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CLINICAL MICROBIOLOGY REVIEWS, JUly 1989. p. 285-314 Vol. 2. No. 30893-8512/89/030285-30$02.00/0Copyright © 1989, American Society for Microbiology

Streptococcal M Protein: Molecular Design and Biological BehaviorVINCENT A. FISCHETTI

The Rockefeller Universit, New York. Newt York 10021

INTRODUCTION ................................................................................. 286ELECTRON MICROSCOPY ................................................................................. 286

Thin Sections ................................................................................. 286Whole Streptococci ................................................................................. 286

ISOLATION AND PURIFICATION ................................................................................. 286Pepsin, Detergent, and Lysin Extraction ................................................................................287

STRUCTURAL ANALYSIS ................................................................................. 288Evidence for the Coiled-Coil Structure of M Protein .................................................................288Structural Analysis of the Complete M Molecule ......................................................................290Basic Model ................................................................................. 291Analysis of the Wall-Associated Region ................................................................................. 293Domain Structure ................................................................................. 293

IMMUNOCHEMISTRY ................................................................................. 293Cross-Reactions ................................................................................. 294Conserved, Variable, and Hypervariable Epitopes ....................................................................294

EVIDENCE FOR TWO DISTINCT CLASSES OF M PROTEIN ...................................................294MAP and Opacity Factor ................................................................................. 294MAP I Antigen and the M6 Protein C-Repeat Domain ..............................................................295C-Repeat Domain and Rheumatic Fever .................................................................................295

SIZE VARIATION IN THE M MOLECULES ...........................................................................295Isolation and Sequence Analysis of Size Mutants ......................................................................295

ANTIGENIC VARIATION AND ITS RELATION TO SIZE VARIATION ......................................297Size Variation Leads to Antigenic Variation ............................................................................297Natural Selection of Size Mutants ................................................................................. 297Variation in the N-Terminal Nonrepeating Segment ..................................................................298

COMPARISON OF M-PROTEIN SEQUENCES ........................................................................298Alignment of Known M-Protein Sequences .............................................................................298Alignment of the Variable Region of Two Different M Proteins ...................................................299

HOMOLOGY OF M PROTEIN WITH OTHER MOLECULES OF GRAM-POSITIVE BACTERIA ....300Group C and G Streptococci ................................................................................. 300Staphylococcal Protein A ................................................................................. 300

STRUCTURAL RELATIONSHIP OF M PROTEIN WITH MAMMALIAN PROTEINS .....................301Tropomyosin ................................................................................. 301Other Coiled-Coil Molecules ................................................................................. 301

IMMUNOLOGICAL CROSS-REACTIVITY BETWEEN M PROTEIN ANDMAMMALIAN PROTEINS ................................................................................. 301

Heart-Reactive Antibodies ................................................................................. 301Immunodeterminants Shared with Myosin and Other Human Proteins .........................................301Renal Tissue-Associated Epitopes Shared with M Protein ..........................................................301Monoclonal Antibodies Cross-Reactive with Coiled-Coil Proteins .................................................302Conformational Determinants ................................................................................. 302

THE emm GENE ................................................................................. 303Relationship of M-Protein Genes ................................................................................. 303Conversion of M- to M+ Phenotype ................................................................................. 303Number of emm Gene Copies ................................................................................. 303Regulation of the emm Gene ................................................................................. 304

HUMAN IMMUNE RESPONSE TO THE M MOLECULE ..........................................................304Immunodominant Region of the M Molecule ...........................................................................304

ANTIPHAGOCYTIC ACTIVITY OF M PROTEIN .....................................................................304N-Terminal Charge Domain ................................................................................. 305Effect on Phagocytic Cells ................................................................................. 305Complement ................................................................................. 305Factor H ................................................................................. 305Fibrinogen ................................................................................. 306

ROLE OF M PROTEIN IN STREPTOCOCCAL ADHERENCE ...................................................306M-PROTEIN VACCINES.306

Type-Specific Protection ................. 306

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CLIN. MICROBIOL. REV.

Non-Type-Specific Protection ...................... 307CONCLUDING REMARKS ...................... 309ACKNOWLEDGMENTS ...................... 309LITERATURE CITED ...................... 309

INTRODUCTION

The group A streptococcus (Streptococcus pyogenes) isresponsible for a number of suppurative human infections, ofwhich acute pharyngitis and impetigo are the most common.As a consequence of ineffective antibiotic therapy or no

therapy, as many as 3 to 5% of individuals who suffer a groupA streptococcal pharyngeal infection may develop acuterheumatic fever (32,204), a disease often resulting in cardiacdamage. While not currently a major problem in developedcountries, rheumatic fever is the leading cause of heartdisease in school-age children in developing nations (1). Forinstance, by one estimate, over 6 million school-age childrenin India are afflicted with rheumatic heart disease (1). Acuteglomerulonephritis, another sequela of group A streptococ-cal disease, is usually the consequence of infection byspecific strains of streptococci (nephritogenic strains) whichinfect either the throat or skin (174). The ability of group Astreptococci to persist in infected tissues is primarily due tothe cell surface M protein, a molecule which confers to thestreptococcus the ability to resist phagocytosis by polymor-phonuclear leukocytes in the absence of type-specific anti-bodies. Resistance to a group A streptococcal infectionappears to be related to the presence of type-specific anti-bodies to the M molecule (128, 129). Since there are >80different serotypes of M protein (i.e., M5, M6, M24, etc.), an

individual may become infected by more than one group Astreptococcal type during a lifetime (129).

Streptococcal M protein was identified over 60 years ago

by Rebecca Lancefield (126). A review by Lancefield in 1962(129) clearly describes the studies carried out for nearly 35years, defining this molecule as a major virulence factor forthe streptococcus due to its antiphagocytic property. In1974, a comprehensive review by Fox (81) delineated studiessince the Lancefield review and underscored the knowledgeto that time of the structure, function, and immunochemistryof the M molecule.The streptococcal M protein is now probably one of the

best-defined molecules of the known bacterial virulencedeterminants. Its structure, function, immunochemistry, andmethod of antigenic variation are unique among knownvirulence molecules and may serve as a model for certainmicrobial systems. To better appreciate the thinking whichled to the current understanding of the M protein, wheneverpossible, attempts will be made to present this review in an

historical perspective, with a chronological account of thestudies over the years, emphasizing the time since 1974.While the antiphagocytic activity and genetics of the Mmolecule will be addressed in this review, the reader is alsodirected to recent reviews by Manjula (131) and Scott (J. R.Scott, in B. Iglewski, ed., The Bacteria, Vol. XIII, in press)on these subjects.

In this review, the basic structure of the M molecule willbe developed. Once established, it then becomes a simplematter to integrate this information into a better awareness

of the biology of the M molecule and its relation to typespecificity, antigenic diversity, and cross-reactivity. Withthese concepts understood, a strategy to control group Astreptococcus-related diseases may become more apparent.

ELECTRON MICROSCOPY

Thin Sections

Thin-section electron micrographs identifying the M pro-tein on the surface of group A streptococci were firstpublished by Swanson et al. (193), using ferritin-labeledM-specific antibodies derived from type-specific rabbit sera.The data suggested that M protein appears as hairlikeprojections on the surface of the streptococcal cell wall (Fig.1) (13, 169). M- streptococci were found to be devoid ofthese surface projections (193).

Whole Streptococci

To better appreciate the features of the proteins on thesurface of the streptococcal cell wall, organisms were pre-pared for electron microscopy by a variety of other tech-niques (68). Whole streptococci mounted on grids, critical-point dried, and viewed under a transmission electronmicroscope exhibit projections which appear to be the resultof interactions among several adjacent M-protein fibrilsforming tuftlike structures (Fig. 2a and b). Interactionsbetween M molecules on adjacent streptococci, previouslyobserved in thin sections (Fig. 1) (169), are also seen in thesepreparations. Whether these surface structures are com-posed exclusively ofM protein or are the result of complexesof M protein and lipoteichoic acid (LTA) or other surfacemolecules was not determined. However, ferritin-labeledanti-M antibodies reveal that the extensions do contain Mprotein (Fig. 3). Surface replica preparations confirm thepresence of these types of projections on the cell surface ofwhole organisms (Fig. 4). Streptococci treated with trypsinto remove the M protein were shown to have a smoothsurface in all of these preparations (not shown).

In experiments designed to answer questions about theplacement ofM protein on the cell wall, Swanson et al. (193)found that, after trypsinization to remove existing M proteinon living streptococci, newly synthesized M protein was firstseen by electron microscopy on the cell in the position of thenewly forming septum. In similar experiments designed afterthe classical experiments of Cole and Hahn and usingfluorescein-labeled anti-M antibodies (44), trypsinized strep-tococci placed in fresh media for 10 min revealed M proteinfirst at the newly forming septum (Fig. 5a). On organismsexamined after extended incubation, M protein was notobserved in the position of the old wall (Fig. 5b), suggestingthat the M molecule is produced only where new cell wall issynthesized (193).

ISOLATION AND PURIFICATION

For many years the method used to prepare crude Mprotein for purification was based on the one developed byLancefield (126) for the preparation of extracts for strepto-coccal typing (for references prior to 1974, see Fox [81] andLancefield [129]). According to this procedure, organismsare suspended in dilute HCI at pH 2.0 and placed in aboiling-water bath for 10 min. After neutralization, theresulting extract is centrifuged and the supernatant is used asstarting material for further characterization of the M mole-

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STREPTOCOCCAL M PROTEIN 287of~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. ,:'

FIG. 1. Electron micrograph of ultrathin sections of group A streptococci exhibiting M-protein fibrils on the cell surface. Junctionsbetween streptococci reveal M-protein fibers from one coccus interacting with those from the adjoining organism. Magnification. x56,000.

cule. While the procedure effectively removes M proteinfrom the cell, it is unlikely that the final purified productrepresents the native M-protein molecule (82). Recognizingthis limitation, other procedures for extracting M proteinfrom whole streptococci or their isolated cell walls wereattempted. These included sonic oscillation (25, 157), alkali(47, 84), group C bacteriophage-associated lysin (43, 79),nonionic detergent (64, 65), nitrous acid (91), cyanogenbromide (202), and guanidine hydrochloride extraction (178),all of which were usually coupled to a particular purificationscheme. However, in nearly all cases the final purified Mprotein exhibited extensive heterogeneity. These results ledto the general conclusion at that time that M protein wascomposed of a heterogeneous group of molecules or ofmultiple subunits (82).

Pepsin, Detergent, and Lysin Extraction

The biochemical features of the M-protein molecule be-came more apparent with the development of an extractionprocedure which specifically cleaves the M protein from thestreptococcal surface. Pepsin digestion at a suboptimal pHof 5.8 releases a biologically active fragment of the M protein(termed PepM) from the cell wall (11, 46). After purification.the fragment is found to be homogeneous by sodium dodecylsulfate-polyacrylamide gel electrophoresis, strongly suggest-ing that the M molecule on the cell surface was also likely tobe homogeneous, contrary to previous belief. Depending on

the serotype, the size of the extracted PepM molecule variesfrom 20,000 to 40,000 in molecular weight (34, 116, 120, 133,152, 169). When injected into rabbits, the pepsin-derivedfragment elicits antibodies capable of initiating phagocytosisof streptococci of the homologous serotype. PepM can alsoabsorb type-specific opsonic antibodies from serum (11,137). Thus, the pepsin-derived M-protein fragment retainssome of the biologically important determinants of the nativeM molecule present on the streptococcal surface. Pepsinextraction, therefore, proved to be one of the better methodsfor obtaining a workable fragment of the M protein and wasused to provide the starting material in a variety of studiesover the ensuing years (14, 18, 22, 117, 133, 137, 152, 158,169). Although it was believed early on that pepsin removedthe M protein in its entirety from the streptococcal surface(13), the nature of the complete molecule was not as yetunderstood.Nonionic detergent was found to remove M protein from

isolated streptococcal cell walls (70). The M molecule re-leased by this method is seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as several closely spacedprotein bands which range from 15,000 to 35.000 in molecu-lar weight. Like pepsin-extracted M protein, the detergent-extracted molecules are able to absorb opsonic antibodiesfrom rabbit antiserum (70) and evoke type-specific opsonicantibodies when injected into rabbits (64). An examination ofthe physicochemical properties of these M molecules re-

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FIG. 2. Critical-point-dried whole group A type 6 streptococci viewed directly by transmission electron microscopy. (a) M, M-proteinprojections on the cell surface. Interactions between cocci seen in Fig. 1 are also observed in these preparations. Magnification, x88,000. (b)Enlargement of the surface of a coccus from part a. Magnification, x330,000.

vealed that they are not globular but asymmetrical, with africtional ratio of 2.2 (70). Thus, the detergent-extracted Mmolecules, though not as homogeneous as the pepsin-de-rived protein, display many of the properties of PepM.

It was not until 1981, when M protein was purified fromthe extracellular supernatants of type 12 group A strepto-coccal L forms and protoplasts, that it became apparent thatthe size of the native M molecule could be as large as 58kilodaltons (kDa) (199). Extraction of M protein from strep-tococci by solubilization of the cell wall with phage lysin(termed lysin, a muralytic enzyme produced during a groupC streptococcal phage infection [69]) also yielded a large M

molecule (43, 79); however, the association of this M proteinwith cell wall fragments, resulting in its high molecularweight, could not be ruled out.

STRUCTURAL ANALYSIS

Evidence for the Coiled-Coil Structure of M Protein

Though sequence and peptide data were limited up to1979, the available information suggested that M moleculesof heterologous serotypes differed in primary structure inspite of a common antiphagocytic activity (18, 65). How-

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STREPTOCOCCAL M PROTEIN 289

FIG. 3. Critical-point-dried whole group A type 6 streptococci pretreated with anti-M6 antibody and identified with ferritin-labeled goatanti-rabbit IgG. Cells were viewed directly by transmission electron microscopy. M. A representative of the M-protein projection on the cellsurface containing the ferritin-labeled antibody. Fibers between cocci also contain bound ferritin particles. Magnification. x96,000.

ever, despite these apparent differences, physical and chem-ical data accumulated to that time revealed that all of the Mmolecules examined shared certain noteworthy properties(81). Their extreme thermal stability (126), elongated shape(70, 84, 166), and appearance as a network of fibers on thecell surface (193) suggested that M molecules resemblepreviously characterized fibrous proteins. When the partialsequence of the PepM24 (terminology used for the M proteinextracted from M24 streptococci with pepsin) protein (18)was compared with that of known proteins, two of the M24cyanogen bromide peptides exhibited up to 40%Y identitywith rabbit skeletal muscle tropomyosin (99). In addition, ahexapeptide, repeated five times in the PepM24 molecule,was identical to a sequence in tropomyosin. This was thefirst direct evidence that M protein may be structurallysimilar to tropomyosin, the prototype molecule for an ot-helical coiled-coil fibrillar protein (145). The isoelectric point(a reflection of amino acid composition), solubility, heatstability, and stability in acid and base of tropomyosin weresimilar to those found for M protein, further confirming thechemical and structural relationship of these two molecules(99).

Additional biochemical support for the probable coiled-coil structure of M protein came when the partial sequencesof the M5, M6, and M24 proteins were compared with that oftropomyosin (136). The sequences within these three mole-

cules contained a repeating seven-residue periodicity ofnonpolar amino acids, a basic characteristic of x-helicalcoiled-coil proteins like tropomyosin. A more detailed studyof the structure and conformation of the PepM6 and lysin-extracted M6 proteins further established the close relation-ship between M protein and tropomyosin (169). In thisanalysis, ultracentrifugation studies revealed that the Mmolecules exist as stable dimers, and circular dichroismspectra indicated that the molecules are 70% a-helical.Electron microscopic images of platinum-shadowed M mol-ecules were seen as rods in which the pepsin- and detergent-extracted forms were approximately half the length of thelysin-extracted protein. Measurements also revealed that theM molecules were similar in width to tropomyosin, and fiberX-ray diffraction indicated that a coiled-coil structure ispresent in the M molecule. Assessment of M protein on thestreptococcal surface compared with isolated M moleculesindicated that each fiber on the cell wall consists of a singledimeric M-protein molecule of about 50 to 60 nm in length(Fig. 1) and is not composed of multimers of smaller subunitsas is the case for pili of gram-negative organisms (33).Though common in mammalian cells, the x-helical coiled-coil design is unique for bacterial surface molecules (41, 42).

Studies by Phillips et al. (169) indicated that purified Mprotein released from the streptococcal cell wall with phagelysin was similar to the size of the native cell-wall-bound M

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dm4, ...P~.~;

"kA.9,f

cl.,.24

.. km t:

f .11'i

FIG. 4. Tantalum and tungsten surface replica of critical-point-dried streptococci revealing the distribution of the projections on the cellsurface. Magnification, x96,000.

molecule. M proteins extracted with pepsin are almost halfthe size of the lysin-extracted molecule and are likelycleaved products derived from the native M molecule. Thisis further supported by electron micrographs of pepsin-treated M-bearing (M F) streptococci depicting shorter sur-face fibers than untreated cells (13, 169). Thus, after pepsindigestion, roughly half of the M molecule remains associatedwith the streptococcal cell.

Structural Analysis of the Complete M Molecule

The completion of the PepM5 sequence (133) allowed forthe first time a thorough analysis of this region of an Mmolecule (141), i.e., the distal half of the native protein (169).The results confirmed what previous partial sequence datahad suggested: except for the N-terminal 11 amino acids, theremainder of the sequence exhibits a seven-residue periodic-ity in the distribution of nonpolar amino acids. In addition, itrevealed that the N-terminal region of this fragment containsa significantly higher net negative charge than the C-terminalregion. While sequence repeats were also identified in thePepM5 molecule (139), they were not as extensive as thosefound in the partial PepM24 sequence (18).

Until 1986, the majority of structural information wasbased on the pepsin fragment of the M molecule. Noinformation, besides the physicochemical analysis on thelysin-extracted M protein (169), was available concerningthe C-terminal half. The successful cloning of the M6 gene(enzin-6) (180) resulted in the ability to determine the com-plete amino acid sequence of an entire M molecule (94). Likethe partial PepM24 sequence (18, 19), the M6 sequence wascomposed of extensive amino acid repeats extending into theC-terminal half of the protein. Approximately 80% of thecomplete M6 molecule is composed of four sequence repeatregions (Fig. 6) (94). Regions A and B contain five directtandem repeat blocks of 14 and 25 amino acids each,respectively. Each A repeat may be further divided into twoseven-residue sub-blocks (not shown). The three centralrepeats in the A and B regions are identical, while therepeats at each end of the five repeat blocks diverge slightly.Region C consists of two and one-half tandem repeats of 42amino acids each, which are not as conserved as the A andB repeats. Region D is composed of four seven-residue"quasirepeats" (77, 94). Recent reports show that aminoacid sequence repeats also occur in the complete M24 (153),

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FIG. 5. Fluorescein-labeled anti-M6 antibody analysis of the appearance of M6 protein on streptococcal cell walls. M6 streptococci weretreated with trypsin to remove surface M protein. Cells, reincubated at 370C, were removed at intervals, fixed, and stained withfluorescein-labeled anti-M6 antibody. (a) After 10 min of incubation, the M protein is located within a thin band at the position of the newlyforming septum. Magnification, x4,000. (b) Location of the M protein after 40 min of incubation. Note that no fluorescein label is seen in theposition of the old wall. Magnification, x4,800.

M5 (148), and M12 (175) proteins, the M5 molecule being theleast repetitive.A C-terminal segment, composed of 19 hydrophobic

amino acids, is predicted to have an a-helical conformationwhich would be sufficient in length to traverse the cytoplas-mic membrane (77). An adjacent region, rich in proline andglycine, is predicted to be located within the cell wallpeptidoglycan (161).

Basic Model

In general, x-helical coiled-coil proteins are constructedfrom a repeating seven-residue amino acid pattern (a-b-c-d-e-f-g)n in which residues in positions a and d are hydro-phobic and form the "core" residues in the coiled-coil andthe intervening residues are primarily helix promoting (145).The arrangement of the amino acids of the M6 protein withinthe seven-residue pattern is shown in Fig. 7. The seven-

residue periodicity found from amino acids 12 through 362strongly suggests that this region is in a coiled-coil confor-mation and forms the helical central rod region of themolecule. Discontinuities in the heptad pattern (seen espe-cially in repeat B), which have been seen in the sequence ofother M proteins (141) and other coiled-coil molecules (144,163, 166), probably account for the flexibility of the Mmolecules observed in electron micrographs (Fig. 1). Basedon these irregularities in the heptad pattern, the central rodregion is divided into three subregions which correlate withthe A-, B-, and C-repeat blocks (77).Based on a detailed analysis of data of M-protein se-

quences, it was concluded that serologically different Mproteins may be built according to a basic scheme: anextended central coiled-coil rod domain flanked by func-tional end domains (77). Because the Q-helical coiled-coil isa structure capable of tolerating a vast number of amino acidsequence changes while preserving its conformation, a large

Wall-associated

Non HelicalRegion Helical Central Rod Region

-iAnchor Region

L-LAAAAAAAAAAAA~~~~~~~~~~~~~~~nAA~~~~~nAAAAAAA~~~~~~~~nAAAAAA~~~~~~~~nAAAAAAAAAAAAAAAAAAA~~~~~~~~~~~~~~~~~~~tI.~~14

N 4-Aj4

25 42

IB 2 B3 B 4B-d;_ C-|-

........... ~~~~~~...... ......

7

Pt

Pepsin

Pro/Gly | Memb. Sc

Chargedtail

FIG. 6. Characteristics of the complete M6 protein sequence (77, 94). Blocks A, B. C, and D designate the location of the sequence repeatblocks. Numbers above the block indicate the number of amino acids per block. Block C3 is half the size of blocks C1 and C2. Shadowedblocks in A and B indicate those in which the sequence diverges from the central consensus sequence. In the C repeats, the sequence in thecenter of C1 and C2 are identical, while the sequence at the ends and block C3 diverge. Pro/Gly denotes the proline-glycine-rich region likelylocated in the peptidoglycan. Membrane anchor is a 19-hydrophobic-amino-acid region adjacent to a 6-amino-acid charged tail. Pepsinidentifies the position of the pepsin-susceptible site after amino acid 228. Wall-associated region designates the segment of the moleculelocated within the cell (this begins at Ala-298 in the C2 block [161]).

u --]ml &A &B &B kB &B &B ILA "%,A ILmmmmmwwuww Li W li w UULJ

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292 FISCHETTI CLIN. MICROBIOL. REV.

I Arg Val Phe Pro Arg Gly ThrVal Clu Asn Pro

a b e d e r 9

I NON HELICAL

REGION

12 Asp Lyo Ala Arg Glu LeuIs Leu Ann Lys Tyr Asp Val Glu25 Aon Ser Met Leu Gin Ala Asn T32 Ann Asp Lys Leu Thr Thr Glu39 Ano Ann Aon Leu Thr Asp Gin w46 Ann Lys Ann Leu Thr Thr Glu iii5:3 Ann Lys Ann Leu Thr Asp Gin T60 Ann Lys Ann Leu Thr Thr GCIun67 Ann Lys Asn Leu Thr Asp Gin 474 Ann Lys Ann Leu Thr Ther Gl u

8I Ann Lys Glu Leu Lys Ala Glu88 Glu Ann Arg Leu Thr Thr Glu95 Ann Lys Gly Leu Thr Lys Lys102 Leu Ser Glu Ala Glu Glu Glu109 AlaIto Ala Ann Lys Glu Arg Glu Ann117 Lys Glu Aa lIle Gly Thr Leu124 Lys Lys Thr Leu Asp Glu Thr131 Val Lys Asp Lyn135 Ile Ala Lys Glu Gin Glu Ser rIn142 Lys Glu Thr Ile Gly Thr Leu r149 Lys Lys Thr Leu Anp Glu Thr mn >156 Val Lys Asp Lyn r0160 Ile Ala Lys Glu Cln Glu Ser 1n167 Lys Glu Thr Ile Gly Thr Leu iii174 Lys Lys Thr Leu Asp Glu Thr w Z181 Val Lys Asp Lys185 Ile Ala Lys Glu Gin Glu Ser192 Lys Glu Thr Ile Gly Thr Leu199 Lys Lys I le Leu Asp Glu Thr 0206 Val Lys Asp Lys o210 Ile Ala Arg Glu Gin Lys Ser217 Lys Gin Asp Ile Gly Ala Leu i224 Lyns Gin Glu Leu Ala Lys Lys_n231 Asp Glu Gly Z234 Asn Lys Val Ser Glu Ala Ser Z241 Arg Lys Gly Leu Arg Arg Asp248 Leu Asp Ala Ser Arg Glu Ala255 Lys Lys Gin Val Glu Lys Asp262 Leu Ala Ann Leu Thr Ala CluG269 Leu Asp Lys Val Lys Glu Glu 15276 Lys Gin Ile Ser Asp Ala Ser 1D283 Arg Gin Gly Leu Arg Arg Asp >290 Leu Asp Ala Ser Arg Glu Ala 4297 Lys Lys Gin Val Glu Lys Ala Cs304 Leu Glu Glu Ala Ann Ser Lys311 Leu Ala Ala Leu Glu Lys Leu318 Ann Lys Glu Leu Glu Glu Ser325 Lys Lys Leu Thr Glu Lys Glu332 Lys Als Glu Leu Gin337 Ala Lys Leu Clu Ala Clu343 Ala Lys Ala Leu Lys Glu Gin o350 Leu Ala Lys Gin Ala Glu Glu357 Leu Ala Lys Leu Arg Ala

363 G4y Lys Ala Ser Asp Ser Gin Thr371 Pro Asp Ala Lys375 Pro Gly Ann lys Val Val331 Pro Gly Lys Gly Gin Ala Z WALL387 Pro Gin Ala Gly Thr Lys C DOMAIN393 Pro Asn Gin Asn lys Ala 0369 Pro Met Lys Glu Thr Lys Arg Gin leu408 Pro Ser Thr Gly Glu Thr Ala Asn Pro

417 Phe P'he Thr Ala Ala Ala leu T424 Thr VaI %let Ala Thr Ala Gly MEMBRANE431 Val Ala Ala \'aiI'ai _

436 Lys Arg Lys CGlu Glu Asn CHARGEDTAIL

FIG. 7. Complete M6 protein sequence arranged to highlight theseven-residue periodicity found in the helical central rod region.Region assignments are based on sequence and conformationalanalyses (77). Arrangement of the sequence is based on the positionof amino acids in a seven-residue periodicity designated by letters athrough g beginning at residue 12 and continues, with interruptionsat residues 109, 131, 156, 181, 206, 231, and 337, through residue362. Alignment from residues 363 to 416 is used essentially tohighlight the regularity of the position of prolines in the sequence.No periodicity is found from residue 363 to the end. Three majorregions are indicated (nonhelical, helical, and anchor). The regionburied within the cell wall is delimited by the wall domain (161). Thepepsin-susceptible site is between Ala-228 and Lys-229.

number of serologically distinct streptococcal M proteinsmay be constructed according to this basic scheme. Al-though the sequence of the heptad may differ between Mtypes, even among strains of the same type, the presence ofpredominantly helix-promoting residues will allow the dif-ferent molecules to attain a common cx-helical conformation.

NH2tr

Hypervariable I

Vorioble

Conserved

COOH

Z-Pepsin

GroupCarbohydrate

Peptidoglycan

Membrane

FIG. 8. Proposed model of the M6 protein from strain D471based on current sequence and structural data (77, 94). The coiled-coil rod region extends about 50 nm from cell wall with a shortnonhelical domain at the N terminus distal from the cell surface. Theproline-glycine-rich region of the molecule is located within thepeptidoglycan, with a short segment of the coiled-coil rod regionpositioned in the group carbohydrate portion of the wall (161). Themembrane anchor segment extends through the cell membrane, withthe charged tail projecting into the cytoplasm. Regions containingconserved, variable, and hypervariable epitopes among heterolo-gous M serotypes are indicated.

Because 3.6 residues are necessary to achieve one turn ofthe helix (102), hydrophobic residues at positions a and d inthe heptad (Fig. 7) will form an inclined stripe around thehelix. For thermodynamic stability, a dimeric, ropelikesuperstructure would be formed as a result of the internal-ization of the hydrophobic residues from two helical chainscoiling around each other (75, 136, 145) (Fig. 8). Thepresence or absence of disruptions in the periodicity ofhydrophobic residues at positions a and d of the heptad

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allows these regions to become more or less flexible (detailedin reference 77).A short 11-residue nonhelical region is found at the

N-terminal end of the coiled-coil rod region, which is distalto the cell surface (Fig. 8). In all M proteins examined thusfar, except M49, which lacks this segment (116), the se-quence of this region is distinct from one M type to another(94, 133, 148, 153, 175) but is nearly 100% identical within anM type (95). It is not certain whether the tertiary conforma-tion of this N-terminal region is similar among the differentM-protein types despite sequence variation. The N-terminalsegment plays an important role in the biological activity ofthe molecule, since antibodies generated to this regioneffectively opsonize the streptococcus of the M type fromwhich it was derived (12, 15, 34, 55, 57, 106, 107) (seebelow). Thus, it seems likely that one purpose for the centralrod region may be to act as a shaft to position the N-terminalregion away from the cell surface (Fig. 8).

Beginning at the C-terminal end of the central rod region isa segment characterized by the presence of regularly spacedprolines and glycines (Fig. 7). This is followed by twophenylalanine residues beginning at residue 417 which signalthe start of a 19-amino-acid stretch of hydrophobic residuesterminated by six charged amino acids. It was suggested thatthe hydrophobic amino acids would serve as a membraneanchor and the adjacent proline-glycine region would belocated in the peptidoglycan and act to stabilize the moleculewithin the cell wall (77). The charged tail, typical of a stoptransfer signal seen in the membrane-associated segments ofseveral membrane-bound proteins (178a), would be expectedto be located on the cytoplasmic side of the membrane.Sequence comparison of the C-terminal region of the M6protein with the C-terminal end of other cell wall proteinsfrom gram-positive bacteria reveals significant homology(see below), suggesting that a common mechanism may existfor the attachment of proteins within the gram-positive cellwall.

Analysis of the Wall-Associated Region

From the translated deoxyribonucleic acid (DNA) se-quence of the M6 C-terminal region, it was implied that thissegment may be responsible for M-protein attachment (94);however, it was not directly determined. Experiments weretherefore designed to isolate and identify the M-proteinregion embedded within the streptococcal cell wall. Trypsinwas used to digest the exposed portion ofM protein from thecell wall, leaving behind the wall-associated region. After thecell wall was solubilized with a muralytic enzyme, antibodiesto synthetic peptides of sequences within the C-terminalregion of the M molecule were used to identify the releasedwall-associated M-protein fragment. A 16-kDa fragment wasidentified as the smallest trypsin-resistant fragment of Mprotein. Because of its location within the cell wall matrix,this fragment was resistant to attack by trypsin despite thepresence of several lysine and one arginine residue withinthe sequence. Amino acid composition of this fragmentrevealed that it was rich in proline and glycine but lacked theamino acids found in the mebrane anchor. Sequence analysisof the purified fragment indicated that its N-terminal end wasat about residue 300, thus localizing a small segment of thehelical central rod region of the M molecule (residues 298 to362) in addition to the proline-glycine region (residues 363 to416), within the cell wall (Fig. 7).

Further evidence suggested that residues 298 to 362 of thehelical rod region are located within the group carbohydrate

moiety of the cell wall, localizing the proline-glycine regionwithin the peptidoglycan. Since no detectable amino sugarswere found with the wall-associated M-protein fragment, it islikely that the C-terminal region of the M6 molecule isintercalated within the group carbohydrate and cross-linkedpeptidoglycan and not covalently linked to them (161).M protein is rarely detected in the spent growth media of

streptococci. Therefore, the incorporation of this moleculeinto the cell wall and membrane appears to be closelyregulated. It is postulated that the C-terminal membraneanchor may be necessary to keep the M molecule attached inthe right orientation within the cell membrane until such timeas the peptidoglycan is cross-linked (156) around the proline-glycine region. Recent studies (V. Pancholi and V. A. Fis-chetti, submitted for publication) have revealed that at pH5.5 in the presence of 30% raffinose, the streptococcal cellwall may be removed with a muralytic enzyme, allowingnearly all the M protein to remain attached to the protoplastmembrane. Shifting the protoplasts to pH 7.4 results in therelease of the M molecule into the supernatant. A mem-brane-bound thiol enzyme was found to be responsible forthe release. The biological significance of such an enzyme iscurrently speculative. Since the released form of the Mmolecule lacks the C-terminal 19 hydrophobic amino acidsand charged tail (161), its attachment to the cell appears to bethrough the cytoplasmic membrane.

Domain Structure

Techniques using limited proteolysis have been effectivein defining functional and structural domains in a variety ofproteins (215), particularly immunoglobulins (88, 167, 185).Similarly, the PepM fragment of the M protein may beregarded as a functional domain and the pepsin-susceptiblesite may be seen as a hinge region of the molecule. Thispreferential site for pepsin at a suboptimal pH is foundbetween the B- and C-repeat blocks in M5 (147), M6 (94),and M24 (153) proteins (Fig. 6) even though the B-repeatblock in M24 is not homologous with that found in M5 andM6. PepM49, however, has a primary pepsin site after aC-repeat block and a secondary site between the B and Crepeats (135). Thus, since the type-specific and antiphago-cytic determinants are located within the PepM fragment,this half of the M molecule may be considered to be afunctional domain of the protein. However, besides the walland membrane anchor domains, the functional attributes ofthe C-terminal domain comprising the C- and D-repeatblocks have yet to be clearly defined.Thus, the streptococcus uses the coiled-coil design to

accomplish a variety of tasks. The rodlike conformationallows the functional antiphagocytic domain to be positionedaway from the cell wall. The plasticity of the sequencesnecessary to form the coiled-coil structure allows the dif-ferent domains of the molecule to be tailored for specificfunctions.

IMMUNOCHEMISTRY

The M-protein molecule is the basis of the Lancefieldserotyping scheme of group A streptococci (126, 177, 195).The typing sera used for this purpose are a set of M-protein-specific rabbit sera prepared against whole organ-isms of specific serotypes. To remove cross-reactive anti-bodies, sera are extensively absorbed with organisms ofselected heterologous serotypes. Nearly 80 to 85 differentserotypes have now been identified, but there exist a large

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number of strains in streptococcal collections for whichtyping sera are not as yet available. The identification of aspecific strain as being a particular serotype requires that itsM-protein extract react in a precipitation reaction with aspecific typing serum. Therefore, these procedures tend toidentify isolates which have not changed immunodetermi-nants responsible for the type-specific precipitation reactionwith absorbed typing sera (177, 195).

Cross-Reactions

Immunochemical studies prior to 1985 were confinedprimarily to either acid- or detergent-extracted M protein orto the N-terminal pepsin fragment. Analysis of the reactivityof antibodies raised to detergent-extracted M6 protein withpeptides representing nearly the complete M6 protein re-veals that the detergent-derived M molecules (DetM) repre-sent the N-terminal half of the native M6 protein (78; V. A.Fischetti, unpublished data) and thus are similar to thepepsin-derived molecule. It is speculated that the nonionicdetergent activates an enzyme within the cell wall prepara-tion which cleaves the M molecule in the region of thepepsin-susceptible site.Acid extracts likely contain fragments of the M protein

derived from both N- and C-terminal regions. Shared anti-genic determinants among M serotypes have been described(81); however, based on the complexity of the acid Mpreparation, the nature of the observed cross-reactions wasnot clearly understood. One hypothesis was that cross-reactive determinants were actually cellular contaminantsassociated with or linked to the M protein (i.e., M-associatedprotein [MAP]) (211, 212), whereas others believed thatsome determinants of the M molecule were shared amongcertain types (80, 83).

It was clear that immunization of rabbits with the PepMfragment resulted in antibodies which were essentially typespecific and opsonic and were free of extensive reactivity tonon-type-specific epitopes found previously with acid-ex-tracted M proteins (21). Peptide map analysis of M protein(derived from the N-terminal half of the molecule) fromcross-reactive and non-cross-reactive serotypes revealedthat cross-reactions correlated with the extent of structuralsimilarity among the M molecules analyzed (65). Limitedcross-reactions were observed when 125I-labeled M6 protein,representing the N-terminal half of the molecule, was used ina radioimmunoassay with unabsorbed rabbit antiserum pre-pared to heterologous M serotypes (64). Using monoclonalantibodies, Dale and Beachey (52) found that some antibod-ies cross-reacted with purified PepM proteins from certainother serotypes; however, the location of these epitopes wasnot defined. Thus, it was clear that, while cross-reactiveepitopes were present in the N-terminal half of the Mmolecules, they were apparently limited in scope.

Conserved, Variable, and Hypervariable Epitopes

Using polyclonal rabbit antisera to M6 protein, Manjula etal. (132) found cross-reactivity with the C-terminal region ofthe PepM5 molecule (located near the center of the nativemolecule), whereas no reactivity was observed with theN-terminal region of the PepM5 molecule. The results sug-gested that the N-terminal 25% of the native M moleculemay represent a hypervariable region of M proteins. Insupport of this, Dale et al. (55, 57) established that syntheticpeptides derived from the N-terminal sequence (residues 1 to20) of the M5 molecule stimulated type-specific antibodies

which did not cross-react with M protein from other sero-types. This was later proven also to be true for M6 (15, 106),M19 (34), and Ml (120) proteins.Monoclonal antibodies directed to the N- and C-terminal

halves of the native M6 protein were used by Jones et al.(109) in an attempt to identify the location of the epitopesaccountable for limited and broad cross-reactions among Mserotypes. Results revealed that an epitope responsible forcross-reactions among 5 of 56 serotypes was located in theN-terminal half of the molecule (within the B repeat) while ahighly cross-reactive epitope (reactive with 20 of 56 sero-types) was located in the C-terminal half (within the Crepeat) (Fig. 6). These studies were extended with two othermonoclonal antibodies, one reacting with an epitope at theN-terminal end within the A-repeat region and one in theC-terminal half within the C-repeat (106, 108). The C-terminal antibody cross-reacted with 30 different serotypestrains, while the new N-terminal monoclonal antibody wasfound to be type specific and reacted only with the M6serotype strain. This monoclonal antibody was the only oneof the four studied that was opsonic for M6 streptococci.

Thus, it is clear from a variety of immunological tech-niques that the C-terminal region of the M molecule isantigenically more conserved among different serotypes. Asthe N-terminal region is approached, the greater the anti-genic variation (Fig. 8). Therefore, the basis of the M-proteintyping scheme is now more clearly understood. In thepreparation of typing sera, absorption of antistreptococcalantisera with heterologous strains was necessary to removethe antibodies reactive with the conserved regions of the Mmolecule, in addition to antibodies to other common anti-gens found on the cell surface. This resulted in what wouldbe expected to be a monospecific antibody population,directed to the type-specific N-terminal region of the Mmolecule (Fischetti, unpublished data).

EVIDENCE FOR TWO DISTINCT CLASSES OFM PROTEIN

MAP and Opacity FactorMAP were identified by Widdowson and co-workers in the

1970s (211, 213). The MAP antigens are present in acidextracts derived from streptococci which express M protein.These antigens lack type specificity but copurify with thetype-specific M protein. At the time MAP antigens werecharacterized, other investigators had identified non-type-specific M antigens in acid extracts which are similar to MAP(83, 201). It was not until M protein was obtained by pepsindigestion of whole streptococci that the type-specific sub-stance could be clearly separated from non-type-specific Mantigens (46).The MAP derived from one serotype shares antigenic

determinants with MAP derived from many heterologousserotypes. Two serologically distinct MAP antigens, MAP Iand MAP II, have been identified, and most isolates of aparticular M serotype contain either MAP I or MAP IIantigen on their surface (211, 214). Expression of MAP II isclosely linked to production of opacity factor (OF), a li-poproteinase whose activity is detected by serum opales-cence; streptococci bearing MAP I lack OF activity. TheMAP I antigen is present on the surface of nearly all Mserotypes associated with outbreaks of acute rheumaticfever. Thus, a link between MAP antigen and the virulenceproperties of group A streptococci is suggested. However,the biochemical composition of MAP I and II remainedelusive.

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MAP I Antigen and the M6 Protein C-Repeat Domain

In a recent antigenic analysis of surface-exposed M-protein epitopes, it became apparent that two basic M-protein structures exist (28). A panel of seven antibodiesdirected to well-defined epitopes located in the B-repeat,pepsin site, and C-repeat regions of M6 protein was testedfor immunoreactivity to over 130 streptococcal isolatesrepresenting more than 60 different serotypes. Isolates ofonly four serotypes (M5, M14, M19, and M36) sharedantigenic determinants located in the B-repeat and pepsinsite regions of M6 protein. In contrast, about half of theserotypes tested displayed strong immunoreactivity withantibody probes directed to C-repeat region epitopes of M6protein. There is a striking correlation between those sero-types possessing the C-repeat region epitopes of M6 andthose previously described as having MAP I antigen (211).Furthermore, nearly all isolates which produce OF (and bydefinition express MAP II antigen) lack immunologicalcross-reactivity with antibodies to the C-repeat region. It islikely that the MAP I antigen represents the C-repeat regionas defined for M6 protein. While it is possible that the MAPI antigen is located on another related surface molecule, theevidence that it is M protein is significant (28, 211, 213). Insummary, the MAP I and MAP II M types closely paralleltwo major structural classes of M-protein molecules, termedclasses I and II, respectively. Streptococci bearing class I Mproteins share surface-exposed antigenic epitopes with theC-repeat region of M6 protein and fail to produce OF,whereas class II serotypes produce OF but are deficient inepitopes cross-reactive with the M6-like C-repeat region(28).The close proximity of the highly conserved C-repeat

region to the poorly homologous B-repeat and pepsin sitesstrongly suggests that the C-repeat region forms a distinctantigenic domain (28) (Fig. 6). In support of the immuno-chemical analysis, structural data suggest that the C-repeatantigenic domain may have unique conformational charac-teristics as well. The B-repeat region through residue 230forms a more flexible coiled-coil structure than the C-repeatregion beginning at residue 231 (77). In addition, amino acidsequence identity between maximally aligned sequences ofM6 (94) and M12 (175) is <27%Y between residues 121 and231, but abruptly increases to 97% complete identity be-tween residues 232 and 298 (extending from the beginning ofthe first C-repeat subblock to the perimeter of the cell wall).(Fig. 7). Thus, the data strongly suggest that the amino-terminal end of the conserved class I M-protein antigenicdomain lies at the beginning of the first C-repeat block (28).

Class I and II M proteins appear to lack immunologicalcross-reactivity in their surface-exposed regions, however.antigenic relatedness between class I and II M proteins canbe demonstrated in their extracted forms (28). Whether theshared antigenic epitopes reside in buried portions of theM-protein molecule remains to be established. The degree ofhomology between M-protein genes of different serotypeshas been addressed in nucleic hybridization studies. Byusing DNA probes corresponding in positions to the C-repeat region and to the cell wall and membrane anchorregions of M6, a class I M protein (147, 182). distinctions canbe made between class I and II serotypes (28). Hybridizationto chromosomal DNA of most class II serotypes is observedonly under conditions of low stringency, whereas class Iserotypes hybridize under high-stringency conditions. Thesefindings suggest that there are fundamental differences be-tween class I and II M proteins at the DNA level (28).

C-Repeat Domain and Rheumatic Fever

Class I streptococcal serotypes are represented amongboth skin and nasopharyngeal isolates. Likewise, both throatand skin strains are found among the OF-producing class IIserotypes. Thus, the conserved C-repeat domain does notappear to be involved in determining the tissue site ofinfection. In addition, serotypes associated with acute glo-merulonephritis are represented by both class I and IIstreptococci. However, there is a strong correlation betweencertain serological M types and outbreaks of acute rheumaticfever (30). Of 16 streptococcal serotypes which have beenassociated with one or more major outbreaks of rheumaticfever, all but one type express the C-repeat region epitopewhich defines class I M protein (28). Rheumatic feverpatients have high complement-fixing titers to MAP I antigen(211). and MAP I-specific antibodies are absorbed by humanheart tissue (213). Thus, the surface-exposed conservedrepeat domain of class I serotypes may be a virulencedeterminant for rheumatic fever (28).

SIZE VARIATION IN THE M MOLECULES

In this and following sections, a direct comparison of theknown M-protein sequences is presented. But, to fullyappreciate the sequence diversity found within M molecules,it is important to understand a recently observed property ofthe M-protein molecule, namely, size variation. By using abroadly cross-reactive monoclonal antibody as a probe, thesize of lysin-extracted M protein from a number of strepto-coccal strains was examined by Western blot (immunoblot)(74). M protein derived from streptococcal isolates of 20different serotypes exhibited variation in size ranging from41 to 80 kDa, depending on the strain. Similar size variationis observed when different M6 streptococcal strains isolatedover a period of 40 years are examined in this way. Thisvariation in size may be explained by the observation thatthe M sequence contains extensive repeats at the DNA level(94). Long reiterated DNA sequences are likely to serve assubstrates for recombinational events or for replicativeslippage (62, 192), generating deletions and duplicationswithin the M-protein gene and leading to the production of Mproteins of different sizes.

Isolation and Sequence Analysis of Size Mutants

To test this hypothesis, it was necessary to isolate clonallyrelated strains of M-protein size mutants in the laboratory,so that the parent and mutants could be compared. AWestern blot system was devised so that M-protein sizemutants could be detected and isolated if they comprised atleast 5% of the culture. From type 6 strain D471, about 1 in2,000 colony-forming units (CFU) were found to produce asmaller size M protein, a rate that seemed too high to be theresult of spontaneous mutation (71). Analysis of the DNAsequence of the M6 genes of four spontaneous M-proteinsize mutants confirmed that they arose by recombinationalevents between reiterated DNA sequences within the struc-tural gene encoding M protein (95). Each of the mutantsbearing smaller-size M protein had a deletion of one or moreof the A- or B-repeat blocks (Fig. 9).Comparison of aligned sequences of the M6 size variants

indicates that extensive changes can occur within this onestrain. While the region on the C-terminal side of the lastC-repeat sub-block remains highly conserved (95), the A-and B-repeat segments have undergone duplications and

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296 FISCHETTI

Cell-Associated

Helical Central Rod RegionAnchor Region

M6.1 N

M6.2 N Al A2 A3 B

M6.3

M6.8

Cl

Cl

N A Cl

N A Cl

C2 C3 |I| Pro/Gly3 Anchor FC

C2 C3 1DD1314 Pro/Gly |ncr FC

C2 C3 DIDID Pro/Gly |emnhb.f .C

C2 C3 2i3 4 Pro/Gly | AnchorFIG. 9. Alignment of M6 sequences of four different M6 strains (95). M6.1 from strain D471 is the prototype M6 protein. M6.2 is from a

deletion mutant derivative of M6.1 in which two A-repeat blocks have been deleted. M6.3 is a deletion mutant derivative of M6.2 in whichone B repeat has been removed. M6.8 from strain M01015 is a natural M6 isolate from a pharyngitis outbreak in the 1980s. This strain differsfrom the prototype M6.1 in the nearly complete loss of A repeats. Region assignments are as those described in the legends to Fig. 6 and 7.

deletions (Fig. 9). The size mutant derivative of the M6.1parent, M6.2, deleted two A-repeat blocks, while mutantM6.3, a derivative of M6.2, was unchanged in the A-repeatblocks but is deleted for one B repeat. However, in spite ofthe extensive variations in the number of repeat blocks,these strains were still typed as M6.

Size variation also occurred in M6 strains isolated fromsingle localized outbreaks of pharyngitis as well as in serialweekly isolates from the throats of individual patients in the1940s, prior to penicillin therapy (Fig. 10) (74, 95). Based onDNA sequence analysis, the size variation of six clinical M6isolates is the result of recombination events within therepeat sequences (95), as was found with the mutantsisolated in the laboratory. For example, when comparedwith M6.1, M6 strain MO1015 producing M protein M6.8(isolated during an M6 outbreak in the 1980s) has only oneand one-half A-repeat segments (Fig. 9), whereas M6.9(isolated in the 1940s) has five A repeats (two of which differin size from the others) and an extra C-repeat segment (95)(data not shown). Sequence analysis of the M6 genes alsoindicated that the isolates from each clinical group areclonally related and not the result of separate acquisitions ofunrelated M6 strains during the course of the infection (95).When the streptococcal strains isolated weekly from thethroats of 15 patients were examined for the size of the Mmolecule (see Fig. 10 for an example of two such patients),8 patients harbored streptococci which exhibited changes inM-protein size but not serotype during the course of theinfection. The observation that size variants occur amongclinical isolates suggests that the size mutant constituted themajor organism in the streptococcal population at the time ofisolation and suggests that a given size mutant has a selectiveadvantage under clinical conditions.

Thus, it is quite clear that variations can occur in both thenumber and size of the repeat blocks within the M-proteinsequence. Even within an outbreak resulting from a singleinfecting strain, the M protein from the progeny of the

IsolIa te2 34 5 67

GL53

Isolate2 3 4 5 6 7

GL44FIG. 10. Western blot of lysin extracts of serial streptococcal

isolates from two patients with pharyngitis illustrating a change inthe size of the M protein. Lanes represent extracts of weeklystreptococcal isolates from the time the patients presented withpharyngitis (week 1). Seven weekly isolates of M17 streptococciwere obtained without patient treatment in 1942. After week 3,patients are usually asymptomatic and are considered to be strep-tococcus carriers. The strains were grown and adjusted to compa-rable optical densities, and the M protein was extracted with phagelysin, which solubilizes the cell wall releasing M protein. Aftersodium dodecyl sulfate-polyacrylamide gel electrophoresis andWestern blot, the nitrocellulose was probed with a monoclonalantibody reactive with M protein. The blots represent the typicalmultiple banding pattern of M protein extracted by this method (74).It can be seen that in patient GL53 the size of the M proteinproduced by the streptococci was the same to the fifth weeklyisolate. At week 6, the streptococci isolated from this individualsynthesized a smaller M protein which changed again in week 7(though all still typed as M17). In patient GL44, the size of the M17molecule remained the same for 4 weeks, at which time thepopulation of streptococci isolated produced a smaller M molecule(weeks 5 to 7). At week 7, the amount of M protein on theseorganisms was reduced, which is typical of organisms isolated in thecarrier state.

Non-HelicalRegion


I I

NV .o 0 001 4

.::::.:: (W at. A* Oa j*I"

!I a *iAi. a

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[ Al ]C A2 ][ A3 ][ A4 1E A5 ]

M6.1 ....MLQANND KLTTENNNLTDONKNLTTENKNLTDQNKNLTTENKNLTDQNKNLTTENKELKAEENRLTTENK....M6. 1 ....MLQANND NLTTENKNLTDQNKNLTTENKELKAEENRLTTENK....

[ KKK l] A4 IL A5 ]L NT Q

Al T D A3

NE TLN N

N KiNL NTD E

0 TTN LK NA2

L Al/ A3 ]L A4 A5 ]

M6.5....MLQANND NLTTENKNLTDQNKNLTTENKELKAEENRLTTENK....FIG. 11. Alignment of the amino acid sequence of the A-repeat region of the M6.1 protein and a deletion mutant derivative, M6.5 (74, 95).

The deletion of two A-repeat blocks in the emm-6.5 gene, which likely arose by a recombination event from the center of the divergent Alrepeat block to the center of the consensus A3 repeat block, has generated a new sequence for this M6 protein mutant (107); the sequenceDK (Asp-Lys) was converted to DN (Asp-Asn).

infecting streptococcus isolated from infected individualsexhibits size differences.

ANTIGENIC VARIATION AND ITS RELATION TO SIZEVARIATION

Antigenic diversity of surface antigens is a mechanism bywhich successful pathogens have been able to survive in theenvironment after infecting immunocompetent hosts (7, 45,146, 194). In bacteria, the best-studied variable antigens arethe gonococcal pilin (146, 194) and the Borrelia variablemajor protein (7). M protein differs from these other anti-genically variable surface proteins in that they undergo geneconversion events involving extragenic recombination be-tween multiple genes, whereas the single copy of emm-6 instreptococci (see below) must involve intragenic recombina-tion events for changes to result.

Clues as to the mechanism of antigenic variation of the Mprotein are based on two previously mentioned observa-tions: size variation of the M molecule (74) and the presenceof repeat sequences within the M-protein gene (94). Basedon these observations, it was hypothesized that changes inthe amino acid sequence may arise as a result of recombi-nation between inexact repeats. Because the end repeatblocks within the A- and B-repeat regions of the M6 se-quence diverge slightly from the consensus sequence of thethree internal blocks, recombination between one of theseterminal blocks and any other central block can generate anew sequence. These changes may then be amplified byrecombination events within these repeated sequences. Se-quences involved in antigenic determinants may be dupli-cated or in some cases deleted by such recombination events(95). Thus, intragenic recombination events would be ex-pected to accelerate the rate at which alterations accumulatein the M-protein gene. Such recombination events werefound to have occurred in the A- and B-repeat regions ofdeletion mutants isolated in the laboratory from strain D471(Fig. 9). In mutant M6.5, derived from M6.1, a deletion ofthe equivalent of two repeat blocks occurred from a recom-bination event beginning from the center of the divergent Al

repeat block to the center of the consensus A3 block. Thisresulted in a sequence change from an Asp-Lys (D-K) to anAsp-Asn (D-N) within this mutant (107) (Fig. 11).

Size Variation Leads to Antigenic Variation

To test the hypothesis that size changes in the emm-6 genecan lead to antigenic variation of the M protein, monoclonaland polyclonal antibodies directed to defined epitopes on theparental M6 molecule were used in competition assays withpurified M protein isolated from the parental M6 strain andsize mutants (107). The results revealed that antigenicchanges occurred in epitopes located at or near the deletionswithin mutant M6 proteins, but not at more distant epitopes.The studies also revealed that an M6 monoclonal antibodyopsonic for the parental M6 strain lost its ability to opsonizeone of the M6 size mutants, supporting the view that sizechanges may also result in changes in antigenic determinantsresponsible for streptococcal survival in an immune environ-ment. Thus, antibodies originally produced in response tosequences present in the parental M6 molecule were unableto bind to mutant M6 protein or opsonize certain M6 sizemutants. It was discovered that, in some size mutants inwhich a direct sequence change had not occurred, changes inconformation were predominant (107). Therefore, a changein the size of the M molecule may be an attempt on the partof the streptococcus to evade immune clearance. While thismay be true for changes in the opsonogenic A-repeat region,antibodies directed to the B- and C-repeat regions have beenshown not to be opsonic for the streptococcus (106). Thus,the pressure for these regions to change may be at a differentlevel, i.e., adherence and colonization.

Natural Selection of Size Mutants

Group A streptococcal pharyngitis is a self-limiting dis-ease in which the production of opsonic antibodies to the Mmolecule results in the elimination of clinical illness (128).However, in some instances, after symptoms subside and inthe presence of opsonic antibodies, the infected individual

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Cell-associated

N A

N A13

Bi B2 B3 B4 B5 |BH

Cl C2 1C3 |Regbn

C2 1C31

Cl C2

D Region

Memb. cPro/Gly Anchor

>98%

Memb. CPro/GIy Anchor

>98%

FIG. 12. Alignment of the sequences of three different serotype M proteins (M6.1 [strain D471], M5 [strain Manfredo], and M24 [strainVaughn]) (94, 148, 153). N terminal to the Pro-Gly region, each M protein differs in the size and number of repeat regions. M24 is lackingA-repeat sequences. Amino acid identity of >98% is found within the Pro-Gly and membrane anchor regions among the three M proteins.Sequence variation increases as the N terminus is approached.

may carry the streptococcus in the throat for several monthswithout clinical signs (termed carrier state) (86, 92, 111). It isclear that more than one opsonogenic epitope is presentwithin the N-terminal region of the M molecule and antibod-ies to only one epitope are adequate for the phagocytosis ofthese organisms (15, 19, 106, 179). Whereas antigenic vari-ation in the gonococcus and Borrelia surface proteins is theresult of the exchange of large genetic blocks, in the strep-tococcus only a few amino acids are changed at a time.Therefore, M-protein mutants created during a streptococcalinfection may not be suitably altered in all opsonogenicepitopes to escape immune recognition and clearance. How-ever, prior to clearance from the infective or carrier state,the streptococcus has the ability to infect other human hosts(203). Thus, M-protein mutants produced in the initial hostduring clinical illness or during the carrier state are able to bedisseminated throughout the population, leading in time tothe accumulation of changes within the M molecule. Conse-quently, through immunological pressure, the antigenicstructure of the M protein is continuously evolving by theselection of organisms producing variant M molecules. Thisprocess is likely to result in a continuum of antigenicallyunique M molecules among the streptococci in the environ-ment.

Variation in the N-Terminal Nonrepeating Segment

The N-terminal nonrepeating segment of the M moleculecontains protective epitopes. While variation within therepeat segments may be attributed to intragenic recombina-tion events, no information is currently available regardingthe method by which sequence changes occur within theshort nonrepeating segment. Sequence analysis of M6 fromseveral strains isolated from the 1940s to the 1980s revealedthat, while variation is seen in the A, B, and C repeats, thesequence from the N terminus to the first A repeat (about 30amino acids depending on the serotype) has been conserved(95); S. Hollingshead, unpublished data). While current datasuggest that no other homologous regions exist outside theemm-6 gene (182, 183), it has been suggested that thisN-terminal segment could arise from recombination betweenextragenic non-emmn-6 segments (148). Attempting to explainthe mechanism of variation of this N-terminal region amongM serotypes, Haanes-Fritz et al. (90) examined the DNAsequence of segments outside the emrn-6 structural gene of

Ml, M6, M12, and M24. They found that, although the 5'sequences of the four mature M proteins exhibited consid-erable variability, extensive similarities were observed in theupstream flanking regions and signal peptides. The authorsproposed that these conserved flanking regions may beinvolved in rare occurrences of homologous recombinationwith regions outside the M gene, resulting in the insertion ofnew variable segments. To date, however, the existence ofthese extragenic regions has not been established, whichmay be a consequence of the use of unsuitable probes.An alternative interpretation of the apparent stability of

N-terminal sequences within a serotype is based on themethod used to type the strains used in sequence studies. Asmentioned earlier, the M typing sera are highly absorbed,containing antibodies that are specific for a given serotype Mprotein; i.e., the typing sera define the serotype. Since theN-terminal end is the most variable region of the M mole-cules, antibodies in these typing sera are naturally directedto epitopes in the N-terminal extreme. Thus, when M6strains are selected for M6 protein analysis, it stands toreason that they all will likely have a similar N-terminalsequence. An M6 strain which has diverged sufficiently inthe N-terminal sequence to prevent the typing serum fromreacting (a single amino acid change could accomplish this)would not be typed as M6 and, thus, would not be analyzedas an M6 protein. Since more than half of the streptococcalstrains isolated in nature are classified as nontypable (173), itis conceivable that many of these organisms are derivativesof typable strains having sequence changes within the non-repetitive N-terminal region. Since the sequences of only afew M molecules have been analyzed to date (none of whichare derived from nontypable streptococci), it is difficult todetermine which types, if any, are related to one anotherwith respect to the sequence in the N-terminal extreme.

COMPARISON OF M-PROTEIN SEQUENCES

Alignment of Known M-Protein SequencesIn addition to the complete M6 sequence from strain D471

(94), sequence data are now available for a number of otherstreptococcal serotypes. The sequences are available for thecomplete M5 (strain Manfredo) (148), M24 (strain Vaughn)(153), and M12 (strain CS24) (175) proteins and the completepepsin fragments of M5 (strain B788) (133) and M49 (strainB915) (116) and partial Ml (strain T1/195/2) (120, 152) and

M6.1

M5

M24 N C3 D Region Pro/Gly Anchor C

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d a d a d a d a d a d a dM6 RVFPRUG-TVEN DKR L SMYQANDKlTfTENNN[TDQKKN[TTNK N[

PepM5 TVT TISD QR KEA DtELE HDLKTK EG| NEG KTENEG KTEN|EG

a d a d a d a d a d a d a d aa dTDQNK TITTENKNrTI1DQ NSNLTTENKE EAEENRLTTENKGLTKKLSEAEEEAANKERKTEKS ERKTAE SE EHEA DKLK

a d a d d a d a d a d d a dENKEAIG L F DETRKDKIAKEQESVETI[TWKITEEVKDKIA1EQQRDTLSWQ ETEREVQNTQYNNETLKIKNGDLTKELN TRQELAN

a d a d d a d a d a d d a d a

E S KE|TIGTKKT[DETVKDKIAKEQESKETI GTLKKILDETVKDKII REQKSKQDESKENEKALNELLEKTVKDKIAKEQEN|KETIGTLKKILDETVKDKLAKEQKSKQN

d a dIGALKQELA 228IGALKQEL 197

FIG. 13. Sequence comparison between the PepM5 protein (strain D788) (134) and the corresponding region of the M6.1 molecule (M6N-terminal half) from strain D471 (77, 94). Identical residues are boxed. Lowercase a and d above the sequence designate the positions ofthese residues in the seven-residue periodicity (a, b, c, d, e, f, and g) based on the alignment in Fig. 7. A 29-residue gap was introduced inthe MS sequence to maximize the number of identities since the MS sequence in this strain is shorter than that represented in Fig. 12.

M19 (unpublished strain) (34) proteins. In addition, theC-terminal one-third of M5 (strain T5B/126/4), M19 (strain(SS400), M24 (strain (C98/135/2), M30 (strain 6GL130), andM55 (strain A92817311) is also available (96). Like the differ-ences observed among the various M6 strains (Fig. 9),alignment of the M-protein sequences from M6, MS, andM24 serotypes reveals differences among the three in thenumber and size of the repeat blocks (Fig. 12). In these threeserotypes, in addition to M19, M30, and M55 (96), a highlyconserved region (>98% identity) is found C terminal to theD repeat (located within the cell wall), and, depending on theserotypes being compared, sequence variation increasesamong these types as the N terminus is approached (Fig. 8).Strong sequence homology is observed among these threetypes within the C-repeat region; however, this homology isgreatly reduced in the B-repeat region and nearly lost in theA-repeat region. The extent of homology depends on theserotypes being compared, some types being more relatedthan others. For instance, the C-repeat blocks between M6and M5 and between M6 and M24 are about 80% identical;however, the B blocks in M6 are not very homologous tothose in M24 (18% identity) but are significantly homologousto those in M5 (54% identity) (Fig. 12). The homology of theB blocks between M5 and M24 is <10%. In the M24sequence, all of the A repeats have been deleted, leaving anextended nonrepetitive region N terminal to the B repeat. Asimilar pattern is seen in the sequence of the pepsin fragmentof the M49 sequence; however, in this sequence, remnantsof an A-repeat region still exist (not shown) (116).The preceding discussion serves to illustrate the differ-

ences in the sequence and repeat blocks for the M proteinfrom these specific serotype strains. M proteins from dif-ferent strains of the same serotype are likely to differ in sizefrom the prototype strain and will exhibit differences insequence as well as immunodeterminants depending on thenature of the size variation. For instance, the major differ-ence between the N-terminal half of M5 from strain B788(112) and M5 from strain Manfredo (148) is a 14-residueinsertion in the A-repeat region of strain B788 and a 25-residue insertion in the B-repeat region of strain Manfredo.Therefore, strain designations are critical when comparisons

are to be made between different M proteins. Although veryfew M sequences are currently known, it is clear that certainserotypes are more closely related than others based on thedegree of homology within the B- and C-repeat segments (28,108, 109). Perhaps, as more data are gathered on other Mproteins, the evolution of the M serotypes will become moreapparent.

Alignment of the Variable Region of Two DifferentM Proteins

The sequence of PepM5 protein from strain B788 (133),comprising the N-terminal half of the native M5 molecule (tothe C-terminal end of the B-repeat region), was comparedwith the corresponding region of the M6.1 sequence (fromstrain D471) (77, 94). Maximal homology is achieved if a gapof 29 residues is introduced in the M5 sequence. This isnecessary since the M molecules from these two strainsdiffer in size, M5 being smaller (Fig. 13) (74). The first 85residues of the M6 sequence show 33 identities (39%) withthe PepM5 molecule. Homology of 56% is observed betweenresidues 115 to 228 of the M6 sequence and 85 to 197 ofPepM5. This increases to 77% identity between residues 164to 228 of M6 and 134 to 197 of M5. In all, 43% identity isobserved between these two segments.Although the number of identities within the hypervariable

region of these two molecules (residues 1 to 163 of M6 and 1to 133 of M5) is far less than those in the region C terminalto these segments, half (22 of 47) of the identities are eitherasparagine or hydrophobic amino acids that occupy coreresidues at positions a and d of the heptad periodicity (Fig.7). It is clear that hydrophobic residues in these positions are

required for the maintenance of the coiled-coil structure ofthe M protein (136, 164). Therefore, in spite of considerablesequence dissimilarity between these two M moleculeswithin the hypervariable region, identities are preserved incritical structural positions. Thus, the maintenance of thecoiled-coil rod region appears to be a biologically significantcharacteristic of the M molecule (76, 77, 141).

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PROTEIN A ANHADANKAQALPETGEENPLIGTTVFGGLSLALGAALLAGRRREL22% (52%)

M PROTEIN NKAPMKETKRQLPSTGETANPFFTAAALTVMATAGVAAVV-KRKEEN56% (63%)

G PROTEIN AKKDDAKKAETLPTTGEGSNPFFTAAALAVMAGAGALAVASKRKED::::.: .. . . .::...: .:.: 35% (60%)

PROTEIN A ANHADANKAQALPETGEENPLIGTTVFGGLSLALGAALLAGRRRELFIG. 14. Alignment of the C-terminal 46 amino acids of staphylococcal protein A, group A streptococcal M protein, and group G

streptococcal protein G. The protein A sequence is positioned on top and bottom to allow comparison with protein G and M protein. Doubledots designate identities, while single dots designate charge or hydrophobic conservation. A gap was introduced in the M-protein sequenceto maximize identities with protein G. Values at right are percent identities (percent identities plus conservative substitutions).

HOMOLOGY OF M PROTEIN WITH OTHERMOLECULES OF GRAM-POSITIVE BACTERIA

Group C and G Streptococci

M protein or M-like proteins of group A streptococci havebeen reported in group B (142), C (142, 217), E (58), and G(142, 143) streptococci. These conclusions are based eitheron serological cross-reactions with typing sera developed toM protein from group A organisms or on the ability of theseorganisms to resist phagocytosis. DNA hybridization analy-sis was performed to define better the relationship of the Mprotein from group A streptococci with that of other strep-tococcal groups (183). By using nearly the complete M6protein gene as a probe, homologous DNA is found in groupC and G streptococci but not other groups (183). Group Cstreptococci are primarily animal pathogens but have beenimplicated in serious human infections (151). Group G or-ganisms are found as part of the normal flora of the skin,upper respiratory tract, and female genital tract. However,these organisms are responsible for an increasing number ofhuman diseases (4, 6, 142).Monoclonal antibodies prepared to the M6 protein from

group A streptococci cross-react with a protein found ongroup G clinical isolates (105). More than half of the group Gstrains examined are resistant to phagocytosis in humanblood. When surface protein is extracted from group G cellswith the muralytic enzyme mutanolysin and reacted inWestern blots with M-protein-specific monoclonal antibod-ies, the protein pattern resembles that seen with M proteinfrom group A organisms. A highly purified, pepsin-derivedprotein fragment from a group G strain was capable ofinducing antibodies in rabbits which opsonized group G cellsin a phagocytosis assay (105). In a separate study, group Gstreptococcal clinical isolates were found to survive inhuman blood and shown to exhibit surface fibers by electronmicroscopy. Rabbit antisera to either whole group G strep-tococci or partially purified pepsin extracts were also op-sonic for homologous isolates (31). Using DNA probesrepresenting the C-terminal and leader sequences of the M12protein gene, Simpson et al. (187) found that DNA fromgroup G strains which infect humans is related to the groupA streptococcal M-protein DNA. This relationship was notseen in DNA from group G strains which cause animaldiseases. Thus, M proteins on group A and G streptococci,especially group G organisms involved in human disease,appear to be functionally and structurally similar.A second protein located on the surface of group G

streptococci (protein G) has sequence homology with groupA M protein. Protein G is both an immunoglobulin G (IgG)-binding protein that binds all four classes of human IgG andanimal IgG classes and an albumin-binding protein (2).Analysis of the DNA sequence reveals that the C-terminal

end of protein G is highly homologous to the M-proteinC-terminal membrane anchor and a small portion of the walldomain, with 56% identical residues (61, 159) (Fig. 14).Although there is some sequence homology between theremaining portion of protein G and M protein, the moststriking feature lies in the presence of sequence repeatblocks. Like M protein, the repeat blocks divide protein Ginto distinct domains. A repeat domain located within theC-terminal half of the molecule, proximal to the cell wall, isresponsible for the IgG-binding activity of protein G (89),while the albumin-binding activity is located in the N-terminal repeat domain (3). Although protein G is rich ina-helix-forming residues, a detailed analysis concerning itsconformation as a coiled-coil structure has not as yet beenperformed.

S. equi, a Lancefield group C organism, is the causativeagent of "strangles," a disease of horses resembling humanpharyngitis. Reports of an M-like molecule with the ability torender these streptococci resistant to phagocytosis are welldocumented (189, 196, 217). The interesting characteristic ofthe M or M-like protein on S. equi is the lack of differentserotypes (8, 9). For instance, Bazeley reports that anantiserum prepared against one strain of S. equi protectedmice from challenge by each of 32 different strains isolatedfrom various parts of Australia (8). Thus, antigenic diversitydoes not appear to exist in this group C antiphagocyticmolecule as it does in the group A M protein, even thoughmany of the characteristics of the cloned S. equi M-likeprotein are shared with group A M protein (85).

Staphylococcal Protein A

Protein A is an IgG-binding protein from staphylococciwhich also has features in common with streptococcal Mprotein. Like protein G, the region of sequence homologywith M protein is located at the C terminus of protein A,where it exhibits 22% identity with M protein (89a) and 35%with protein G (Fig. 14) (61, 159). The homology amongthese three proteins is even more striking when conservationof charge or hydrophobicity is considered. The conservationof sequence and chemical properties of amino acids withinthe C-terminal region suggests that there likely exists acommon mechanism by which protein A, protein G, and Mprotein are anchored to the gram-positive cell wall. Althoughprotein A does not exhibit (x-helical coiled-coil potential(159), the repetitive nature of the sequence (198) is reminis-cent of the repeats found in both M protein (94) and proteinG (61). While serological differences between protein Amolecules on various staphylococcal strains has not yet beenreported, the presence of size variation among clinicalisolates has recently been described (38).

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TABLE 1. Sequence homology of M proteinwith mammalian proteins

(4 IdentityM protein Mammalian protein (amino acid

overlap)"

M24 Myosin heavy chain (rabbit cardiac) 34 (44)Tropomyosin (human cardiac) 33 (42)Keratin (type Il bovine) 40 (30)Lamin (human) 36 (45)

MS Tropomyosin (human fibroblast) 32 (28)Myosin heavy chain (rabbit cardiac) 39 (41)Keratin (type 1. bovine) 43 (30)Tropomyosin (human cardiac) 33 (45)

M6 Myosin (rat cardiac) 38 (50)Keratin (type 1. bovine) 44 (32)Tropomyosin (human cardiac) 38 (50)

M12 Keratin (type 1. human) 41(32)Myosin heavy chain (rabbit cardiac) 31 (32)

Identity based on fastp analysis of known sequences (130) and inspection.

STRUCTURAL RELATIONSHIP OF M PROTEIN WITHMAMMALIAN PROTEINS

Tropomyosin

M protein resembles muscle oQ-tropomyosin on the basis ofphysicochemical properties and sequence homologies (99).As described previously, N-terminal sequences of certainM24 peptides have up to 40% identity with tropomyosin, anda hexapeptide of tropomyosin appears five times in the M24protein (99). In subsequent studies, homologies with tropo-myosin were also observed with other M sequences, namely.MS and M6 (136). Many of the observed identities corre-spond to the core hydrophobic amino acids in positions a andd in the seven-residue repeat pattern necessary to maintainthe coiled-coil (refer to Fig. 7 and section on structure) (77.136, 164). However, in certain regions, identities are alsofound in externally positioned amino acids. It was suggestedthat the correlation between sequences in M protein andtropomyosin provides direct evidence, at a molecular level,of a structural similarity between a streptococcal and amammalian protein, and this similarity may be relevant inthe pathogenesis of streptococcal diseases (99, 150).

Other Coiled-Coil Molecules

An analysis of the PepM5 sequence revealed that theperiodicity and sequence of this M protein more closelyresemble the coiled-coil rod region of myosin than tropomy-osin (138, 141). Sequence comparison of the complete M5(148), M6 (77, 94), M12 (175), and M24 (153) proteins withknown protein sequences in a data base reveals the besthomology to be primarily with myosin and tropomyosin(Table 1). Other coiled-coil proteins such as keratin andlamin also show significant homology with M proteins.The basic structure of the M6 protein (77) is remarkably

close to that of eukaryotic intermediate filaments (keratin.desmin, and vimentin), whose elemental structures are alsobased on a coiled-coil central rod region with structurallydifferent end regions (166, 190). Its superstructure is com-

posed of two to three ot-helices wrapped around each other,and, like M protein, intermediate filaments from differentsources vary immunologically as well as in size and compo-

sition (190). While it would be tempting to speculate on thepossible role of these homologies in the pathogenesis ofcertain streptococcus-related diseases, it would be best toawait the results of future experiments.

IMMUNOLOGICAL CROSS-REACTIVITY BETWEEN MPROTEIN AND MAMMALIAN PROTEINS

Heart-Reactive Antibodies

Cross-reactions have been observed between streptococ-cal components and mammalian tissue. Kaplan and Meyes-erian (113) described a component of M5 and M19 strepto-coccal cell walls which induces antibodies reactive withmuscle tissue and correlates with M protein. Zabriskie andFreimer (219) observed that purified streptococcal mem-branes evoked cross-reactive antibodies which reacted withhuman cardiac muscle sarcolemma. While subsequent at-tempts were made to identify these cross-reactive antigensfrom the streptococcus (200). they have not been character-ized sufficiently to determine their identity.

Immunodeterminants Shared with Myosin and OtherHuman Proteins

Dale and Beachey (54) discovered that rabbits immunizedwith PepM5 protein produced antibodies which cross-reactwith myosin but not tropomyosin or actin. By enzyme-linkedimmunosorbent assay, the cross-reactive epitope is alsofound in the M6 and M19 proteins and is exposed on thesurface of the streptococcus. Furthermore, these investiga-tors found that the M5 molecule contains at least threeepitopes which stimulate antibodies to human sarcolemmalmembranes and are partially shared with M6 and M19proteins (53). One of the myosin cross-reactive epitopes waslocalized to amino acids 84 to 116 of the M5 sequence (56).More recently. Sargent et al. (179). using antibodies to

eight synthetic peptides representing the entire PepM5 pro-tein, determined that peptide 164-197 stimulates antibodiesin rabbits which react with isolated cardiac sarcolemmalmembranes but not with purified myosin. The reactivity islocalized to a 40-kDa protein in the sarcolemmal membrane.The results indicate that the epitope located in the 164-197sequence is shared with M6 but not M24 or M19. Thissequence is located close to the center of the native M5molecule (within the B-repeat region), N terminal to thepepsin-susceptible site. This segment is highly conservedbetween the M5 and M6 proteins (77) (Fig. 13) but not M24.

Synthetic peptides representing the N-terminal 20 aminoacids of M5 (57) and M6 (15) proteins were found to evoketype-specific protective but not heart-reactive antibodies.These studies suggested that, by using sequences from theN-terminal end of the M molecule, heart-reactive epitopesmight be avoided. However, subsequent experiments (34)revealed that PepM19 and synthetic peptides representingresidues 1 to 24 of M19 evoked both opsonic antibodies andheart-reactive antibodies to a sarcolemmal protein, indicat-ing that epitopes stimulating autoimmunity may also belocalized within the type-specific N-terminal region.

Renal Tissue-Associated Epitopes Shared with M Protein

Sera from patients with poststreptococcal glomerulone-phritis are shown to have antibodies against basement mem-brane antigens (63). The antigens involved in these reactionsare glomerular heparin-sulfate proteoglycan and basement

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M6 .1

Tropomyosin

bc e ef c e bc11I r I I I 1 1

310 320 330 340abcdefgabcdefgabcdefgabcdefgabcdefgabcdefgLEEANSK KLEKLNKELEESKKLTEKELQAKLEAEAA

YEEVARKLVIIESDLERAEERAELSEGKCAELEEELKTVTNN170

c

180 190 200

q

f

e 9FIG. 15. Alignment of the sequence of M6 protein and human cardiac tropomyosin in a region exhibiting significant homology. Lowercase

letters a through g directly above the sequence designate the positions of these amino acids within the seven-residue pattern (a-b-c-d-e-f-g)in both segments based on previous analysis (77, 191). Lowercase letters b, c, g, e, and f identify these external locations in the heptad repeat.Double dots indicate identities and single dots are conservative substitutions. Below, a cross sectional view of a coiled-coil (looking down thehelices from the N terminus) revealing the relative position of residues a to g in the structure (adapted from McLachlan and Stewart [145]).Positions a and d are nonpolar and, thus, internalized in the formation of the coiled-coil. This reveals that the identities in positions b, c, g,e, e, f, c, e, b, c would be clustered nearly contiguously along the outer face of the coiled-coil.

membrane type IV collagen and laminin. It is speculated thatthe cross-reactivity may be related to structural similaritybetween the streptococcal M protein and basement mem-brane antigens such as laminin. More recently, it has beenshown that monoclonal antibodies prepared to renal cortexcross-react with M proteins from type 6 and 12 streptococci(87). These studies were extended to type 1 M protein, inwhich antisera raised in rabbits to a synthetic peptiderepresenting the N-terminal 1 to 26 amino acids and a similarpeptide lacking residues 20 to 22 both were able to opsonizeMl streptococci and react with human renal glomeruli byimmunofluorescene (119). The cross-reactive epitope waslocalized to the tetrapeptide Ile-Arg-Leu-Arg located atresidues 23 to 26 in the Ml peptide, while the regionresponsible for eliciting the opsonic antibodies was residues1 to 20. The Ile-Arg-Leu-Arg sequence has not been found inother M-protein sequences such as MS, M6, and M24, whichare not considered nephritogenic serotypes. However, it wasalso not part of the M49 sequence, a nephritis-associatedserotype (116). The authors state that this result indicatesthat the observed tetrapeptide may not be the only sequencedefining a renal autoimmune epitope (119).

Monoclonal Antibodies Cross-Reactive with Coiled-CoilProteins

Cunningham and Russell (49) prepared monoclonal anti-bodies to streptococcal membranes from M5 streptococciwhich cross-react with detergent-extracted human heartsarcolemmal antigens. The monoclonal antibodies reactmore strongly with heart antigens than with kidney orskeletal muscle antigens. Some of the monoclonal antibodiesare directed to skeletal muscle myosin, and one is reactivewith ventricular myosin. The myosin-reactive antibodycould be absorbed with either myosin or whole streptococci(121). Two monoclonal antibodies reactive with M5 protein

and a streptococcal membrane protein were found to bepolyspecific. One reacts with DNA, polyinosinic acid,polydeoxythymidylic acid, and cardiolipin, and its reactivitywith the cytoskeleton could be blocked with antivimentin(50); the other reacts with the cytoskeleton and is inhibitedby the a-helical proteins myosin, actin, and keratin. Furthercharacterization of these monoclonal antibodies revealedthat they are specific for epitopes in the heavy chain ofmyosin: one for light meromyosin and the other for both lightand heavy meromyosin. Both of these myosin fragmentscontain a portion of the coiled-coil rod region. The authorsspeculate that the induction of antibody-producing clonesagainst myosin by a streptococcal antigen may explain atleast some of the cross-reactive responses seen duringstreptococcal sequelae. Recently, Fenderson et al. (62a)described the production of monoclonal antibodies whichreact with M5 and M6 proteins and human and rabbittropomyosin. At least one of these cross-reactive monoclo-nal antibodies also exhibits strong reactivity with humanheart tissue by immunofluorescence.

Conformational Determinants

Since many of the M-protein cross-reactive antibodies (48,50, 56, 119, 121) also react with other coiled-coil proteins(i.e., myosin, tropomyosin, keratin, vimentin, etc.), it islikely that in several cases the immunological recognition isbased on a common conformational determinant. Sequencealignments between M protein and tropomyosin (Fig. 15), aswell as between other cross-reactive coiled-coil molecules,do not reveal a common linear sequence (Fischetti, unpub-lished data). Identities are usually distributed among severalnonadjacent amino acids within a localized region of thesemolecules. This is not surprising based on the molecularconstraints imposed on a coiled-coil structure, i.e., whereevery first and fourth amino acid in the heptad repeat are

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internalized in the formation of the coiled-coil, leaving theintervening residues in external positions (145). For exam-ple, an analysis of the position of the identities within theseven-residue repeats found in an alignment between M6protein and tropomyosin. reveals that they appear primarilyin positions b, c, g, e, and f, all of which are located nearlycontiguous to one another on the outer surface of thecoiled-coil structure (Fig. 15). Thus, it is possible that thespatial distribution of these residues in two conformationallysimilar molecules could form the basis of a common immu-nodeterminant.

Consequently, since streptococci are capable of varyingthe M-protein sequence (95, 107), certain sequences foundwithin this changing molecule are likely to be homologouswith sequences located in relatively stable mammaliancoiled-coil proteins. Thus, as seen with the analysis of theM5, M6, M12, and M24 proteins, each shares homology withmyosin, tropomyosin, and other coiled-coil proteins to dif-fering degrees as well as to different regions of the respectivemolecules (Table 1). While some homologous regions mayform cross-reactive determinants, the significance of thesedeterminants in the pathogenesis of streptococcal diseases isjust beginning to be understood.

THE emm GENEAs noted previously, no information regarding the C-

terminal half of the M molecule was available until the genefor M6 protein was first cloned from streptococcal strainD471 (emm-6.1) into Esclewrhia (/oli (94, 180). The Mprotein produced in E. coli (ColiM6) was shown to react withboth monoclonal and polyclonal antibodies prepared to thestreptococcus-derived M6 protein. The cloned moleculeaccumulates in the periplasmic space of E. coli, and no Mprotein is detected on the surface of E. co/i or in the growthsupernatant (73). A small amount of M protein is present inthe cytoplasm, where its molecular size is larger than theperiplasmic form. This is consistent with the idea that thelarger molecule retains the hydrophobic leader sequenceprior to export to the periplasm.The M-protein genes from M5 (115), M12 (175), and M24

(153) streptococcal serotypes have also been cloned andshown to produce immunologically active M proteins. Onsodium dodecyl sulfate-polyacrylamide gel electrophoresis,the cloned M6 molecule appears as a close triplet of bands.a characteristic also seen with the cloned M5 protein (171).The reason for the multiple banding pattern is not entirelyunderstood, but seems to be associated with the C-terminalregion, since pepsin cleavage of ColiM6 results in a C-terminal fragment which retains the triple banded structure(72) while the N-terminal half (or PepM) is homogeneous (21,34, 72, 137).

Relationship of M-Protein GenesThe relationship of the emmn-6 gene in M6 streptococci to

emm genes in other streptococcal serotypes was determinedby hybridization experiments, using the enmm-6.1 gene as aprobe (183). The results revealed that DNA from all of the 56different serotypes tested contain homologous cmiui genes.Among the group A streptococci tested in these studies wereseveral which were functionally M- (i.e., those phagocy-tized in human blood). An e(mm gene could be identified in allbut one M- strain. The reason for the M- phenotype inemm1117-containing strains is not known, but may reflect adefect at the level of translation or transcription of Mprotein.

Localization of the region in the emin gene responsible forhybridization with all tested serotypes was accomplished byhybridizing DNA from 10 different serotypes with smallerprobes derived from the emm-6.1 gene. This investigationrevealed that the 3' region of the emim-6. / gene is conservedamong these serotypes while the 5' segment is variable (182).A similar result was also reported when DNA probes corre-sponding to the 5' and 3' ends of the einmn-5 gene (their snp5notation for the gene) were used in hybridization studieswith DNA from heterologous M serotypes (114). Thesefindings corroborated earlier results of studies which usedmonoclonal antibodies with defined epitopes to identify thevariable and conserved segments of the M molecule (109).

Ribonucleic acid sequence analysis of emm gene tran-scripts primed with specific oligonucleotide probes fromemnm-6.I was used to determine the degree of homologywithin the 3' end of the emmn-5, emm-19, emmn-24, emnm-30.and emmn-55 genes (96). The results revealed that the C-terminal one-third of these genes are >95% identical toemnmn-6.1. This was later confirmed with the reported se-quence from other strains of M5 (148) and M24 (153)proteins.

Conversion of M- to M+ Phenotype

DNA hybridization studies demonstrated that M- strainT28/51/4 lacked an emm gene (183), and electron microscopyrevealed that this strain has no surface fibrils associated withthe presence of M protein (193). In addition, this strain iseasily phagocytized in normal human blood (193). With aspecially constructed shuttle vector, experiments were de-signed to move the cloned emm-6.I gene into this emim-deficient streptococcus (181). The resultant transconjugantwas converted to M6', resistant to phagocytosis in humanblood, and opsonized by anti-M6 antiserum. Immunofluo-rescence studies revealed that M6 protein was located on thecell surface of the transconjugant. When inoculated intorabbits, the transconjugant induced opsonic antibodies toM6 streptococci. In a similar set of experiments, the emnin-5gene was transformed into S. sanguis, and M5 protein wasdemonstrated on the cell surface by immunofluorescence(171). Although producing less M protein than authentic M5streptococci, these organisms removed opsonic antibodiesfrom anti-M5 serum, indicating that the antiphagocytic de-terminants are expressed on the surface of S. sanguis. Thus.the emm-6.1 and emnm-5 genes contain all information nec-essary to convert an M- streptococcus to a biologically andimmunologically MX organism, further emphasizing the roleplayed by M protein in the antiphagocytic capacity ofstreptococci.

Number of emm Gene Copies

As stated earlier, one copy of the emin gene is detected inan M6 strain (183) and in the chromosome of nine differentserotypes as determined by Southern hybridization with M6gene probes (182). This supports the hypothesis that recom-bination leading to size and antigenic variation is an intra-genic event. However, in contrast, two bands were found tohybridize with a digest of the M5 chromosomal DNA. Thisresult may be due to restriction site heterogeneity for this M5strain or the presence of two einmn-5 gene copies in the M5organisms. The latter possibility is supported by Kehoe et al.(115). who identified two copies of an emmn-5 gene in a straindifferent from the one used in the studies by Scott et al.(182). Since these were the only two reported studies of

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emmn-5 genes, it is not certain whether the presence of twoeinin genes in M5 streptococci is the rule or whether thesestrains are unusual.

Regulation of the emm Gene

Lancefield observed that streptococcal strains maintainedin the laboratory for extended periods of time (several weeksto a few months) lose their ability to survive in human blood(129). Quantitative measurements of the M-protein contentsuggest that these streptococci do not contain sufficient Mprotein for survival (24, 129). Passage of these organismsthrough mice or human blood results in the surviving strep-tococci producing higher quantities of M protein (24), sug-gesting that a regulatory mechanism may be responsible forM-protein expression.

Cleary et al. (40) found that M12 strain CS44 produced M-mutants at high frequency which correlated with a variationin colony morphology. Spanier et al. (188) isolated two M-mutants from this population, which, by Southern blotanalysis, were found to contain a small deletion in a regionupstream from the M12 structural gene promoter. In a recentanalysis of these strains and derivatives thereof, it wasdetermined that the deletion was about 500 base pairsupstream of the emm-12 gene (186). Although this charac-teristic could not be confirmed in other M serotypes, theseresults suggest that a positive regulator may be locatedupstream from the M-protein structural gene.A positive regulator for the production of M protein was

also suggested by the experiments of Scott et al. (181), inwhich a plasmid containing the emm-6.1 gene and its pro-moter was transferred to a strain lacking an emm gene (T28/51/4). Even though the plasmid was present in a high copynumber, the production of M6 protein was about 30- to50-fold less than in the parental M6 strain from which theemm-6.1 gene was derived. They suggested that the emrn-deleted strain may also be deleted for a positive regulatornecessary for wild-type expression. By using Tn9l6 muta-genesis in D471, the strain from which the emm-6.1 gene wasderived, a mutant was found which produced 50-fold less Mprotein than the parental strain. By Southern blot analysis, itwas shown that the Tn insertion was about 2 kilobasesupstream from the emm-6.1 structural gene. Northern (ribo-nucleic acid) blot analysis revealed that the mutant did notproduce any detectable emm-6.1-specific messenger ribonu-cleic acid. Thus, the authors named the gene responsible forthis effect mry, for M-protein ribonucleic acid yield.

In support of the hypothesis that the emrn-deleted strainT28/51/4 was unable to sustain full emm expression, South-ern blot hybridization revealed that this strain was in factdeleted for the may gene (35). Thus, a positive regulatorappears to be necessary for high M-protein expression.Whether environmental factors play a role in this expressionor whether, like certain positive regulators in other patho-gens (149, 206), mnry controls other virulence determinants inthe streptococcus requires further study.

HUMAN IMMUNE RESPONSE TO THE M MOLECULE

In earlier experiments designed to examine the humanimmune response to the M protein as a result of a strepto-coccal infection, it was clear that, once infected with aspecific streptococcal serotype, an individual remained pro-tected from subsequent infection by strains of that serotypebut was susceptible to other streptococcal types (see reviewby Lancefield [1291). This limited protection was based on

the development of type-specific opsonic antibodies to the Mmolecule which were found to persist years after a strepto-coccal infection (128). However, after such an infection,measurable amounts of opsonic antibodies are usually de-layed when compared with the response to other streptococ-cal antigens (124, 125, 176). In more recent studies withcompetitive inhibition assays with purified M protein, it wasdiscovered that type-specific opsonic sera contained anti-bodies directed to a majority of the immunodeterminants onthe M molecule while nonopsonic sera did not (64). Thus, itwas suggested that, for some reason, opsonic antibodies areproduced late during an immune response to the M protein.

Immunodominant Region of the M Molecule

To define further the development of an immune responseto the total M6 protein, synthetic peptides representing 82%of the native molecule were examined for reactivity withrabbit and human sera by kinetic enzyme-linked immunosor-bent assay (78). The results reveal that rabbits immunizedwith the native M protein or whole streptococci respond byreacting first and predominantly to one of the three sequencerepeat regions of the molecule, namely, the B repeat. Anti-bodies to this region have recently been shown to benonopsonic (106). Antibodies to peptides representing thevariable N-terminal and adjacent A-repeat regions appearonly when opsonic antibodies are detected in the serum.Antibodies to peptides located within the conserved C-terminal half of the molecule are limited even after severalimmunizations. By the same assay, an examination of hu-man sera revealed that those sera opsonic for M6 strepto-cocci contain antibodies reactive predominantly to the N-terminal and A-repeat regions. Nonopsonic human sera toM6 streptococci show low reactivity to most peptides. ByWestern blot analysis of the N- and C-terminal halves of theM6 molecule, all human sera tested contain antibodies to theconserved C-terminal half, while only sera opsonic for M6streptococci react with the N-terminal variable half. Theauthors suggest that antibodies to the C-terminal half areprimarily directed to conformational determinants and thusmay not react with small synthetic peptides (78).The immunodominant nature of the B-repeat region could

explain the observed delay in the appearance of opsonicantibodies in human sera after exposure to M-protein-pos-itive streptococci (124, 125, 176). It is possible that anexaggerated response to the nonopsonogenic B repeat mayin some way affect the response to the adjacent regions. Italso explains the observed presence of high levels of bindingantibodies to the M molecule in the absence of opsonicactivity. That M6 antibodies to the N-terminal and A-repeatsegments were elevated in the M6 opsonic human sera is inkeeping with the observation of Lancefield (128) that lifelongtype-specific immunity usually occurs after a streptococcalinfection.

ANTIPHAGOCYTIC ACTIVITY OF M PROTEIN

The capacity of the group A streptococcus to causedisease may be attributed partially to its ability to resistphagocytosis by human neutrophils. This capacity can easilybe demonstrated in the laboratory, using a bactericidal oropsonophagocytosis assay developed by Lancefield (127) inwhich a few log-phase M t streptococci are added to a smallamount of heparinized human blood from a donor lackingantibodies to the streptococcal serotype being tested. Thetube is sealed and rotated end over end at 370C to allow

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maximal contact between phagocytes and streptococci. Atthe end of 3 h. the contents of the tube are analyzed forsurviving streptococci. In this assay, the M+ streptococcusgrows unabated, whereas streptococci with insufficientquantities of M protein are easily cleared from the system. Inthe case of an M- organism, several thousand may be addedto this system and be phagocytized effectively, while <10streptococcal chains of an M+ strain are adequate to allowgrowth to several hundred chains at the end of the incubationperiod. The addition to the assay tubes of rabbit antiserum.prepared against either purified M protein or whole strepto-cocci of a specific serotype, neutralizes the antiphagocyticactivity of the M molecule, allowing streptococci of thatsame serotype to be phagocytized. Antisera prepared toother streptococcal serotypes which cross-react with the Mprotein of the organisms in the assay have no opsonic effecton these streptococci.A different assay has been used by Beachey and co-

workers (21) in which streptococci are mixed with humanblood much the same as described in the Lancefield assay(127); however, a higher bacterial inoculum is used. After3-h rotation, a smear is made of the blood and the phago-cytes are observed under the microscope and scored fortheir association with streptococci. This assay does notdistinguish between live attached organisms and ingestedkilled streptococci. In many cases, however, good correla-tion is seen with the Lancefield method (127); nevertheless,a different parameter is likely being measured by this assay(i.e., at the level of attachment and ingestion).

N-Terminal Charge Domain

The mechanism by which the M molecule exerts itsantiphagocytic effect has been elusive since its discoverynearly 60 years ago. Despite sequence variation within theN-terminal hypervariable region of the M molecules, acommon feature found in the sequence of M5 (139), M6 (77),M24 (153), and M49 (116) is a concentration of acidic aminoacids within this segment. These residues occur predomi-nantly in external positions within the coiled-coil structure(77). Some of these amino acids likely enter into salt bridgeformation between the two M-protein chains and function tostabilize the coiled-coil (165), while others become hydratedand contribute to the net negative charge in this region.While the exact function of this conserved charge domain isnot known, it has been suggested that it may contribute tothe antiphagocytic property of the molecule (66, 136, 139) byhampering contact between the streptococcus and the phago-cyte by electrostatic repulsion. In support of this idea, it wasfound that, when opsonic IgG was fractionated into acidicand basic components, the opsonic activity was localized inthe IgG population that was most basic in charge whilenonopsonic binding antibodies were located in the othercharged IgG populations (66). This suggests that the basicantibodies bind to the more acidic regions of the M molecule(i.e., the N-terminal region) and thereby neutralize thenegative charge.

Effect on Phagocytic Cells

In attempts to determine whether the M protein on thewhole streptococcus impairs the phagocytic capacity ofneutrophils, M streptococci were rendered nonviable withmitomycin and used at 150-fold excess over phagocytes in abactericidal assay containing a small number of viable phago-cytosis-susceptible streptococci (140). The results revealed

that the phagocytes could effectively seek out and destroythe relatively few susceptible streptococci in the presence ofthe excess M' cells. Thus, the nonviable M+ streptococcidid not interfere with the phagocytic activity of the neutro-phils. When these experiments were repeated with livestreptomycin-susceptible M + and streptomycin-resistantM- streptococci, to select for M- survivors on streptomycinplates, similar results were obtained (Fischetti, unpublisheddata). This helped to rule out the possible effects of mitomy-cin or nonviable organisms in the previous experiments.Hence, in the presence of M ' streptococci, the phagocytesare not preoccupied by attempting to phagocytize the M+cells: they simply ignore them.

Complement

The alternative complement pathway is considered to bean important part of the nonspecific defense system againstinfection (155). The deposition of a large number of comple-ment component C3b fragments on the surface of microbialparticles, termed opsonization, results in the uptake of theseparticles by phagocytic cells bearing C3b receptors. Severallines of evidence have indicated that M protein exerts itsantiphagocytic effect by interfering with the alternativecomplement pathway. It has been shown that M- strepto-cocci are quickly phagocytized after being opsonized via thealternative pathway while M+ streptococci resist phagocy-tosis in the absence of type-specific antibodies (29, 168).That M streptococci fail to bind effectively to phagocyticcells suggests that C3b was either insufficiently deposited onthe streptococcal surface or inacessible to the C3b receptorsof the phagocyte. When Jacks-Weis et al. (103) quantitatedthe amount of C3 deposited on the streptococcal surface,they found that M- organisms bound up to four times moreC3 than M+ cells. They concluded that, while reduced, theamount of C3 deposited on the M+ cell should be sufficientfor phagocytosis and, thus, could not be the sole reason forthe antiphagocytic effect of the M molecule.

Factor H

The deposition of C3b on the surface of particles isamplified by particle-bound C3 convertase, also termedC3b,Bb, which is a labile enzyme complex that activates C3(154). Specific inhibitors such as decay-accelerating factorand factor H prevent C3 activation by rendering C3b sus-ceptible to inactivation by factor I. Factor H is a 150-kDa,3-globulin present in serum (500 jg/ml) and functions tocontrol fluid-phase as well as surface-bound C3b (205).

While the studies of Jacks-Weis et al. (103) demonstratethat M' streptococci bind less C3b than M- cells, thedeposition on M' cells was found to be uneven comparedwith the more uniform distribution of C3b on M- organisms.Horstmann et al. (98), using purified complement compo-nents instead of serum, did not find any difference in eitherthe deposition or the inactivation of C3b or the dissociationof the C3b,Bb complex on the surface of M+ and M-streptococci when complement regulatory proteins wereomitted. In fact, both strains exhibited a spontaneous decayof the C3b,Bb complex similar to zymosan-bound C3b,Bbwhich served as a control. However, when purified factor Hwas added to streptococcus-bound C3b, the cofactor activityof factor H for the cleavage of C3b by factor I was six toeight times higher when C3b was bound to M+ streptococciinstead of M- organisms. M+ streptococci were found tobind factor H selectively. Furthermore, M+ streptococci of

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five different serotypes, as well as purified M protein, werefound to bind factor H with dissociation constants as high as6 x 10' M, emphasizing the functional significance of thisbinding reaction.

It was proposed that the antiphagocytic action of the Mprotein may be explained by this finding. When a strepto-coccus contacts serum, the M protein specifically bindsfactor H to its surface. Any C3b fixed in the vicinity of thiscomplex is controlled by factor H, which inhibits or reversesthe formation of C3b,Bb complexes and serves as a cofactorin the conversion of C3b to iC3b by factor I. Thus, the lownumbers of C3b molecules deposited on the M streptococ-cus will prevent C3b-dependent phagocytosis. The regula-tion of complement deposition by binding factor H to abacterial surface appears to be a novel route by which apathogen is able to evade alternative pathway activation andphagocytic clearance.

FibrinogenKantor (110) found that a component in partially purified

acid extracts of group A streptococci precipitated fibrinogen.In a number of studies, the active component was subse-quently found to be M protein (101, 110, 152, 207, 209).Similar binding characteristics for fibrinogen were found forother streptococcal groups (C and G), suggesting that M-protein analogs are also present on these organisms (122). Inattempts to examine the role of this interaction with regardto the antiphagocytic action of the M protein, Whitnack andBeachey (207) measured the association of streptococci withpolymorphonuclear leukocytes in the presence of serum andplasma. In this study, bound and ingested streptococci werenot differentiated. Their results indicate that fibrinogenblocks the association of streptococci with the polymorpho-nuclear leukocytes. Immunofluorescence analysis with anti-C3 revealed that streptococci pretreated with fresh serumbound this complement component evenly on their surfaceswhereas streptococci treated with plasma exhibited limitedcomplement deposition. The authors suggested that therestricted deposition of C3 prevents complement-mediatedopsonization of these organisms. This finding differed fromthat of Jacks-Weis et al. (103), who detected an unevendeposition of C3 on streptococci pretreated with nonimmuneserum. Chhatwal et al. (39) also demonstrated, by immuno-fluorescence, that fibrinogen inhibited C3 fixation on thesurface of group A, B, C, and G streptococci.Subsequent experiments by Whitnack et al. (210) were

designed to examine the influence of fibrinogen on opsoniza-tion of streptococci by type-specific and cross-reactive anti-sera. Rabbit antisera against PepM protein of the homolo-gous type overcame the antiopsonic effect of the fibrinogen.Using cross-reactive sera, these authors found that, in thetwo M proteins (M5 and M6) studied in detail, antibodiesinhibiting fibrinogen binding to M protein accounted for alarge part of the cross-reacting anti-M antibody in the sera.In all, these studies indicate that fibrinogen binds to bothtype-specific and cross-reactive regions of the M molecule.To explain the common antiphagocytic function of M pro-teins from different serotypes, it was suggested that thefibrinogen binding site may be conserved among differentserotypes. That the CR3 receptor on human polymorphonu-clear leukocytes binds both complement C3b and fibrinogen(218) may explain why fibrinogen-coated streptococci (208)adhere to but are not ingested by these phagocytic cells.Thus, since the assay used in many of these studies mea-sures phagocyte association, the role of fibrinogen in strep-tococcal clearance is not completely defined.

R. D. Hortsmann and V. A. Fischetti (manuscript inpreparation) examined the effect of fibrinogen-bound Mprotein on factor H binding and on the regulation of C3b onthe streptococcal surface. Results revealed that, while thepresence of fibrinogen on the streptococcal surface slightlyreduced the binding of factor H to M protein, it did notinterfere with complement inhibition. They found that C3b isequally well regulated by factor H when it is bound tofibrinogen. Thus, factor H reduces the deposition of C3b onthe streptococcal surface with or without fibrinogen boundto the M protein.

ROLE OF M PROTEIN IN STREPTOCOCCALADHERENCE

Group A streptococci colonize the nasopharyngeal mu-cosa of humans, leading to an inflammatory response fol-lowed by clinical illness or an asymptomatic carrier state. Itwas first shown by Ellen and Gibbons (60) that streptococcibearing the M-protein molecule on their surface can adherebetter to epithelial cells in vitro than M-deficient organisms.There is also strong evidence that LTA secreted from thestreptococcus mediates direct contact with the epithelial cellsurface. M protein or another surface molecule might facil-itate this process by anchoring and orienting the LTAadhesin so that the lipid moiety is able to make contact withthe epithelial cell (20). Studies also suggest that M-positivestreptococci can attach to epithelial cells by a mechanismindependent of LTA. When M+ and M- streptococci aretested for their binding capacity to human pharyngeal,buccal, and tongue epithelial cells, the M ' bacteria attach insignficantly higher numbers to the pharyngeal than to thebuccal or tongue cells (197). The M- organisms bind inequivalent low numbers to all three oral cell types. WhileM I streptococci could be inhibited from binding to pharyn-geal cells by using the complete M6 protein molecule as wellas LTA, 1,000-fold more LTA than M protein is required toattain equivalent inhibition.M protein binds specifically to F actin, forming regular

parallel bundles of actin filaments in solution (36). In addi-tion to isolated M protein, whole M' streptococci also bindspecifically to F-actin filaments. While M protein does notcompete with tropomyosin for binding to actin, it doescompete with myosin subfragment 1 as a result of thisinteraction. While the biological implication of this binding isunclear at this time, it is becoming more apparent that actinis present on the surface of a number of cell types such asfibroblasts (184), lymphocytes (160), and human B cells (5).Therefore, the interaction of streptococcal M protein withsurface actin on a host cell may be a possible event. Thus,while there may be multiple mechanisms by which strepto-cocci can attach to epithelial cells, it seems apparent that Mprotein provides the organism with an adherence advantage;consequently, it is considered to be an attachment factor.

M-PROTEIN VACCINES

Type-Specific Protection

Since the observation by Lancefield that antibodies to theM protein have the capacity to opsonize streptococci inpreparation for phagocytic clearance (126), the M proteinhas been a prime candidate for a vaccine to prevent group Astreptococcal infections. Fox has reviewed the trials andtribulations of the early attempts in M-protein vaccine de-velopment (81). Since the early 1970s, few human trials have

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been attempted (recently reviewed [D. Bessen and V. A.Fischetti, in M. M. Levine and G. Wood, ed., The MolecularApproach to Newt and Improved Vaccines, in press]). This ispartially based on previous problems with hypersensitivityreactions found with the acid-extracted M-protein prepara-tions (81); essentially, only type-specific protection wasobserved. In addition, attempts to separate heterologousprotein contaminants from the type-specific determinantswere disappointing. Except for one investigation (22), allvaccine development since the 1970s has been based onanimal studies and on an analysis of the immune response toa variety of M-protein preparations with and without adju-vants (10, 26, 27, 37, 78, 104, 216) and in combination withother antigens (37).

In 1979, Beachey et al. (22) used pepsin-extracted M24protein (the N-terminal half of the native M molecule) toimmunize human volunteers. This highly purified prepara-tion was found to be free of non-type-specific reactivity.Unlike earlier acid-extracted products (81), skin delayed-type hypersensitivity tests in 37 adults proved to be negativewith this PepM protein. Immunization with alum-precipi-tated PepM24 protein led to the development of type-specificopsonic antibodies in 10 of 12 volunteers and the formationof positive delayed cutaneous hypersensitivity to PepM24 in11 individuals. None of the volunteers developed heart-reactive antibodies as determined by immunofluorescence.These studies clearly indicated that M-protein vaccines freeof sensitizing antigens could be produced, but also illustratedthe type specificity of the immune response.Encouraged by these studies, Beachey and co-workers

embarked on a systematic approach to develop methods bywhich a type-specific epitope-based vaccine could be devel-oped to protect against streptococcal disease. It was quicklylearned that peptides representing the first 20 or so N-terminal amino acids of M24 protein evoked type-specificopsonic antibodies to M24 streptococci (17). Experimentswith synthetic peptides of the N terminus of MI, MS, M6,and M19 proteins resulted in the same conclusions (12, 15,23, 34, 107, 179). When a hybrid peptide was synthesizedfrom the sequence of the M24 and MS proteins and injectedinto animals in complete Freund adjuvant, opsonic antibod-ies were produced to both MS and M24 streptococci (12).Opsonic antibodies to three M proteins were obtained whenthe N-terminal sequences of three M-protein sequences(MS-M6-M24) were synthesized in tandem and injected intorabbits (16).

Attenuated Salmonella typhimarium has also been usedeffectively for delivering the M-protein antigen in a mousemodel (174). The cloned gene for the complete MS proteinwas introduced into the aroA strain of S. tvphimarium bytransformation with a plasmid vector. The transformedsalmonella expressed the MS protein in the cytoplasm andhad the capacity to induce serum and salivary IgA, IgG, andIgM to MS protein when fed orally to BALB/c mice. Theorally immunized mice were protected from challenge by MSbut not heterologous M24 streptococci.An important factor to consider for the development of a

type-specific epitope-based vaccine is the potential of thestreptococcus to generate new M serotypes by changing theamino-terminal portion of M protein. In the type 6 M-proteinmolecule of strain D471, type-specific opsonizing antibodiesare directed against epitopes located both at the amino-terminal end (residues 1 through 21) and within the A-repeatblock (106), which begins at amino acid 27 and continues toresidue 96 (77, 94). As mentioned previously, high-frequencyintragenic recombinational events within the A-repeat block

region can lead to a significant loss in the opsonizing abilityof monospecific antibody (107). However, to date littleinformation is available on mechanisms for generating anti-genic variation at the extreme amino-terminal nonrepeatregion. A second major concern for type-specific epitope-based vaccines centers on the antigenic diversity of the morethan 80 serological types of M protein. Certain serotypes aremore likely than others to be associated with severe cases ofpharyngitis or outbreaks of rheumatic fever; thus, the num-ber of distinct type-specific determinants incorporated into avaccine can be limited to some extent (28). A type-specificstreptococcal vaccine against upper respiratory infectionand acute rheumatic fever would require a multivalentantigen corresponding to stable immunodeterminants ofthose serotypes which together account for the majority ofnasopharyngeal isolates.A clear understanding of the molecular basis of antigenic

variation within the hypervariable nonrepeating segment ofthe M protein should enable investigators to make decisionson how best to circumvent the evasive mechanisms used bygroup A streptococci.

Non-Type-Specific Protection

The incidence of group A streptococcal respiratory infec-tion rises sharply at age 4, peaks at age 6, and rapidlydeclines above age 10, reaching adult levels by 18 years (32).At its peak incidence, 50% of children between the ages of 5and 7 suffer from streptococcal infection each year. Thedecreased incidence of streptococcal pharyngitis in adultsmight be explained by an age-related host factor. Alterna-tively, protective antibodies directed to antigens common toall group A streptococcal serotypes might arise as a conse-quence of multiple infections experienced during childhood,resulting in an elevated response to conserved protectiveepitopes of the M molecule. In exploring the second hypoth-esis, it was found that sera of most adults examined have astrong antibody response to the conserved regions of M6protein (27a, 78). While antibodies directed to conservedepitopes fail to neutralize the antiphagocytic property of Mprotein (106), they might afford protection by an alternativemechanism.

Kurl et al. (123) found that whole-cell vaccines consistingof type 12, 18, 30, 49, 50, or 55 streptococci were equally asefficient at protecting mice immunized intranasally fromlethal infection after challenge with M50 streptococci. Theauthors suggest that local immunity against M50 strepto-cocci could be achieved by a nonspecific mechanism; how-ever, the molecular components of the streptococcus in-volved in eliciting this protection were not defined.

It should be emphasized at this juncture that humans arethe only reservoir for group A streptococci, and except forM type 50, which is naturally virulent for mice (97), all otherserotypes need to be adapted to the animal being consideredas a possible model for streptococcal disease. For instance,group A streptococci isolated fresh from human infection arenot virulent for mice, and nearly 107 CFU need to beadministered intraperitoneally to cause death within 1 to 2days (129). However, after such a passage in the mouse(usually several passages are required), a strain is isolatedwhich will cause death with a dose of <10 CFU. For somegroup A strains, a mouse-virulent organism may not beisolated. Although the amount of M protein increases aftermouse passage, its role in mouse virulence is not completelydefined (93). However, it is clear that immunization with Mprotein or passively administered type-specific M-protein

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VO 3- rP 0u0

30-(N)

25- *

0

° 20-

X 15-

10-

-

FIG. 16. Phachallenged withimmunized intra

synthetic pepticof the M6 mole(cultures were ta5 x 105 streptculture-positiveseparate experisame number ol4, with a totalin colonized anm6, 8, 9/10, and:

antibodies willenge with theThe C-repe

among strept(28, 106, 108,:minants localito the surfacethan half of thfindings, Mill'served M-prothe pepsin ckand, thus, artbased on antisequence is loC terminal tothe peptide us

ogous to the s

antibodies toported by thimolecule witlin the M mcbecome react

In view ofexposed cons

CTB Only (n=52) 106, 108, 109), it should be possible to generate antibodiesPeptide-CTB (nm52) reactive to the majority of serological types by using only a

1.05 * * * few distinct antigens for immunization. If a conserved-regionvaccine is to be effective in preventing nasopharyngealinfection by group A streptococci, it may require stimulation

s * of the secretary immune response.Secretory IgA is a major first-line defense against bacterial

infections of the mucosal epithelium (118), and it has been$ 9 9 6 ~~~demonstrated that M-protein-specific secretary IgA can pro-

vide significant protection against streptococcal infection ina mouse model (26, 27a, 67). Passive immunization at themucosa allowed for a more precise evaluation of the roleplayed by secretary IgA in preventing streptococcal infec-

1 2 4 6 8 9/10 14/15 human anti-M-protein secretary IgA administered intrana-

Days Post-Challenge sally protected mice against systemic infection after intrana-

sal challenge with group A streptococci. In contrast, anti-aryngeal colonization of peptide-immunized mice M-protein-specific IgG administered intranasally had no

l group A streptococci. Mice (52 per group) were protective effect at this site, although it was highly opsonic

anasally with either cholera toxin B-subunit (CTB) or and promoted streptococcal phagocytosis in whole blood.

les derived from sequences in the conserved region.lcope to chlr oi -uui. Phrnga An oral or intranasal route of immunization has been used

cu.le coupled to cholera toxin Bsubunit. Pharyngeal

iken up to 15 days following intranasal challenge with for several group A streptococcal vaccines, with the intent of

tococci. Results are expressed as the number of evoking a strong secretary IgA response (27, 51, 123, 170,plus dead mice at each time point from three 172). To determine whether the conserved exposed epitopes

iments. Although colonization differed, nearly the ofM protein influence the course of mucosal colonization byf mice died each day in each group beginning on day group A streptococci, peptides corresponding to these re-

f 15 dead per group by day 15. Statistical difference gions were used as immunogens in a mouse model (27).d dead mice was significant (*P < 0.05) on days 1, 2, Synthetic peptides corresponding to residues 216 to 235, 248

14/15 by chi-square analysis. to 269, and 275 to 284 of the M6 sequence (Fig. 7) were

covalently linked to the mucosal adjuvant cholera toxin B11 protect mice from lethal streptococcal chal- subunit and administered intranasally to the mice. About 1homologous serotype (59). month later, animals were challenged intranasally with live

at region of MG protein is highly conserved streptococci and pharyngeal colonization was monitored forcoccii of many distinct serological types (27a, 15 days. It was found that mice immunized with the cholera109). Monoclonal antibodies directed to deter- toxin B subunit-peptide complex showed a significant reduc-ized to the C-repeat region of M6 protein bind tion in colonization compared with mice receiving choleraof whole streptococci which represent more toxin B subunit alone (Fig. 16). Thus, despite the fact that

e serotypes examined (108, 109). Despite these conserved-region peptides are unable to evoke an opsonic

er et al. (147) suggest that antibodies to con- antibody response (106), these peptides have the capacity tostein epitopes located on the C-terminal side of induce antibodies capable of influencing the colonization ofsavage site do not bind to intact streptococci group A streptococci at the nasopharyngeal mucosa in thise of no value in a vaccine. Their findings are model system.bodies prepared to a synthetic peptide whose The successful cloning of the emm-6.1 gene into vaccinia)cated within the conserved region immediately virus and its high expression in mammalian cells infectedthe pepsin site. Perhaps the conformation of with the recombinant vaccinia virus may prove to be a

ied to raise antibodies is not sufficiently homol- powerful vector for delivery of the M molecule to mucosal;ame region in the native molecule to enable the surfaces (100). In an effort to express only the conserved-react with the intact molecule. This is sup- region epitopes located in the C-terminal half of the mole-

eir finding that, after cleavage of the native cule, genetic engineering methods were used to truncate theh pepsin (resulting in a conformational change N-terminal half of the M6 gene and recombine the C-terminal)lecule [1071), the peptide-specific antibodies gene fragment into the vaccinia genome (VV:M6'). Western

ive. blot analysis revealed that the cells infected with theoverwhelming evidence for the presence of VV:M6' recombinant produce a molecule of about 30 kDa

;erved epitopes on the native M molecule (28, that is reactive with a monoclonal antibody specific for the

TABLE 2. Throat culture results of mice vaccinated intranasally with either wild-type or recombinant vaccinia virus

No. culture positive"/total no. challenged (%. colonized or dead) at given days postchallenge"Vaccinia virus vaccine

1 2 3 6 8 10

Wild type 19/25 (76) 18/25 (72) 19/25 (76) 18/25 (72) 21/25 (84) 19/25 (76)

Recombinant (VV:M6')" 3/28 (11) 9/28 (32) 7/28 (25) 8/28 (29) 8/28 (29) 8/28 (29)

Animals culture positive for group A streptococci or dead after streptococcal challenge.' Significant difference (P < 0.005) at all time points between wild-type and recombinant virus-vaccinated mice by chi-square analysis.Recombinant vaccinia virus contains the gene for the C-terminal half (conserved region) of the M6 molecule.

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C-terminal half of the M protein (V. A. Fischetti, W. M.Hodges, and D. E. Hruby, Science, in press). Mice wereimmunized intranasally with either the VV:M6' recombinantor wild-type vaccinia virus and challenged intranasally andorally 1 month later with 5 x 106 streptococci. Pharyngealcultures taken up to 10 days postchallenge revealed that theVV:M6'-immunized animals differed significantly from con-trols. Of the VV:M6'-immunized animals, only 16% of 152total swabs taken were streptococcus positive, with 10 (6%)yielding >100 CFU, whereas 69% of 115 swabs were posi-tive in the wild-type group and 40 (35%) displayed >100CFU. On average, >70% of the animals immunized withwild-type virus were culture positive for group A strepto-cocci at every swab day up to 10 days after challenge (Table2). This is compared with <30% colonization of mice immu-nized with the VV:M6' recombinant. These results confirmand extend the previous studies that used synthetic peptidesfrom conserved regions of the M molecule to protect againststreptococcal colonization (27).The achievement of non-type-specific protection in animal

models (27, 123, 216) and the evidence that protection can bemediated by nonopsonic antibody (26) hold promise for analternative to type-specific M-protein vaccines. The greatestchallenge will lie in identifying those common peptideswhich elicit the broadest protection without stimulatingantibodies cross-reactive with host tissue.

CONCLUDING REMARKS

The group A streptococcus utilizes a coiled-coil moleculardesign for the M protein, a pattern commonly found ineucaryotes and viruses (41, 42) which has not been thor-oughly explored in bacteria. This type of structure offers anumber of distinct advantages to the organism. For instance,the rodlike coiled-coil structure allows functional domains tobe located sufficiently away from the difficulties inherent inmicrobial surfaces which in many cases actively stimulatethe alternative complement pathway. Because the coiled-coil design may tolerate a large number of sequence changes,yet maintain a common conformation, the molecule has thecapacity to change its immunodeterminants readily, allowingthe organism to avoid or delay immune recognition. Further-more, the rodlike motif coupled with the plasticity of thesequence allows this type of structure to be tailored forseveral specific functions. This is accomplished by dividingthe molecule into discrete functional domains directed byimmunological and colonization pressures. Because of thesewide-ranging characteristics, the coiled-coil pattern is likelyto be found as commonly in bacteria as it is in eucaryotes.

ACKNOWLEDGMENTS

For their help in many of the studies on the structure, function.and genetics of the M molecule, I acknowledge all of my collabora-tors over the years. Discussions with Maclyn McCarty. JohnZabriskie, Emil Gotschlich, and the late Rebecca Lancefield havehelped shape my current understanding of the group A streptococ-cus. Their time and patience are greatly appreciated. I thank DebraBessen, Emil Gotschlich. Kevin Jones. Belur Manjula, VijaykumarPancholi, Olaf Schneewind. and June Scott for critical review andsuggestions for the manuscript.This work was supported by Public Health Service grant A111822

from the National Institutes of Health.

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