Isolation and characterization of group A streptococcal Fc receptors

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Isolation and characterization of group A streptococcal Fc receptors
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Yarnall, Michele S., 1959-
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Thesis (Ph. D.)--University of Florida, 1985.
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Includes bibliographical references (leaves 82-90).
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by Michele S. Yarnall.
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Typescript.
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Vita.

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ISOLATION AND CHARACTERIZATION OF GROUP A
STREPTOCOCCAL Fc RECEPTORS







By

MICHELE S. YARNALL


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1985















ACKNOWLEDGEMENTS


I wish to express my thanks to Dr. Michael D.P. Boyle for giving

me the chance to work in his laboratory. I appreciate all of his help

and guidance and have enjoyed working with him.

I would like to thank my committee members, Dr. E.M. Ayoub, Dr.

A.P. Gee, Dr. J.W. Shands, Dr. A.S. Bleiweis, Dr. J.B. Flanegan, and my

external examiner, Dr. A.D. O'Brien, for their helpful suggestions

throughout this study.

I would also like to thank Dr. Frank Muzopappa for his

encouragement and advice throughout my years at Kutztown State

College.

I wish to give my appreciation to my family who has been

constantly supportive of me. I would especially like to thank Kim to

whom I can always talk and who has been great company on many

vacations.

Finally, I would like to thank David C. Palmer. His love,

patience, and understanding has helped me through some difficult

times.















TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ............................................... ii

LIST OF TABLES ................................................. v

LIST OF FIGURES ................................................ vi

ABSTRACT ......................... ............................ v iii

CHAPTER

ONE INTRODUCTION ............ .............................. 1

Staphylococcal Protein A The Type I Fc Receptor ....... 1
Streptococcal Fc Receptors Type II Through Type V ..... 4
Fc Receptors as Virulence Factors ................. 7
Practical Applications of Bacterial Fc Receptors ........ 8
Summary ................................................. 9

TWO A TECHNIQUE FOR THE DETECTION OF BOUND AND SECRETED Fc
RECEPTORS ................................................. .11

Introduction ................................ ...... 11
Materials and Methods ................................... 12
Results .......................................... ...... 14
Discussion ..................................... .... 24

THREE ISOLATION AND PARTIAL CHARACTERIZATION OF THE TYPE II Fc
RECEPTORS FROM A GROUP A STREPTOCOCCUS .................. 27

Introduction ............................................ 27
Materials and Methods ................................. 28
Results ................................................. 36
Discussion ............................................. 60

FOUR DISTRIBUTION OF THE TYPE II Fc RECEPTORS ON NEPHRITOGENIC
AND NON-NEPHRITOGENIC GROUP A STREPTOCOCCI ............. 65

Introduction .......................................... 65
Materials and Methods ............................... 66
Results ................................................. 67
Discussion ............................................. 72









Page

FIVE CONCLUSION ........................................ ..... 79

REFERENCES ...................................................... 82

BIOGRAPHICAL SKETCH ..................................... ... 91















LIST OF TABLES


TABLE Page

1-1. Species and Subclass IgG Reactivities of Bacterial
Fc Receptors ......................................... 5

3-1. Fc Receptor Activity in Streptococcal Extracts ......... 38

4-1. Binding of 1251 Human IgG to Nephritogenic and
Non-Nephritogenic Group A Streptococci ................. 70

4-2. Interaction of Nephritogenic and Non-Nephritogenic
Group A Streptococci with Human IgG Subclasses ......... 73














LIST OF FIGURES


FIGURE Page

2-1. Schematic representation of the immunoblotting
procedure for detection of surface Fc receptors ......... 16

2-2. Detection of Fc receptors on the surface of
staphylococcal strains .................................. 17

2-3. Sensitivity of assay for secreted Fc receptors .......... 19

2-4. Fc receptor expression of a group A streptococcal
strain ........................................... 20

2-5. Fc receptor expression of two bacterial subpopulations
selected from the original strain 64/14 ................. 22

2-6. Fc dependent uptake of 1251-labeled human IgG by
increasing numbers of bacteria .......................... 23

3-1. Western blot autoradiograph of affinity purified
extractions of 64/14/HRP ................................ 39

3-2. Sodium dodecyl sulfate polyacrylamide gel
electrophoresis of the type II Fc receptor .............. 41

3-3. The effect of dipeptides on the binding of
1251-labeled human IgG subclasses to an Fc
receptor-positive group A streptococcus ................. 43

3-4. The effect of glycyl-histidine on the binding of
1251-labeled human IgG to three bacterial strains ....... 44

3-5. Human immunoglobulin G subclass reactivity of
bacterial Fc receptors .................................. 46

3-6. Reactivity of three human myeloma IgG3 samples with
bacterial Fc receptors ................................. 47

3-7. Inhibition of binding 1251-labeled IgG3 to a
group A streptococcus by unlabeled human IgG
subclasses ..................................... ...... 49

3-8. Separation of the type IIa Fc receptor from the type
IIb Fc receptor ........................................ 51











3-9. Inhibition of 1251 human IgG or 1251 hunan
IgG3 to group A strain 64/14/HRP by immunogloublin G
from a variety of nar.malian species ..................... 53

3-10. Reactivity of IgG front a variety of species with the
type Ila or type IIb Fc receptor ........................ 55

3-11. Inhibition of binding of 1251-labeled hunan IgG
or 1251-labeled human IgG3 to 64/14/HRP by
monospecific antibodies against the purified type IIa
or type IIb Fc receptors ............................... 58

3-12. Inhibition of binding of 1251 hunan IgG to
64/14/HRP by antibody against the type I receptor,
the type II receptor, or the type III receptor .......... 59

4-1. Binding of 1251 human IgG to nephritogenic and
non-nephritogenic yroup A streptococci .................. 69

4-2. Proposed mechanism of the pathogenesis of
post-streptococcal glomerulonephritis ................... 75


FIGURE


Page












Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


ISOLATION AND CHARACTERIZATION OF GROUP A
STREPTOCOCCAL Fc RECEPTORS

By

Michele S. Yarnall

August 1985

Chairman: Michael D.P. Boyle, Ph.D.
Major Department: Immunology and Medical Microbiology

An immunoblotting technique was developed to select a group A

streptococcal strain rich in Fc receptors. A high Fc receptor-positive

strain was selected and used as the source for the isolation of two

functionally active Fc receptors. A variety of extraction techniques

were compared including 1) heat extraction at neutral, acid or alkaline

pH, 2) treatment with the enzymes mutanolysin, hyaluronidase, trypsin,

papain or phage lysin, or 3) autoclaving or heating in the presence of

sodium dodecyl sulfate. The most homogeneous receptor was recovered

following heat extraction and contained two molecular weight forms.

One form had a molecular weight of 56,000 daltons and the other form

had a molecular weight of 38,000 daltons. The 56,000 dalton Fc

receptor was capable of reacting with human IgG subclasses 1, 2, and 4,

pig IgG and rabbit IgG. The 38,000 dalton Fc receptor could only bind

human IgG subclass 3. The two Fc receptors could be separated by

binding to and elution from a column of immobilized human IgG3 which

resulted in the isolation of the 38,000 dalton Fc receptor. The


viii









unbound material from the immobilized IgG3 column was applied to an

immobilized column of human lyG and the 56,000 dalton Fc receptor was

recovered following elution. Both Fc receptors were antigenically

related. Monospecific antibodies prepared against either the 56,000

dalton Fc receptor or the 38,000 dalton Fc receptor demonstrated

reactivity with both molecular weight forms. Both group A

streptococcal Fc receptors were found to be antigenically and

physicochemically distinct from either the type I receptor found on the

majority of Staphylococcus aureus strains or the type III Fc receptors

found on the majority of group C streptococcal strains.

The distribution of Fc receptors on group A streptococci recovered

from patients who developed post-streptococcal glomerulonephritis was

tested. A high incidence of Fc receptor positive nephritogenic strains

were found, but an absolute correlation between Fc receptor expression

and pathogenic potential could not be established.














CHAPTER ONE
INTRODUCTION


Certain bacteria are capable of interacting with immunoglobulins

in two distinct ways. One involves a specific antigen-antibody

reaction in which the F(ab) portion of the immunoglobulin molecule

participates. This interaction mediates the clearance of bacteria from

the host. The second interaction involves the Fc portion of the

immunoglobulin molecule. It has been reported that certain

staphylococci (Jensen, 1958; Forsgren and Sjoquist, 1966) and

streptococci (Kronvall, 1973a) have surface receptors that are capable

of reacting with the Fc region of certain classes and subclasses of

immunoglobulins. Functional studies of their reactivity with different

species and subclasses of IgG have suggested that five distinct

bacterial Fc receptor activities exist (Myhre and Kronvall, 1981).

These are designated as types I through V.

Staphylococcal Protein A The Type I Fc Receptor

Physicochemical Properties

The most extensively studied Fc receptor is the type I receptor

isolated from Staphylococcus aureus and designated protein A. Protein

A has been reported to be produced and or secreted by approximately 90%

of all staphylococci studied (Langone, 1982a; Sperber, 1976); however,

marked quantitative differences exist between different strains. The

most widely studied Staphylococcus aureus Cowan I strain is rich in

surface protein A whereas the Staphylococcus aureus Wood 46 strain









expresses very low levels (Freirer et al., 1979; Reis et al., 1984a).

Isolation of protein A by lysostaphin extraction results in a

homogeneous product which is composed of a single polypeptide chain

with a molecular weight of 42,000 daltons (Sjoquist et al., 1972, Bjork

et al., 1972). The Fc binding part of the protein consists of four

repetitive subunits. Recently, the gene for protein A has been

identified and cloned (Uhlen et al., 1984; Duggleby and Jones, 1983;

Lofdahl et al., 1983). The DNA sequence has revealed a fifth region,

homologous to the four repetitive subunits. This region, however, does

not appear to bind the Fc region of immunoglobulins. Protein A has an

elongated structure which results in anomalous estimates of molecular

weight by gel filtration (Bjork et al., 1972). It has been purified in

high yields fror. bacterial culture supernatants, and extracts of

staphylococci, by affinity chromatography, using columns of immobilized

rabbit or human IgG (Kronvall, 1973b).

Biological Properties

The binding of protein A to the Fc region of IgG mediates a

variety of biological activities. Complexes between protein A and IgG

are capable of activating the classical complement pathway (Stahlenheim

et al., 1973). Addition of protein A to human (Kronvall and Gewurz,

1970; Stahlenheim et al., 1973), guinea pig (Sjoquist and Stahlenheim,

1969), rabbit (Stahlenheim et al., 1973), dog or pig serum (Kronvall

and Gewurz, 1970) was shown to deplete complement activity. This

depletion depended on the amount of protein A relative to IgG. Langone

et al. (1978a,1978b) demonstrated that protein A and IgG formed

complexes that behaved functionally like IgM in their interaction with

both whole complement and purified C1. The molecular formula of these









IgM-like complexes is [(IgG)2PA]2 (Langone et al., 1978b; Hanson

and Schumaker, 1984). Several studies have shown that formation of

these complexes between protein A and IgG inhibited phagocytosis of

Staphylococcus aureus, by suppressing the opsonizing ability of

complement (Dossett et al., 1969; Forsgren and Quie, 1974; Peterson et

al., 1977; Verhoef et al., 1977; Musher et al., 1981).

Protein A is also capable of stimulating T- and B-cell

mitogenesis. The nature of this response, however, depends on whether

protein A is added in a soluble or insoluble form. Several studies

have shown that soluble protein A can act as a potent T-cell mitogen,

but can only activate B-cells in the presence of helper T-cells

(Gugliemi and Preud'Horine, 1980; Schuurman et al., 1980; Dosch et al.,

1980). Smith et al. (1983) suggested that the T-cell mitogenic

activity was not associated with protein A, but was due to a bacterial

exotoxin.

In contrast to soluble protein A, protein A on intact bacteria, or

bound covalently to sepharose or sephadex particles, is primarily a

T-cell-independent, B-cell mitogen (Romagnani et al., 1980; Ruuskanen

et al., 1980; Pryjma et al, 1980a,b). Insoluble protein A can

stimulate the secretion of IgM, IgG, and IgA by both peripheral blood

lymphocytes (Muraguchi et al., 1980; Romagnani et al., 1980; Gausset et

al., 1980), and spleen cells (Ringden et al., 1977).

In vivo studies have shown that protein A mediates immune

reactions in a manner similar to that of antigen-antibody complexes.

Some of these effects appear to involve complement activated by the

protein A-IgG complexes (Lawman et al., 1984). Anaphylaxis-like

reactions and Arthus reactions are seen when as little as 10 pg of

protein A is injected intradermally in guinea pigs (Gustafson et al.,









1968). Larger amounts of protein A (500-1000 iy) cause fatal

anaphylactic shock. In humans, an intradermal injection of 10 Ug of

protein A causes wheal and erythema reactions (Martin et al., 1967).

Streptococcal Fc Receptors Type II Through Type V

Certain strains of group A, C, and G streptococci have been

reported to have Fc receptors analogous to protein A (Kronvall,

1973a). This was first recognized by the ability of these organisms to

agglutinate red blood cells sensitized with a sub-agglutinating dose of

antibody (EA), indicating the presence of an IgG Fc receptor on the

surface of these bacteria (Kronvall, 1973a). This Fc reactivity was

further confirmed by measuring non antigen-specific binding of

radiolabeled human myeloma IgG to these bacteria. Based on the ability

of sera from different animal species to inhibit the binding of

radiolabeled human IgG to a variety of bacteria, several distinct types

of streptococcal Fc receptors (FcR) have been identified (Myhre and

Kronvall, 1977). The receptor characteristic of group A streptococci

is the FcR type II. Groups C and G streptococci carry a common, or

related receptor, designated type III. Class IV FcR is found only on

bovine group G B-hemolytic streptococci, and type V is found on certain

strains of Streptococcus zooepidemicus (Myhre and Kronvall, 1981). See

Table 1-1.

Only one of the four types of streptococcal Fc receptors has been

purified and characterized. This is the type III receptor found on

certain group C and group G streptococci. Heat treatment (Freimer et

al., 1979), hot acid extraction (Havlicek, 1978; Muller and Blobel,

1983), phage lysis (Reis et al., 1984a; Christensen and Holm, 1976),

papain (Bjorck and Kronvall, 1984), trypsin or mutanolysin digestion






5



TABLE 1-1

Species and Subclass IgG Reactivities of Bacterial Fc Receptorsa



IgG Fc Receptor Typeb
Immunoglobulin
I II III IV V


Human




Mouse




Cow


Sheep


Goat


Horse



Rabbit

Guinea Pig

Rat

Dog

Cat

Pig

Chicken


IgGi
IgG2
IgG3
IgG4

IgGi
IyG2a
IgG2b
IgG3

IgG1
IgG2

IgG1
IgG2

IgG1
IgG2

IgG(ab)
IgG(c)
IgG(T)

IgG

IgG

IgG

IgG

IgG

IgG

IgG


+
+++
+++
+++


+
+++
+++
+++


NTC
NT
NT
NT


+++
+++
(+)


-++ NT +++

-++ NT NT

NT

NT

NT

-++ NT +++

NT NT


+++ = Indicates strong reactivity
+ = Indicates low reactivity
(+) = Weak, atypical reactivity
a = Sunnarized from Kronvall, 1973a; Myhre and Kronvall, 1977,
1980a,1981; keis, 1984c.
b = See text
c = NT, Not tested


~+
~+

+++
~+

tt+
+++










(Reis et al., 1985) have been used to isolate this Fc receptor.

Digestions with papain or trypsin yield a homogeneous product with

molecular weights of 29 K with papain, or 40 K with trypsin.

Considerable heterogeneity in size of the type III Fc receptor, ranging

from 30,000 to 100,000 daltons, is observed with the other extraction

procedures; however, all molecular weight species are antigenically

related (Reis et al., 1984a), indicating that they are oligmers or that

cell wall components are attached. The streptococcal FcR type III has

a very broad reactivity, including all four subclasses of human IgG and

many other mammalian classes and subclasses (Myhre and Kronvall, 1980b;

Myhre and Kronvall, 1981; Reis et al., 1984b; Boyle, 1984). The

biological properties of the type III receptors have not been

extensively characterized.

Little information is available on the type II FcR found on

certain group A streptococci. Attempts to isolate this receptor have

met with limited success for several reasons. Less than 50% of the

group A streptococcal strains tested have detectable Fc receptors

(Kronvall, 1973a; Freimer et al., 1979), whereas, the type III Fc

receptor is found on greater than 80% of the group C and group G

streptococcal strains tested (Myrhe and Kronvall, 1979; Myrhe and

Kronvall, 1980b). The type I Fc receptor is found on approximately 90%

of all Staphylococcus aureus strains tested (Langone, 1982a; Sperber,

1976). The amount of Fc receptor on the surface of positive strains of

group A streptococci is much less than that found of the surface of

Staphylococcus aureus or group C streptococci (Christensen and Oxelius,

1974; Freiner et al., 1979; Reis et al., 1983). In addition to low

amounts of Fc receptors on group A streptococci, these Fc receptors









have been reported to be unstable during subculture (Freirer et al.,

1979). Havlicek (1978) has reported that Fc receptors are found more

frequently on fresh isolates than on freeze-dried strains. Only 30% of

the freeze-dried strains had Fc receptors, while all of the fresh

isolates tested in this study had detectable Fc receptor activity.

The type II Fc receptor, unlike protein A or the type III Fc

receptor, is restricted in its reactivity with mammalian immuno-

globulins, reacting only with the four subclasses of human IgG, rabbit

IgG, and pig IgG (Myhre and Kronvall, 1977;1980a,b) (see Table 1-1).

It has recently been reported that, based on morphological evidence,

different types of IgG-Fc receptors exist on group A streptococci

(Wagner et al., 1983). Wagner's electron microscopy studies show that

ferritin-conjugated IyG from various species result in different

labeling patterns. Studies on the inhibition of binding of homologous

versus heterologous IgGs are also in agreement with the existence of

more than one type of IgG Fc receptor on the same strain.

The group A streptococcal IgG Fc receptors are distinct from

several cell wall constituents, including the M-protein, group carbo-

hydrate, peptidoglycan (Christensen et al., 1979), and lipoteichoic

acid (Schalen et al., 1980). In addition to IgG Fc receptors, some

strains of group A streptococci are capable of binding the Fc region of

human IgA through a distinct receptor (Christensen and Oxelius, 1975;

Kronvall et al., 1979; Schalen et al., 1980).

Fc Receptors as Virulence Factors

The involvement of Fc receptors in the pathogenicity of disease is

unknown, although some evidence suggests that there is a correlation

between the virulence of certain ,roup A streptococci and their ability

to bind the Fc portion of human IgG (Burova et al., 1980,1981). Serial









mouse passages of various group A strains have been reported to result

in the selection of highly virulent variants. A majority of these

strains have enhanced expression of certain surface proteins, including

IgG Fc receptors, indicating the possibility of an association between

Fc receptor activity and virulence (Burova et al., 1980,1981). Recent

evidence indicates that group A streptococcal FcR activity can be

induced, or its expression enhanced, in association with other factors

that are known to relate to virulence. The role of plasmids in the

expression of anti-phagocytic activity, opacity factor, and IgG and IgA

Fc receptors has been explored (Burova et al., 1983). Burova has found

that these factors may be triggered by insertion of plasmid DNA into

the bacterial chromosome. Plasmid control of the group A Fc receptors

offers an explanation of why these receptors are lost during subcultur-

ing. Before the importance of Fc receptors as virulence factors can be

critically assessed, however, this receptor will have to be isolated,

and methods developed to quantify then on fresh isolates and within

immune complexes.

Practical Applications of Bacterial Fc Receptors

The ability of certain bacterial surface proteins to react

selectively with the Fc portion of immunoglobulin molecules has been

utilized in a variety of immunological procedures primarily for

purifying and quantifying reactive classes and subclasses of IgG

(Lanyone, 1982b; Boyle, 1984; Goding, 1978). These receptors can be

labeled to high specific activity or immobilized without loss of Fc

binding activity arid can be used to detect and quantify antigens,

antibodies, or antigen-antibody complexes (Langone et al., 1979; Gee

and Lanyone, 1981). Currently, by using type I and type III bacterial









Fc receptors, virtually any antibody of the IgG class, with the excep-

tion of those raised in birds or rats, can be detected. This enables a

single tracer to be used in many assays for different antigens. In

addition, binding of an electron dense ligand to Fc receptors can be

used in a variety of techniques for locating and quantifying antigen-

antibody complexes (Goding, 1978; Gee and Langone, 1981). Isolation of

the type II, type IV, and type V bacterial Fc receptors with restricted

reactivities would enable techniques to be developed that can focus on

a narrower range of immunoglobulin species, classes and subclasses.

Summary

The binding of imunoglobulins via the Fc region to certain

bacteria has been known for several years. Several studies involving

the binding of various species' classes and subclasses of immuno-

globulins to staphylococci and streptococci have determined that at

least five distinct bacterial Fc receptors exist. Only two of these

bacteria Fc receptors, however, have been purified to homogeneity and

characterized. These receptors are the type I Fc receptor from

Staphylococcus aureus and the type III Fc receptor, which is found on

certain strains of group C and group G streptococci. The scant amount

of information on the other bacterial Fc receptors is due in large part

to the failure to identify bacterial strains with high levels of stable

Fc receptors type II, IV or V on their surface.

The involvement of these Fc receptors in the pathogenicity of

disease is unknown. Protein A has been shown to activate complement

and cause B-cell mitogenesis in vitro and immediate-type hypersensitiv-

ity reactions in vivo. Little is known, however, about the biological

properties of the streptococcal Fc receptors and whether they are









involved in the pathogenicity of disease. Some evidence suggests that

there is a correlation between the virulence of certain group A

streptococci and their ability to bind the Fc region of human IgG.

Until the group A streptococcal Fc receptors are purified and the

biological activities explored, however, the importance of these

receptors in the pathogenesis of group A streptococcal infections, and

post-infection sequelae, will be difficult to assess.

The purpose of this study was to isolate and characterize the type

II Fc receptors found on certain group A streptococci and to examine

the distribution of the type II Fc receptors and their relationship in

the pathogenesis of streptococcal diseases. The specific aims were

to:

1. Develop a method to screen bacterial isolates for Fc receptor

proteins, in particular the type II Fc receptors (Chapter

Two).

2. Isolate and characterize the type II Fc receptors front group A

streptococci (Chapter Three).

3. Determine the distribution of the type II Fc receptors on

nephritoyenic and non-nephritogenic group A streptococci

(Chapter Four).















CHAPTER TWO
A TECHNIQUE FOR THE DETECTION OF BOUND AND SECRETED
BACTERIAL Fc RECEPTORS


Introduction

The type II Fc receptor on certain group A streptococci has proved

difficult to study because it is only found at low levels on positive

strains and is frequently lost during subculturing. The initial aim of

this study was, therefore, to establish a rapid method to screen group

A streptococcal strains for Fc receptors and to identify and expand

individual colonies that expressed high levels of these receptors.

Previous studies have shown that passage of streptococci in nice

resulted in enhanced Fc receptor expression (Burova et al., 1980,

1981). This approach has been used to isolate a group A streptococcal

strain with increased Fc receptor expression (Reis et al., 1984d).

Until a method is developed to screen fresh isolates of group A

streptococci without subculturing, it will be difficult, however, to

assess the role that this receptor may play in group A streptococcal

infections and post-infection sequelae.

This chapter describes a semi-quantitative procedure that enables

isolates of group A streptococci to be screened rapidly for Fc receptor

expression. Using this technique, a mouse-passaged group A strepto-

coccal strain that was selected for its high levels of surface Fc

receptors (Peis et al., 1984d) was shown to be heterogeneous in the

level of expression among individual colonies. Colonies expressing

high levels of surface Fc receptors were selected from replica plates









and were shown to maintain a higher average level of Fc receptor

expression on repeated subculture.

Materials and Methods

Bacterial Strains, Media, and Growth Conditions

Laboratory strains and fresh isolates of p-henolytic streptococci,

obtained from Dr. Elid Ayoub at the University of Florida, College of

Medicine, and Staphylococcus aureus strains, obtained from the American

Type Culture Collection, were used in these studies. B-hemolytic

streptococci were grouped by the Phadebact Streptococcus Test,

Pharmacia Diagnostics, Piscataway, NJ. All strains were grown in

Todd-Hewitt broth (DIFCO) for 18-24 hr at 370C, harvested by

centrifugation, and washed in phosphate-buffered saline (PBS), pH 7.2.

The optical density at 550 mm was determined to standardize the

concentration of organisms used in subsequent tests.

Staphylococcus aureus Cowan I and Staphylococcus aureus Wood 46

served respectively as high and low protein A (PA)-producing strains.

IgG and F(ab')2 Fragments

Stock human IgG was prepared by chromatography of normal human

serum on DEAE cellulose (Boyle and Langone, 1980). Aliquots were stored

at -700C until use. Human IgG F(ab')2 fragments were prepared by

pepsin digestion of the stock IgG preparation by the method described

by Reis et al. (1983). The IgG and F(ab')2 fragments were prepared

from an individual donor and therefore the IgG and F(ab')2 fragments

displayed the same distribution of antigenic reactive antibodies.

Iodination of IgG

Purified human IgG was iodinated by mild lactoperoxidase method

using enzyme beads (Bio-Rad) as described previously (Reis et al.,

1983). The IgG routinely had a specific activity of 0.3 mCi/mg.









Detection of Surface Bacterial Fc Receptors

An overnight suspension of bacteria was diluted in Todd-Hewitt

broth to give 10-100 colonies when 0.1 ml was plated on Todd-Hewitt

agar (Todd-Hewitt broth containing 1.5% agar). The plates were

incubated at 370C for 16 h and replica plated onto blood agar plates

(BBL Microbiology Systems, Cockeysville, MD 21030) as described by

Lederbery and Lederberg (1952). A circular piece of nitrocellulose,

previously soaked in 25 mM Tris, 192 mM glycine (pH 8.3) and 20% v/v

methanol followed by a circular piece of Whatman 3 m paper was placed

on top of the colonies. The agar was removed from the petri dish and a

piece of 3 Gm paper was placed on the bottom of the agar. The

bacterial colonies were transferred to the nitrocellulose by

electrophoresis at 70 V for 3 h in the above buffer. This procedure

resulted in quantitative transfer of the bacterial colonies, as judged

by comparing photographs of plates before transfer with the stained

nitrocellulose membrane after the electroblotting procedure.

After electrophoresis, the nitrocellulose was washed in veronal

buffered saline (VBS) containing 0.25% gelatin and 0.25% Tween-20 for

one hour with four 250 ml changes. The nitrocellulose was probed for

3 h in the washing buffer containing 2 x 105 cpm/nl 1251-labeled

hunan IgG and a two-fold excess of unlabeled F(ab')2 fragments.

Inclusion of F(ab')2 fragments in the probing mixture eliminates any

binding of IgG through antigenic recognition sites (Reis et al., 1983).

After probing, the nitrocellulose was washed four times in 0.01 M EDTA,

1 II NaC1, 0.25% gelatin, and 0.25% Tween-20 for 15 min each and allowed

to air dry. The nitrocellulose blots were autoradiographed by exposing









to Kodak XAR-5 film with intensifying screen for 3 days at -700C. This

procedure is summarized in Figure 2-1.

Absorption of 1251-Labeled IgG by Bacteria

The detection of Fc-reactive proteins on the surface of bacteria

was determined by the ability of bacteria to bind 1251-labeled

human IgG using a modification of the method of Nyhre and Kronvall

(1977). Briefly, a standard number of bacteria was incubated with

20,000 cpm 1251-labeled human IgG and a twofold excess of unlabeled

F(ab')2 fragments for 1.5 h at 370C. The bacteria were pelleted by

centrifugation at 1000 x g for 5 min and washed twice with 2 ml of VBS

containing 0.01 M EDTA and 0.1% gelatin (EDTA-gel). The radioactivity

associated with the bacteria was determined in an LKB autogamma

counter. The cpm bound to the bacterial pellet were expressed as

percent of added radioactivity.
Results

Fc Receptor Expression of Staphylococcus aureus

The purpose of this study was to develop a procedure that could

rapidly screen isolated colonies of bacteria for Fc receptor

expression. Initially I used the protein A-rich Staphylococcus aureus

Cowan I strain as a reference high level positive control and the

Staphylococcus aureus Wood 46 strain as a reference low level positive

control. In these experiments, a dilution of Staphylococcus aureus

Cowan I or Wood 46 strain was seeded onto Todd-Hewitt agar plates to

yield approximately 50 colonies following overnight incubation at 370C.

The colonies were transferred to nitrocellulose and probed with

1251-labeled human IgG containing a two-fold molar excess of









unlabeled F(ab)2 fragments. The F(ab')2 fragments were prepared

from the same source of human IgG as that used to prepare the labeled

tracer. Previous studies have shown that inclusion of the F(ab')2

fragments in the probing mixture eliminates any binding of IgG through

antigenic recognition sites (Reis et al., 1983). Consequently, only

binding via the Fc region is measured using this probing mixture. The

procedure is detailed in the Methods and summarized in Figure 2-1. The

results of a typical experiment using Staphylococcus aureus strains are

presented in Figure 2-2. The Staphylococcus aureus Cowan I strain

demonstrated a greater expression of Fc receptors than Wood 46 strain.

The surface expression of Fc receptors on an individual plate was

consistent from colony to colony, for both the Cowan I and the Wood 46

strain.

Quantification of Secreted Fc Receptors

Secreted Fc receptors were measured using a modification of the

blotting procedure described in Figure 2-1. After removal of the agar

from the petri dish, the nitrocellulose was placed on the underside of

the agar away from the bacteria rather than directly in contact with

the colonies. Fc receptors that were secreted by the bacteria were

electroblotted through the agar and bound to the nitrocellulose while

the colonies were unable to pass through the agar. The nitrocellulose

was probed as before with 1251 human IgG containing unlabeled

F(ab')2 fragments, and then autoradiographed. Figure 2-2 illustrated

the secretion of Fc receptors by the Cowan I and Wood 46 strain. As

expected, the Cowan I strain secreted high levels of Fc receptor

activity. By contrast, the Wood 46 strain did not secrete sufficient

protein A to be detected by this procedure, see Figure 2-2.





















I. REPLICA
PLATE


(+)-(







NITRO-
CELLULOSE


4. PROBE WITH- 5. AUTO-
25I-PROTEIN RADIOGRA
RADIOGRAPH


AGAR


Schenatic representation of the ir.inunoblotting procedure for
detection of surface Fc receptors.


Fig. 2-1.


I
















'2"IIgG Binding to Staphylococcal Strains


Cell Associated


Secreted




al. 4
dk %*


B













Fiy. 2-2. Detection of Fc receptors on the surface of staphylococcal
strains.
A, Staphylococcus aureus Cowan I strain; B, Staphylococcus aureus
Wood strain. The colonies of staphylococci were blotted onto
nitrocellulose and probed with 123I-labeled human IgG in the
presence of unlabeled F(ab')2 fragments as described in the text.
Autoradiography was carried out by exposure of the blot for 3 days at
-70C to X-ray film using an intensifying screen.









In order to determine the sensitivity of this method for detecting

secreted Fc receptors, differing concentrations of purified protein A

(0.05-500 ng/5 il) were applied directly to agar plates. The agar was

removed and treated as described above. The applied protein A was

electroblotted through the agar onto nitrocellulose and probed for

binding of labeled 1251 human IgG. The blot was autoradiographed

at -70C for one day and the diameter of the resulting dot was

measured. The area of the dot was proportional to the square of its

radius (r). When the value of r2 for each concentration of protein A

originally applied to the agar was plotted against the concentration of

protein A added, a linear relationship was observed over the range 6.25

to 50 ng, see Figure 2-3.

Fc Receptor Expression on a Group A Streptococcal Strain

In the initial experiments a mouse-passed strain of group A

streptococci, 64/14, which has been shown to have high levels of

surface Fc receptor (Reis et al., 1984d), was selected to study the

expression of Fc receptors on the surface of streptococci. A single

colony of the mouse-passed strain 64/14 was grown overnight in

Todd-Hewitt Broth. Bacteria were then diluted as described in the

Methods to yield 10-100 colonies per plate and tested for Fc receptor

expression. The Fc receptor expression on the individual colonies of

strain 64/14 is illustrated in Figure 2-4. Unlike the uniform pattern

observed with Staphylococcus aureus strains (Figure 2-2), the intensity

of the spots on the autoradioyraph varied considerably, indicating

heterogeneity in expression of Fc receptors on individual colonies.

This effect was not due to variation in the size of individual

colonies, since, as shown on the replica plated sanple, the colony size

















60

50-

40

30



S20-
E
E

CIA

10





5-
| I I I I I I
5 10 20 30 40 5060


ng PROTEIN A ADDED






Fig. 2-3. Sensitivity of assay for secreted Fc receptors.
Protein A front 0.05 to 500 ng was applied in 5 1l of VBS, to a
culture plate, electroblotted through the ayar onto nitrocellulose and
then probed with 1251-labeled human IgG in the presence of
unlabeled F(ab')2 fragments. The blot was autoradiographed by
exposure for 1 day at -700C to X-ray film using an intensifying screen
and the diameter of the resulting dot on the X-ray film was measured.
The value of r2, which is proportional to the area of the dot, was
plotted against the concentration of protein A applied to the plate.


















0-I.~
0TI
6*
S)(
I 0


..

.4C


*


*.
*


A'A












Fig. 2-4. Fc receptor expression of a group A streptococcal strain.
The panel on the left of the figure shows colonies of strain 64/14
on a blood agar replica plate. The panel on the right is an autoradi-
ograph of a replica plate following the probing with 1251-labeled
human IgG in the presence of unlabeled F(ab')2 fragments. Autoradi-
ography was carried out by exposure of the blot for 3 days at -700C to
X-ray filn using an intensifying screen.









was remarkably uniform. A representative high intensity (Fc receptor-

rich) and low intensity (Fc receptor-poor) colony was selected from the

replica plate and subcultured. The expression of surface Fc receptor

on the progeny of a low-producer colony and a high-producer colony are

shown in Figure 2-5. The progeny of the high producing colony

maintained greater levels of Fc receptor expression than the progeny of

the low producing colonies; however, heterogeneity in surface

expression of Fc receptors within the selected strains was still

observed. On repeated subculturing, however, the low and high

producers remained readily distinguishable, and none of these strains

secreted measurable quantities of the Fc receptor.

The differences in average Fc receptor expression of these two

bacterial subpopulations were also tested in a direct binding assay.

In this assay the indicated number of bacteria was incubated at 370C

for 1.5 h with 2 x 104 cpn/O.1 ml 1251 human IgG and a two-fold

molar excess of unlabeled F(ab')2 fragments in a total volume of

0.2 ml VBS-gel buffer. The bacteria were pelleted by centrifugation at

1000 x g for 5 min and washed twice with 2 ml of EDTA-gel buffer and

the number of cpm bound to the bacteria determined. The results in

Figure 2-6 demonstrate greater IgG binding to the high producing

population than to the low producing population, with the parental

64/14 strain binding an intermediate level. These results are in

agreement with the findings in Figure 2-5 and would suggest that the

blotting assay is accurately reflecting surface Fc receptor expression

on these bacteria.



















































Fig. 2-5. Fc receptor expression of two bacterial subpopulations
selected front the original strain 64/14.
The autoradiograph in the left panel was obtained by probing a
high Fc receptor producing substrain and the autoradiograph in the
right panel was obtained by probing a low Fc receptor-producing colony.
These colonies were selected and subcultured front individual colonies
in the replica plate shown in Figure 2-4. Auturadiography was carried
out by exposure of the blot for 3 days at -70C to X-ray film using an
intensifying screen.
intensifying screen.








































I06 107


Number of


Bacteria


Fig. 2-6. Fc dependent uptake of 1251-labeled human Ig by
increasing numbers of bacteria.
.--, parental strain 64/14; A---, high Fc receptor producing
substrain from 64/14; ----o, low Fc receptor producing substrain from
64/14.









Discussion

In this chapter, a method for rapidly screening fresh isolates of

streptococci for Fc receptors has been described. This technique can

measure both cell-associated and secreted Fc receptors and is capable

of identifying individual colonies that express high levels of these

receptors. Initially, the method was developed using Fc receptor

positive Staphylococcus aureus strains that express stable levels of

the type I Fc receptor on their surface. These were the protein A-rich

Cowan I strain and the protein A-poor Wood 46 strain (Freimer et al.,

1979; Reis et al., 1984c). The conditions for electroblotting

bacterial colonies onto nitrocellulose and probing for Fc receptor

expression were established using these strains (Fig. 2-2). The

125I-labeled human IgG probe was made Fc receptor-specific by

including a two-fold molar excess of unlabeled F(ab')2 fragments from

the same lyG pool used to prepare the labeled tracer. Under these

conditions the unlabeled F(ab')2 fragments inhibit the binding of any

specific anti-bacterial antibody (Reis et al., 1983). In agreement

with previous reports, all of the staphylococcal colonies expressed

surface protein A, and there was a marked quantitative difference

between the levels expressed on the Cowan and Wood strains (Freimer et

al., 1979; Reis et al., 1984c).

When this approach was applied to a mouse-passaged group A

streptococcal strain, the intensity of individual autoradiographed

streptococcal colonies showed wide variation (Fig. 2-4). In order to

ensure that these findings were reproducible, one colony that expressed

high levels of Fc receptors and a second colony, with a low level of Fc

receptor expression, were subcultured from the replica plate.









Following subculture, each colony was plated, electroblotted and probed

as described. The results indicated that the intensity of the progeny

colonies were markedly different (Fig. 2-5). Differences in surface Fc

receptor expression of the two substrains were confirmed by direct

Fc-mediated binding of labeled human lyG (Fig. 2-6). In the two days

required to expand a high or low expressing population, considerable

heterogeneity in Fc receptor expression on the resulting colonies was

readily detected (Fig. 2-4). These findings suggest that the

expression of these type II Fc receptors on group A streptococci is

constantly shifting and could explain many of the reports of changes

in, or loss of, Fc receptor expression during subculture of group A

streptococcal strains (Kronvall, 1973a; Christensen and Oxelius, 1974;

Freimer et al., 1979).

The presence of extrachromosomal DNA in certain group A strepto-

cocci has been reported (Clewell, 1981; Burova et al., 1983;

Ravdonikas, 1983; Ravdonikas et al., 1984). Conjugation experiments

between group A streptococcal strains with Fc receptors and group A

streptococcal strains without Fc receptors, suggest that either Fc

receptor expression is controlled by a plasmid, or that the gene for Fc

receptors is encoded on a plasmid (Burova et al., 1983; Ravdonikas et

al., 1984). No plasmids have been isolated, however, from the mouse-

passaged group A streptococcal strain 64/14 (data not shown). The

possibility still exists that the plasmid has integrated into the

genome, but more studies are needed in order to understand the control

of Fc receptor expression that is expressed on group A streptococcal

strains.









In addition to providing a rapid, semi-quantitative assay for

surface Fc receptor expression on individual colonies of bacteria, the

method described was readily modified to measure secreted Fc receptors.

This was achieved by carrying out the electroblotting stage of this

assay with the nitrocellulose placed on the opposite side of the agar

from the bacterial colonies. The approach readily detected nanogram

quantities of secreted protein A from the staphylococcal strains (Fig.

2-3). To date, no Fc receptor secreting group A streptococcal strain

has been identified.

The method described in this chapter for screening individual

colonies of bacteria for Fc receptors has enabled me to monitor type II

Fc receptor expression on a group A streptococcal strain. By continual

selection, I can now maintain a strain rich in the type II receptor

from which to isolate this receptor.

This method can also be used to screen bacteria for other

receptors. Using a modification of the procedure P2-microglobulin,

fibrinogen, fibronectin, and collagen type I and type III receptors

were found on certain isolates of staphylococci and streptococci (see

Chapter Four). In addition, this method can be applied to the study of

fresh isolates of bacteria, to determine if the presence or absence of

a particular protein, or receptor, correlates with subsequent disease

course.














CHAPTER THREE
ISOLATION AND PARTIAL CHARACTERIZATION OF THE TYPE II Fc RECEPTORS
FROM A GROUP A STREPTOCOCCUS


Introduction

Although many group A streptococci have surface receptors for the

Fc region of all four subclasses of human IgG (Myhre and Kronvall,

1977,1979,1980b,1981; Reis et al., 1984d), these receptors are

frequently lost during subculturing (Christensen and Oxelius, 1974;

Freimer et al., 1979), making the isolation and characterization of

these proteins difficult. Recently Reis et al. (1984d) have described

the isolation of a stable Fc receptor-rich group A streptococcal

strain. This strain, which was recovered following fourteen sequential

passages of a group A streptococcus in mice (Reis et al., 1984d),

demonstrated Fc receptor expression approaching that of the protein A

rich Staphylcoccus aureus Cowan I strain (Reis et al., 1984d).

Although it has maintained a high level of Fc receptor expression for

over two years, heterogeneity of expression was observed between

individual colonies when monitored using an immunoblotting assay

(Chapter Two). The immunoblotting technique has enabled me to monitor

Fc receptor expression continuously on individual colonies and to

select substrains with high levels of Fc receptor expression. In this

Chapter, I describe the isolation and characterization of the Fc

receptors from such an Fc receptor-rich group A streptococcal

substrain.









Materials and Methods

Bacterial Strains, Media, and Growth Conditions

The mouse-passaged group A streptococcal strain 64/14 was shown in

Chapter Two to contain colonies with different levels of surface

expression of Fc receptors. Using the immunoblotting technique

described in Chapter Two, a single colony expressing high surface Fc

receptor activity (64/14/HRP) was selected and used as the source for

the isolation of the type II Fc receptor. Staphylococcus aureus Cowan

I strain served as a representative type I Fc receptor-positive strain

and the group C streptococcal strain 26RP66 served as a type III

receptor-positive strain. All strains were grown in Todd-Hewitt broth

(UIFCO) as stationary cultures for 18-24 hr at 370C, harvested by

centrifugation and resuspended in phosphate-buffered saline (PBS), pH

7.2. The optical density at 550 nm was determined to standardize the

concentration of organisms used in subsequent tests. An 00550 of

0.3 corresponded to approximately 2 x 109 organisms/ml.

Dipeptides

Glycyl-L-tyrosine, glycyl-L-histidine, and glycylglycine were

purchased from Sigma Chemical Company, St. Louis, MO.

Extraction of Fc Receptors

The selected group A streptococcal strain 64/14/HRP was grown

overnight at 37C in Todd-Hewitt broth. Approximately 6 g (wet weight)

of bacteria were recovered from 3 liters of Todd-Hewitt broth.

Mutanolysin extraction. Approximately 6 g (wet weight) of group A

strain 64/14/HRP were suspended in 30 ml of 20 mM Tris-HC1, pH 7.5, 1

mM iodoacetic acid, and 1 mrl benzamidine HC1. To this suspension, 100

pg/nl pancreatic DNase (Sigra), 100 pg/ml pancreatic RNase (Signa), and









100 ug/ml mutanolysin were added. A commercial mutanolysin preparation

(Sigma) was further purified to remove protease activity using the

method described by Siegel et al. (1981). The enzyme and bacteria were

incubated at 370C in a shaking water bath for 4 hr. The mixture was

then centrifuged at 10,000 g for 10 min and the resulting supernatant

filtered through a 0.2 pm filter to remove the remaining bacteria. The

filtrate was dialyzed against 20 mM Tris-HC1, pH 7.5, containing 1 mM

iodoacetic acid, 1 mM benzamidine-HC1, and 1 mM phenylmethyl sulfonyl

fluoride (PMSF).

Hyaluronidase extraction. Approximately six grams (wet weight) of

the group A strain, 64/14/HRP, were suspended in 30 ml of 0.15 M PBS,

pH 7.2. To this suspension, 10 mg type IV hyaluronidase (Sigma) was

added and incubated at room temperature for 30 min. The bacteria-free

supernatant was recovered as described above.

Papain digestion. Group A strain 64/14/HRP, approximately 2 g

(wet weight), was suspended in 20 mis of 10 mM Tris-HC1, pH 8.0 and

0.02% NaN3. Two milliliters of 0.4 M cysteine and 1.6 mg papain were

added to this suspension and allowed to incubate at 370C for 1 hr. The

reaction was stopped by addition of iodoacetic acid to a final

concentration of 30 mM. The bacteria-free supernatant was recovered as

described above.

Trypsin digestion. Approximately 2 grams (wet weight) of

group A strain 64/14/HRP in 50 mM KPO4, 5 mM EDTA, 0.02% NaN3, pH

6.1 (20 ml) was incubated at 370C for 1 hr. with 80 ug pancreatic DNase

and 400 Ug trypsin (Sigma). Addition of benzamidine-HC1 to a final

concentration of 100 mM stopped the reaction. Bacteria were removed as

described above.









Phage lysin treatment. Approximately 2 g (wet weight) of the

group A strain 64/14/HRP were suspended in 20 ml of 50 mM KP04, 5 mM

EDTA, 0.02% NaN3, pH6.1. Phage lysin (0.2 ml), previously activated

by incubation at room temperature for 15 min. in dithiothreitol (DTT)

at a final concentration of 50 mM, was added to the 10% suspension and

incubated at 370C for 1 hr. The phage lysin was prepared as previously

described by Fischetti et al. (1971). The bacteria-free supernatant

was recovered as described above.

SDS treatment. A 10% (w/v) suspension of group A strain 64/14/HRP

in 1% sodium dodecyl sulfate was incubated at 80C for 10 min.

Bacteria were removed as described above and SDS was removed by

dialysis against 20 mM Tris-HC1, pH 7.5.

Autoclave treatment. A 10% (w/v) suspension of group A strain

64/14/HRP was autoclaved at 1240C for 15 min, with or without the

addition of 1% SDS.

Hot acid/hot alkaline extractions. Extractions were carried out

according to the method of Lancefield (1928). Bacteria were suspended

in 0.15 M PBS to form a 10% suspension and the pH was adjusted to 2 (or

10) with 0.5 M HC1 (or 0.5 M NaOH). The bacterial suspension was

boiled for 10 min and the pH neutralized. The bacteria-free super-

natants were recovered as described previously.

Heat extraction. This was carried out as described above, but at

neutral pH using PBS, pH 7.0.

IgG and IgG Subclasses

Stock human IgG was prepared by chromatography of normal human

serum on DEAE cellulose (Boyle and Langone, 1980). Aliquots were

stored at -700C until use. Human IgG3 (K) cryoglobulin was a gift









from Dr. Richard Weber, National Institutes of Health, Bethesda, MD.

The cryoglobulin was isolated as described in (Saluk and Clem, 1971).

Human IgG subclasses were provided by the WHO/IUIS Immunoglobulin

Subcommittee.

IgG1 (K) Lot No. 0781 and IgG1 (x) Lot No. 0180;

IgG2 (K) Lot No. 0380 and IgG2 (A) Lot No. 0981;

IgG3 (K) Lot No. 0282 and IgG3 (K) Lot No. 0784 and

IgG3 (X) Lot No. 0381;

IgG4 (K) Lot No. 0981 and IgG4 (X) Lot No. 0880

Purified rabbit, cow, sheep, goat, rat, dog, and pig IgG was purchased

from Cappel Laboratories, Inc., Cochranville, PA.

Immobilized IgG Preparations

Antigens were coupled to immunobeads (Bio-Rad) by the method

described in Reis et al. (1983). The ligand to be immobilized was

covalently bound to the immunobead matrix by peptide bond formation

between the carboxylic groups on the beads and amino acids groups of

the ligand. This reaction is catalyzed by carbodiimide and the

resulting beads can be stored for up to six months at 4C in the

presence of 0.02% sodium azide without loss of reactivity.
Immobilized IgG for affinity purification of the streptococcal Fc

receptor was prepared by covalently coupling human IgG to the high

capacity Affi-gel 15 activated beads (Bio-Rad) as described in (Reis et

al., 1984b).
iodination of IgG and Protein A

Purified Protein A (Pharmacia, Piscataway, NJ) or purified IgG or

IgG subclasses were iodinated by mild lactoperoxidase method using

enzyme beads (Bio-Rad) as described previously (Reis et al., 1983).

The IgG or protein A routinely had a specific activity of 0.3 mCi/mg.










Preparation of Fc Specific Probe
1251 labeled human IgG was made Fc specific by the inclusion

of a twofold molar excess of unlabeled F(ab')2 fragments in the

probing mixture (Reis et al., 1983). Human IgG F(ab')2 fragments

were prepared by pepsin digestion of the stock IgG preparation by the

method described by Reis et al. (1983). The IgG and F(ab')2

fragments were prepared from an individual donor and consequently the

IgG and F(ab')2 fragments contained the same distribution of

antigenic reactive antibodies. Therefore, only binding via the Fc

region is measured using this probing mixture.

Competitive Binding Assay For Soluble Bacterial Fc Receptor

Fc receptor activity in extracts were quantified using the

competitive binding assay of Reis et al. (1983). This assay was

carried out using VBS-gel as the diluent. In this assay 1.0 ml of a

test sample or buffer was mixed with 0.1 ml of a standard suspension of

agarose beads with covalently coupled human IgG (Bio Rad Laboratories,

Richmond, CA), and 0.1 ml of 1251 protein A (approximately 20,000

cpm) and incubated at 37C for 90 min. Two milliliters of veronal

buffered saline containing 0.01 M trisodium ethylenediamine tetra-

acetate and 0.1% gelatin (EDTA-gel) was added to each tube and

centrifuged at 1,000 g for 5 minutes and the supernatant fluid

decanted. After an additional wash, the radioactivity associated with

the beads was determined in an LKB Gamma Counter.

Gel Electrophoresis and Western Blotting

Proteins were analyzed by electrophoresis under denaturing

conditions in polyacrylamide gels containing sodium dodecyl sulfate

according to Laem li (1970). Gels for staining were fixed in a









solution of 40% ethanol and 10% acetic acid, stained with Coonassie

brilliant blue R-250 (0.25% w/v in 40% ethanol and 10% acetic acid) for

1 hr, and destined by soaking in several changes of 10% ethanol and

10% acetic acid. Gels used for blotting were equilibrated in 25 nM

Tris, 192 mM glycine, pH 8.3 and 20% v/v methanol (transfer buffer) for

30 nin. A piece of nitrocellulose, previously soaked in the transfer

buffer, was placed on top of the gel. The gel and nitrocellulose were

sandwiched between 2 pieces of Whatman 3 mm paper and placed in a

Bio-Rad Trans Blot apparatus with the nitrocellulose oriented between

the anode and the gel. Electrophoresis was at 70 volts for 3 hr in the

above buffer.

After electrophoresis, the nitrocellulose was washed in veronal

buffered saline (VBS) containing 0.25% gelatin and 0.25% Tween-20 for 1

hr with four 250 nl changes. The nitrocellulose was probed for 3 hr in

the washing buffer containing 2 x 105 cpm/ml of the appropriate

species or subclass of IgG. After probing the nitrocellulose was

washed four times in 0.01 M EDTA, 1 M NaCl, 0.25% gelatin, and 0.25%

Tween-20 for 15 r.iin each and allowed to air dry. The nitrocellulose

blots were autoradiographed by exposing to Kodak XAR-5 film with

intensifying screen for 1 to 3 days at -700C.

Dot-Blot Procedure to Test Species Reactivity

Dot blots were performed using the bio-Rad bio-dot n;icrofiltration

apparatus and a modification of the Bio-Rad procedure. A standard

number of group A strain 64/16/HRP was incubated at 370 for 1 hr with

increasing concentrations of dog, goat, pig, sheep, rabbit, rat,

bovine, or human IgG. A piece of nitrocellulose, previously soaked in

25 rmM Tris, 192 r. i glycine, pH 8.3, and 20% V/V methanol (wash buffer),









was placed in the apparatus. Following incubation, the mixture was

pipetted into the wells and washed with the above buffer. The

nitrocellulose was removed from the apparatus, washed and probed as

described above in the Western blotting procedure.

Preparation of Monospecific Antibodies to a Single Species of Affinity
Purified Fc-Reactive Material

Monospecific antibodies were prepared in chickens whose non-immune

IgG does not react with the Fc-receptor protein being studied. The

choice of a non-reactive host to immunize is important to avoid compli-

cations with hypersensitivity and Arthus reactions (Gustafson et al.,

1968). Immunoglobulins were isolated from egg yolks as described

below. Eggs from a white Leghorn chicken were collected prior to

injection with isolated Fc-reactive protein. This provided a source of

pre-immunization IgG from an individual animal. This was used as a

control for later studies. The chicken was then injected with an

immunogen containing approximately 50 pg of Fc-reactive material

intramuscularly or subcutanously in complete Freund's adjuvant. The

imm unogen used was a single form of the Fc-receptor protein that was

isolated first by affinity purification by binding to, and elution from

a column of irnobilized IgG, and then further purified by SDS

polyacrylanide gel electrophoresis. The gel was stained with Coomassie

blue, and a single stained band was cut from the gel, emmulsified in

adjuvant and used.as the immunogen. The chickens were allowed to rest

for three weeks and then injected with approximately 50 pg of the

immunogen prepared as described above that had been emulsified in

incomplete Freund's adjuvant. Eggs were collected from the chickens,

immunoglobulins were extracted, and the production of antibody was

monitored as described below.










Chloroform Extraction of Egg Yolks

Eggs from the chicken were extracted using a modification of the

procedure described by Aulisio and Shelokov (1967). Briefly, the yolks

were separated from the albumin and adhering membrane, diluted with an

equal volume of PBS, and shaken several times. The suspension stood at

room temperature for 10 min. The extraction was repeated a total of 4

times before centrifugation at 10,000 g for 20 min. To the super-

natant, an equal volume of chloroform was added and the mixture was

shaken at room temperature every 30 min for 2 hr before incubating at

4C overnight. The extraction was centrifuged at 5,000 g for 10 min.

The resulting clear supernantant was assayed for antibody production by

measuring the inhibition of 1251 labeled human IgG binding to the

type II receptor-rich group A bacterial strain, 64/14/HRP.

Chicken Anti-Type I and Anti-Type III Antibodies

Monospecific antiserum to the staphylococcal type I Fc receptor

was prepared as described in (Reis et al., 1984c). Monospecific

antiserum to the streptococcal type III Fc receptor was prepared as

described by Reis et al. (1984a).

Imnobilized Anti-Type II Fc Receptor

Antibodies raised against the affinity purified 38,000 dalton

(type IIb) Fc receptor present in the affinity purified heat extract of
64/14/HRP was prepared as described above. This anti-type IIb Fc

receptor antibody was isolated by chloroform extraction of the yolks of

eggs from inmunized chickens. The antibodies were concentrated by

anr.ionium sulfate precipitation (40%) and covalently coupled to Affi-gel

15 activated beads (Bio-Rad) as described in (Reis et al., 1984a). The

immobilized anti-type lib Fc receptor was used for the affinity

purification of the type II Fc receptors.










Purified Type I and Type III Fc Receptors

Purified type I Fc receptor (protein A) was obtained from

Pharmacia Fine Chemicals, Piscataway, NJ. The type III Fc receptor was

purified as described by Reis, et al. (1985).

Results

Solubilization of The Type II Fc Receptor

A mouse-passaged group A streptococcal strain, 64/14/HRP, was

selected because of its high levels of surface Fc receptors (Chapter

Two) and its stability on subculture (Reis et al., 1984d). Several

extraction procedures were compared, including: 1) hot-acid

extraction, alkaline extraction, or heat extraction at neutral pH;

2) treatment with the enzymes hyaluronidase, mutanolysin, papain,

trypsin, or phage lysin; or 3) heating, or autoclaving the bacteria in

the presence of sodium dodecyl sulfate (SDS). The resulting cell-free

extracts were tested for soluble Fc receptor activity using two assays.

The first was a competitive binding assay, that measures the inhibition

of 1251 labeled protein A (the type I Fc receptor) to immobilized

human IgG and is capable of detecting nanogram quantities of

Fc-reactive proteins (Reis et al., 1983). The second assay was a

Western blotting procedure. In this technique, the extractions were

electrophoresed on an SDS polyacrylamide gel, electroblotted onto

nitrocellulose, and probed with the 1251 labeled human IgG Fc

specific probe. By running a duplicate gel and staining with Coomassie

brilliant blue, Fc receptor activity can be matched to specific protein

bands. The Western blotting procedure has a number of advantages.

First, it enabled size heterogeneity of Fc receptors to be detected and

second, it detected Fc reactivities with sites on IgG not related to









the binding site for staphylococcal protein A. Details of these

procedures are described in the Materials and Methods.

The only treatments that resulted in the solublization of

significant quantities of Fc receptor activity were heat extraction

at neutral pH, and treatment with the enzymes mrutanolysin or

hyaluronidase (Table 3-1). Extraction with SDS, or autoclaving in the

presence of SUS, also resulted in solubilizing Fc receptor activity,

but with lower specific activity. Extraction of the group A

streptococci with acid, alkali, papain, trypsin, phage lysin, or by

autoclaving did not solubilize detectable quantities of a functionally

active Fc receptor in either assay. Comparison of the extracts by

Western blot analysis showed that the extract with the least

heterogeneity resulted from heating the bacteria at neutral pH (Fig

3-1). This extraction procedure was consequently used to isolate the

type II Fc receptor.

Isolation of the Type II Fc Receptor

The type II Fc receptor-rich, group A streptococcus was heat

extracted as described in the Methods. The bacteria-free extract was

dialyzed against 20 rn1 Tris-HC1, pH 7.5 containing protease inhibitors

at a concentration of 1 rrM phenylmethyl sulfonyl fluoride (PMSF), 1 mM

iodoacetic acid, and 1 mM benzamidine-HC1 to prevent degradation of the

Fc receptors (Grubb et al., 1982).

The dialyzed extract was applied to a column of human IgG

immobilized on Affi-gel 15 which had been prewashed with 3 M MgC12

and then equilibrated in 20 rV~ Tris-HC1, pH 7.5, containing protease

inhibitors. The extract was applied to the column, and unbound

material was eluted by washing with the Tris-HCl buffer. Fc receptor

was eluted by addition of 3 M MgC12 to the 20mM Tris-HCl, pH 7.5


























C C C C
o 0 0 0
e *r- --
*e= *"- 0r- *r-

n *- *- -- N *4- -r-
* c c 0
a C C 0o C C


o o o0 o


+


C14 C C
o o A 0 0 0 f 0
Z z Z z z z


(A
4-)

0o

4--
x
LUJ


0
u
U
0


04-


-I S


LU

CO *r-
c:
I-
o4-









C t-
Ce-







o



y

u-


c-
0

ro *-


C -

C)


C LO

co S-
0
G 4-
4- a0)

*.- S-


o-


V)
>) -o





CI- C
4-)
0 -
C r



4-'

CO
4 -





4- O


s-
1i

.r- U -O I- CId-





















*i-- **- *- -
0 ( I c C> > W
>-


cl
;M











0
01 +



fu -.\C *0 a M ( a


a O O 0 > 3 c > >
+ t t0 3 : =r CL ( ) <1
o -k r- na n ao
... "-ua "










23 4 5 6


56k- I. -
^^~ ^-


38k-


Fig. 3-1. Western blot autoradiograph of affinity purified extractions
of 64/14/HRP.
Lanes 1 and 2 contain 1.8 ug and 4.2 ug of heat extracted Fc
receptor. Lanes 3 and 4 contain 4 ug and 12 ug of hyaluronidase
extracted Fc reactive material. Lanes 5 and 6 contain .5 ug and 1.2 ug
mutanolysin extracted Fc receptor. The affinity purified samples were
electrophoresed on an SDS polyacrylamide gel, electroblotted onto
nitrocellulose, and probed with 1251 Fc specific probe as described
in Midterials and Methods. Autoradiography was for 20 hr at -700C with
an intensifying screen.


- -0 e









buffer. Other eluting buffers, including 1% SDS solution, 0.1 M

glycine-HCl, pH 2.0 or 0.2 M glycyl tyrosine, were tested, but only

MgC12 resulted in the recovery of significant quantities of

functionally active Fc receptors.

After elution from inmobilized IgG, each fraction was dialyzed

overnight against PBS containing 10 rfi EDTA to facilitate the removal

of Mg++ and all samples were finally dialysed into PBS without

EUTA. Each fraction was assayed for Fc receptor activity using a

competitive binding assay. Fractions containing Fc receptor activity

were pooled and concentrated by Amicon Ultrafiltration using a PM-10

membrane with a molecular weight cut off of 10,000 daltons. Aliquots

were stored at -700C and maintained their Fc receptor activity for at

least 1 yr.

The size heterogeneity of the affinity purified Fc receptor

was determined by electrophoresis on an SDS polyacrylamide gel followed

by staining with Coomassie brilliant blue (Fig. 3-2A). Two protein

bands were observed. The major protein band had a molecular weight of

56,000 daltons and a minor band was observed at 38,000 daltons. To

determine if both bands had Fc receptor activity, a parallel SDS

polyacrylamide gel was run, the separated proteins were electroblotted

onto nitrocellulose and probed with the 1251 labeled Fc specific

probe as described in Materials and Methods. The results of the

Western blot demonstrated that the 56,000 dalton protein reacts

strongly with the 1251 labeled Fc specific probe, whereas the

38,000 dalton protein demonstrated only weak reactivity (Fig. 3-2B).


























S-56KI


-38KI1












Fig. 3-2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of
the type II Fc receptor.
Panel A: Lane 1 contains 5 ug of the type I receptor (staphylo-
coccal protein A). Lane 2 contains 4 ug of the type II Fc receptor.
Samples were electrophoresed under denaturing conditions and stained
with Coomassie brilliant blue as described in Materials and Methods.
Panel B is the autoradiograph of a duplicate gel which was electro-
blotted onto nitrocellulose and probed withl25I Fc specific probe.
Autoradiography was for 2 days at -70C using an intensifying screen.
Lane 1 contains 30 ng of the type I receptor. Lane 2 contains 4000 ng
of the type II receptor.









The Effect of Dipeptides on the Binding of IgG Subclasses to the Type
II Fc Receptor

Certain amino acid residues in the Fc region of human IgG have

been reported to be important in the binding of human IgG or certain

human IgG subclasses to the type I Fc receptor (protein A). Tyrosine

residues in the Fc region of human IgG (Deisenhofer et al., 1978) and

in protein A (Sjoholm et al., 1973) have been shown to be involved in

this interaction. The ability of protein A to bind certain allotypes

of IgG3 has been shown to be associated with a single amino acid in

the heavy chain (Recht et al., 1981; Haake et al., 1982; Shimizu et

al., 1983). Comparison of amino acid sequences of immunoglobulins that

bind protein A with those that do not, has implicated the importance of

histidine residue at position 435 of the heavy chain of the IgG3

molecule (Haake et al., 1982). Furthermore, Bywater (1978,1983)

reported that glycyl-tyrosine could elute human IgG bound to a protein

A sepharose column. When glycyl-tyrosine was tested for its ability

to displace the bound type II Fc receptor from a human IgG affinity

column, no Fc receptor activity was eluted. Glycyl-tyrosine and a

second dipeptide, glycyl-histidine, however, did inhibit the binding of

certain human IgG subclasses to the type II receptor-rich group A

strain 64/14/HRP (Fig. 3-3). The binding of human subclasses 1, 2 and

4 to 64/14/HRP was inhibited by both glycyl-tyrosine and glycyl-

histidine, whereas the effect of these two dipeptides on the binding of

IgG3 was minimal (Fig. 3-3). Glycylglycine had no effect on any of

the subclasses and none of these dipeptides inhibited the uptake of

labeled human IgG to the type I Fc receptor-positive Staphylococcus

aureus Cowan strain, or to the type III Fc receptor-positive yroup C

streptococcal strain 26RP66 (Fig. 3-4).





















20 -



3 C D

loo



80-

60


40-

20-


100 200 300 400 100 200 300 400

DIPEPTIDE CONCENTRATION (iM)

Fig. 3-3. The effect of dipeptides on the binding of 1251-labeled
human IgG subclasses to an Fc receptor-positive group A
streptococcus.
A standard number of group A streptococci was incubated for 1.5
hours at 370C with 20,000 cpm of the appropriately 1251-labeled
human IgG subclasses in the presence of varying concentrations of
dipeptide. The bacteria were pelleted by centrifugation at 1000 x g
for 10 min and washed twice with 3 nl of veronal buffered saline
containing 0.01 M EDTA and 0.1% gelatin. The radioactivity associated
with the bacteria was determined in an LKB autogamma counter. The cpm
bound to the bacterial pellet were expressed as percent of maximum cpm
bound.
Panel A represents the results obtained from the binding of 1251 IgG1
Panel B represents the results obtained from the binding of 1251 IgG2
Panel C represents the results obtained from the binding of 1251 IgG3
Panel D represents the results obtained from the binding of 1251 IgG4
*-*_ Glycylglycine
*--_ Glycyl-L-histidine
A--^ Glycyl-L-tyrosine






44










100



80

z

m 60

01.
0-
.-j
S40-
0
I.-


20-




100 200 300 400 500

GLYCYL- HISTIDINE (mM)









Fig. 3-4. The effect of glycyl-histidine on the binding of 1251.
labeled human IgG to three bacterial strains.
A standard number of Staphylococcus aureus Cowan I strain ( -A),
group C streptococcal strain 26RP66 (*-e), or the group A streptococcal
strain 64/14/HRP (n-n) was incubated for 1.5 hr at 37*C with 20,000 cpn
of 1251 labeled human IgG in the presence of the indicated amounts
of gycyl-histidine. The bacteria were pelleted by centrifugation at
1000 x g for 10 min and washed twice with 2 nl of veronal buffered
saline containing 0.01 M EUTA and 0.1% gelatin. The radioactivity
associated with the bacteria was determined in an LKB autoganma
counter. The cpm bound to the bacterial pellet were expressed as
percent of maximum cpm bound.









Reactivity of the Type II Fc Receptor With Human IgG Subclasses

The differences in the observed reactivity between the human IgG

subclasses and the dipeptides (Fig. 3-3) raised the possibility that

more than one Fc receptor existed on the surface of the group A strain

64/14/HRP. To test this possibility, samples containing the affinity

purified heat extracted type II Fc receptor were electrophoresed on SDS

polyacrylamide gels, transferred to nitrocellulose by electroblotting

and parallel gels were probed with 1251 labeled human IgG1,

IgG2, IgG3, or IgG4. The type I Fc receptor and the type III Fc

receptor were included on each gel as reference positive controls (Fig.

3-5).

The type I Fc receptor, protein A, contained one major protein

band with a Mr of approximately 52,000 that bound IgG1, IgG2, and

IgG4 but not IgG3 (Fig. 3-5). The heat extracted, affinity

purified type II receptor contained a major protein band with an Mr

of approximately 56,000, and a minor band with a Mr of approximately

38,000. The major 56,000 dalton band reacted with human IgG1,

IgG2, and IgG4 but failed to react with human IgG3. The minor

38,000 dalton protein reacted only with human IgG3. The type III Fc

receptor demonstrated a single protein band with a Mr of

approximately 40,OUO and this protein reacted with all four human IgG

subclasses (Fig. 3-5).

A major concern with the results obtained in Figure 3-5, was the

possibility that the observed reactivity of the type II receptor with

IgG3 was unique to the labeled myeloma probe being used. Conse-

quently these studies were repeated using two other human IgG3

myeloma proteins. The results shown in Figure 3-6 indicate that all





46


A B C D

1 2 3 2 3 2 3 I 2 3


.- *



56 K--














Fig. 3-5. Human imunoglobulin G subclass reactivity of bacterial Fc
receptors.
Lane 1 contains 20 ng of the type I receptor (Staphylococcal
Protein A). Lane 2 contains 100 ng of the type III Fc Receptor, and
Lane 3 contains 4,000 ng of the type II Fc Receptor. Samples were
electrophoresed under denaturing conditions in polyacrylamide gels
containing sodium dodecyl sulfate according to Laemmli (13). Gels were
equilibrated in 251,14 tris, 192 mM glycine, pH 8.3 and 20% v/v methanol
(transfer buffer) for 30 min. A piece of nitrocellulose previously
soaked in the transfer buffer was placed on top of the gel. The gel
and nitrocellulose were sandwiched between 2 pieces of Whatman 3mm
paper and placed in a Bio-Rad trans blot appparatus with the nitro-
cellulose oriented between the anode and the gel. Electroblotting was
at 70 volts for 3 hours in the above buffer. After electroblotting,
the nitrocellulose was washed in veronal buffered saline containing
0.25% gelatin and 0.25% Tween-20 for 1 hour with four 250 ml changes.
The nitrocellulose was probed for 3 hours in the washing buffer
containing 2X105 cpn/nl 1251 human IgG of the appropriate
subclass. After probing the nitrocellulose was washed four times in
0.011 EDTA, IM NaCl, 0.25% gelatin, and 0.25% Tween-20 for 15 min each
and allowed to air dry. Autoradiography was carried out by exposure of
the blot for 3 days at -700C to Kodak XAR-5 film using an intensifying
screen.
Panel A is the result from blots probed with 1251 IgG1.
Panel B is the result from blots probed with 1251 IgG2.
Panel C is the result from blots probed with 1251 IgG3.
Panel 0 is the result from blots probed with 1251 IgG4.



















A


I 2 3


1 2 3 1 2 3


Fig. 3-6. Reactivity of three hunan myeloma IgG3 samples with
bacterial Fc receptors.
Lane 1 contains 20 ng of the type I receptor. Lane 2 contains 100
ny of the type III receptor and Lane 3 contains 4,000 ng of the type II
receptor. Samples were electrophoresed on parallel SDS polyacrylamide
gels, electroblotted onto nitrocellulose, and each blot was probed with
a different 1251 labeled myeloma IgG3 protein as described in
the legend to Fig. 1. Autoradioyraphy was carried out by exposure of
the blot for 24 hours at -700C to x-ray film using an intensifying
screen.
Panel A represents a blot probed with 1251 IgG3 (K) Lot No. 0784.
Panel B represents a blot probed with 1251 IgG3 (K) Lot No. 0282.
Panel C reDresents a blot nrobed with 1251 Tnr, t i n- n ri


38 K-


gJJ


I .


S --- .. .


\ "/ *- "




II,









three human IgG3 samples reacted with the minor Mr 38,000 protein

in the affinity purified heat extracts of the group A streptococcus.

One of the IgG3 samples was found to react with the type I receptor

staphylococcall protein A) and this particular sample of IgG3 also

reacted with the major Mr 56,000 protein as well as the minor Mr

38,000 protein in the extract of the group A streptococcus (Fig. 3-6,

Panel A).

The IgG3 reactivity was further confirmed in inhibition studies

using unlabeled subclass standards. Only the sample containing IgG3

could efficiently inhibit the binding of the 1251 labeled human

IgG3 to the type II receptor-positive group A streptococcus, while

equimolar concentrations of IgG1, IgG2 or IgG4 showed minimal

inhibition (Fig. 3-7). All of the samples of unlabeled IgG3 tested

were capable of inhibiting the binding of the labeled IgG3, with the

most efficient inhibitor being the sample that was also used to prepare

the labeled probe.

Separation of Two Functionally Distinct Type II Fc Receptors

The 56,000 dalton Fc receptor which reacted with IgG1, IgG2,

and IgG4 (designated type IIa), was separated from the 38,000 dalton

IgG3-specific Fc receptor (designated type IIb) by use of an

immobilized column of IgG3. Heat extracts of 64/14/HRP containing

both Fc receptors were applied to a column of an isolated human IgG3

(K) myeloma immobilized on Affi-gel 15. The column was pre-

equilibrated in 20 mM Tris-HC1, pH 7.5. The type IIa Fc receptor

failed to bind to the IgG3 column and was recovered in the void

volume of the column by washing with the Tris-HC1 buffer. The type lIb

receptor, which bound to the IgG33 column, was eluted with 3 M


















z
0 60-
i-

z
40-



20-




0.06 0.125 Q25 0.5 1.0 2.0

IgG SUBCLASS ADDED (pg)




Fig. 3-7. Inhibition of binding 125I labeled IgG3 to a group A
streptococcus by unlabeled human IgG subclasses.
Approximately 1 x 107 of the Fc receptor-rich group A
streptococci, were incubated at 37C for one hour with the indicated
amounts of each human IgG subclass. Following incubation, each mixture
was dotted onto a piece of nitrocellulose previously soaked in 25 mM
tris, 192 mM glycine, pH 8.3 and 20% v/v methanol using the Bio-rad
bio-dot microfiltration apparatus. After each well was washed with the
above buffer, the nitrocellulose was removed and washed in veronal
buffered saline containing 0.25% gelatin and 0.25% Tween-20 (wash
buffer) for one hour with four 250 ml changes. The nitrocellulose was
probed for three hours in the wash buffer containing 2 x 105 cpm/ml
251 labeled human IgG3. After probing the nitrocellulose was
washed four times in 0.01H EDTA, li Na C1 0.25% gelatin and 0.25%
Tween-20 and allowed to air dry. The nitrocellulose was cut into
sections that contained an individual well and the cpm associated with
each section of nitrocellulose was determined using an LKB autogamma
counter and the percent inhibition of 1251 IgG3 binding to the
bacteria was calculated.
*--* IgG1
A--A IgG2
o--o IgG3
A--A IgG4









MgC12 in 20 nM Tris-HC1, pH 7.5, dialyzed, and concentrated as

described above.

The unbound material from the IgG3 column (the type IIa

receptor) was further purified by binding to, and eluting from a column

of immobilized human IgG. The affinity purified Fc receptors were

analyzed by the Western blotting procedure. The type IIa and type IIb

Fc receptors were electrophoresed on SDS polyacrylamide gels,

electroblotted onto nitrocellulose and parallel gels were probed with

either 1251 labeled human IgG to detect the type IIa Fc receptor,

or 1251 labeled human IgG3 to detect the type IIb Fc receptor

(Fig 3-8). 1251 labeled human IgG was used as a probe for the type

IIa Fc receptor because of the distribution of subclasses 1, 2, and 4

in relation to subclass 3 found in normal human serum. Human IgG

subclass 3 comprises only 8% of the total human IgG in the serum

(Lewis et al., 1970). Therefore, the contribution made by IgG subclass

3 in the labeled human IgG probe is minimal.

In Fig. 3-8A, the type IIa Fc receptor reacted strongly with the

human IgG probe and no contamination with the type lib Fc receptor was

seen on the gel probed with 1251 labeled IgG3 (Fig. 3-8B).

Similarly, the isolated type IIb Fc receptor reacted strongly with the

IgG3 probe (Fig. 3-8B) and no contamination of the type IIa Fc
receptor was seen (Fig. 3-8A). The type I and type III Fc receptors

were included on each gel as reference positive controls.

Species Immunoglobulin Reactivity With the Type IIa and Type IIb Fc
Receptors

Five distinct bacterial Fc receptors have been classified based on

the reactivity of whole bacteria with different sources of

















A B

1 2 3 4 1 2 3 4





56 K-




-38K

















Fig. 3-8. Separation of the type Ila Fc receptor from the type IIb Fc
receptor.
Lane 1 contains 30 ng of the type I Fc receptor (protein A). Lane
2 contains 30 ny of the type III Fc receptor. Lane 3 contains 3 pg of
the type IIa Fc receptor, and Lane 4 contains 2 ug of the type IIb Fc
receptor. Samples were electrophoresed on SDS polyacrylamide gels,
electroblotted onto nitrocellulose, and probed with either 1251
labeled human IgG or IgC3. Panel A represents a blot probed with
1251 huran IgG. Panel B represents a blot probed with 125
human IgG3.









immunoglobulins (Myhre and Kronvall, 1981). In this classification,

the type II Fc receptor associated with group A streptococci Fhould
bind all four subclasses of human IgG, and IgG from rabbit'-'ad pig

(Myhre and Kronvall, 1977) (see Table 1-1). In the next-sertig of

experiments, the IgG species binding profile for the grouptP strain
64/14/HRP which was used to isolate the type Ila and type T1'i c
receptors was determined. A standard number of group A stfrfi
64/14/HRP was incubated with increasing concentrations of dod, goat,
sheep, rat, pig, rabbit, bovine, or human IgG. After -hicubatra n, the
mixture was .dotted onto nitrocellulose and the nitrocellfffise&'was then
probed with either 1251 labeled human IgG, or 1251 labeled' ei
human IgG3. Details are described in Materials and Methods. %e
results indicate that the Fc receptors on the surface ofgrouiptA strain
64/14/HRP could only bind pig, rabbit, and human IgG. Dog,- 6't,
sheep, rat or bovine IgG could not inhibit the binding of:12I ;
labeled human IgG (Fig. 3-9A) or 1251l'abeled human IgG3(Ffgl I
3-9B) to strain 64/14/HRP. -:ain
The ability of pig and rabbit IgG to inhibit the binding' b both
1251 labeled human IgG and 1251 labeled human IgG3 indicate: u:

that either the type I:Ia and type lib Fc receptors on tho'surf~i e of
group A strain 64/14/HRP are both capable of reacting, or _d)W Fr
receptors are linked.on the bacterial surface and the bindfri fofi human,

pig, or rabbit IgG to one Fc receptor sterically hinders- the'iability of
the other Fc receptor to bind. -" :-ee

The purpose of the next experiment was to determine if botWethe
type IIa and type lib Fc receptors reacted equally with rabbit :nd pig
IgG, or if the inhibition seen in Fig. 3-9 with both 125i latibed
human IgG and human IyG3 was due to steric hindrance.









IgG ADDED (jg)


In 0
SIo 0

O 0



*


o
0 0
C6 C (\


* 0*


8 5
oo


* 0
0O
0*
* *


HUMAN 0* *


B IgG ADDED (pg)


0 00 OO O
0 L2 c 0 0 0
GOATO O O O O 0

PIG @*******
DOG O
RABBIT O OO O
SHEEP @00 0000
RAT OO O 0
cowHUMAN *OO
HUMAN &


ssee







0@$$


Fig. 3-9. Inhibition of 1251 human IgG or 1251 human IgG3 to
group A strain 64/14/HRP by immunoglobulin G from a variety
of mammalian species.
A standard number of group A strain 64/14/HRP was incubated at
370C for 1 hr with the indicated amount of goat, pig, dog, rabbit,
sheep, rat, cow, or human IgG. Following incubation, each mixture was
dotted onto nitrocellulose. The nitrocellulose was washed and probed
with 1251 labeled human IgG or human IgG3 as described in
Materials and Hethods. Autoradiography was for 16 hr at -700C with an
intensifying screen.
Panel A represents the blot probed with 1251 labeled human IgG.
Panel B represents the blot probed with 1251 labeled human IgG3.


0 0
00
0.o
*


0 .
0
* 0

*


0*0
000
0**-


GOAT
PIG
DOG
RABBIT
SHEEP
RAT
COW


000090
OOO.O0
OO0. O*









Samples containing both the type IIa and type IIb Fc receptors

were electrophoresed on SUS polyacryladmide gels, electroblotted onto

nitrocellulose and probed with 1251 labeled human IgG, pig IgG,

rabbit IgG, dog IgG, or bovine IgG (Fig. 3-10). The results show that

only the type IIa Fc receptor could bind 1251 labeled pig or rabbit

IgG. Reactivity with the type IIb Fc receptor was only seen with
1251 labeled hunan IgG (Fig. 3-10). This suggests that the two Fc

receptors must be closely linked on the surface of group A strain

64/14/HRP to account for the partial inhibition of 1251 labeled

IgG3 that is seen in Fig. 3-9B.

Antigenic Relationship Between the Type IIa and Type lIb Fc Receptors

In order to test whether both the type IIa and type lIb Fc

receptors were antigenically related, antibodies were prepared in

chickens against the type IIa and type IIb Fc receptors. The affinity

purified heat extract, containing both Fc receptors, was electro-

phoresed on an SDS polyacrylamide gel and both the 56,000 dalton Fc

receptor and 38,000 dalton Fc receptor were identified by Coomassie

blue staining. Each band was cut from the gel and used as the

imnunogen to prepare the monospecific antibodies using the immunization

schedule described in Materials and Methods. Antibody was isolated

from the egg yolk by chloroform extraction and activity was monitored

by the ability of the isolated extract to inhibit binding of 1251

hunan IgG or human IgG3 to the group A strain 64/14/HRP. The results

in Fig. 3-11A indicate that the antibody prepared against the type IIa

(Mr = 56,000) Fc receptor can efficiently inhibit the binding of both
1251 labeled human IgG and 1251 labeled human IgG3 to the

group A strain 64/14/HRP. A similar result was observed using the

























56K- 1


38 K-


















Fig. 3-10. Redctivity of lyG from a variety of species with the type
IIa or type lib Fc receptor.
Affinity purified heat extract (4 ug) containing both the type IIa
and type lib Fc receptors was electrophoresed on five parallel SDS
olyacrylamide gels, electroblotted onto nitrocellulose and probed with
251 labeled hunan IgG (Lane 1), pig IgG (Lane 2), rabbit IgG (Lane
3), dog IgG (Lane 4), or bovine IgG (Lane 5) as described in Materials
and Methods. Autoradiography was for 30 hr at -700C using an
intensifying screen.









anti-type lib Fc receptor (Fig. 3-11B). No inhibition was observed

when normal chicken immunoglobulins were added. The shapes of the

inhibition curves of the IgG binding or the IgG3 binding were similar

for each antibody (compare Figures 3-11A and 3-11B). This finding

suggests that either each Fc receptor was present at an approximately

equal density and recognized with equivalent affinity, or that the two

Fc receptors were closely linked on the bacterial surface. When the

heat extract was affinity purified by binding to a column of

immobilized chicken anti-type lib Fc receptors and then eluting with 3

M MgC12, both the type IIa and type IIb Fc receptors were recovered

in a functionally active form (data not shown). This provides evidence

that the two distinct Fc receptors are antigenically related and that

the observed inhibition by the anti-type IIa and type IIb antibodies on

the intact group A organism could not solely be attributed to steric

hindrance.

Antigenic Relationship of the Type I, Type II and Type III Fc
Receptors

The antigenic relationship between the type I, II and III

bacterial Fc receptors was tested using monospecific chicken antibodies

to each receptor type. Each antibody was tested at a number of

concentrations for its ability to inhibit the binding of 1251

labeled human lyG to a fixed concentration of the group A streptococcal

strain 64/14/HRP. The results, presented in Fig. 3-12, indicate that

only the antibody against the type II Fc receptor could inhibit the

binding of 1251 labeled human IgG to strain 64/14/HRP. Antibodies

against the type I Fc receptor could not inhibit the binding of
1251 labeled human IgG to the group A strain 64/14/HRP or to the

























Fig. 3-11. Inhibition of binding of 1251-labeled human IgG or
1251-labeled human IgG subclass 3 to group A
streptococcal strain 64/14/HRP by monospecific antibodies
against the purified type IIa Fc receptor or the type IIb
Fc receptor.
A standard number of group A streptococcal strain 64/14/HRP was
incubated 1.5 hr at 37C with 20,000 cpn of 1251 labeled human IgG
or 1251 labeled human IgG3 in the presence of the indicated
amounts of the monospecific chicken anti-type IIa (Panel A) or
anti-type lib Fc receptor (Panel B). The bacteria were pelleted by
cenrrifugation at 1000 x g for 10 min and washed twice with 2 ml
veronal buffered saline containing 0.01 M EDTA and 0.1% gelatin. The
radioactivity associated with the bacteria Was determined in an LKB
autogamma counter and the percent inhibition calculated.
o__o 1251 labeled human IgG3
*--* 1251 labeled human IgG


















80o


401-


201-


10 100
RELATIVE ANTI-TYPE Ira Fc RECEPTOR


_ I


80o-


601


40k-


20-


I O RECEPTOO
10 100
RELATIVE ANTI-TYPE IIb Fc RECEPTOR


In '





















100 -



80-



5 60-



40



20



10 100
RELATIVE ANTIBODY CONCENTRATION









Fig. 3-12. Inhibition of binding of 1251 hunan IgG to 64/14/HRP by
antibody against the type I receptor (0-a), the type II
receptor (*-*), or the type III receptor (o-o).
A relative antibody concentration of 100 for the type I antibodies
inhibits the binding of 125I human IgG to the Staphylococcus aureus
Cowan I strain by 94% and inhibits the binding of l' I hunan IgG to
the group C streptococcal 26RP66 strain by less than 10%.
A relative antibody concentration of 100 for the type III antibody
inhibits the binding of 1251 human IgG to the Staphylcoccus aureus
Cowan Strain by less than 10% and inhibits the binding of 125I
human IgG to the group C streptococcal 26RP66 strain by 92%.









group C Fc receptor-rich strain 26PP66, but could completely inhibit

the binding of the labeled immunoglobulin to the Staphylococcus aureus

Cowan strain. Similarly, the antibody to the type III receptor failed

to inhibit binding of 1251 human IgG to either the group A

streptococcal strain 64/14/HRP or to the Staphylococcus aureus Cowan

strain while totally inhibiting the binding of labeled immunoglobulin

to the Fc receptor-rich group C streptococcus 26RP66. No inhibition of

binding was observed to any bacterial strain when normal chicken

immunoglobulins were added.

Discussion

In this Chapter I describe the isolation and characterization of

two functionally active Fc receptors from an Fc receptor-rich substrain

of a group A streptococcus. This bacterial substrain has been selected

from a mouse-passaged group A strain by use of an immunoblotting

technique that measures Fc receptor expression on individual colonies

(Chapter Two). Several methods for solubilizing the Fc receptor from

this bacteria were tested, including: 1) heat extraction at neutral,

acid or alkaline pH, 2) digestion with the enzymes mutanolysin,

hyaluronidase, papain, trypsin, or phage lysin, or 3) autoclaving or

heating the bacteria in the presence of SDS. Soluble Fc receptor

activity was detected after heat extraction at neutral pH, treatment

with mutanolysin or hyaluronidase, or following autoclaving or heating

in the presence of SDS, Table 3-1. No activity was recovered following

any of the other extraction procedures (see Table 3-1). Heat

extraction at neutral pH resulted in the most homogeneous form of the

receptor, (see Fig. 3-1) and this also had one of the highest yields of

any of the extraction procedures tested (see Table 3-1).









Functionally active Fc receptor activity could be isolated from

the heat extract by binding to and elution from a column of immobilized

human IgG. A variety of different agents were tested for their ability

to remove the bound receptor from the immobilized human IgG column and

the most efficient was found to be 3 M MgC12. Analysis of the

affinity purified material by SDS polyacrylamide gel electrophoresis

revealed two bands with molecular weights of 56,000 and 38,000 daltons

(Fig. 3-2). When the proteins were electroblotted onto nitrocellulose

and probed with a 1251 human IgG Fc specific probe, both bands were

shown to be active, although the 38,000 dalton band had much less

activity. These two proteins bands could be shown to be antigenically

related, since both were recovered when the heat extract was affinity

purified on a column of immobilized chicken antibody that was directed

against a single form of the affinity purified Fc receptor. Although

these findings would be consistent with the 38,000 dalton protein being

a degradation product of the larger 56,000 dalton receptor, protease

inhibitors were included in all buffers used during the purification.

Another possibility was that these two molecular weight forms were

distinct Fc receptors. Recent studies by Wagner et al. (1983) using

immunoelectronmicroscopic approaches suggest that several distinct Fc

receptors can be present on the surface of a single group A

streptococcus. Analysis of the effects of various dipeptides (Fig.

3-3) and the reactivity of different human IgG subclasses (Fig. 3-5)

confirmed the existence of at least two distinct Fc receptors on the

group A strain being studied.









The identification of two distinct Fc receptors from group A

strain 64/14/HRP was shown by probing four parallel nitrocellulose

blots containing the affinity purified type II Fc receptors with each

human IgG subclass. The 56,000 dalton protein (designated as type IIa

Fc receptor) could bind human IyG subclasses 1, 2, and 4, whereas the

38,000 dalton protein (designated as type lib Fc receptor) only bound

hunan IgG subclass 3 (Fig. 3-5). Two other IgG3 myeloma proteins

were also tested and both reacted with the 38,000 dalton receptor (Fig.

3-5). Only one of the IgG3 myeloma proteins tested was capable of

reacting with the type I Fc receptor and this immunoglobulin also

demonstrated a low level of reactivity with the 56,000 dalton (type

IIa) receptor (Fig. 3-6A). This suggests that the amino acid residues

which are important in the binding of the Fc region of human IgG1,

IgG2, IgG4, and certain allotypes of IgG3, to the type I Fc

receptor, may also be part of the recognition site on the IgG molecule

for the type IIa Fc receptor. In addition, the type IIa Fc receptor

was capable of binding pig and rabbit IgG in contrast to the type IIb

Fc receptor which could only bind human IgG3 (Fig. 3-10).

The studies on the effect of dipeptides on the binding of certain

human IgG subclasses to the Fc receptors on the surface of group A

strain 64/14/HRP provided further evidence for two distinct

functionally active forms of Fc receptor. Significant inhibition of

binding of IgG1, IgG2 and IgG4 to 64/14/HRP was observed with

glycyl-histidine and glycyl-tyrosine, while IgG3 binding was not

markedly effected (Fig. 3-3). Tyrosine and histidine are known to be

important residues in the binding of IgG to the type I Fc receptor









(Deisenshofer et al., 1978; Recht et al., 1981; Haake et al., 1982;

Shimizu et al., 1983), but in these studies only the type IIa receptor

was inhibited. No effects were observed on the interaction of IgG with

either the type I or type III Fc receptors (Fig. 3-4).

Although the type IIa and type Ilb Fc receptors are functionally

and physicochemically distinct, they were shown to be antigenically

related (Fig. 3-11). Monospecific antibodies prepared against the type

IIa or type IIb Fc receptors showed similar patterns of inhibition of

the binding of both 1251 labeled human IgG and 1251 labeled

human IgG3 to the group A strain 64/14/HRP. Also, both Fc receptors

were recovered when the heat extract was affinity purified on a column

of immobilized anti-type IIb Fc receptor, indicating that the similar

inhibition curves produced with either monospecific antibody could not

solely be attributed to steric hindrance of closely linked Fc receptors

on the bacterial surface. based on the results of the inhibition study

using pig and rabbit IgG (Fig. 3-9), however, the type IIa and type IIb

Fc receptors are probably located close to each other on the cell

surface.

To date, there have been two other reports describing the

isolation of an Fc receptor from group A streptococci. Havlicek (1978)

described the isolation of an Fc receptor from Streptococcus pyogenes

which had a molecular weight of approximately 100,000 daltons and was

recovered from the bacteria following acid extraction. Grubb et al.

(1982) reported the isolation of a type II receptor from a group A

streptococci, type 15, following alkaline extraction. This receptor

could only be isolated to homogeneity in the presence of high

concentrations of protease inhibitors and had an apparent molecular









weight of 29,500. The results presented in Table 3-1 indicate that I

was unable to recover Fc receptor activity following either acid or

alkaline extraction of the group A strain used. These findings would

suggest that the Fc receptors on group A streptococci may represent a

heterogeneous group of molecules which are antigenically related.

The group A streptococcal Fc receptors I have described are anti-

genically and physicochemically distinct from Fc receptors isolated

from other bacteria. Antibodies prepared against the type I staphylo-

coccal or type III group C streptococcal Fc receptors failed to react

with the group A Fc receptor and all three receptors had different

molecular weights, see Figures 3-5 and 3-12.

The studies reported here suggest that at least two functionally

and physicochemically distinct Fc receptors are present on the surface

of certain group A streptococci. To date, using an immunoblotting

technique to study expression of Fc receptors on individual bacterial

colonies (Chapter Two) I have been unable to find a strain that

expressed only the IgG3 selective Fc receptor. The ability to isolate

such a subclass selective receptor has a variety of important practical

applications for the isolation and quantitation of human IgG3. The

importance of such receptors in the pathogenesis of certain strepto-

coccal infections is unclear. The interaction between bacterial

products and components of the host immune system may explain some of

the post infection sequelae associated with infection by certain group

A streptococci.















CHAPTER FOUR
DISTRIBUTION OF THE TYPE II Fc RECEPTORS ON NEPHRITOGENIC AND
NON-NEPHRITOGENIC GROUP A STREPTOCOCCI


Introduction

The importance of Fc receptors in the course of bacterial

infections and post-infection sequelae is not clear. Bacterial Fc

receptors have been postulated as virulence factors (Ginsberg, 1972;

Schalen, 1982; Christensen et al., 1977,1978,1981) and a correlation

has been reported between the virulence of certain group A streptococci

and their ability to bind the Fc portion of human IgG (Burova et al.,

1980). Post-streptococcal glonerulonephritis, a complication that

occasionally occurs following a group A streptococcal infection, is

believed to result from deposition of complement fixing immune

complexes in the glomeruli which initiate a complex series of reactions

that result in the development of renal lesions (Levinsky, 1981).

In the studies described in this chapter, a series of

nephritogenic and non-nephritogenic group A streptococci were screened

for Fc receptor expression. This study was designed to determine

whether Fc receptors are more frequently associated with group A

strains with nephritogenic M serotypes, than with those of M serotype.

not associated with nephritogenic potential.

In Chapter Two, I have described a sensitive blotting technique

that detects Fc receptor expression on bacterial surfaces. In this

chapter I have applied a modification of this technique to study the









interaction of human lyG or human IgG subclasses with group A

streptococci recovered from patients who either did, or did not develop

post-streptococcal glormerulonephritis.

tlaterials and Methods

Streptococcal Strains

Eighteen of the streptococcal strains used in this study were

obtained frno the Rockefeller University collection and were a gift

from Dr. Vincent Fischetti. Fifteen of these strains were isolated

from patients with post-streptococcal glomerulonephritis. The

characteristics of each of these strains have been reported in detail

by Villarreal et al. (1979). Other group A streptococal strains were

obtained from Dr. Elia Ayoub at the University of Florida, College of

Medicine and were either throat or skin isolates.

Dot-Blotting Procedure

Dot blots were performed using the Bio-Rad bio-dot microfiltration

apparatus and a modification of the Bio-Rad procedure. A piece of

nitrocellulose previously soaked in 25 nM tris, 192 mM glycine, pH 8.3

and 20% v/v methanol (wash buffer) was placed in the apparatus. Two-

fold serial dilutions of the bacteria were pipetted into the wells.

The bacteria were diluted in the wash buffer, starting with approxi-

mately 1 x 108 bacteria. The concentration of organisms was

standardized by measuring the optical density at 550 nm. After washing

the bacteria in each well with the above buffer, the nitrocellulose was

removed and washed four times in veronal buffered saline (VBS), pH

7.35, containing 0.25% gelatin and 0.25% Tween-20. Each wash was

carried out for a period of 15 minutes using 250 ml of buffer. The









nitrocellulose was then probed for three hours in the washing buffer

containing 2 x 105 cpm/ml of the appropriate 1251 labeled human

IgG or human IgG subclass. After probing, the nitrocellulose was

washed four times in 0.01 M EDTA, 1 M NaCI, 0.25% gelatin, and 0.25%

Tween-20 (15 minutes each wash) and allowed to air dry. All washing

and probing steps were performed at ambient temperature. The

nitrocellulose blots were autoradiographed by exposing to Kodak XAR-5

film with an intensifying screen for 3-5 days at -700C.

lodination

Human IgG and human IgG subclases were iodinated as described in

Chapter Three.

Results

The distribution of Fc receptors on the surface of 35 strains of

group A streptococci was studied using a dot-blotting procedure. The

results of the experiment probing nephritoyenic and non-nephritogenic

group A strains with 1251 labeled human IgG are presented in Fig.

4-1 and the corresponding radioactive counts for the individual sample

wells are presented in Table 4-1. A comparison of the results in Fig.

4-1 with the counts in Table 4-1 indicate that the intensity of the

spot on the autoradiograph correlates closely with the number of
1251 counts bound to the bacteria attached to the nitrocellulose.

Binding was shown to be dependent on the concentration of bacteria used

in the assay, and non-specific binding was found, under the

experimental conditions chosen, not to be significant (Table 4-1). A

similar relationship between the intensity of the spot on an

autoradiograph and the counts bound to the bacteria was observed with

all of the labeled probes tested.

















Fig. 4-1. Binding of 1251 human IgG to nephritogenic and
non-nephritogenic yroup A streptococci.
The indicated numbers of bacteria were dotted onto nitrocellulose
and probed with 1251 human IgG as described in Materials and
Methods.


Panel A


Strain
B920
B512
11434
A992*
A995
A207
A928
B281
A547
B931*
B905
A374
B438
D897*
B923
B915
F2030
B515


Panel B


M-type
49+
4
12+
18
57+
2
55+
12+
NT
2
2
12+
18
12+
12+
49
1
NT


Strain
646
647
648
650
652
653
654
SHS1
SHS4
SHS7
SHS8
SHS9
SHS10
SHS14
SHS16
SHS17
SHS18


M-type
49+
49+
1
22
NT
"787"
11
NT
12+
NT
NT
NT
NT
NT
1
12+
12+


1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
-13.
14.
15.
16.
17.


NT Non-typable
+ Nephritogenic


M serotypes (Stollerman, 1971)


Panel A contains fifteen nephritogenic strains and three
non-nephritogenic strains (*). Autoradiography was at -70C for 3 days
with an intensifying screen.
Panel B contains seventeen non-nephritogenic strains. Autoradiography
was for 1 day at -70C with an intensifying screen.










Ix 108

5 x 107

2.5 x 107

1.25 x 107

I xl08

5 x 107

2.5 x 107

1.25 x 107


A
I


i0


*

g


2


13 14 15


3 4















16 17


Ix 108
5 x 107
2.5 x 107
1.25 x 107
I x 108

5 x 107
2.5 x I07
1.25 x 107


I 2

* g
* g




0


*


4


5 6

* *
0@


7 8 9 10



*0

0


13 14 15 16 17


8 9


5


6 7






9


10


1




18


11 12

























0
4-)
S-
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C.



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t-0



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en
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20


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w- o 0 nLO L L






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(- 4-)



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qt 00 C) t
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(MO m CMCM cN c )
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in O-4-4-4-4i-i-i r


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,-- < c_ '-
-..U.+-> I I
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J= C









Since the results obtained in Chapter Three indicate the presence

of at least two distinct Fc receptors on group A strains, the

experiments described above using 1251 labeled human IgG as probe

were repeated on the nephritogenic strains with each 1251 labeled

human IgG subclass. The results are presented in Table 4-2. In

general, the nephritogenic strains which were able to bind the 1251

labeled human IgG probe, showed no particular preference for any one

subclass. One strain, A547, did bind IgG3 to a greater extent than

any other subclass, while another strain, A928, had opposite binding

characteristics. These two strains might be useful in isolating the

type lib and type IIa Fc receptors, respectively (see Chapter Three).

Discussion

The role of streptococcal products in generating post-

streptococcal glonerulonephritis is not well understood. In 1982,

Boyle proposed that the Fc receptors and the H protein were critical

components and proposed the following hypothesis which contains these

elements:

1) Nephritogenic strains of streptococci produce an Fc-reactive

protein.

2) This protein is released alone, or associated with other cell

wall constituents and forms complexes with normal IgG which

efficiently bind and activate complement.

3) These complexes of Fc-reactive protein and IgG are either not

cleared or inefficiently cleared by the reticuloendothelial

syster,i and lodge in the kidney. The presence of the

anti-phagocytic M protein within such a complex could inhibit

its efficient clearance.






73



TABLE 4-2

Interaction of Nephritogenic and Non-nephritogenic
Group A Streptococci with Hunan IgG Subclasses



STRAIN M-TYPE TOTAL IgG1 IgG2 IgG3 IgG4
IgG


B920

B512

11434

A992*

A995

A207

A928

B281

A547

B931*

B905

A374

B438

D897*

B923

B915

F2030

B515


49

4

12

18

57

2

55

12

NT

2

2

12

18

12

12

49

1

NT


+1-+

+++

+/

++





++f

+1-


++


+1 -


++


+1 -


+1 -


+++

++



++



+/-

+/-



++++



++++

+

++





+



++


* = non-nephritis causing strains
NT = nontypable strain
- = no detectable binding
+ = binding detectable with 1 x 108 bacteria
++ = binding detectable with 5 x 107 bacteria
-++ = binding detectable with 2.5 x 107 bacteria
++++ = binding detectable with 1.25 x 107 bacteria


++

+++

+/-

+

+

+



+/-

+/-



+
-



+







+/-

+++









4) Once in the kidney, these complexes are trapped in the

vicinity of the basement membrane and tissue damage results

both through the action of complement itself and by other

components of the host's cellular immune system recruited by

chemotactic split products of complement (e.g., C5a).

This hypothesis is summarized in Fig. 4-2.

To test the first element of this hypothesis, a rapid dot-blotting

procedure was developed which enabled me to compare the Fc receptor

expression on nephritogenic vs. non-nephritogenic group A streptococcal

strains. Fifteen out of the eighteen nephritogenic strains tested

(73%) were positive for Fc receptor expression (Fig. 4-1A), whereas 65%

of the non-nephritoyenic strains also had Fc receptors on their surface

(Fig. 4-1B). Since the nephritogenic strains tested were library

strains, the possibility exists that these strains have lost Fc

receptor expression during subculture (Kronvall, 1973a; Christensen and

Oxelius, 1974; Freimer et al., 1979). Although the majority of

non-nephritogenic stains tested were fresh isolates, non-nephritogenic

library strains were also tested and 71% of these library strains still

maintained Fc receptor expression.

In Chapter Three, two distinct Fc receptors were isolated from a

group A streptococcal strain. One Fc receptor was capable of binding

human IgG subclass 1, 2, and 4, while the other Fc receptor was

specific for human IgG subclass 3. Lewis et al. (1970) have reported

that the immunoglobulin subclass composition of the glomerular deposits

in human renal diseases was selective arnd did not reflect the normal

serum concentration of these proteins. Patients who had granular

deposits of immunoglobulin, which suggest an inmune complex
















CLEARED
BY RES


TRANSPORTED
TO KIDNEY


ATTRACT PMNs


C5a COMPLEMI
DERIVED CHEM


COMPLEMENT COMP
ACTIVATION ACTIVE

ENT
OTATIC


ELEMENT
ATION


INNOCENT BYSTANDER


Proposed mechanism of the pathogenesis of post-streptococcal
gl orierul onephri tis.


~~3 ".^
-^*~ ^


Fig. 4-2.


X


X


X









pathogenesis, tended to have selective deposits of innunoglobulin

composed of a single or dominant subclass, usually IgG2 (Lewis et

al., 1970). In light of these studies, the nephritogenic group A

strains were tested for reactivity with each human IgG subclass (Table

4-2), however, none of the Fc receptor-positive nephritogenic strains

showed selective binding to any particular human IgG subclass.

Since Fc-reactive proteins were found on the majority of both

nephritogenic strains and non-nephritogenic strains, these results

indicate that nephritogenicity probably requires other factors in

addition to the ability to produce Fc-reactive proteins. One factor

which has been shown to be important to this pathogenic process is the

anti-phagocytic M protein (Jacks-Weis et al., 1982). The presence of

the M protein in these complexes might prevent their clearance from the

circulation. Such uncleared complexes could lodge in the kidney,

activate complement, and initiate the pathogenic process that leads to

kidney destruction. By contrast, complexes lacking this protein, e.g.,

staphylococcal protein A IgG complexes, would be effectively cleared

from the circulation and consequently an absolute correlation between

Staphylococcus aureus infections and nephritis would not be expected.

Certain M types of group A streptococci are correlated with

nephritogenic potential (Stullerman, 1971), however, not all group A

streptococci with those nephritogenic serotypes cause glomerulo-

nephritis. Also, post-streptococcal glomerulonephritis is not always

caused by a strain with a nephritogenic M serotype. In these studies,

only 53% of the strains isolated from patients with glonerulonephritis

had a nephritogenic M serotype. Of these strains, 63% had Fc receptors

on their surface. These results are similar to those obtained from the









strains isolated from patients without glomerulonephritis, in which 65%

of the strains have Fc receptors on their surface. Consequently, the

results of this study suggest that no absolute correlation exists

between Fc receptor expression and nephritogenicity. Although both the

M~ protein and Fc receptors have been proposed as virulence factors

(Ginsberg, 1972; Schalen, 1982; Christensen et al., 1977,1978,1981),

more studies need to be done in order to determine all the factors

required for post-streptococcal glomerulonephritis. Villarreal et al.

(1979) has reported the occurrence of an extracellular protein isolated

from patients with post-streptococcal glomerulonephritis, however, this

protein, like the type II Fc receptor and the M protein, was sometimes

produced by streptococci obtained from patients without the disease.

No biological properties of this extracellular protein could be

determined, but based on its reactivity with normal and specific rabbit

antiserum, its properties were distinct from those of the type II Fc

receptor (Ohkuni et al., 1983).

The dot-blotting procedure described in this chapter can be

applied to the study of other bacterial diseases. In a recent study, I

have applied this technique to study the expression of a variety of

different receptors on clinical bacterial isolates recovered from

patients suffering from endocarditis (Yarnall et al., 1985). Receptors

for collagen type I and type III, Clq, fibrinogen, fibronectin, and

human IgG were studied. The results of this study failed to identify a

common surface receptor that could account for the ability of these

bacteria to colonize damaged heart tissue.

Since the early studies of Koch (1903), it has been necessary to

fulfill a certain number of criteria before it is possible to establish









a cause-effect relationship between any infectious agent and a disease

process. In carrying out the studies reported in this chapter, I was

aware that it would not be possible to evaluate critically the

hypothesis summarized in Figure 4-2. The results I have obtained would

indicate that the ir.nunoblotting technique I have developed can be used

as an efficient approach for more fully characterizing individual

strains of bacteria isolated from patients with a specific disease.

While this approach will never lead to elucidating a definitive

pathogenic mechanism, the information obtained from these studies is

helpful in determining if any correlation exists between a particular

receptor or surface protein and an infectious agent isolated from

individuals with a common disease state.















CHAPTER FIVE
CONCLUSION


Bacterial Fc receptors have been known for several years. Myhre

and Kronvall (1981) have characterized the functional activity of these

receptors on the bacterial surface, identifying five basic types. The

type I staphylococcal Fc receptor (Langone, 1982a) and the type III

group C streptococcal Fc receptor (Reis et al., 1984c,1984d,1985;

Bjorck and Kronvall, 1984) have been purified and extensively

characterized. Little information is available on the other three

types of Fc receptors. A type II Fc receptor which is found on certain

strains of group A streptococci has been isolated (Grubb et al., 1982),

but with low yield, and the functional activities of the isolated type

II Fc receptor were not characterized.

In this study, I have described the isolation and characterization

of the type II Fc receptors from a mouse-passaged group A

streptococcus. A method was developed which enabled me to select an

individual Fc receptor-rich substrain from which to isolate the type II

Fc receptors. The type II Fc receptors were recovered in high yield

and were composed of two molecular weight forms that were antigenically

related, but functionally distinct. The 56,000 dalton receptor was

capable of binding hur.,an IgG subclasses 1,2 and 4, pig, and rabbit IgG.

The 38,000 dalton receptor could only bind the Fc region of human IgG

subclass 3. This is the first report of a unique receptor for a

particular subclass of human IgG.









The isolation of an Fc receptor which binds the Fc region of human

IgG3 has several practical applications. It can be used for

separating or depleting IgG3 from serum or secretions by immobilizing

the Fc receptor on sepharose. The IgG3-specific Fc receptor can be

radiolabeled or enzyme-linked for use in assays to detect and quantify

IgG3. This would be beneficial in diagnosing diseased states in

which the production of an IgG subclass is restricted to IgG3. For

example, Beck (1981) observed that antibodies directed against the

rubella virus were primarily of the IgG3 subclass. Antibodies

against thrombocytes in the serum of patients with idiopathic

thrombocytopenic purpura were demonstrated by Karpatkin et al. (1973)

to be limited to IgG3. In addition, an extensive study by Natvig et

al. (1967) using Gm-specific antisera showed that the Rh antibodies in

the sera of mothers after an incompatible pregnancy belong to the

IgG3 subclass. The IgG3 Fc receptor might be useful in exploring

the mechanism of the IgG3 restriction in these diseases.

The role of group A streptococcal Fc receptors in the pathogenesis

of infection or post-infection sequelae is not clear. The distribution

of the Fc receptors on nephritogenic and non-nephritogenic group A

streptococci was studied, but no absolute correlation could be

established. The biological activities of the type II Fc receptors can

now be explored using the purified Fc receptors in both in vivo and in

vitro systems. Complement activation, mitogenesis, and the nature of

Fc receptor-IgG complexes can be examined to clarify the role of Fc

receptors as virulence factors.

The methods described in this study to detect secreted and cell

associated Fc receptors, and to solubilize Fc receptors can be used in









future studies to explore new directions. First, streptococcal strains

can be treated with various mutagens or antibiotics to create a strain

which can secrete Fc receptors. Methicillin has been used to isolate

strains of Staphylococcus aureus that produce only extracellular

protein A (Winblad and Ericson, 1973). An enhancement of the formation

of extracellular protein A was also achieved by growing Staphylococcus

aureus in the presence of puromycin (Movitz, 1976). These approaches

can be applied to streptococci to produce a mutant strain which

secretes Fc receptors.

With the discovery of a unique receptor for the Fc region of

IgG3, it is possible that different strains of streptococci have Fc

receptors that are specific for other human IgG subclasses. Using the

techniques described in Chapter Three, one can search for new unique Fc

receptors. Identification of Fc receptors for each human subclass

would be of great value in identifying and isolating IgG subclasses for

use in immunoanalytical and immunodiagnostic assays and for studying

the fine structure of IgG constant domains.

Finally, the ability to screen large numbers of individual

bacterial colonies would be useful in identifying clones carrying a

specific gene that codes for surface receptors or proteins. Isolation

of a bacterial vector with the gene for a group A streptococcal Fc

receptor could be accomplished using this technique. Once the gene is

identified and cloned, several studies could be done to clarify the

loss of Fc expression observed on subculture. Also, specific mutations

of the cloned gene could determine if group A streptococcal Fc

receptors are virulence factors and whether or not they are involved in

the pathogenesis of post-infection sequelae.









REFERENCES


Aulisio, C.G., and A. Shelokov. 1967. Substitution of egg yolk for
serum in indirect fluorescence assay for Rous Sarcoma virus
antibody. Proc. Soc. Exp. Biol. Med. 126:312.

Beck, 0. 1981. Distribution of virus antibody activity among human
IgG subclasses. Clin. Exp. Immunol. 43:626.

Bjorck, L., and Kronvall, G. 1984. Purification and some properties
of streptococcal protein G, a novel IgG-binding reagent. J.
Immunol. 133:969.

Bjork, L., B.A. Peterson, and J. Sjoquist. 1972. Some physiochemical
properties of protein A from Staphylococcus aureus. Eur. J.
Biochem. 25:579.

Boyle, M.D.P. 1982. Fc-reactive proteins and their potential role in
poststreptococcal nephritis. Grant application to the National
Institutes of Health.

Boyle, M.D.P. 1984. Applications of bacterial Fc receptors in
imnunotechnology. Biotechniques Nov/Dec:334.

Boyle, M.D.P., and J.J. Langone. 1980. A simple procedure to use
whole serumx as a source of either IgG- or IgM-specific antibody.
J. Immunol. Methods. 32:51.

Burova, L.A., P. Christensen, R. Grubb, A. Jonsson, G. Samuelsson, C.
Schalen, and M. Svensson. 1980. Changes in virulence, M protein
and IgG Fc receptor activity in a type 12 group A streptococcal
strain during mouse passages. Acta Path. Microbiol. Scand. (B)
88:199.

Burova, L., P. Christensen, R. Grubb, I.A. Krasilnikov, G. Samuelson,
C. Schalen, M.L. Svensson, and U. Zatterstrom. 1981. IgG-Fc
receptors in T-type 12 group A streptococci from clinical
specimens: Absence from M-type 12 and presence in M-type 22.
Acta. Path. Microbiol. Scand. Sect. (B) 89:433.

Burova, L.A., L.E. Ravdonikas, P. Christensen, C. Schalen, and A.
Totolian. 1983. The genetic control of virulence in group A
streptococci. II. Trigger effects by plasmids on anti-phagocytic
activity, opacity factor and IgG and IgA Fc-receptors. Acta Path.
Microbiol. Immunol. Scand. (B) 91:61.

Bywater, R. 1978. Elution of immunoglobulins from protein A-sepharose
CL-4B columns. In: Chromatography of Synthetic and Biological
Polymers. (R. Epton, ed.) Ellis Horwood, Chichester, U.K., pp.
337-340.

Bywater, R., G.-B. Eriksson, and T. Ottosson. 1983. Desorption of
imunoglobulins from protein A-sepharose CL-4B under mild
conditions. J. Imnunol. Methods 64:1.









Christensen, P., and S.E. Holm. 1976. Purification of imunoglobulin
G Fc-reactive factor from Streptococcus azgazardah. Acta. Path.
Microbiol. Scand. Sect. (C) 84:196.

Christensen, P., and V.A. Oxelius. 1974. Quantitation of the uptake
of human IgG by some streptococci groups A, B, C, and G. Acta.
Path. Microbiol. Scand. (B) 82:475.

Christensen, P., and V.A. Oxelius. 1975. A reaction between some
streptococci and IgA myeloma proteins. Acta Path. Microbiol.
Scand. (C) 83:184.

Christensen, P., A. Grubb, R. Grubb, G. Samuelsson, C. Schalen, and
M.L. Svenson. 1979. Demonstration of the non-identity between
the Fc-receptor for human IgG from group A streptococci type 15
and M protein, peptidoglycan and the group specific carbohydrate.
Acta. Path. Microbiol. Scand. (C) 87:257.

Christensen, P., A.G. Sjoholm, and S.E. Holm. 1977. Binding of
aggregated IgG to nephritogenic type 12 streptococci: Influence
of serum, C1 and C4. Acta Path. Microbiol. Scand. 85:359.

Christensen, P., A.G. Sjoholm, S.E. Holm, B. Hovelius, and P.A. Mardh.
1978. Binding of aggregated IgG in the presence of fresh serum by
group A streptococci producing pharyngeal infection: Possible
connection with types frequently involved in acute nephritis.
Acta Path. Microbiol. Scand. 86:29.

Christensen, P.J., J. Sramek, and U. Zatterstrom. 1981. Binding of
aggregated IgG in the presence of fresh serum: Strong association
with type 12 group A streptococci. Acta. Path. Microbiol. Scand.
89:87.

Clewell, D.B. 1981. Plasmids, drug resistance, and gene transfer in
the genus Streptococcus. Microbiol. Rev. 45:409.

Deisenhofer, T., A. Jones, R. Huber, J. Sjodahl, and J. Sjoquist.
1978. Crystalization, crystal structure analysis and atomic model
of the complex formed by a human Fc fragment and fragment B of
protein A from Staphylococcus aureus. Hoppe-Seyler's Z. Physiol.
Chem. 359:975.

Dosch, H.M., R.K.B. Schuurman, and E.W. Gelfand. 1980. Polyclonal
activation of human lymphocytes in vitro. II. Reappraisal of T
and B cell-specific mitogens. J. Immunol. 125:827.

Dossett, J.H., G. Kronvall, R.C. Williams, Jr., and P.G. Quie. 1969.
Antiphagocytic effects of staphylococcal protein A. J. Inmunol.
103:1405.

Duggleby, C.I., and S.A. Jones. 1983. Cloning and expression of the
Staphylococcus aureus protein A gene in Escherichia coli. Nucleic
Acids Res. 11:3065.









Fischetti, V.A., E.L. Gotschlich, and A.W. Bernheimer. 1971.
Purification and physical properties of group C streptococcal
phaye-associated lysin. J. Exp. Med. 133:1105.

Forsgren, A., and P.G. Quie. 1974. Effects of staphylococcal protein
A on heat labile opsonins. J. Immunol. 112:1177.

Forsgren, A., and J. Sjoquist. 1966. "Protein A" from Staphylococcus
aureus. I. Pseudo-immune reaction with human yammaglobulin. J.
Immunol. 97:822.

Freimer, E.H., R. Raeder, A. Feinstein, J. Herbert, B.W. Burner, and
R.R. Coombs. 1979. Detection of protein A-like substances on
hemolytic streptococci prior to use in mixed reverse passive
antiglobulin hemagglutination (MRPAH). J. Immunol. Methods
31:219.

Gausset, P.H., G. Delespesse, J. Duchateau, and H. Collet. 1980. In
vitro response of human peripheral blood lymphocytes to protein A.
DNA synthesis and generation of cells synthesizing the three major
classes of IgG. Immunology 41:891.

Gee, A.P., and J.J. Langone. 1981. Immunoassay using 1251-iodine
or enzyme-labeled protein A and antigen-coated tubes. Analyt.
Biochem. 116:524.

Ginsburg, I. 1972. Mechanisms of cell and tissue injury induced by
group A streptococci: Relation to poststreptococcal sequelae. J.
Infect. Dis. 126:419.

Goding, J.W. 1978. Use of staphylococcal protein A as an
immunological reagent. J. Immunol. Methods 20:241.

Grubb, A., R. Grubb, P. Christensen, and C. Schalen. 1982. Isolation
and some properties of an IgG Fc binding protein from group A
streptococci type 15. Int. Arch. Allergy Appl. Immunol. 67:369.

Gugliemi, P., and Preud'Homme. 1980. Stimulation of T lymphocytes by
protein A from Staphylococcus aureus in B-derived chronic
lymphocytic leukaeriia. Clin. Exp. Immunol. 41:136.

Gustafson, G.T., G. Stalenheim, A. Forsgren, and J. Sjoquist. 1968.
Protein A from Staphylococcus aureus. II. Production of
anaphylaxis-like cutaneous and systemic reactions in non-immunized
guinea pigs. J. Immunol. 100:530.

Haake, D.A., E.C. Franklin, and B. Frangione. 1982. The modification
of human immunoglobulin binding to staphylococcal protein A using
diethylpyrocarbonate. J. Immunol. 129:190.

Hanson, D.C., and V.N. Schumaker. 1984. A model for the formation and
interconversation of protein A-immunoglobulin G soluble complexes.
J. Imrunol. 132:1397.









Havlicek, J. 1978. Occurrence of Fc-reacting factor in acid extracts
of Streptococcus pyogenes and its relationship to M protein. Exp.
Cell. Biol. 46:146.

Jacks-Weis, J., Y. Kim, and P.P. Cleary. 1982. Restricted deposition
of C3 on M+ group A streptococci: Correlation with resistance
to phagocytosis. J. Immunol. 128:1897.

Jensen, K. 1958. A normally occurring staphylococcus antibody in
human serum. Acta. Path. Microbiol. Scand. 44:421.

Karpatkin, S., P.H. Schur, N. Strick, and G.W. Siskind. 1973. Heavy
chain subclass of human anti-platelet antibodies. Clin. Immunol.
Immunopathol. 2:1.

Koch, R. 1903. Festschrift Zum Sechzigsten Geburtstage. Verlag Von
Gustav Fischer, Jena, Germany.

Kronvall, G. 1973a. A surface component in group A, C, and G
streptococci with non-immune reactivity for immunoglobulin G. J.
Immunol. 111(5):1401.

Kronvall, G. 1973b. Purification of staphylococcal protein A using
irmmunoabsorbants. J. Scand. Immunol. 2:31.

Kronvall, G., and H. Gewurz. 1970. Activation and inhibition of
lyG-mediated complement fixation by staphylococcal protein A.
Clin. Exp. Imrunol. 7:211.

Kronvall, G., L. Bjorck, E.B. Myhre, and L.W. Wannamaker. 1979.
Immunoglobulin binding to group A, C, and G. streptococci. In:
Pathogenic Streptococci. (M.T. Parker, ed.) Reedbooks Limited,
Chertsery, Surrey, U.K., pp. 74-76.

Laemmli, U.K. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature (Lond.)
227:680.

Lancefield, R.C. 1928. The antigenic complex of Streptococcus
haemolyticus. I. Demonstration of a type specific substance in
extracts of Streptococcus haemolyticus. J. Exp. Med. 47:91.

Langone, J.J. 1982a. Protein A from Staphylococcus aureus and related
immunoglobulin receptors produced by streptococci and pneumococci.
Adv. Immunol. 32:157.

Langone, J.J. 1982b. Use of labeled protein A in quantitative
imunochemical analysis of antigens and antibodies. J. Inmunol.
Meth. 51:3.

Langone, J.J., M.D.P. Boyle, and T. Borsos. 1978a. Studies on the
interaction between protein A and imunoglobulin G. I. Effect of
protein A on the functional activity of IgG. J. Imnunol.
121:327.









Langone, J.J., M.D.P. Boyle, and T. Borsos. 1978b. Studies on the
interaction between protein A and immunoglobulin G. II.
Composition and activity of complexes formed between protein A and
IgG. J. Immunol. 121:333.

Langone, J.J., M.D.P. Boyle, and T. Borsos. 1979. 1251-protein A:
Application to the quantitative determination of fluid phase and
cell-bound IgG. J. Immunol. Methods 18:128.

Langone, J.J., C. Das, R. Mainwaring, and W.T. Shearer. 1984.
Complexes prepared from protein A and human serum, IgG, or Fc
fragments: Characterization by irimmunochemical analysis of
ultracentrifugation fractions and studies on their
interconversion. Molec. Cell. Biochem. 65:159.

Lawman, M., M.D.P. Boyle, A.P. Gee, and M. Young. 1984. A rapid
technique for measuring leukocyte chemotaxis in vivo. J. Immunol.
Methods 69:197.

Lederberg, J., and E.M. Lederberg. 1952. Replica plating and indirect
selection of bacterial mutants. J. Bacteriol. 63:399.

Levinsky, R.J. 1981. Role of circulating immune complexes in renal
diseases. J. Clin. Pathol. 34:1214.

Lewis, E.J., G.J. Busch, and P.H. Schur. 1970. Gamma G globulin
subgroup composition of the glomerular deposits in human renal
diseases. J. Clin. Invest. 49:1103.

Lofdahl, S., B. Suss, M. Uhlen, L. Philipson, and M. Lindburg. 1983.
Gene for staphylococcal protein A. Proc. Natl. Acad. Sci. USA
80:697.

Martin, R.R., J.G. Crowder, and A. White. 1967. Human reactions to
staphylococcal antigens. A possible role of leukocyte lysosomal
enzymes. J. Immunol. 99:269.

Movitz, J. 1976. Formation of extracellular protein A by
Staphylococcus aureus. Eur. J. Biochem. 68:291.

Muller, H.P., and H. Blobel. 1983. Purification and properties of a
receptor for the Fc-component of immunoglobulin G from
Streptococcus dysgalactiae. Zbl. Bakt. Hyg. 1. Abt. Orig. A.
254:352.

Muraguchi, A., T. Kishinoto, T. Kuritani, T. Watanabe, and Y. Yamamura.
1980. In vitro immune response of human peripheral lymphocytes.
V. PHA- and protein A-induced human R colony formation and
analysis of subpopulations of B cells. J. Immunol. 125:564.

Musher, D.M., H.A. Verbrugh, and J. Verhoef. 1981. Suppression of
phagocytosis and chemiotaxis by cell wall components of
Staphylococcus aureus. J. Immunol. 127:84.









Myhre, E.B., and G. Kronvall. 1977. Heterogeneity of non-immune
immunoglobulin Fc-reactivity among gram positive cocci:
Description of three major types of receptors for human
immunoglobulin G. Infect. Immun. 17:475.

Myhre, E.B., and G. Kronvall. 1979. Immunoglobulin binding to
group-A, -C and -G streptococci. In: Pathogenic Streptococci.
(M.T. Parker, ed.) Reedbooks Ltd., Chertsey, Surrey, U.K.,
pp. 76-78.

Myhre, E.B., and G. Kronvall. 1980a. Demonstration of a new type of
inmunoglobulin G receptor in Streptococcus zooepidemicus strains.
Infect. Immun. 27:808.

Myhre, E.B., and G. Kronvall. 1980b. Immunochenical aspects of
Fc-mediated binding of human IgG subclasses to group A, C, and G
streptococci. Molec. Immunol. 17:1563.

Myhre, E.B., and G. Kronvall. 1981. Inmunoglobulin specifications of
defined types of streptococcal Ig receptors. In: Basic Concepts
of Streptococci and Streptococcal Diseases. (S.E. Holm and P.
Christensen, eds.) Reedbooks Ltd., Chertsey, Surrey, U.K.,
pp. 209-210.

Natvig, J.B., H.G. Kunkle, and S.D. Litwin. 1967. Genetic markers of
the heavy chain subgroups of human IgG. Cold Spring Harbor Symp.
Quant. Biol. 32:173.

Ohkuni, H., J. Friedman, I. Van de Rijn, V.A. Fischetti, T. Poon-King,
and J.B. Zabriskie. 1983. Immunological studies of post-
streptococcal sequelae: Serological studies with an extracellular
protein associated with nephritogenic streptococci. Clin. Exp.
Immunol. 54:185.

Peterson, P.K., J. Verhoef, L.D. Sabath, and P.G. Quie. 1977. Effect
of protein A on staphylococcal opsonization. Infect. Immun.
15:760.

Pryjna, J., J. Munoz, R.M. Gallbraith, H.H. Fudenberg, and G. Virella.
1980a. Induction and suppression of immunoglobulin synthesis in
cultures of human lymphocytes: Effects of pokeweed mitogen and
Staphylococcus aureus Cownan I. J. Immunol. 124:656.

Pryjna, J., J. Munoz, G. Virella, and H.H. Fudenberg. 1980b.
Evaluation of IgM, IgG, IgA, IgD, and IgE secretion by human
peripheral blood lymphocytes in culures stimulated with pokeweed
mitogen and Staphylococcus aureus Cowan I. Cellular Immunol.
50:115.

Ravdonikas, L.E. 1983. The genetic control of virulence in group A
streptococci. I. Conjugal transfer of plasmids and their effect
on expression of some host cell properties. Acta Path. Microbiol.
Immunol. Scand. (B) 91:55.









Ravdonikas, L.E., P. Christensen, L.A. Burova, K. Grabovskaya, L.
Bjorck, C. Schalen, M-L. Svensson, and A.A. Totolian. 1984. The
genetic control of virulence in group A streptococci. III.
Plasmid-induced >>switch-off<< -effect on some pathogenic
properties. Acta Path. Microbiol. Immunol. Scand. (B) 92:65.

Recht, B., B. Frangione, E. Franklin and E. Van Loghem. 1981.
Structural studies of a human 3 myeloma protein (GOE) that
binds staph protein A. J. Immunol. 127:917.

Reis, K.J., E.M. Ayoub, and M.D.P. Boyle. 1983. Detection of
receptors for the Fc region of IgG on streptococci. J. Immunol.
Methods. 59:83.

Reis, K.J., E. M. Ayoub, and M.D.P. Boyle. 1984a. Streptococcal Fc
receptors. I. Isolation and partial characterization of the
receptor from a group C streptococcus. J. Immunol. 132:3091.

Reis, K.J., E.M. Ayoub, and M.D.P. Boyle. 1984b. Streptococcal Fc
receptors. II. Comparison of the reactivity of a receptor from a
group C streptococcus with staphylococcal protein A. J. Immunol.
132:3098.

Reis, K.J., M.D.P. Boyle, and E.M. Ayoub. 1984c. Identification of
distinct Fc receptor molecules on streptococci and staphylococci.
J. Clin. Lab. Immunol. 13:75.

Reis, K.J., M. Yarnall, E.M. Ayoub, and M.D.P. Boyle. 1984d. Effect
of mouse passage on Fc receptor expression by group A
streptococci. Scand. J. Immunol. 20:433.

Reis, K.J., E.M. Ayoub, and M.D.P. Boyle. 1985. A rapid method for
the isolation and characterization of a homogeneous population of
streptococcal Fc receptors. J. Micro. Methods, in press.

Ringden, 0., B. Rynnel-Dagoo, E.M. Waterfield, E. Moller, and G.
Moller. 1977. Polyclonal antibody secretion in human lymphocytes
induced by killed staphylococcal bacteria and by
lipopolysaccharide. Scand. J. Immunol. 6:1159.

Romagnani, S., M.G. Guidizi, F. Almerigogna, and M. Ricci. 1980.
Interaction of staphylococcal protein A with membrane components
of IgM- and/or IgD-bearing lymphocytes from human tonsil. J.
Immunol. 124:1620.

Ruuskanen, 0., W.B. Pittard, III., K. Miller, G. Pierce, R.U. Sorensen,
and S.H. Polmor. 1980. Staphylococus aureus Cowan I-induced
immunoglobulin production in human cord blood lymphocytes. J.
Immunol. 125:411.

Saluk, P.H., and L.W. Clem. 1971. The unique molecular weight of the
heavy chain from human IgG3. J. Immunol. 107:298.









Schalen, C. 1982. Immunoglobulin receptors of group A streptococci:
Their specificity and importance for virulence. Ph.D. Thesis,
University of Lund, Lund, Sweden.

Schalen, C., P. Christensen, A. Grubb, G. Samuelson, and M.L. Svensson.
1980. Demonstration of separate receptors for human IgA and IgG
in group A streptococci type 4. Acta Path. Microbiol. Scand. (C)
88:77.

Schuurman, R.K.B., E.W. Gellfand, and H.M. Dosch. 1980. Polyclonal
activation of human lymphocytes in vitro. I. Characterization of
the lymphocyte response to a T cell-independent B cell mitogen.
J. Immunol. 125:820.

Shimizu, A., M. Honzawa, S. Ito, T. Miyazaki, H. Matumoto, H. Nakamura,
T.E. Michaelsen, and Y. Arata. 1983. H NMR studies of the Fc
region of human IgG1 and IgG3 immunogloublins: Assignment of
histidine resonances in the CH3 domain and identification of
IgG3 protein carrying G3m(st) allotypes. Mol. Immunol.
20:141.

Siegel, J.L., S.F. Hurst, E.S. Libermann, S.E. Coleman, and A.S.
Bleiweis. 1981. Mutanolysin-induced spheroplasts of
Streptococcus mutans are true protoplasts. Infect. Immun.
31:808.

Sjoholm, I., A. Bjerkenm, and J. Sjoquist. 1973. Protein A from
Staphylococcus aureus. XIV. The effect of nitration of protein A
with tetranitromethane and subsequent reduction. J. Immunol.
110:1562.

Sjoquist, J., and G. Stalenheim. 1969. Protein A from Staphylococcus
aureus. IX. Complement-fixing activity of protein A-IgG
complexes. J. Immunol. 103:467.

Sjoquist, J., B. Meloun, and H. Hjelm. 1972. Protein A isolation from
Staphylococcus aureus after digestion with lysostaphin. Eur. J.
Biochem. 29:572.

Smith, E.M., H.M. Johnson, and J.E. Blalock 1983. Staphylococcus
aureus protein A induces the production of interferon- in human
lymphocytes and interferon-a/B in mouse spleen cells. J. Immunol.
130:773.

Sperber, W.H. 1976. The identification of staphylococci in clinical
and food microbiology laboratories. C.R.C. Crit. Rev. Clin. Lab.
Sci. 7:121.

Stahlenheim, G., 0. Gotze, N.R. Cooper, J. Sjoquist, and H.J.
Muller-Eberhard. 1973. Consumption of human complement
components by complexes of IgG with protein A of Staphylococcus
aureus. Immunochemistry 10:501.









Stollerman, G.H. 1971. Rheumatogenic and nephritogenic streptococci.
Circulation 43:915.

Uhlen, M., M. Lindberg, and L. Philipson. 1984. The gene for
staphylococcal protein A. Immunol. Today 5(8):244.

Verhoef, J., P.K. Peterson, Y. Kim, L.D. Sabath, and P.G. Quie. 1977.
Opsonic requirements for staphylococcal phagocytosis:
Heterogeneity among strains. Immunology 33:191.

Villarreal, H., Jr., V.A. Fischetti, I. Van de Rijn, and J.B.
Zabriskie. 1979. The occurrence of a protein in the
extracellular products of streptococci isolated from patients with
acute glomerulonephritis. J. Exp. Med. 149:459.

Wagner, B., M. Wagner, and M. Ryc. 1983. Morphological evidence for
different types of IgG-Fc receptors in group A streptococci. Zbl.
Bakt. Hyg. A 256:61.

Winblad, S., and C. Ericson. 1973. Sensitized sheep red cells as a
reactant for Staphylococcus aureus protein A. Acta Path.
Microbiol. Scand. (B) 81:150.

Yarnall, M., K.J. Reis, E.M. Ayoub, and M.D.P. Boyle. 1984. An
immunoblotting technique for the detection of bound and secreted
bacterial Fc receptors. J. Microbiol. Methods 3:83.

Yarnall, M., E.M. Ayoub, and M.D.P. Boyle. 1985. Analysis of surface
receptor expression on bacteria isolated from patients with
endocarditis. Submitted.











BIOGRAPHICAL SKETCH


Michele S. Yarnall was born on October 27, 1959, in Allentown,

Pennsylvania. She lived in Allentown until 1977 when she went to

Kutztown State College, Kutztown, Pennsylvania, and majored in biology.

After graduating in 1981, she headed south to the University of Florida

where she started graduate school in the Department of Immunology and

Medical Microbiology. She plans to continue studying possible

virulence factors and their relationship in the pathogenesis of

disease.