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Localization of the Myc-Tagged IPF15632 Protein

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Title:
Localization of the Myc-Tagged IPF15632 Protein
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Folkner, Brie
Lewin, Al ( Mentor )
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Gainesville, Fla.
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University of Florida
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English

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University of Florida
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Localization of the Myc-Tagged IPF15632 Protein

Brie Folkner


ABSTRACT


Candida albicans gene IPF15632 has been identified by IVIAT technology as a gene expressed during

oropharyngeal candidiasis. This gene encodes a protein with a molecular weight of 42 kiloDaltons. The goal of

this project was to localize this protein in the yeast cell. To begin, a peptide "tag" derived from the Myc oncogene

was added to the C-terminus of the protein using PCR. The Myc-tagged IPF15632 was cloned into the pMB7-

1 plasmid, which contains bacterial HisG and yeast URA3 sequences. The plasmid was linearized by SphI and

PvuII and used to transform different strains of C. albicans, which have copies of HisG at the IPF15632

loci. Recombination of the Myc-tagged IPF15632 occurred due to the presence of HisG in both the plasmid and

the yeast strains. The URA3 acted as a selectable marker for the recombinants by growing transformants on

medium lacking uracil. A mouse anti-Myc antibody followed by a FITC anti-mouse antibody were used to identify

the Myc-tagged IPF15632 protein by immunofluorescence microscopy. The localization of this protein may allow for

a greater understanding of the protein's functions within the yeast cell and its involvement in C.

albicans pathogenicity. This knowledge may one day benefit the development of antifungal targets or C.

albicans vaccines.



INTRODUCTION


Candida albicans is a fungus which grows on mucosal membranes of the mouth, intestinal tract, and

genitourinary tract of healthy humans. This commensal organism's growth is kept in check in healthy hosts by

the normal microbiota that share the same habitats. However, C. albicans causes disease in

immunocompromised individuals and in those hosts whose competing microbiota are suppressed by antibiotics

or other factors.1 A wide range of diseases can be caused when C. albicans is able to invade and damage

underlying tissues in a given mucocutaneous site.2, 3



Infants lack a fully developed immune system and when first infected with C. albicans they may

exhibit oropharyngeal candidiasis, or thrush. The use of oral contraceptives or antibiotics, pregnancy, and

diabetes cause an increased amount of glycogen in the vagina and this leads to vulvovaginal candidiasis in

women. Immunocompromised individuals, such as those with AIDS or receiving chemotherapy for cancer, also have

a greater incidence of both types of candidiasis.1 In addition, immunocompromised individuals have a greater
4




chance of candidemia, the fourth leading blood stream infection in the United States. As the yeast infect

organs, candidemia leads to disseminated or systemic candidiasis.5 Systemic candidiasis when treated

with appropriate antifungal therapy has between a 30 and 60% fatality rate; when left untreated this disease

is usually fatal.6



An immunogenic antigen encoded by the C. albicans gene IPF15632 was detected by using In Vivo Induced

Antigen Technology (IVIAT). A genomic expression library for C. albicans was screened with serum from patients

with active oral thrush; IPF15632 protein was recognized by this serum. This gene was knocked, and the null

mutant displayed a delayed virulence in a murine model of disseminated candidiasis.8 This gene has very

weak homology to a cell wall protein from Saccharomyces cerevisiae (baker's yeast).9 Based on this homology

we hypothesize that IPF15632 protein (42 kiloDaltons) is part of the cell wall. My project's goal is to

localize IPF15632 in C. albicans by using a protein tag derived from the Myc oncogene (Myc)

and immunofluorescence.



By localizing this protein, we may gain insight into the protein's function in the pathogenesis of candidiasis. In

the future this information may be useful for developing targets for antifungal therapy or C. albicans vaccines.



METHODS AND MATERIALS


Cloning IPF15632 with a Myc tag


IPF15632 is a gene of 1185 base pairs encoding a protein of 395 amino acids. The Myc tag encodes an epitope of

11 amino acids. A reverse primer (Myc reverse primer shown in Figure 1) was made with the reverse

complement Myc sequence to incorporate it at the 3' end of IPF15632 by PCR. The PCR product (Myc-

tagged IPF15632, shown in Figure 1) was cloned into a plasmid and then sent to a core lab for sequence

analysis. The Myc-tagged IPF15632 was then inserted into a plasmid vector (pMB7-1), which was used

for homologous recombination in a C. albicans IPF15632 null mutant. This vector has two copies of HisG gene from

S. typhimurium flanking the URA3 gene from C. albicans.



Homologous Recombination of Myc-tagged IPF15632 into null mutant


The vector was digested with SphI and SacI so that the Myc-tagged IPF15632 gene, the URA3 gene, and one copy

of the HisG insert will be transformed into the null mutant. Recombinants will be selected for by growing on

Sabourad Dextrose plates without uracil. The null mutant lacked both copies of IPF15632 and the URA3 gene so

that these cells cannot grow in absence of uracil. The URA3 allowed for selection of recombinants on plates

lacking uracil.



Screen for recombinants


Recombinants were screened by Southern Blot (Figure 2) using their genomic DNA digested with PvuII and SphI.





A Myc-tagged IPF15632 sequence (labeled F2 on Figure 1) was labeled with a-[32P]-ATP by random priming using
a kit from New England Biolabs. This labeled probe was used for hybridization to the Southern Blot

nitrocellulose membranes.






PF P1562Foiwur Prmer I ravem Pridmer
I n e r t IF f 5 0 ? F o( w a Brd I = B 5S6j R ew r se /7


SI / Y

Myc-tagged IPF 15632



Figure 1. Schematic of Myc-tagged IPF15632 (1,196bp) represented by the orange arrow. The

Myc reverse primer used in cloning PCR and RT-PCR is shown by the yellow arrow. Other primers used

for cloning are shown in green. F1 and F2 are primer sequences used that were radioactively labeled

and hybridized to Southern blots. The Myc tag is shown at the end of the gene in black.



Total RNA was isolated from two recombinants (1 and 9 on Southern blot Figure 2) with the correct reinsertion of

the Myc-tagged IPF15632. RNA was extracted using Qiagen's RNeasy Mini Kit with a beadbeater. The RNA

samples were treated with DNAse (deoxyribonuclease) using Ambion's DNA-free kit. The concentration of RNA in

the samples was measured by spectrophotometry by taking the OD260 reading and using the following

formula: micrograms of RNA/ microliter = (A260)*(Dilution Factor)*(40 micrograms/microliter)/1000. RT-PCR

was performed in order to verify presence of the Myc-tagged IPF15632 mRNA (Figures 3 and 4). Amersham's

cDNA kit was used to make cDNA from the tagged gene's specific mRNA. PCR was then performed using

the IPF15632 forward and Myc reverse primers. A negative control (the IPF15632 null mutant) was used which

does not have the Myc tag.






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Figure 2. Southern Blot of genomic DNA extracted from C. albicans null mutant for IPF15632

transformed with the Myc-tagged IPF15632 gene and hybridized with probe F2 (Fig. 1). Lane C is the

non-transformed IPF15632 null mutant (a control), lane L is the 1kb ladder, and lanes 1-10 are

the transformants. The presence of a 6.2kb band or a 5.8kb band signifies that the recombination

has occurred. All of the samples show recombination.






L 1 2 3 4 5 6 7 1


Figure 3. Control PCR of cDNA from recombinants (lanes 4-6) using EFBetal primers. This PCR

shows that the recombinants are making mRNA. Lane 1 is a negative control of the PCR with no

DNA added. Lanes 2-3 and 7-8 are positive controls using cDNA and DNA from SC5314 (wild type

C. albicans) and the null mutant for IPF15632, respectively.



L 1 2 3 4 5


1 kb trnd


Figure 4. Results of RT-PCR using Myc reverse primer for RT and PCR with cDNA. The gel shows

that mRNA is being made by the recombinants with the Myc-tagged IPF15632 gene due to the

presence of a 1kb band in lanes 4-6. Lanes 1, 2, and 3 are negative controls (water, SC5314, and the

null mutant); SC5314 shows some background which accounts for nonspecific binding of the Myc

reverse primer.



Total protein was extracted by lysing cells in a French Pressure Cell from two recombinants (1 and 9 on Southern

blot Figure 2). Protein expression of Myc-tagged IPF15632 was verified by western blot, and a negative

control (IPF15632 null mutant) was used which does not have the Myc tag.



The Western blot was performed with both log and stationary phase extracted total protein grown at 300 and 370

C. Protein was separated on an SDS polyacrylamide gel (5% stacking and 15% separating gels). The gels

were transferred to nitrocellulose membranes and then hybridized with antibodies. A mouse anti-Myc





antibody (1:1000 dilution in PBS) was used to bind Myc, followed by a secondary anti-mouse antibody

(1:5000 dilution in PBS) linked with a horseradish peroxidase and enhanced chemiluminescence for detection using

X-ray film (Figure 5).




L 1 2 3 4 S 6 7 8 9




42l












Figure 5. Western blot of protein extracted from recombinants at 370 log phase (lanes 2, 3 and 4)

and 370 stationary phase (lanes 6, 7 and 8). The negative controls are SC5314 (lane 9) and the

IPF15632 null mutant for (lanes 1 and 5). The anti-Myc antibody shows nonspecific binding and the

Myc-tagged IPF15632 protein is expected to be seen at the 42 kD band.




Imm unofluorescence1


The recombinants with the correct reinsertion of the Myc-tagged IPF15632 were grown to log phase and then

diluted to OD600 of 0.8 to 1.0. This dilution of cells was fixed with 4% paraformaldehyde. 1500 microliters of the

fixed cell suspension were treated with 10 units Zymolyase 100T in a 1.2M Sorbitol phosphate buffer at pH 7.4

(10 minutes at 350C with gentle shaking) in order to remove the cell wall so that the cells are permeable. The

cells were resuspended in phosphate buffered saline (PBS) and twenty microliters were placed in a well on a

poly-lysine coated slide. Slides were allowed to dry and then flattened with -200C methanol and acetone. The

wells were blocked for one hour and then incubated overnight at 40C with primary rabbit anti-Myc antibody

(1:200 dilution in PBS). A secondary antibody (goat anti-rabbit IgG, 1:40 dilution in PBS) labeled with FITC

(a fluorophore) was used to detect the primary antibody. A drop of DAPI (4, 6 diamino-2 phenylindole) was added

to each well in order to stain the nuclei before attaching a cover slide. A Zeiss Axioplan 2 Fluorescence

Microscope was used at 100x magnification to detect the fluorescence of the DAPI stain and FITC. Pictures of

the slide wells were taken and the same frames were merged in order to view DAPI and FITC fluorescence

together (Figure 6).



















Figure 6. Results of immunofluorescence for C. albicans null mutant for IPF15632 and two

recombinants labeled 1 and 9 (also on Southern blot Figure 2). The FITC fluorescence is green and

stains the primary anti-Myc antibody. The nuclei are blue and stained by DAPI. The null mutant

shows background of antibody staining which is smooth and evenly dispersed across the yeast cells.

The recombinants show punctate staining by antibody and is not localized strictly to the nucleus.

The antibody staining of recombinants suggests that Myc-tagged IPF15632 protein is localized to

the cytosol or cell membrane.




RESULTS



The sequence of the Myc-tagged IPF15632 gene was correct as shown by sequence analysis. After transforming

the C. albicans IPF15632 null mutants with the Myc-tagged IPF15632 gene, only ten recombinant colonies grew

on the media lacking uracil. These colonies were screened by Southern blot for proper recombination of the

Myc-tagged IPF15632. If the Myc-tagged IPF15632 recombined in one of the original loci for the gene, we expected

a 6.2kb band or a 5.8kb band when hybridizing with the F2 probe to Southern blot; these two bands represent

the two alleles of IPF15632. As shown in Figure 2, lanes 1 and 3 have the 6.2kb band and lanes 2 and 4-10 have

the 5.8kb band. All ten had the Myc-tagged IPF15632 inserted.



Total RNA was extracted from two of the ten positive recombinants (1 and 9 on Southern blot Figure 2). The

control PCR of cDNA and DNA using elongation factor betal primers gave a 600bp (without Intronl) and 900 bp

band (with Intronl), respectively, when run on an agarose gel (Figure 3) which shows that mRNA is being

recovered from the yeast. The original reverse transcriptase (RT) using an oligo-dt primer did not give enough

cDNA of the Myc-tagged IPF15632 gene because level of message for this gene could have been low. When the

RT was performed a second time using the reverse primer specific for Myc, enough cDNA was made to do a

PCR which showed that the Myc-tagged IPF15632 recombinants were making message and that the null mutant

was not making message (Figure 4).



Because the RT-PCR was positive for mRNA of Myc-tagged IPF15632, a Western blot was performed in order

to ensure that protein was being made. The Western blot showed more than one band, implying that the primary

anti-Myc antibody was not very specific (Figure 5). A 42 kD band which would represent the protein of the

Myc-tagged IPF15632 gene is not clearly seen on the blot.


Null







The immunofluorescence experiments were performed with a different monoclonal anti-Myc antibody in order to

gain a greater degree of specific binding. After fixing the yeast cells and digestion with Zymolyase, cells were

checked under a microscope at 40x magnification in order to ensure that over-digestion or some other error had

not occurred. Analyzing the pictures taken by the Zeiss Axioplan 2 Fluorescence Microscope at 100x

magnification shows the yeast cell in green and the nuclei in blue. Pictures (Figure 6) taken from the same

frame were merged and a blue nucleus is visible within each yeast cell. The control used for this experiment was

the IPF15632 null mutant; any FITC fluorescence for this yeast is background. The pictures of the recombinants

(1 and 9 on Southern blot Figure 2) show that the FITC fluorescence is punctate and brighter, whereas on the

null mutant the FITC fluorescence is smooth and more evenly dispersed throughout the cell. The FITC fluorescence

of the recombinants is not localized strictly to the nucleus, but seems spread out within the cytosol.

However, because confocal microscopy was not performed, it is possible that the fluorescence is localizing to

the cell's surface.



DISCUSSION


C. albicans expresses many as yet unidentified proteins during pathogenesis in humans. IPF15632 is one such

protein which shows very low homology to any other yeast proteins. The results of these experiments provide

greater insight into the location of this protein within the cell.



Southern blot analysis of the recombinant's DNA proves we were successful in creating mutants with the Myc-

tagged IPF15632 into each allele of the gene. The results of the Western blot show that the anti-Myc antibodies

used had low specificity for the Myc tag. Because the anti-Myc antibodies and the secondary antibodies used

were made in animals (rabbit, mouse, donkey, and goat) and these animals have been exposed to C. albicans,

this could result in a possible polyclonal antibody contamination with some endogenous antibodies to C. albicans.

The Western blot (Figure 5) does not give a clear idea if the protein is being made or how much of it is being

made. However, the immunofluorescence pictures show that FITC fluorescence differs between the IPF15632

null mutant and the recombinants (Figure 6); there is more fluorescence in certain locations of the recombinants

that does not appear in the IPF15632 null mutant. Based on the current location of the highly fluorescing areas, it

is possible that the Myc-tagged IPF15632 protein localizes to the cytosol or cell wall of the yeast. Without

further electron and confocal microscopy techniques, the exact location of Myc-tagged IPF15632 cannot

be determined.



By repeating some experiments, the data already obtained may strengthen and clarify our findings. A better anti-

Myc antibody should be used if possible for the Western blot and immunofluorescence or the same antibodies

already used could be first preabsorbed to C. albicans in order eliminate any contamination. The extracted

protein could also first be purified by column chromatography, which would concentrate the Myc-tagged

IPF15632 protein before verifying by Western blot that the protein is being produced. Since it is possible that the

Myc-tagged IPF15632 protein could be part of the cell wall, the immunofluorescence assay should be





performed without digesting cells with Zymolyase.


In conclusion, this project has attempted to localize the IPF15632 protein expressed in C. albicans

during oropharyngeal infections in humans. Based on immunofluorescence, we have found that this protein is

likely localized to the cytosol or cell membrane of the yeast cell. This information may eventually help to identify

the protein's function in C. albicans pathogenesis. Once the function has been determined, this protein might be

used as an anti-fungal target in drug therapy or in a vaccine for C. albicans.






REFERENCES



1. Tortora GJ, Funke BR, Case CL. Microbiology: An Introduction 7th ed. 2002. San Francisco, CA: Benjamin

Cummings; 331-340, 594-595, 734-735.

2. Calderone RA, Fonzi WA. Virulence factors of Candida albicans. Trends Microbiol. 2001;9:327-335.

3. Smith H. Questions about the behaviour of bacterial pathogens in vivo. Trans R Soc Lond B Biol Sci.

2000;355:551-564.

4. Edmond MB, Wallace SE, McClish DK, et al. Nosocomial bloodstream infections in United States hospitals: a

three year analysis. Clin Infect Dis. 1999;29:239-244.

5. Nguyen MH, Cheng S, Clancy CJ. Assessment of Candida albicans genes expressed during infections as a tool

to understand pathogenesis. Med Mycol. 2004;00:1-12.

6. Ben-Abraham R, Keller N, Teodorovitch N, et al. Predictors of adverse outcome from candidal infection in a

tertiary care hospital. J Infect. 2004;49(4):317-23.

7. Handfield M, Brady LJ, Progulske-Fox A, Hillman JD. IVIAT: a novel method to identify microbial genes

expressed specifically during human infections. Trends in Microbiol. 2000;8(7):336-339

8. Checkley MA. Identification of C. albicans virulence factors expressed only during oropharyngeal

candidiasis. Dissertation Research Proposal, University of Florida College of Medicine; September 2000.

9. Candida albicans Database (Candida DB, Pasteur Institute, France)

Gene: IPF15632

10. Inglis DO, Johnson AD. Ashi protein, an asymmetrically localized transcriptional regulator, controls

filamentous growth and virulence of Candida albicans. Molec and Cell Biol. 2002;22(24):8669-8680.





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