigureS2).Partialanalysisofthe Ap Dog2454reads spanningR4(estimatedat~8kbbyPCR;Figure3C) showedthattheorderofthe50-and30-mostthreerepeat unitsdifferedfromeithertheHZorDog1/rodentstrain R4repeatpatterns(Additionalfile2:FigureS2A).Notably,ourpreliminaryanalysesofthehorseandruminant 454readssuggesttheabsenceofdistinctR3andR4 regionsin virB6 4 inthesestrains.Rather,thefewrepeat unitsidentifiedtodateappeartobeacombinationofR3 andR4repeats(datanotshown).Itisalsounclearifthe ~17kband~25kbPCRproductsgeneratedwithprimersAB1393/1466in Ap Var-1and Ap NorLamb-V2,respectively(Figure3C),arecomposedmainlyofrepeats, oralternativelyifafifth virB 6 geneparalogexistsin thesestrains.Takentogether,thedatapresentedhere clearlydemonstratetheextremevariabilityoftheT4SS VirB6-4subunitamong A phagocytophilum strains.Althoughthedifferencesbetweenthemorecloselyrelated human,dogandrodentUSstrainsweremainlywithin repeat-ladenregions,thefactthatanextensive,distinct repeatpatternwasmaintainedintwostrainswould speakagainstthepossibilitythatthevariabilitymaybe attributedsolelytothehighlyrecombinogenicnatureof suchstructures.Worthnoting,CampRipley,wherethe infectedjumpingmousewascaptured(2001)isonly~20 milesawayfromthecityofBaxter,MN,whereDog1 resides.Althoughtherearenorecordsofwherethisdog mayhaveactuallyacquiredtheinfection,itpresented withsevereclinicaldiseasein2007. Theunusualstructureandlikelyantigenicityofthe C-terminalregionofthe A phagocytophilum VirB6-40sis apparentinhydrophobicityplots(Figure4).Whatspecificpropertiesthesedistinctrepeatpatternsmayconfer ontoeachstrainawaitsfunctionalanalysisoftheseproteinsin A phagocytophilum .ThecorrectedVirB6-4 translatedproteinhadapredictedmolecularweightof 470,695Dacontaining4,322aminoacidresiduescomparedtomolecularweightsof90,742,103,204and 158,321DafortheHZstrainVirB6-1,VirB6-2and VirB6-3,respectively.Interestingly,thepredictedacidity oftheVirB60salsoincreasedfromVirB6-1toVirB6-4 (pI ’ sof8.4,6.8,5.1and4.0forthe Ap HZVirB6-1,VirB62,VirB6-3andVirB6-4,respectively).The Ap Dog1/ Ap JM VirB6-4polypeptideshadapredictedmolecularweight of603,529Dacontaining5,550aminoacids,andapIof 3.96.Despitethesedissimilarities,atleasteighttransmembranesegmentswerepredictedforallVirB6 paralogs.Periplasmic/outermembranechannelsubunits:VirB2, VirB7andVirB9SeveralotherT4SSsubunitscontributetothesecretion channelacrosstheperiplasmandoutermembrane. VirB7subunitsaretypicallysmalllipoproteinsthatmay stabilizeVirB9[38,39].In A phagocytophilum strainsa putativeVirB7isabsolutelyconservedbetweenstrains andmaybelipidmodifiedthroughanN-terminalcysteineonthematuremolecule.VirB9ishydrophilicand alsolocalizestotheperiplasmandoutermembrane.In A tumefaciens theC-terminalregionofVirB9ispartof theoutermembraneproteinchannelandissurfaceaccessible[40].Thereisalsoevidenceforsurfaceexposure ofVirB9in Ehrlichiachaffeensis and A phagocytophilum [18,19,41].VirB9-1,whichisencodedonthepolycistronic virB8 1 virD4 transcript[31],hasastronglypredictedsignalpeptideandtwotransmembranehelices.Of allthepotentiallyexposedcomponentsoftheT4SS, VirB9of A phagocytophilum appearstobetheleastdiverseamongstrains.Therearesomeaminoacidsubstitutionsinruminantandhorsestrains(2 – 6total comparedto Ap HZ)butintheotherstrainsVirB90sare unchanged(datanotshown). UnlikeVirB90s,VirB20sarethemostdiverseofall T4SSsubunitsin A phagocytophilum ,intermsofboth copynumberandsequence.VirB2proteinsaretypically constituentsofpiliandofthesecretionchanneland theirdiversityin Anaplasma suggeststhepossibilityof exposed,antigenicallyvariablestructures.In A marginale ,VirB2isexpressedtogetherwiththemajorouterAl-Khedery etal.BMCGenomics 2012, 13 :678 Page5of15 http://www.biomedcentral.com/1471-2164/13/678

PAGE 6

membraneproteinMSP3onasequence-variablepolycistronictranscript[25,42].Themechanismofexpressionin A phagocytophilum isnotknown.VirB20sof othergeneraaretypicallysmallhydrophobicproteins withalongsignalpeptidesequenceandtwohydrophobicalphahelicesforintegrationintothecytoplasmic membrane.Thisalsoappearstobethecasefor A phagocytophilum .TheVirB2paralogsinthedifferent strainsarepredictedtohavetwohydrophobicalphahelicesoflengths22+/ 3and20+/ 0.2aminoacidsand signalpeptidesoflength27+/ 2aminoacids.Thisis truedespitetheirsequencediversity(Figure5).Aswith manyotherT4SScomponents,theruminantandhorse strainsaremoredistanttaxonomicallyinVirB2 Figure4 HydrophobicityplotsofVirB6-4proteinsfrom A.phagocytophilum HZ(top)orDog1(bottom)strains. Al-Khedery etal.BMCGenomics 2012, 13 :678 Page6of15 http://www.biomedcentral.com/1471-2164/13/678

PAGE 7

sequencecomparedtoVirB20sofhumananddog strains.AlignmentofallVirB2paralogsandorthologs showsthatsequencediversityisprimarilylocalizedto twohypervariableregionseitherprecedinganNterminalcysteineorclosetotheC-terminus(Figure6). Thisissimilartothehypervariableregionsfoundamong VirB2paralogsof A marginale [25].Energeticsubunits:VirB4andVirB11ATPasesaretypicallyusedinT4SStoenergizesubstrate transferandhavebeenfoundineveryT4SSdescribed. Ingram-negativebacteriathesearetypicallyintegral membraneproteinsencodedbygenesresidingupstream of virB2 (encodingpilin).Thisistrueforallstrainsof A phagocytophilum andithasbeensuggestedthatthisarrangementofmultiple virB2 paralogsand virB4 2 may allowassemblyofanantigenicallyvariablesurfaceorganelle[20].Theenergeticsubunititself,VirB4-2,is however,wellconservedbetweenstrains.Themostdistanttaxonomicrelationshipwasfoundbetweenhuman andruminantstrains(29totalaminoacidsubstitutions in Ap NorLamb-V1comparedto Ap HZ,Figure7).The otherenergeticsubunit,VirB11,wasalsowell-conserved betweenstrains(6aminoacidsubstitutionsbetween Ap NorLamb-V1and Ap HZ;datanotshown).Type4couplingprotein:VirD4Type4couplingproteinssuchasVirD4areATPases thatfunctioninsubstraterecognitionandtranslocation usingtheT4SS.Theyareassociatedwithmosteffector translocatorsystems.Theytypicallypossessaminimum oftwoN-terminaltransmembranedomains.Oftenmost heterogeneityexistsintheseN-terminalregions[20]. The A phagocytophilum VirD40sconformsomewhatto thisstereotypewiththreestronglypredictedN-terminal transmembranesegments.AswiththeotherATPasesof the A phagocytophilum T4SS,thereislittlevariationin VirD4,atotalof17aminoacidsubstitutionsofwhich4 areN-terminalbutmore(12)areC-terminal.Again,the evolutionaryrelationshipsamongVirD4sequencespositiontheruminantandhorsestrainsmoredistantlyto theU.S.dog,humanandrodentstrains(Figure8).ConclusionsA phagocytophilum representsarecentreclassificationof intracellularorganismsinfectingdifferentanimalspecies Figure5 PhylogenetictreestoshowtherelationshipofsyntenicVirB2proteinsfromdifferentstrainsof A.phagocytophilum Al-Khedery etal.BMCGenomics 2012, 13 :678 Page7of15 http://www.biomedcentral.com/1471-2164/13/678

PAGE 8

andhumansandcausingdiversediseasesymptomatology [43].Thesebacteriawerepreviouslyknownas Ehrlichia phagocytophila Ehrlichiaequi ,andtheagentofhuman granulocyticehrlichiosis.Despitethedifferenceswithin thisspecies,theoverallgenomestructureandsyntenyof theT4SSismaintained.However,genestructuralanalysis revealsevidenceofgeneduplicationandconsiderablediversityofT4SScomponentsinstrainsinfectingdifferent animals.Taxonomictreessuggestacloseevolutionaryrelationshipof A phagocytophilum strainsinfectingU.S. humans,miceanddogsandamoredistantrelationship withruminantandhorsestrains.Thisrelationshipisnot uniquetotheT4SSbutisalsosupportedbysimilartaxonomictreesofother A phagocytophilum proteinsofconservedmetabolicfunction(Figure9).WithintheT4SS multicomponentmembranecomplex,theenergeticand internalscaffoldingproteincomponentsarethemostconserved.Incontrast,componentsthatformtheproposed exposedstructuresoftheT4SS,suchasVirB2andVirB6, aremorevariable.T4SSareimportantvirulence determinantsofbacteria,thereforethesedifferencesmay resultinthedifferentinfectivityandvirulenceprofiles observedwithdifferentstrains.ItwillbeofinteresttodeterminethemoleculararchitectureofVirB6paralogsin differentstrains,includinginteractionswithotherT4SS componentsandeffectors.Oftheknownsurfaceexposed componentsoftheT4SS,VirB9isthemostconserved. Thisproteinhasbeenproposedasavaccinecomponent against A marginale andmayalsobesuitableagainst A phagocytophilum .MethodsA.phagocytophilum strains,cellculture,andexperimental infectionThe A phagocytophilum U.S.strainsHZ(human-origin, NY),MRK(horse-origin,CA),JM(rodent-origin,MN) andDog1(dog-origin,MN)werepropagatedinHL-60 cellsinRPMI-1640medium(ThermoFisherScientific, Inc.,Waltham,MA)supplementedwithfinal10%heatinactivatedfetalbovineserum(ThermoScientific)and ApDog1-VirB2-1 M-----------------M S N L T G F V A VL S V IMMF ------G V A G A ---IDAC-GVEPTAEKDHTVA V PI K -----GDV A V K SVSG V LQT V R R FC L P V M I G V VS G AV I ITV F G R S A W F A I A ML I VF SC IF L G GS EF I Q K F T E G V G D ----S A G T K H S R V I ASRL-----ApHz-VirB2-1 M-----------------M S N L T G F V A VL S V IMMF ------G V A G A ---IDAC-GVEPTAEKDHTVA V PI K -----GDV A V K SVSG V LQT V R R FC L P V M I G V VS G AV I ITV F G R S A W F A I A ML I VF SC IF L G GS EF I Q K F T E G V G D ----S A G T K H S R V I ASRL-----ApDog2-VirB2-1 M-----------------M S N L T G F V A VL S V IMMF ------G V A G A ---IDAC-GVEPTAEKDHTVA V PI K -----GDV A V K SVSG V LQT V R R FC L P V M I G V VS G AV I ITV F G R S A W F A I A ML I VF SC IF L G GS EF I Q K F T E G V G D ----S A G T K H S R V I ASRL-----ApJM-VirB2-1 M-----------------M S N L T G F V A VL S V IMMF ------G V A G A ---IDAC-GVEPTAEKDHTVA V PI K -----GDV A V K SVSG V LQT V R R FC L P V M I G V VS G AV I ITV F G R S A W F A I A ML I VF SC IF L G GS EF I Q K F T E G V G D ----S A G T K H S R V I ASRL-----ApVar1-VirB2-1 M-----------------M S N L T G F V A VL S V IMMF ------G V A G A ---IDAC-GVEPTAEKDHTVA V PI K -----GDV A V K SVSG V LQT V R R FC L P V M I G V VS G AV I ITV F G R S A W F A I A ML I VF SC IF L G GS EF I Q K F T E G V G D ----S A G T R H S R V I ASRL-----ApMRK-VirB2-1 I-----------------M S N L T G F V A VL S V IMMF ------G V A G A ---IDAC-GVEPTAEKDHTVA V PI K -----GDV A V K SVSG V LQT V R R FC L P V M I G V VS G AV I ITV F G R S A W F A I A ML I VF SC IF L G GS EF I Q K F T E G V G D ----S A G T R H S R V I ASRL-----A pNorV1-VirB2-1 M-----------------M S N L T G F V A VL S V IIMF ------G V A G A ---IDAC-GVEPTAEKDHTVA V PI K -----GDV A V K SVSG V LQT V R R FC L P V M I G V VS G AV I ITV F G R S A W F A I A ML I VF SC IF L G GS EF I Q K F T E G V G D ----S A G T R H S R V I ASRL-----A pNorV2-VirB2-1 M-----------------M S N L T G F V A VL S V IMMF ------G V A G A ---IDAC-GVEPTAEKDHTVA V PI K -----GDV A V K SVSG V LET V R R FC L P V M I G V VS G AV I ITV F G R S A W F A I A ML I VF SC IF L G GS EF I Q K F T E G V G D -----P A G T R H S R V I ASRL-----ApDog1-VirB2-2 ------------------M FG L T R F M A VL A L VVA L V G F GT S A F A S ---------------------T TG S -----DDV A A K VIC N VV V F V Q R L G L P IM T G VIL G A S I M A IF G K L A W A A I V ML V VF T A IFF G AG K L I Q K F A A G V G S DIIG K A D S F E C K GNGATTLS---ApJM-VirB2-2 ------------------M FG L T R F M A VL A L VVA L V G F GT S A F A S ---------------------T TG S -----DDV A A K VIC N VV V F V Q R L G L P IM T G VIL G A S I M A IF G K L A W A A I V ML V VF T A IFF G AG K L I Q K F A A G V G S DIIG K A D S F E C K GNGATTLS---ApHz-VirB2-2 ------------------M FG L T R F M A VL A L VVA L V G F GT S A F A S ---------------------T TG S -----DDV A A K VIC N VV V F V Q R L G L P IM T G VIL G A S I M A IF G K L A W A A I V ML V VF T A IFF G AG K L I Q K F A A G V G S DIIG G N A E S F E C K GNGATTLSS--ApDog2-VirB2-2 ------------------M FG L T R F M A VL A L VVA L V G F GT S A F A S ---------------------T TG S -----DDV A A K VIC N VV V F V Q R L G L P IM T G VIL G A S I M A IF G K L A W A A I V ML V VF T A IFF G AG K L I Q K F A A G V G S DIIG G N A E S F E C K GNGATTLSS--ApVar1-VirB2-2 ------------------M FG L T R F M A VL A L VVA L V G F GT S A F A N A ---STA--------------S AG S -----DDV A A K VIC N VV V F V Q R L G L P IM T G VIL G A S I M A IF G K L A W A A I V ML V VF T A IFF G AG K L I Q K F A A G V G S DIVG D TN S F E C K G G GGTVLK---ApMRK-VirB2-2 ------------------M F S L T R F M A VL A L VVA L V G VGT SD F A S A ---SAP--------------A TG S -----DDV A A K VIC N VV V F V Q R L G L P IM T G VIL G A S I M A IF G K L A W A A I V ML V VF T A IFF G AG K L I Q K F A A G V G S DIIG N TD S F E C K G G GQTVLGK--ApNorV2-VirB2-2 ------------------M FG L T R F M A VL A L VVA L V G F GT S A F A T------A---------------QYA S -----DDV A A K VIC N VV V F V Q R L G L P IM T G VIL G A S I M A IF G K L A W A A I V ML V VF T A IFF G AG K L I Q K F A A G V G S DIIG D A N S F E C R GNGETKLGG-SK ApNorV1-VirB2-2 ------------------M FG L T R F M A VL A L VVA L V G F GT S A F A T T ---GST---------------QYA S -----DDV A A K VIC N VV V F V Q R L G L P IM T G VIL G A S I M A IF G K L A W A A I V ML V VF T A IFF G AG K L I Q K F A A G V G S DIIG D A N S F E C R GNGETKLGGVSK ApDog1-VirB2-4 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G E ---I K A N D F D C K E V AEK------ApHz-VirB2-4 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G E ---I K A N D F D C K E V AEK------ApDog2-VirB2-4 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G E ---I K A N D F D C K E V AEK------ApJM-VirB2-4 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G E ---I K A N D F D C K E V AEK------ApDog1-VirB2-5 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A D ---TA----TN----------EEH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A N G V G D ---L K A TE F D C K E V TK-------ApJM-VirB2-5 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A D ---TA----TN----------EEH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A N G V G D ---L K A TE F D C K E V TK-------ApHz-VirB2-5 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A N G V G D ---L K A TE F D C K E V TK-------ApDog2-VirB2-5 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A N G V G D ---L K A TE F D C K E V TK-------ApMRK-VirB2-5 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A N G V G D ---L K A TE F D C K E V TK-------ApNorV2-VirB2-5 ------------------M A K V V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D DH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G D ---L N A N D F D C K T V TG-------ApDog1-VirB2-9 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G E ---L N A N D F D C K T V TG-------ApJM-VirB2-9 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G E ---L N A N D F D C K T V TG-------ApDog2-VirB2-9 ------------------M A K I V R F F T ST A G MFLL L L L CS H G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G E ---L N A N D F D C K T V TG-------ApMRK-VirB2-9 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------A EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G E ---L N A N D F D C K T V TG-------ApMRK-VirB2-4 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A D ---TA----TN---------A EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G E ---L N A N D F D C K E V AEK------ApNorV2-VirB2-4 ------------------M A K V V R F F T ST A G MFLL L L L CS Q G V A A G ---ASAG-TAN----------EEH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G E ---L N A N D F D C K S V SDK------ApNorV2-VirB2-9 ------------------M E K I V R F F T NT A G MFLL L L L CS Q G I A V G ---VAADQKAN----------EEH K -----KEE T S K VIC N A V S F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G D ---L N A N D F D C K E V AEK------ApVar1-VirB2-novel2 ------------------M V R I V R F F T ST A S MFLL L L L CS Q G V A A G ---VSAG-SPS---------V DH K -----NED T S K VIC N VV T F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A N G V G D ---L N A K D F D C K S V SNGK-----ApNorV1-VirB2-novel1 ------------------M A R I V K F L T HT T G MFLL L L L CS Q G V A A G ---ASTG-AQS---------A EH K -----NED T S K VIC N VV S F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G G---V N A D G F D C K D V ATKP-----ApNorV1-VirB2-novel5 ------------------M A R I V K F L T HT T G MFLL L L L CS Q G V A A G ---ASTG-AQS---------A EH K -----KED T S K VIC N VV M F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A E G V G G---V K GN D F D C K S V SDVK-----ApNorV1-VirB2-4 ------------------M V R I V R F F T ST A G MFLL L L L CS Q G V S A G ---ASAG-SLD---------D GH K -----NED T S K VIC N VV T F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G E ---I K A D G F D C K K V TG-------ApVar1-VirB2-novel6-partial ------------------M V R I V R F L T RT T G MFLL L L L CS Q G V A A G ---ASAD-KAN---------T DH K -----KED T S K VIC N VV T F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I ---------------------------------ApVar1-VirB2-4 ------------------M V R T V R F W T RI T G MFLL L L L CS Q G V A A G --------------------A EE H -----KGD T S K VIC N VV E F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G D ---L N A DK F D C K D V KGEGQHN--ApVar1-VirB2-novel4 ------------------M V K V V R F F T ST A G MFLL L L L CS Q G V A A ----DGA---------------KTD H -----DGA T S R VIC N VV E F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A D G V G N ---L D A K G F D C K N V KGDN-----ApVar1-VirB2-novel7-partial -----------------------------------------------------------------------------------------------P IM T G EF L G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A D G V G N ---L D A K G F D C K N V KGDN-----ApNorV1-VirB2-novel2 ------------------M V R I V R F L A RT T G MFLL L L L CN Q G I A S ----AVS--------------A DD H -----KGD T S R VIC N VV E F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A D G V G N ---L N A K G F D C K T V KGDN-----ApNorV1-VirB2-novel4 ------------------M A R I V K F L T RT T G MFLL L L L CN Q G I A V G --------------------A EE H -----KED T S K VIC N VV G F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A D G V G N ---L N A K G F D C K D V KGDK-----ApNorV1-VirB2-novel6 ------------------M A R I V K F L T RT T G MFLL L L L CN Q G I A V G --------------------A EE H -----KED T S K VIC N VV G F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A D G V G N ---L N A K G F D C K T V KGDN-----ApNorV1-VirB3-novel3 ------------------M A K V V R F F T ST T W MFLL L L L CN Q G I A G A VSPAVG---------------QGD H -----KGD T S K VIC N VV E F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A D G V G N ---L N A K G F D C K T V KGDN-----ApVar1-VirB2-5 ------------------M E K I V R F L MRT T G M C FL L L L CS Q G V A G A --GAAS--------------T DD H -----KGD T S K VIC N VV M F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G D ---L K A N D F D C K K V TT-------ApNorV1-VirB2-6 ------------------M E K I V R F L MRT T G M C FL L L L CS Q G V A V A --GAAL--------------A DD H -----KGD T S K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G D ---L K A N D F D C K K V TS-------A pVar1-VirB2-novel5 ------------------M V R I V R F L A RT T G M C FL L L L CS Q G V A E P --GTVS--------------A GD H -----KGD T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G D ---L K A D N F D C K T V QGDK-----ApVar1-VirB2-novel3 ------------------M V R I V R F L T RT T G MFLL L L L CS Q G V A V A SLGGTS--------------T DD H -----KGD T S K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G D ---L N A EK F D C K D V TKQ------ApNorV1-VirB2-5 ------------------M E K I I R F L MRT T G M C F V L L L CS H G I V A S --AAAA--------------A TD H -----NGA T S K VIC N VV E F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A K G V G G---L D A D N F D C S K V KDDASGSSSP ApVar1-VirB2-novel1 ------------------M V R I V R F L MRT T G M C F V L L L CS H G I A S A ---AAE--------------A TK H -----DGA T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G E ---L E A D N F D C S K V KDDASGSSSL ApDog1-VirB2-6 ------------------M A K I V R F F T ST V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G I G E ---L D A D N F D C S K V QAEESSSV-ApHz-VirB2-6 ------------------M A K I V R F F T ST V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G I G E ---L D A D N F D C S K V QAEESSSV-ApDog2-VirB2-6 ------------------M A K I V R F F T ST V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G I G E ---L D A D N F D C S K V QAEESSSV-ApJM-VirB2-6 ------------------M A K I V R F F T ST V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G I G E ---L D A D N F D C S K V QAEESSSV-ApMRK-VirB2-6 ------------------M A K V V R F F T ST V G MFLL L L L CS N G I A S A ---AAA--------------A TK H -----DGA T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G I G E ---L D A D N F D C S K V KAEESNSV-ApNorV2-VirB2-6 ------------------M A K V V R F F T ST V G MFLL L L L CS H G I A S A ---AAAV-------------A TK H -----DGA T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G I G E ---L D A D N F D C S K V KAEESNSVLP ApVar1-VirB2-6 ------------------M S K I V R F F T HT T C MFLL L F L CN Q G I A A A ---AEA----------------TK H -----DGA T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G I G E ---L D A D N F D C S K V KAEESNSV-ApDog1-VirB2-7 ------------------M A K V V R F F T ST V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A Q G V G E ---W E A EK F D C K D I KAG------ApJM-VirB2-7 ------------------M A K V V R F F T ST V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A Q G V G E ---W E A EK F D C K D I KAG------ApHz-VirB2-7 ------------------M A K V V R F F T ST V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P S I A ML I VF T A IFF G SS K I I G K F A Q G V G E ---W E A EK F D C K D I KAG------ApDog2-VirB2-7 ------------------M A K V V R F F T ST V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P S I A ML I VF T A IFF G SS K I I G K F A Q G V G E ---W E A EK F D C K D I KAG------A pMRK-VirB2-7 ------------------M A K V V R F F T ST V G MFLL L L L CS N G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P S I A ML I VF T A IFF G SS K I I G K F A Q G V G E ---W E A EK F D C K D I KAG------ApVar1-VirB2-7 ------------------M A K V V R F L T GT V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV I F I Q K L G L P IM T G VI M G SS I M A IF G R L A W P S I A ML I VF T A IFF G SS K I I G K F A Q G V G E ---W E A EK F D C K D I KAG------ApNorV2-VirB2-7 ------------------M G R I V R F L T RT V G MFLL L L L CS Q G I A S A ---AAA--------------D TD H -----SGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P S I A ML I VF T A IFF G SS K I I G K F A Q G V G E ---W E A EK F D C K D I KAG------ApNorV1-VirB2-7 ------------------M A K I V K F F MHT T C MFLL L F F CN Q G I A A A ---AT----------------HIEP-----KDPI S R VIC N VV I F I Q K L G L P IM T G VI M G SS I M A IF G R L A W HT I A T L V VF T A IFF G AG K I I S K I A S G I G G---L N A EK F D C K P G KAKKQEIWII ApDog1-VirB2-8 M-----------------F T N I L R S C V ISI I FFIF L I L PA V S V S A A ---PVAH-------------A AG D -----GEVI S K VIC N VV V F V Q R L G L P IM T G VIL G SS I M A V F G R L A W P A I V ML V VF T A IFF G AG K L I S K F A G G I S E ---L G A E D F D C R V L AGKNI----ApDog2-VirB2-8 M-----------------F T N I L R S C V ISI I FFIF L I L PA V S V S A A ---PVAH-------------A AG D -----GEVI S K VIC N VV V F V Q R L G L P IM T G VIL G SS I M A V F G R L A W P A I V ML V VF T A IFF G AG K L I S K F A G G I S E ---L G A E D F D C R V L AGKNI----ApJM-VirB2-8 M-----------------F T N I L R S C V ISI I FFIF L I L PA V S V S A A ---PVAH-------------A AG D -----GEVI S K VIC N VV V F V Q R L G L P IM T G VIL G SS I M A V F G R L A W P A I V ML V VF T A IFF G AG K L I S K F A G G I S E ---L G A E D F D C R V L AGKNI----ApMRK-VirB2-8 M-----------------F T N I L R S C V ISI I FFIF L I L PA V S V S A A ---PVAH-------------A AG D -----GEVI S K VIC N VV V F V Q R L G L P IM T G VIL G SS I M A V F G R L A W P A I V ML V VF T A IFF G AG K L I S K F A G G I S E ---L G A E D F D C R V L AGKNI----ApNorV2-VirB2-8 M-----------------F T N I L R S C V ISI I FFIF L I L PA V S V S A A ---PVAH-------------A AG D -----GEVI S K VIC N VV V F V Q R L G L P IM T G VIL G SS I M A V F G R L A W P A I V ML V VF T A IFF G AG K L I S K F A G G I S E ---L G A E D F D C R V L AGKNI----ApNorV1-VirB2-8 M-----------------F T N I L R S C V ISI I FFIF L I L PA V S V S A A ---PVAH-------------A AG D -----GEVI S K VIC N VV V F V Q K L G L P IM T G VIL G SS I M A V F G R L A W P A I V ML V VF T A IFF G AG K L I S K F A G G I S E ---L G A E D F D C R V L AGKNI----ApVar1-VirB2-8 M-----------------F T N I L R S C V ISM I FFIF L I L PA V S V S A A ---PVAH-------------A AG D -----GEVI S K VIC N VV V F V Q K L G L P IM T G VIL G SS I M A V F G R L A W P A I V ML V VF T A IFF G AG K L I S K F A G G I S E ---L G A E D F D C R V L AGKNI----ApHz-VirB2-8 M-----------------F T N I L R S C V ISI I FFIF L I L PA V S V S A A ---PVTH-------------A AG D -----GEVI S K VIC N VV V F V Q R L G L P IM T G VIL G SS I M A V F G R L A W P A I V ML V VF T A IFF G AG K L I S K F A G G I S E ---L G A E D F D C R V L AGKNI----ApDog1-VirB2-3 MDTQGRAIAEDRRSFARTF F N K K V F F L IIQ G S LFF V L L L IL D E A Y A G V ---AESN-LFPAVAQHG---S AT N -----EDV T S K VIC N VV K F V R G I G L P IM T G VIL G SS V M A IF G R L A W P A I A A L V I F T A V FF G A E K V I S K F T D G I S V---M Q TG N C D T I-----------ApHz-VirB2-3 MDTQGRAIAEDRRSFARTF F N K K V F F L IIQ G S LFF V L L L IL D E A Y A G V ---AESN-LFPAVAQHG---S AT N -----EDV T S K VIC N VV K F V R G I G L P IM T G VIL G SS V M A IF G R L A W P A I A A L V I F T A V FF G A E K V I S K F T D G I S V---M Q TG N C D T I-----------ApDog2-VirB2-3 MDTQGRAIAEDRRSFARTF F N K K V F F L IIQ G S LFF V L L L IL D E A Y A G V ---AESN-LFPAVAQHG---S AT N -----EDV T S K VIC N VV K F V R G I G L P IM T G VIL G SS V M A IF G R L A W P A I A A L V I F T A V FF G A E K V I S K F T D G I S V---M Q TG N C D T I-----------ApJM-VirB2-3 MDTQGRAIAEDRRSFARTF F N K K V F F L IIQ G S LFF V L L L IL D E A Y A G V ---AESN-LFPAVAQHG---S AT N -----EDV T S K VIC N VV K F V R G I G L P IM T G VIL G SS V M A IF G R L A W P A I A A L V I F T A V FF G A E K V I S K F T D G I S V---M Q TG N C D T I-----------ApMRK-VirB2-3 MDTQGRAIAEDRRSFARTF F N K K V F F L IIQ G S LFF V L L L IL D E A Y A G V ---AESN-LFPAVAQHG---S AT N -----EDV T S K VIC N VV K F V R G I G L P IM T G VIL G SS V M A IF G R L A W P A I A A L V I F T A V FF G A E K V I S K F T D G I S G---M Q TG N C D T I-----------ApNorV2-VirB2-3 MDTQGRAMAEDRRSFARTF F N K K V F F L IIQ G S LFF V L L L IL D E A Y A G V ---AESN-LFPAVAQHG---S AT N -----EDV T S K VIC N VV K F V R G I G L P IM T G VIL G SS V M A IF G R L A W P A I A A L V I F T A V FF G A E K V I S K F T D G I S G---M Q TG N C D T I-----------ApNorV1-VirB2-3 MDTQGRAMAEDRRSFARTF F N K K V F F L IIQ G S LFF V L L L IL D E A H A G V ---AESN-LFPAVAQHG---S AA N -----EDV T S K VIC N VV K F V R S I G L P IM T G VIL G SS V M A IF G R L A W P A I A A L V I F T A V FF G A E K V I S K F T D G I S G---M Q TG N C D T I-----------ApVar1-VirB2-3 MNTQGRAMAEDRRSFARTF F N K K V F F L IIQ G S LFF V L L L IL D E A H A G V ---SESN-LFPAVAQHG---S AA N EDVTSEDV T S K VIC N VV K F V R S I G L P IM T G VIL G SS V M A IF G R L A W P A I A A L V I F T A V FF G A E K V I S K F T D G I S G---M Q TG N C D T I-----------Consensus/80% ...................bsp lsbFhs..shbbbblhb..s.thAts.....................s..p........stKVICNVV.Fsp+lGLPIMTGVILGSSlMAIFGRLAWsAIAMLlVFTAIFFGttKlI.KFs.Gltp. ...hpA.sb-sp.h.......... Figure6 MultiplesequencealignmentofVirB2aminoacidsequencesfromdifferentstrainsof A.phagocytophilum Al-Khedery etal.BMCGenomics 2012, 13 :678 Page8of15 http://www.biomedcentral.com/1471-2164/13/678

PAGE 9

4mML-glutamine(Lonza,Rockland,ME),andinthe absenceofantibiotics. Ap HZand Ap MRKhavebeen describedpreviously[15,44].The Ap JMstrain(CR011258)originatedfromameadowjumpingmouse( Zapus hudsonius )trappedatCampRipley,MN[45].The Ap Dog1strainoriginatedfromthebloodofadogfrom Baxter,MNnaturallyinfectedwith A phagocytophilum asevidencedbythedetectionofdistinctivemorulaeina diagnosticbloodsample,andsequencingoftheExpressionSite-linked msp2 / p44 gene.Briefly,wholebloodwas collectedfromtheanimalwithEDTAasananticoagulant.Thebuffycoatlayerwascollectedafterlow-speed centrifugationofthewhole-blood,washedin1xphosphatebufferedsaline(PBS,Hyclone,cat.no.SH30256.01), thenaddedtoacultureofuninfectedHL-60cells.The culturewasleftundisturbedfor3days,afterwhichmorulaebegantoappear.The Ap Dog2strainalsooriginated fromaMNdogandwaspassagedtoandmaintainedin the Ixodesscapularis ISE6tickcelllineasdescribed[46]. TheApvariant1CRT35strain(tick-origin,MN), maintainedinISE6cells,hasbeendescribed[47].For DNAisolation,culturesweremaintaineduntil90-100% ofcellswereinfectedwithmaturemorulae.Cellswere pelletedbycentrifugationat2500xgfor20minat4C. Pelletsweregentlyresuspendedin1.5mlcoldPBS,transferredtoscrew-capmicrofugetubes,andcentrifugedat Figure7 PhylogenetictreestoshowtherelationshipofsyntenicVirB4proteinsfromdifferentstrainsof A.phagocytophilum Figure8 Phylogenetictreetoshowtherelationshipofsyntenic VirD4proteinsfromdifferentstrainsof A.phagocytophilum Al-Khedery etal.BMCGenomics 2012, 13 :678 Page9of15 http://www.biomedcentral.com/1471-2164/13/678

PAGE 10

1500xgfor20minat4C.Supernatantswereremoved andthecellpelletsstoredat 80Cuntilfurtheruse. TwonaturallyoccurringNorwegianlamb A phagocytophilum strainsdifferinginthe 16S rRNAgeneanddegreeofvirulencewereusedtoexperimentallyinfect lambsraisedinanindoorenvironmentwithbarriers againsttickentryandtickinfestation.Lamb00186was infectedwiththemorevirulentvariant1(identicalto GenBankM73220)andlamb0054withvariant2(identicaltoGenBankAF336220)[48],tobereferredtoas Ap NorLamb-V1and-V2fromhereon.Infectionswere monitoredbymicroscopyandbloodwasharvestedat maximumparasitemia.Topurifybuffycoatscontaining theinfectedneutrophils,approximately2.5lofNacitratedbloodwascollectedfromeachanimal.The bloodwastransferredto1lcentrifugebottlesandcentrifugedat2,500-3,000xginaswing-outbucketrotor for30minat4C.Afterremovingmostoftheplasma layer,thebuffycoatlayerwascollectedwithminimal contaminationofredbloodcells.Thecellswerediluted 1:3withPBS,mixedgentlyandcentrifugedat1,500xg for20minat4C.FollowingthreePBSwashes,supernatantswereremovedandthecellpelletsstoredat 80C. Theexperimentalstudyinsheepwasapprovedbythe NorwegianAnimalResearchAuthority.Purificationofhostcell-free A phagocytophilum and genomicDNA(gDNA)isolationFortheHZ,JM,Dog1,MRKandNorLamb-V1and-V2 strains,intact,hostcell-freeorganismswithminimal Figure9 Phylogenetictreetoshowtherelationshipofotherconservedproteinsfromdifferentstrainsof A.phagocytophilum. These proteinsare:PolA,DNApolymeraseI;LeuS,leucyl-tRNAsynthetase;AtpA,ATPsynthaseF1,alphasubunit;ValS,valyl-tRNAsynthetase;RecG, ATP-dependentDNAhelicase;LigA,NAD-dependentDNAligase. Al-Khedery etal.BMCGenomics 2012, 13 :678 Page10of15 http://www.biomedcentral.com/1471-2164/13/678

PAGE 11

hostcellgDNA/RNAcontaminationwerepurifiedfrom frozenPBSpelletsofinfectedcellspreparedasabove. Samplesandreagentsweremaintainedonicethroughouttheentireprocedure,andallcentrifugationsperformedat4C.Followingaquickthaw,hostcellswere disruptedbyvigorousvortexingfor5min.AnequalvolumeofPBSwasaddedandvortexingcontinuedfor 3min.Cellulardebriswasremovedbycentrifugationat 200xgfor15min.Afterremovingmostofthesupernatantstofreshtubes,thesewerepassedseveraltimes througha31Gneedleandsavedonice.Pelletswere resuspendedwellinfinal500 lPBSthenpassedserially through22G,25G,28Gand,whenpossible,31Gneedlesattachedtoa1mlsyringe.3 – 5volumesPBSwere addedandmixedbyvortexing.Debriswasremovedby centrifugationat200xgfor10min.Supernatantswere pooledtothosefromthepreviouscentrifugationstep. RNaseAwasaddedtoafinal250 – 300 g/mlandthe samplesincubated45 – 60minat37C.Sampleswere centrifugedat21,000xgfor30minandthesupernatantsremovedcompletely.Pelletswereresuspendedin 50 – 100 lPBSeachandtransferredtofreshtubes.To ensurehomogeneityofthesuspension,initiallyadrawnout10 lpipettetipwasusedtodisruptthepelletby swirlingfollowedbyup/downpipettingandgentlevortexing,beforeswitchingtoalargertip.Thesamplewas furtherhomogenizedbyseveralpassesthrougha2831Gneedle.PBSwasaddedtofinal500 – 700 land DNaseItofinal250 g/ml.Following45 – 60minincubationat37Cthesampleswerecentrifugedat21,000xg for30min.Pelletswerehomogenizedasaboveandthe DNaseItreatmentrepeated.EDTA(pH8.0)wasadded tofinal25mMandthesamplescentrifugedasabove. TubeswerewashedtwicewithPBSwithoutdisturbing thepelletsandresidualPBSwasremovedafter3min centrifugationat21,000xg.Pelletswerehomogenized asabovein600 – 800 lRPMIculturemedium(containing10%fetalbovineserum)addedincrementallyand transferredtoa50mltube.Culturemediumwasadded toafinalvolumeof6mlbeforepassagethroughaprewet,2 mpore-size,25mm,GMF-150glassmicrofiber syringefilter(Puradisc25GD;WhatmanInc.,Florham Park,NJ).Thefilterwaswashed3-4xwithculture medium.Washeswerepooledtothefiltrateandcentrifugedat22,000xgfor30min.Thepellets,comprisedof free,non-viableorganismsandhostcellmitochondria, wereresuspendedinPBS,transferredtomicrofugetubes andre-pelletedat21,000xgfor30min.Supernatants wereremovedcompletelyandthepelletswereprocessed immediatelyorstoredat 20C.Forevery108hostcells usedat90-100%infectivity,enoughorganismswere recoveredtoyieldonaverage1 – 1.5 ghigh-quality DNAusingeithertheGentraPuregeneYeast/Bact.kit (QiagenInc.,Valencia,CA)ortheQIAGENBlood& CellCultureDNAminikitfollowingthemanufacturer ’ s protocols. FortheDog2andApvariant1strains,organismswere culturedandisolatedfromISE6tickcellsasdescribed [49].Hostcell-freebacteriawerepreparedfromtwoculturesin25cm2flasks,collectedbycentrifugationfor 10minat11,000xgat4C,andlysedinGentraPuregenelysisbuffer(Qiagen)at80Cfor5min.Sincethese DNAsamplesalsocontainedaconsiderableamountof small(<500bp)DNAspeciesnaturallyassociatedwith theISE6hostcellline,the A phagocytophilum gDNA wasfurtherpurifiedbyelectroelutionfromagarosegels, followedbyphenol/chloroformextractionandEtOH precipitationusingconventionalprotocols.Preparationofhostcell-free A phagocytophilum agarose plugsforopticalmappingAp Dog1wasinitiallyselectedforcompletegenomesequencingtocomparewiththepublishedHZstrain. Whenadraftgenomewasassembledfor Ap Dog1itwas largelysyntenicwithHZexceptforthe vir B6locus,indicatingapossibleerrorinthesequenceofoneorbothof thestrains.Accordingly,the Ap Dog1draftgenomesequencewasverifiedbyOpticalMapping.Inpreparation forOpticalMapping(performedbyOpGenInc., Gaithersburg,MD),hostcell-freeorganismswereembeddedin0.5%low-meltingpointagaroseplugsand subsequentlylysed,allowingaccesstotheintact, ~1.48Mbcircular A phagocytophilum chromosome.A procedurerecommendedbyOpGenwasfollowed.All solutionsweremadefreshusingOpGensuggested reagents.Intact Ap Dog1organismswerepurifiedas above,exceptthatthepelletoffreeorganismsobtained followingcentrifugationofthefiltratewasresupended andwashedincellsuspensionbuffer[200mMNaCl, 100mMEDTA-Na2(pH8.0),10mMTris(pH7.2)]. Plugsweremadeimmediatelyoncompletionoftheisolationprocedure.Briefly,followingthefinalcentrifugationofthepurifiedorganisms,thepelletwas resuspendedincellsuspensionbufferusing40 – 50 lfor every108hostcellsusedat>95%infectivity.Thesample waspassed2xthrougha31Gneedle(3/10mlcapacity InsulinSyringewithfused8mmlongneedle,BD #328438;Becton,Dickinson&Co.,FranklinLakes,NJ) toensurehomogeneityofthethicksuspension,andan equalvolumeof1%lowmeltingpointSeaPlaqueGTG agarose[(Lonza#50111)dissolvedinDEPC-treated water(Invitrogen#750023;Carlsbad,CA)andheldat55C] wasimmediatelyadded.Followingmixing,100 laliquots weredispensedintoplugmolds(Bio-Rad#170-3713; Hercules,CA)andallowedtosetfor1hrat4Cpriorto transferintoa50mltubecontaining5 – 10ml,50CNDSK solution[filtersterileNDSsolu tion(1%N-lauroylsarcosine (Sigma#L5000;St.Louis,MO)in0.5MEDTA-Na2Al-Khedery etal.BMCGenomics 2012, 13 :678 Page11of15 http://www.biomedcentral.com/1471-2164/13/678

PAGE 12

(pH9.5),supplementedwithfinal2mg/mlproteinase K(Pierce#17916;Rockford,IL)immediatelypriorto use].Thetubewasincubateduprightat50Cwith mildshaking(40rpm)for8 – 24hrs,untiltheplugs turnedclearandcolorless.Plugsweregentlywashed 3xin5ml0.5MEDTA-Na2(pH9.5),thentransferred toafreshtubeandstoredinEDTAat4C.Optical MappingdatageneratedfromtheBamHI-digested Ap Dog1chromosomewasanalyzedusingtheOpGen MapSolversoftware.454GenomesequencingandbioinformaticsIsolatedDNAwasprovidedtotheInterdisciplinaryCenterforBiotechnologyResearch(ICBR)corefacilities, UniversityofFloridaforlibraryconstructionandpyrosequencingontheRoche/454GenomeSequenceraccordingtostandardmanufacturerprotocols.Regularread librariesweregeneratedforallstrains.Additionally,3kb pairedendlibrariesweremadefor Ap HZ, Ap Dog1and Ap MRK.Genomecoveragerangewas31.3xto72.1x. Foreachstrain,theSFFformatflowfiles,returnedby ICBRforbioinformaticsanalysis,werefirstcombined andconvertedto.fastaand.qualfiles(orthetwocombinedin.fastqformat)usingRoche/454GenomeSequencerFLXSystemsoftware.Genomedraftswere assembledusingtheCLCGenomicsWorkbenchsoftwaresuite(version4.0-4.9)bymappingreadsinitially againstthefullyannotated,Sangersequenced Ap HZ genome(GenBankCP000235),thenagainstthecompleted Ap Dog1genome.Defaultparameterswereused: lengthfraction,0.5;similarity,0.8;andforpairedend reads,minimumdistance,1500/maximumdistance, 4500.Toobtainthe vir loci,theresultingconsensussequenceandunderlyingalignedreadswereinspectedfor conflictsandmismatchedpairedendssuggestingthe presenceofinsertionsand/ordeletionsnotmirroredin theconsensus.Theseweremanuallycorrected.Gaps werealsomanuallyclosedwherepossible.Briefly,overlappingreadscoveringatleast2kbofsequenceonboth sidesofagapandextendingintoitwereindividually extractedfromthealignment.Anewconsensusforeach sidewasobtainedbyassemblingthereadsagainsteach other,and250N ’ swereaddedtoitsends.Thesewere individuallyusedasthereferencesequenceagainst whichallthe454readswerere-mappedtopullout novelreadsextendingintotheunknownregion.The processwasrepeatedmultipletimes,allowingfortheincrementalfillingofthegap.PCR,followedbysequencingwasperformedwhensequencesextrapolatedinthis fashionspannedcomplextandemrepeatregionssuchas repeatregions1and3(R1andR3inFigure3A)ofthe virB6 4 gene,orwhengapclosurecouldnotbecompletedduetosuchstructures,aswasthecasewiththe extremelylong virB6 4 R4(Figure3A)region. AminoacidsequenceswerealignedwithMAFFT[50] anddisplayedwithCHROMA[51].Taxonomicrelationshipsusedaneighbor-joiningtreeandtheITTsubstitutionmodel[52]andweredisplayedusingArchaeopteryx (http://www.phylosoft.org/archaeopteryx).Hydrophobicity analyseswereconductedusingthemethodofHoppand Woods[53,54]atweb.expasy.organdtransmembranesegmentswerepredictedwithTMpredathttp://www.ch. embnet.org/software/TMPRED_form.html.PCRamplificationofvirB6-4generepeatregions,cloning, andPacificBiosciencessequencingDuetodifficultiesinamplifyi ngtandemrepeat-containing DNA,allPCRreactionsspanningthe virB6 4 generepeat regionswereperformedinthepresenceof1.5-1.7M Betaine(Sigma).The8.36kbPCRproductspanningR3 andR4inthe Ap HZstrain(Figure3A,3C,andAdditional file2:FigureS2A)wasamplifiedusingtheiProof High-FidelityDNAPolymerasesystemwithGCbuffer (Bio-Rad).Reactionstotaled50 lwith5ngpurified A phagocytophilum gDNA,1.0Upolymerase,1.5mMMgCl2, 200 MeachdNTP,and250nMeachprimer(AB1393: 50-CGGGATCTAAGACAGATGATGATTC-30,forward; AB1466:50-CTCATCCTGATGCGTCTCCTTAG-30,reverse;Figure3A).35cyclesof30secdenaturingat98C, 20secannealingat67C,and5minextensionat72Cwere performed.PCRproductsspanningR4in Ap JMand Ap Dog1(both~10.3kb;Figure3C)werederivedusing Takara ’ sPrimeSTARGXLDNAPolymerasesystem(ClontechLaboratories,MountainView,CA).Reactionscontained5nggDNA,1.25Upolymerase,1.0mMMgCl2, 200 MeachdNTP,and200nMeachprimer(AB1395:50CACCAGAGGATGCAGCATTAG-30,forward;AB1466, reverse;Figure3A)intotal50 l.Followingthemanufacturer ’ srecommendations,2-stepPCRwasperformedwith 30cyclesof10secdenaturingat98Cand10minannealing/extensionat68C.PCRproductswereanalyzedon 0.5%agarosegelsalongsidethe1kbPlus(Invitrogen)and theGeneRulerHighRange(Fe rmentas,Inc.,GlenBurnie, MD)DNAladders.InordertoTA-clonetheamplicons,Aoverhangswereaddedtotheendsusing0.5-1.0units AmpliTaqDNApolymerase(AppliedBiosystems,Foster City,CA)ina10 – 15minreactionat72C.Productspurifiedfromagarosegels(beforeorafterA-overhangaddition) wereclonedintothepCR-XL-TOPOvector(Invitrogen) andtransformedinto E coli Stbl2(Invitrogen),whichis morepermissivetorepeat-ladenforeignDNA.Recombinantscontainingthecorrectsizeinsertwereendsequenced toverifytheiridentity. Inpreparationforsequencingwiththelong-read lengthPacificBiosciences(PacBio)next-generationsequencingRSinstrument,constructswerelinearizedwith restrictionenzymeswhichcutthevectoronly,buton oppositesidesoftheinsertwithintheMultipleCloningAl-Khedery etal.BMCGenomics 2012, 13 :678 Page12of15 http://www.biomedcentral.com/1471-2164/13/678

PAGE 13

Site.For Ap HZ,equimolaramountsoftheTAclone werecutwitheitherHindIIIorEcoRV.FollowingpoolingandEtOHprecipitation,thelinearizedDNAmix wassubmittedtoICBR/UFforSMRTbelllibraryconstructionandsequencing.Librarieswereconstructed usingacommercialstrobelibrarypreparationkit (#001-326-530;PacificBiosciences,MenloPark,CA) followingstandardmanufacturerprotocols.Tofurther increasethelikelihoodoffullcoverage,thestrobesequencingrunwasperformedusingtwodifferent conditions:I)45minlightperiod(continuouscollectiontime);andII)(5minlightperiod,10mindark period),followedby(45minlightperiod,10mindark period).The Ap JMand Ap Dog1constructswere double-digestedwithHindIII/XbaItoexcisethe~10.3kb inserts.Followingseparationon0.5%agarosegels,the insertswererecoveredfromagaroseslicesbyelectroelutionandfurtherpurifiedandconcentratedbypassage overQIAquickspincolumnsfollowingthePCRPurificationkitprotocol(Qiagen).SMRTbelllibrariesweremade asabovethensequencedusingasingle75minmovie timerun. Duetotherepetitivenatureoftheclonedgenefragments,combinedwiththerelativelyhigherror-rateof thePacBiosystem,allattemptstoassemblethereads de novo failedtoyieldasequenceoftheexpectedsize. Therefore,foreachconstruct,reads>3kbwereselected fromthemulti-fastafilesusingtheGalaxysuite[55], andimportedintotheCLCGenomicsWorkbenchfor assemblyandfurtheranalysis.Thesewereassembledat lowstringencyinitiallyagainstaconsensussequence representinganentire(vectorandinsertsequence)linearconstructtowhichsufficientN ’ swereaddedbased ontheestimatedgap-size.Startingwithreadsinitiating outsidetherepeatregion,thelongestoftheassembled readswerevisuallyinspectedforthepresenceof virB6 4 R4repeatsignature-sequences(Additionalfile2:Figure S2)andtheirsequencemanuallycorrectedwherenecessary.TheextendedsequenceswereusedtoreplaceN ’ s intheconsensusandtheprocessrepeatedseveraltimes untilsufficientreadswith>2kbsequenceoverlapwere recoveredspanningtheentireinsertregion.Forverification,thecompletedsequenceforeachstrainwasusedas thereferencetore-mapalltherespective>3kbPacBio readsandtheRoche/454readsathigherstringency.GenBankAccessionNumbers:foreachisolate,the vir genesarelistedinorderThesequencesof vir lociarecompleteforstrains Ap Dog1and Ap JM.Thesequenceoftherepetitive virB6 4 locuswasincomplete( Ap Dog2)ornotdeterminedfortheotherstrainsexcept Ap Hz.Weprovidea revisedsequenceof virB6 4 forthepreviouslysequenced [15] Ap HZstrain. Ap Dog1:JX415845-JX415868 B2-1B2-2,B2-3,B2-4,B2-5,B2-6,B2-7,B2-8,B2-9, B3,B4-1,B4-t1,B4-2,B6-1,B6-2,B6-3,B6-4,B8-1,B82,B9-1,B9-2,B10,B11,D4 Ap JM:JX415869-JX415892 B2-1,B2-2,B2-3,B2-4,B2-5,B2-6,B2-7,B2-8,B2-9, B3,B4-1,B4-t1,B4-2,B6-1,B6-2,B6-3,B6-4,B8-1,B82,B9-1,B9-2,B10,B11,D4 Ap Dog2:JX415893-JX415915( virB6-4 submitted separatelyasgapped) B2-1,B2-2,B2-3,B2-4,B2-5,B2-6,B2-7,B2-8,B2-9, B3,B4-1,B4-t1,B4-2,B6-1,B6-2,B6-3,B8-1,B8-2,B91,B9-2,B10,B11,D4 Ap NorLambV2:JX415916-JX415938 B2-1,B2-2,B2-3,B2-4,B2-5,B2-6,B2-7,B2-8,B2-9, B3,B4-1,B4-t1,B4-2,B6-1,B6-2,B6-3,B8-1,B8-2,B91,B9-2,B10,B11,D4 Ap NorLambV1:JX415939-JX415966 B2-1,B2-2,B2-3,B2-4,B2-5,B2-6,B2-7,B2-8,B2novel1,B2-novel2,B2-novel3,B2-novel4,B2-novel5, B2-novel6,B3,B4-1,B4-t1,B4-2,B6-1,B6-2,B6-3,B81,B8-2,B9-1,B9-2,B10,B11,D4 Ap HZ virB6-4 :JX415967 Ap Var1:JX415968-JX415996 B2-1,B2-2,B2-3,B2-4,B2-5,B2-6,B2-7,B2-8,B2novel1,B2-novel2,B2-novel3,B2-novel4,B2-novel5, B2-novel6,B2-novel7,B3,B4-1,B4-t1,B4-2,B6-1, B6-2,B6-3,B8-1,B8-2,B9-1,B9-2,B10,B11,D4 Ap MRK:JX415997-JX416019 B2-1,B2-2,B2-3,B2-4,B2-5,B2-6,B2-7,B2-8,B2-9, B3,B4-1,B4-t1,B4-2,B6-1,B6-2,B6-3,B8-1,B8-2,B91,B9-2,B10,B11,D4 ApDog2 virB6-4 Gapped:JX416020.AdditionalfilesAdditionalfile1:FigureS1. Multiplesequencealignmentof VirB6-3aminoacidsequencesfromdifferentstrainsofA. phagocytophilum.ArrowsindicatethelocationsofC-terminal41-mer repeats. Additionalfile2:FigureS2. StructureofthevirB6-4repeatregionsR3 andR4infourUSA.phagocytophilumstrains.A.Comparativemapsof AB1393/AB1466PCRproductsdetailingtherepeatunitcontentofR3and R4inthehuman,rodentanddogstrains. Ap JMand Ap Dog1have identical virB6 4 genesandare,therefore,representedbyonemap. ModeratevariabilityinthenumberandsequenceoftheR3405bp repeatunits(lightbluearrows)isapparent.Thesmallbarattheendof R3correspondstothe30-mostpartialrepeatunitpresentinallstrains.Al-Khedery etal.BMCGenomics 2012, 13 :678 Page13of15 http://www.biomedcentral.com/1471-2164/13/678

PAGE 14

ThecoloredarrowswithinR4representthefiverepeattypesT1a(yellow), T1b(green),T2a,(red),T2b(darkblue)andT2c(grey).Therepeatpattern in Ap HZshowsnorelationshiptothatof Ap JM/ Ap Dog1,whichisalso 2.87kblarger,totaling9.76kb.Thisregionwasnotfullycharacterizedin Ap Dog2asindicatedbyabrokenline,buttherepeatpatternofthe50and30-mostrepeatsisclearlydifferentfromthatoftheotherstrains.The smallbardownstreamofthesecondrepeatunitrepresentsapartially characterizedtype2repeatunit.Linesaboveandbelowthe Ap HZand Ap JM/ Ap Dog1mapsdelineatesegmentsofsequenceidentitywithinthe respectiveR4regions.Theirsizesarespecified. B .Alignmentofthe nucleicacidsequenceofall virB6 4 R4repeatunittypesidentifiedto date.Type1repeatsareshowninblack,type2inblue.Differences betweensub-typesarehighlighted.AsingleBamHIsitepresentinall type2repeatsisunderlined.Withtheexceptionofonlyafew nucleotidesateachend,type1andtype2repeatunitsdonotshareany sequences. C .Alignmentoftheaminoacidsequencesoftherepeatunits showninB.Thesinglenucleotidedifferencesbetweensub-typesdonot leadtochangesinaminoacidsequence. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions BAKandAFBconceivedthestudy,performedbioinformaticsanalysesand draftedthemanuscript.BAKgrewinfectedHL-60cellcultures,purified organisms,isolatedgDNA,designedandsupervisedPCRandsubmitted sequencestoGenBank.AMLperformedPCRanalysesandcloningand superviseddatatransferbetweenunits.SSandEGGisolatedtheEuropean sheepstrains,infectedandmonitoredsheep,andpreparedorganismsat maximalparasitemia.UGMandCMNisolatedandcultured invitro theJM, MRK,Dog2andApvariant1strains,andpreparedDog2andApvariant1 straingDNA.ARAandSMMestablishedtheDog1strain.Allauthorsreadand approvedthefinalmanuscript. Acknowledgements TheresearchdescribedherereceivedsupportfromgrantsRO1GM081714 andGM081714-03S1andfromPfizerAnimalHealth.WethankDr.Roberta Veluci-Marlow,SusanBendaandAdamWebsterforhelpwithculturingcells infectedwith A phagocytophilum ,andDr.SavitaShankerforhighthroughputDNAsequencing. Authordetails1DepartmentofInfectiousDiseasesandPathology,CollegeofVeterinary Medicine,UniversityofFlorida,Gainesville,FL,USA.2Departmentof ProductionAnimalSciences,SectionofSmallRuminantResearch,Norwegian SchoolofVeterinaryScience,Sandnes,Norway.3DepartmentofEntomology, UniversityofMinnesota,StPaul,MN,USA.4PhysiologicalSciences,Collegeof VeterinaryMedicine,UniversityofFlorida,Gainesville,FL,USA.5PfizerAnimal Health,Kalamazoo,MI,USA. Received:10July2012Accepted:20November2012 Published:29November2012 References1.DumlerJS,ChoiKS,Garcia-GarciaJC,BaratNS,ScorpioDG,GaryuJW, etal : Humangranulocyticanaplasmosisand Anaplasmaphagocytophilum EmergInfectDis 2005, 11 :1828 – 1834. 2.JinH,WeiF,LiuQ,QianJ: Epidemiologyandcontrolofhuman granulocyticanaplasmosis:asystematicreview. VectorBorneZoonoticDis 2012, 12 :269 – 274. 3.BakkenJS,DumlerJS: Clinicaldiagnosisandtreatmentofhuman granulocytotropicanaplasmosis. AnnNYAcadSci 2006, 1078 :236 – 247. 4.DahlgrenFS,MandelEJ,KrebsJW,MassungRF,McQuistonJH: Increasing incidenceof Ehrlichiachaffeensis and Anaplasmaphagocytophilum inthe UnitedStates,2000 – 2007. AmJTropMedHyg 2011, 85 :124 – 131. 5.WeilAA,BaronEL,BrownCM,DrapkinMS: Clinicalfindingsanddiagnosis inhumangranulocyticanaplasmosis:acaseseriesfromMassachusetts. MayoClinProc 2012, 87 :233 – 239. 6.LiH,ZhouY,WangW,GuoD,HuangS,JieS: Theclinicalcharacteristics andoutcomesofpatientswithhumangranulocyticanaplasmosisin China. IntJInfectDis 2011, 15 :e859 – e866. 7.EbertsMD,BeallMJ,StillmanBA,ChandrashekarR,BreitschwerdtEB: Typical andatypicalmanifestationsof Anaplasmaphagocytophilum infectionin dogs. JAmAnimHospAssoc 2011, 47 :e86 – e94. 8.BowmanD,LittleSE,LorentzenL,ShieldsJ,SullivanMP,CarlinEP: Prevalenceandgeographicdistributionof Dirofilariaimmitis Borrelia burgdorferi Ehrlichiacanis ,and Anaplasmaphagocytophilum indogsin theUnitedStates:resultsofanationalclinic-basedserologicsurvey. Vet Parasitol 2009, 160 :138 – 148. 9.FoleyJ,NietoNC,MadiganJ,SykesJ: Possibledifferentialhosttropismin Anaplasmaphagocytophilum strainsintheWesternUnitedStates. AnnN YAcadSci2008, 1149 :94 – 97. 10.FoleyJE,NietoNC,MassungR,BarbetA,MadiganJ,BrownRN: Distinct ecologicallyrelevantstrainsof Anaplasmaphagocytophilum EmergInfect Dis 2009, 15 :842 – 843. 11.MassungRF,MatherTN,PriestleyRA,LevinML: Transmissionefficiencyof theAP-variant1strainof Anaplasmaphagocytophila AnnNYAcadSci 2003, 990 :75 – 79. 12.MassungRF,PriestleyRA,MillerNJ,MatherTN,LevinML: Inabilityofa variantstrainof Anaplasmaphagocytophilum toinfectmice. JInfectDis 2003, 188 :1757 – 1763. 13.MassungRF,CourtneyJW,HiratzkaSL,PitzerVE,SmithG,DrydenRL: Anaplasmaphagocytophilum inwhite-taileddeer. EmergInfectDis 2005, 11 :1604 – 1606. 14.StuenS: Anaplasmaphagocytophilum -themostwidespreadtick-borne infectioninanimalsinEurope. VetResCommun 2007, 31 (Suppl1):79 – 84. 15.DunningHotoppJC,LinM,MadupuR,CrabtreeJ,AngiuoliSV,EisenJ, etal : Comparativegenomicsofemerginghumanehrlichiosisagents. PLoS Genet 2006, 2 :e21. 16.RikihisaY,LinM,NiuH: TypeIVsecretionintheobligatoryintracellular bacterium Anaplasmaphagocytophilum CellMicrobiol 2010, 12 :1213 – 1221. 17.WaksmanG,FronzesR: MoleculararchitectureofbacterialtypeIV secretionsystems. TrendsBiochemSci 2010, 35 :691 – 698. 18.NiuH,RikihisaY,YamaguchiM,OhashiN: DifferentialexpressionofVirB9 andVirB6duringthelifecycleof Anaplasmaphagocytophilum inhuman leucocytesisassociatedwithdifferentialbindingandavoidanceof lysosomepathway. CellMicrobiol 2006, 8 :523 – 534. 19.GeY,RikihisaY:Identificationofnovelsurfaceproteinsof Anaplasma phagocytophilum byaffinitypurificationandproteomics. JBacteriol 2007, 189 :7819 – 7828. 20.Alvarez-MartinezCE,ChristiePJ: BiologicaldiversityofprokaryotictypeIV secretionsystems. MicrobiolMolBiolRev 2009, 73 :775 – 808. 21.LopezJE,PalmerGH,BraytonKA,DarkMJ,LeachSE,BrownWC: Immunogenicityof Anaplasmamarginale typeIVsecretionsystem proteinsinaprotectiveoutermembranevaccine. InfectImmun 2007, 75 :2333 – 2342. 22.MorseK,NorimineJ,PalmerGH,SuttenEL,BaszlerTV,BrownWC: AssociationandevidenceforlinkedrecognitionoftypeIVsecretion systemproteinsVirB9-1,VirB9-2,andVirB10in Anaplasmamarginale InfectImmun 2012, 80 :215 – 227. 23.MorseK,NorimineJ,HopeJC,BrownWC: BreadthoftheCD4(+)Tcell responseto Anaplasmamarginale VirB9-1,VirB9-2andVirB10andMHC classIIDRandDQrestrictionelements. Immunogenetics 2012, 64 :507 – 523. 24.AraujoFR,CostaCM,RamosCA,FariasTA,SouzaII,MeloES, etal : IgGandIgG2antibodiesfromcattlenaturallyinfectedwith Anaplasma marginale recognizetherecombinantvaccinecandidateantigens VirB9,VirB10,andelongationfactor-Tu. MemInstOswaldoCruz 2008, 103 :186 – 190. 25.SuttenEL,NorimineJ,BearePA,HeinzenRA,LopezJE,MorseK, etal : Anaplasmamarginale typeIVsecretionsystemproteinsVirB2,VirB7, VirB11,andVirD4areimmunogeniccomponentsofaprotective bacterialmembranevaccine. InfectImmun 2010, 78 :1314 – 1325. 26.DarkMJ,Al-KhederyB,BarbetAF: Multistraingenomeanalysisidentifies candidatevaccineantigensof Anaplasmamarginale Vaccine 2011, 29 :4923 – 4932. 27.ParkJ,KimKJ,ChoiKS,GrabDJ,DumlerJS: Anaplasmaphagocytophilum AnkAbindstogranulocyteDNAandnuclearproteins. CellMicrobiol 2004, 6 :743 – 751.Al-Khedery etal.BMCGenomics 2012, 13 :678 Page14of15 http://www.biomedcentral.com/1471-2164/13/678

PAGE 15

28.Garcia-GarciaJC,Rennoll-BankertKE,PellyS,MilstoneAM,DumlerJS: SilencingofhostcellCYBBgeneexpressionbythenucleareffector AnkAoftheintracellularpathogen Anaplasmaphagocytophilum Infect Immun 2009, 77 :2385 – 2391. 29.NiuH,Kozjak-PavlovicV,RudelT,RikihisaY: Anaplasmaphagocytophilum Ats-1isimportedintohostcellmitochondriaandinterfereswith apoptosisinduction. PLoSPathog 2010, 6 :e1000774. 30.GillespieJJ,BraytonKA,WilliamsKP,DiazMA,BrownWC,AzadAF,Sobral BW: Phylogenomicsrevealsadiverse Rickettsiales typeIVsecretion system. InfectImmun 2010, 78 :1809 – 1823. 31.OhashiN,ZhiN,LinQ,RikihisaY: Characterizationandtranscriptional analysisofgeneclustersforatypeIVsecretionmachineryinhuman granulocyticandmonocyticehrlichiosisagents. InfectImmun 2002, 70 :2128 – 2138. 32.BergerBR,ChristiePJ: Geneticcomplementationanalysisofthe Agrobacteriumtumefaciens virBoperon:virB2throughvirB11are essentialvirulencegenes. JBacteriol 1994, 176 :3646 – 3660. 33.YuanQ,CarleA,GaoC,SivanesanD,AlyKA,HoppnerC, etal : Identification oftheVirB4-VirB8-VirB5-VirB2pilusassemblysequenceoftypeIV secretionsystems. JBiolChem 2005, 280 :26349 – 26359. 34.JuddPK,KumarRB,DasA: Spatiallocationandrequirementsforthe assemblyofthe Agrobacteriumtumefaciens typeIVsecretionapparatus. ProcNatlAcadSciUSA 2005, 102 :11498 – 11503. 35.KumarRB,XieYH,DasA: Subcellularlocalizationofthe Agrobacterium tumefaciens T-DNAtransportporeproteins:VirB8isessentialforthe assemblyofthetransportpore. MolMicrobiol 2000, 36 :608 – 617. 36.CascalesE,ChristiePJ: Agrobacterium VirB10,anATPenergysensor requiredfortypeIVsecretion. ProcNatlAcadSciUSA 2004, 101 :17228 – 17233. 37.RancesE,VoroninD,Tran-VanV,MavinguiP: Geneticandfunctional characterizationofthetypeIVsecretionsysteminWolbachia JBacteriol 2008, 190 :5020 – 5030. 38.AndersonLB,HertzelAV,DasA: Agrobacteriumtumefaciens VirB7and VirB9formadisulfide-linkedproteincomplex. ProcNatlAcadSciUSA 1996, 93 :8889 – 8894. 39.SpudichGM,FernandezD,ZhouXR,ChristiePJ: Intermoleculardisulfide bondsstabilizeVirB7homodimersandVirB7/VirB9heterodimersduring biogenesisofthe Agrobacteriumtumefaciens T-complextransport apparatus. ProcNatlAcadSciUSA 1996, 93 :7512 – 7517. 40.BaylissR,HarrisR,CoutteL,MonierA,FronzesR,ChristiePJ, etal : NMR structureofacomplexbetweentheVirB9/VirB7interactiondomainsof thepKM101typeIVsecretionsystem. ProcNatlAcadSciUSA 2007, 104 :1673 – 1678. 41.GeY,RikihisaY: Surface-exposedproteinsof Ehrlichiachaffeensis Infect Immun 2007, 75 :3833 – 3841. 42.MeeusPF,BraytonKA,PalmerGH,BarbetAF: Conservationofagene conversionmechanismintwodistantlyrelatedparaloguesof Anaplasma marginale MolMicrobiol 2003, 47 :633 – 643. 43.DumlerJS,BarbetAF,BekkerCP,DaschGA,PalmerGH,RaySC, etal : Reorganizationofgenerainthefamilies Rickettsiaceae and Anaplasmataceae intheorder Rickettsiales :unificationofsomespecies of Ehrlichia with Anaplasma Cowdria with Ehrlichia and Ehrlichia with Neorickettsia ,descriptionsofsixnewspeciescombinationsand designationof Ehrlichiaequi and ‘ HGEagent ’ assubjective synonymsof Ehrlichiaphagocytophila IntJSystEvolMicrobiol 2001, 51 :2145– 2165. 44.GribbleDH: Equineehrlichiosis. JAmVetMedAssoc 1969, 155 :462 – 469. 45.JohnsonRC,KodnerC,JarnefeldJ,EckDK,XuY: Agentsofhuman anaplasmosisandLymediseaseatCampRipley,Minnesota. VectorBorne ZoonoticDis 2011, 11 :1529 – 1534. 46.MunderlohUG,JauronSD,FingerleV,LeitritzL,HayesSF,HautmanJM, et al : Invasionandintracellulardevelopmentofthehumangranulocytic ehrlichiosisagentintickcellculture. JClinMicrobiol 1999, 37 :2518 – 2524. 47.MassungRF,LevinML,MunderlohUG,SilvermanDJ,LynchMJ,GayweeJK, KurttiTJ: IsolationandpropagationoftheAp-Variant1strainof Anaplasmaphagocytophilum inatickcellline. JClinMicrobiol 2007, 45 :2138 – 2143. 48.GranquistEG,BardsenK,BergstromK,StuenS: Variantandindividual dependentnatureofpersistent Anaplasmaphagocytophilum infection. ActaVetScand 2010, 52 :25. 49.FelsheimRF,HerronMJ,NelsonCM,BurkhardtNY,BarbetAF,KurttiTJ, MunderlohUG: Transformationof Anaplasmaphagocytophilum BMC Biotechnol 2006, 6 :42. 50.KatohK,TohH: RecentdevelopmentsintheMAFFTmultiplesequence alignmentprogram. BriefBioinform 2008, 9 :286 – 298. 51.GoodstadtL,PontingCP: CHROMA:consensus-basedcolouringof multiplealignmentsforpublication. Bioinformatics 2001, 17 :845 – 846. 52.JonesDT,TaylorWR,ThorntonJM: Therapidgenerationofmutationdata matricesfromproteinsequences. ComputApplBiosci 1992, 8 :275 – 282. 53.HoppTP,WoodsKR: Acomputerprogramforpredictingprotein antigenicdeterminants. MolImmunol 1983, 20 :483 – 489. 54.HoppTP: Useofhydrophilicityplottingprocedurestoidentifyprotein antigenicsegmentsandotherinteractionsites.MethodsEnzymol 1989, 178 :571 – 585. 55.GoecksJ,NekrutenkoA,TaylorJ: Galaxy:acomprehensiveapproachfor supportingaccessible,reproducible,andtransparentcomputational researchinthelifesciences. GenomeBiol 2010, 11 :R86.doi:10.1186/1471-2164-13-678 Citethisarticleas: Al-Khedery etal. : StructureofthetypeIVsecretion systemindifferentstrainsof Anaplasmaphagocytophilum BMC Genomics 2012 13 :678. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Al-Khedery etal.BMCGenomics 2012, 13 :678 Page15of15 http://www.biomedcentral.com/1471-2164/13/678


xml version 1.0 encoding utf-8 standalone no
mets ID sort-mets_mets OBJID sword-mets LABEL DSpace SWORD Item PROFILE METS SIP Profile xmlns http:www.loc.govMETS
xmlns:xlink http:www.w3.org1999xlink xmlns:xsi http:www.w3.org2001XMLSchema-instance
xsi:schemaLocation http:www.loc.govstandardsmetsmets.xsd
metsHdr CREATEDATE 2013-01-26T16:11:16
agent ROLE CUSTODIAN TYPE ORGANIZATION
name BioMed Central
dmdSec sword-mets-dmd-1 GROUPID sword-mets-dmd-1_group-1
mdWrap SWAP Metadata MDTYPE OTHER OTHERMDTYPE EPDCX MIMETYPE textxml
xmlData
epdcx:descriptionSet xmlns:epdcx http:purl.orgeprintepdcx2006-11-16 xmlns:MIOJAVI
http:purl.orgeprintepdcxxsd2006-11-16epdcx.xsd
epdcx:description epdcx:resourceId sword-mets-epdcx-1
epdcx:statement epdcx:propertyURI http:purl.orgdcelements1.1type epdcx:valueURI http:purl.orgeprintentityTypeScholarlyWork
http:purl.orgdcelements1.1title
epdcx:valueString Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum
http:purl.orgdctermsabstract
Abstract
Background
Anaplasma phagocytophilum is an intracellular organism in the Order Rickettsiales that infects diverse animal species and is causing an emerging disease in humans, dogs and horses. Different strains have very different cell tropisms and virulence. For example, in the U.S., strains have been described that infect ruminants but not dogs or rodents. An intriguing question is how the strains of A. phagocytophilum differ and what different genome loci are involved in cell tropisms and/or virulence. Type IV secretion systems (T4SS) are responsible for translocation of substrates across the cell membrane by mechanisms that require contact with the recipient cell. They are especially important in organisms such as the Rickettsiales which require T4SS to aid colonization and survival within both mammalian and tick vector cells. We determined the structure of the T4SS in 7 strains from the U.S. and Europe and revised the sequence of the repetitive virB6 locus of the human HZ strain.
Results
Although in all strains the T4SS conforms to the previously described split loci for vir genes, there is great diversity within these loci among strains. This is particularly evident in the virB2 and virB6 which are postulated to encode the secretion channel and proteins exposed on the bacterial surface. VirB6-4 has an unusual highly repetitive structure and can have a molecular weight greater than 500,000. For many of the virs, phylogenetic trees position A. phagocytophilum strains infecting ruminants in the U.S. and Europe distant from strains infecting humans and dogs in the U.S.
Conclusions
Our study reveals evidence of gene duplication and considerable diversity of T4SS components in strains infecting different animals. The diversity in virB2 is in both the total number of copies, which varied from 8 to 15 in the herein characterized strains, and in the sequence of each copy. The diversity in virB6 is in the sequence of each of the 4 copies in the single locus and the presence of varying numbers of repetitive units in virB6-3 and virB6-4. These data suggest that the T4SS should be investigated further for a potential role in strain virulence of A. phagocytophilum.
http:purl.orgdcelements1.1creator
Al-Khedery, Basima
Lundgren, Anna M
Stuen, Snorre
Granquist, Erik G
Munderloh, Ulrike G
Nelson, Curtis M
Alleman, A Rick
Mahan, Suman M
Barbet, Anthony F
http:purl.orgeprinttermsisExpressedAs epdcx:valueRef sword-mets-expr-1
http:purl.orgeprintentityTypeExpression
http:purl.orgdcelements1.1language epdcx:vesURI http:purl.orgdctermsRFC3066
en
http:purl.orgeprinttermsType
http:purl.orgeprinttypeJournalArticle
http:purl.orgdctermsavailable
epdcx:sesURI http:purl.orgdctermsW3CDTF 2012-11-29
http:purl.orgdcelements1.1publisher
BioMed Central Ltd
http:purl.orgeprinttermsstatus http:purl.orgeprinttermsStatus
http:purl.orgeprintstatusPeerReviewed
http:purl.orgeprinttermscopyrightHolder
Basima Al-Khedery et al.; licensee BioMed Central Ltd.
http:purl.orgdctermslicense
http://creativecommons.org/licenses/by/2.0
http:purl.orgdctermsaccessRights http:purl.orgeprinttermsAccessRights
http:purl.orgeprintaccessRightsOpenAccess
http:purl.orgeprinttermsbibliographicCitation
BMC Genomics. 2012 Nov 29;13(1):678
http:purl.orgdcelements1.1identifier
http:purl.orgdctermsURI http://dx.doi.org/10.1186/1471-2164-13-678
fileSec
fileGrp sword-mets-fgrp-1 USE CONTENT
file sword-mets-fgid-0 sword-mets-file-1
FLocat LOCTYPE URL xlink:href 1471-2164-13-678.xml
sword-mets-fgid-1 sword-mets-file-2 applicationpdf
1471-2164-13-678.pdf
sword-mets-fgid-3 sword-mets-file-3
1471-2164-13-678-S2.PDF
sword-mets-fgid-4 sword-mets-file-4
1471-2164-13-678-S1.PDF
structMap sword-mets-struct-1 structure LOGICAL
div sword-mets-div-1 DMDID Object
sword-mets-div-2 File
fptr FILEID
sword-mets-div-3
sword-mets-div-4
sword-mets-div-5