Group A Streptococcus (GAS) is one of the most prevalent bacterial pathogens causing human disease worldwide, ranging from the fairly mild to highly severe. Organisms of this species are remarkable for the extensive diversity in their spectrum of virulence factors and surface antigens. Importantly, those variant features can be used to organize GAS strains into subgroups that, in turn, correlate with distinct clinical features.
The primary ecological niche of GAS is the superficial epithelium of the oropharynx or skin. It is at these tissues that GAS can cause mild infections (>700 million/year) or persist in an asymptomatic carrier state (at the throat) [1,2]. Impetigo predominates in tropical and subtropical regions (transmission via skin contact), whereas pharyngitis spikes when the weather drives people indoors (transmission via respiratory droplets) and thus, geospatial distances arise because of environmental factors that favor one mode of transmission over the other. It has been long recognized that there exist discrete ‘throat strains’ and ‘skin strains’ and in more recent years the molecular basis for this distinction has been delineated to some extent.
The molecular underpinnings for the tendency of a given GAS strain to preferentially infect either the throat or skin is not completely understood. However, recent discoveries advance our knowledge of the molecular markers and virulence factors which define the clinically distinct subgroups of GAS strains causing pharyngitis vs. impetigo.
MOLECULAR CORRELATES OF GROUP A STREPTOCOCCUS DISEASE
Classical throat and skin strains  were recognized as such based on their M protein serotype, defined by the N-terminal amino acid sequence at the tips of the M protein cell surface fibril . Some 20 years ago a nucleotide-based typing scheme arose based on the extreme 5’ end of the emm gene, with more than 230 emm types characterized to date [5,6]. Around that same time, three major emm pattern groups were defined based on sequence differences in the more highly conserved 3’ end of emm and flanking emm-like genes (i.e., mrp and enn), encoding the cell wall-spanning domain of M and M-like proteins (Fig. 1a) [7–9,10▪]. emm type is highly predictive of emm pattern group based on the vast majority (97%) of GAS strains measured for both features  and thus, reasonably good inferences can be made for emm pattern based solely on knowledge of emm type.
The emm pattern group – known as emm patterns A–C, D, and E – displays highly significant associations with pharyngitis and impetigo [10▪,13,14]. A meta-analysis of 23 pharyngitis and six impetigo population-based surveys conducted throughout the world [10▪,11] shows that pattern A–C strains represent ∼47% of pharyngitis isolates but only 8% of impetigo isolates (Fig. 1b); emm pattern A–C strains represent the classical throat strains and are designated ‘throat specialists’. In sharp contrast, pattern D strains represent ∼50% of impetigo isolates, but only ∼2% of pharyngitis isolates; they are classical skin strains and designated ‘skin specialists.’ In contrast, emm pattern E isolates account for almost equal fractions of throat and skin infections and as a group, they are designated ‘generalists.’
The M proteins discussed in this review belong to emm pattern groups A–C (M1, M3, M12, M18) and E (M89). A new molecular typing scheme for GAS developed in late 2014 is based on the phylogeny of the sequence of the entire surface-exposed portion of mature M protein, which contains the determinants of M serotype plus functional binding domains. The scheme is known as ‘emm cluster’ typing whereby 97% of emm types fall into a major clade (X or Y), and 82% of emm types are assigned to one of 16 well supported sequence clusters [12▪▪]. Since its introduction, emm cluster analysis has been widely adopted for molecular epidemiology studies [15–20].
The emm cluster typing scheme can be applied to the emm typing and emm pattern data for the 29 population-based surveillance studies on GAS pharyngitis and impetigo infections, as reported in [10▪,11]. Figure 1c shows that more than 99% of emm pattern A–C isolates (so-called throat specialists) have an emm type belonging to clade Y, largely within clusters AC1–5. Similarly, the vast majority of emm pattern D isolates (so-called skin specialists) also belong to clade Y, but largely in emm clusters D1–5, with D4 being most dominant. In contrast, the emm pattern E isolates (generalists) are restricted to clade X, although cluster E6 contains ∼14% of the total pattern D isolates. The fractional ratio of pharyngitis to impetigo isolates is highly skewed for clusters AC1–5 (high) and D1–5 (low) (Fig. 1d). Overall, the emm cluster scheme, based on the entire surface-exposed portion of M protein, is highly consistent with the emm pattern scheme that is based on the C-terminal peptidoglycan-spanning domain of emm and emm-like flanking genes.
MULTIFUNCTIONAL M PROTEIN
The strong correlations between emm genotypes and clinical phenotypes provide a molecular handle for elucidating the basis of tissue site preferences for infection at either the throat or skin. Several functional domains within M proteins, and their predicted binding motifs, strongly correlate with emm clusters, including plasminogen-binding in emm cluster D4 and fibrinogen-binding in most A–C clusters within clade Y [12▪▪].
The differential binding activities for human host proteins are potential determinants of tissue tropism. In particular, the plasminogen-binding M protein of emm cluster D4 (known as PAM) is associated with a very strong preference for impetigo over pharyngitis (Fig. 1e). Many recent studies have enriched our knowledge of the molecular specificity of PAM and its interaction with streptokinase [21–25]. Despite advances on M protein-based GAS–host interactions, precisely how differential modulation of the coagulation–fibrinolysis pathways  may translate into bacterial tissue tropisms remains elusive.
FCT-REGION PRODUCTS AND ADHERENCE
Ten years ago the discovery was made that T-antigens, which form the basis for a second major serotyping scheme for GAS, are comprised of thin, elongated pili . This finding had a profound impact on our recent understanding of GAS epidemiology and pathogenesis [28–40]. The fibronectin- and collagen-binding proteins and T-antigen loci, known as the FCT-region, harbors the T-antigen genes and lies ∼300 kb from emm on the GAS chromosome. It encodes pilus proteins and additional adhesins and has at least nine major forms [41–44]. There are significant correlations between several genes within the FCT-region and emm pattern and/or emm type [10▪,45–48] (Fig. 2a–c).
A signature feature of GAS pili is that the backbone and accessory pilin proteins spontaneously form covalent intramolecular isopeptide bonds promoting mechanical stabilization to counteract the shearing forces that occur during bacterial adherence to the host epithelium [49–55]. A more recent, major advance lies in the finding that internal thioester bond formation leads to covalent linkage of GAS adhesins to host ligands, whereby GAS adhesins act as ‘chemical harpoons’ [53,56,57▪,58▪▪,59]. Motifs for thioester domains (TEDs) are present in the pilin tip adhesin (Cpa) and adhesins encoded by other FCT-region genes (PrtF1/SfbI and the FbaB form of PrtF2) (Fig. 2a).
The highly reactive thioester bonds of PrtF1/SfbI and PrtF2/FbaB (but not Cpa) bind human fibrinogen present in purified form and plasma, and fibrinogen-associated with epithelial cell surfaces mimicking an inflammatory state [58▪▪]. Furthermore, thioester binding to fibrinogen involves only a single Lys residue, highlighting the specificity of the binding reaction. If indeed GAS pili and other FCT-region products are determinants of tissue tropism, as supported by strong epidemiological correlates, how might TEDs confer tissue-specific binding? Do there exist other physiologically relevant host ligands for GAS-mediated thioester bond formation, in addition to fibrinogen?
Sequence heterogeneity is particularly high among pilin backbone proteins (e.g., FctA), pilin tip proteins (e.g., Cpa) and the N-terminal half of PrtF1/SfbI [44,45,60]. Some structural forms of Cpa have two distinct TEDs, whereas other Cpa forms are devoid of putative TEDs [56,57▪]. Is thioester bond formation and host target ligand specificity influenced by the adjacent polypeptide sequence and conformation of FCT proteins? Insofar as the fibrinogen ligand, fibrinogen binding by M1 protein blocks phagocytosis of GAS by neutrophils, but also inhibits GAS adherence to epithelial cells [61▪]. Do M1 protein–fibrinogen interactions counteract processes mediated by TEDs and FCT-region proteins? The PrtF1/SfbI and PrtF2/FbaB proteins that bind fibrinogen via TEDs in the N-terminal region are multifunctional and also act as adhesins by binding fibronectin through C-terminal repeat domains [58▪▪]. PrtF1 and PrtF2 promote cellular invasion by GAS into epithelial and/or endothelial cells, whereby the mechanism of cellular uptake is dictated by the specific PrtF1/PrtF2 molecular form [62,63]. Differential transcription and posttranslational modifications of FCT-region proteins, often influenced by the microenvironment of the infection site within the host, likely add further levels of complexity to the possible mechanisms by which thioester bond formation might confer host tissue specificity for throat (pharyngitis) vs. skin (impetigo) infections caused by GAS.
NEW INSIGHTS ON GROUP A STREPTOCOCCUS PATHOGENESIS AT THE UPPER RESPIRATORY TRACT
One of the challenges to understanding the pathogenesis of GAS on a molecular level derives from the fact that GAS is strictly a human pathogen. Mice engrafted with human skin provide a highly valid model for impetigo, whereby low physiological inoculum doses of GAS yield histopathological alterations that capture many of the key features of human disease (reviewed in ). In contrast, animal models for GAS infection at the upper respiratory tract (URT) have, historically, had many shortcomings, partly because of the very high inoculum doses of GAS required.
GAS can cause acute pharyngitis, but far more often GAS persists at the URT in a semidormant carrier state wherein the human host has no disease symptoms and transmission rates are probably quite low. The strong association between emm genotypes and the throat (Fig. 1) is limited to pharyngitis, and does not include asymptomatic carriage. One major challenge has been to experimentally distinguish between these two forms of GAS–host interactions (carriage vs. pharyngitis) at the URT.
M3 GAS recovered from the URT of humans lacking signs of infection underwent whole genome sequencing in an effort to identify nucleotide polymorphisms that are unique to carriage strains, via comparison to invasive disease isolates [32–35]. Phenotypes favorable to carriage (as measured by adherence and/or invasion of epithelial cells), and less favorable for invasive disease (e.g., survival in blood, soft-tissue infection), include decreased hyaluronic acid capsule production, decreased transcription of the gene encoding Mga (an activator of emm gene transcription), increased cell surface expression of the collagen-like protein SclA, and pleiotropic effects resulting from an amino acid change in the sensor kinase LiaS [32–35]. However, nasopharyngeal colonization by GAS in the mouse does not strictly parallel these particular carriage vs. invasive genotypes [65–67], perhaps indicative of complex relationships involving multiple factors.
It appears that a high bacterial burden during nasopharyngeal GAS colonization of mice better represents pharyngitis as opposed to carriage. However in humans, acute pharyngitis is often accompanied by a purulent exudate that is typically not observed in mice. Also in humans, a specific immune response acts to clear a GAS URT infection by ∼7–10 days, whereas asymptomatic carriage can linger for weeks or even months [2,68,69]. The duration of GAS colonization in mice can extend well beyond what is typical for acute pharyngitis in humans and it is not known whether long-term mouse colonization is a better mimic of pharyngitis or throat carriage in this particular regard.
Consistent with the findings of a classical study , several recent reports show that hyaluronic acid capsule production enhances nasopharyngeal colonization in the mouse. Truncation of the multifunctional regulator RocA, which modulates the CovRS two-component system, leads to hyperencapsulation of GAS of several M-types [71–73,74▪]. In M18 strains, truncated rocA leads to increased longevity of nasopharyngeal carriage of GAS in mice and enhanced GAS transmission by a respiratory route, as evidenced by bacterial shedding . Similarly, the truncated RocA gene confers hyperencapsulation in M3 strains [74▪,75,76]; its effect on GAS behavior in URT models has not yet been reported. The hyaluronic acid capsule facilitates GAS adherence to the oropharyngeal epithelium by binding human CD44 . Recent mouse studies show that capsular hyaluronic acid is also essential for GAS dissemination from the URT to locally draining lymph nodes, via interaction with lymphatic vessel endothelial receptor-1 [78▪▪]. The authors speculate that hyperencapsulated M18 GAS get trapped in the lymphoid tissue, thereby limiting GAS spread to the bloodstream and perhaps offering an explanation for why M18 strains are rarely recovered from invasive disease but are often associated with acute rheumatic fever.
A recently emerged M89 clade of GAS associated with a high incidence of invasive disease lacks hyaluronic acid capsule but displays enhanced production of the exotoxins nicotinamide adenine dinucleotide (NAD)-glycohydrolase (NGA, which is also known as SPN) and streptolysin O (SLO) [79▪,80▪▪]. The nga-slo locus of the new M89 clade is most similar in sequence to that of M12 and modern M1 strains, suggesting acquisition from a recent common ancestor [79▪,80▪▪,81▪▪,82]. In a mouse model for soft-tissue infection, the capsule is dispensable if NGA/SPN and SLO toxin levels are high [80▪▪]. The precise evolutionary history of the epidemic M89 clade remains somewhat controversial for European vs. North American clones [83–85]. The modern M1 strain, which is frequently recovered from cases of pharyngitis , yields a higher bacterial burden and longevity in URT colonization in a nonhuman primate model as a direct consequence of its newly acquired nga-slo locus [81▪▪]. An accessory gene region (AGR-3) that lies immediately downstream of nga-slo is differentially distributed among GAS strains in accordance with the emm pattern group, as are isoforms of the spn/nga-ifs (ifs, gene encoding immunity factor for SPN) gene products [10▪,48,87].
A hallmark feature of GAS is the production of superantigens (SAgs), which act as virulence factors in severe invasive disease [88,89]. Since invasive disease is rare, the ecological viewpoint begs the question: What purpose do SAgs serve the organism within its primary niche? A recent study examined the role of SAgs in GAS–nasopharyngeal interactions using transgenic mice expressing human leukocyte antigen (HLA) class II antigens [90▪▪]. Using an M18 strain, a 10 000-fold increase in bacterial burden in nasal tissue was evident for HLA-humanized mice as compared with unmodified mice (although GAS inoculum doses were quite high in these experiments). Importantly, deletion of select SAg genes abrogated this effect. Furthermore, vaccination with a toxoid form of SAg protected mice from nasopharyngeal challenge with GAS, paving a novel path toward GAS disease control [90▪▪]. There exists more than 10 variant SAg genes that have a differential presence/absence among GAS strains [91▪,92,93▪], and correlations between SAg alleles and emm pattern group have been noted . The degree to which recent acquisition of SAg genes leads to emergence of new clones as a consequence of enhanced URT colonization and/or transmission is a topic for future inquiries.
GAS induces a robust Th17 response in nasal-associated lymphoid tissue (NALT) in mice, which is the functional equivalent of human palatine tonsils [95–97]. In a recent advance, intranasal challenge of mice with live GAS promoted migration of GAS-specific Th17 cells from NALT into the brain, with breakdown of the blood–brain barrier, extravasation of IgG, and loss of excitatory synaptic proteins within the central nervous system [98▪▪]. This animal model holds great promise for better understanding the induction of autoimmune disease triggered by GAS infection at the URT, such as acute rheumatic fever (specifically, Sydenham's chorea) and possibly other neuropsychiatric disorders . The M-types of the classical rheumatogenic GAS [100,101] are recognized as a subset of the throat specialist strains, although that concept continues to be challenged .
A gap in our understanding remains between the molecular correlates of GAS disease and the pathogenic mechanisms of key virulence factors mediating superficial throat vs. skin infection. More precise characterization of the genes and nucleotide sequences that distinguish the populations of throat and skin strains, combined with improved animal models for GAS disease, are likely to strengthen the connections between epidemiology correlates and tissue tropism mechanisms.
The author thanks Andrew Steer for providing the emm typing data used in the meta-analysis.
Financial support and sponsorship
Supported by NIH (AI-117088) and a Bridge Funding grant from NYMC/Touro.
Conflicts of interest
There are no conflicts of interest.
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76. Cao TN, Liu Z, Cao TH, et al. Natural disruption of two regulatory networks in serotype M3 group A streptococcus isolates contributes to the virulence factor profile of this hypervirulent serotype. Infect Immun 2014; 82:1744–1754.
77. Cywes C, Stamenkovic I, Wessels MR. CD44 as a receptor for colonization of the pharynx by group A Streptococcus
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78▪▪. Lynskey NN, Banerji S, Johnson LA, et al. Rapid lymphatic dissemination of encapsulated group A streptococci via lymphatic vessel endothelial receptor-1 interaction. PLoS Pathog 2015; 11:e1005137.
The study describes a mechanism for dissemination of GAS from the URT into draining lymph nodes, mediated by a specific interaction between hyaluronic acid capsule and lymphatic vessel endothelial receptor-1; potentially important implications for rheumatic fever are discussed.
79▪. Turner CE, Abbott J, Lamagni T, et al. Emergence of a new highly successful acapsular group A streptococcus clade of genotype emm89 in the United Kingdom. MBio 2015; 6:e00622.
The study describes the genetic steps leading to the emergence of a new, highly prevalent M89 strain, involving loss of hyaluronic acid capsule and increased production of the toxins NADase and SLO.
80▪▪. Zhu L, Olsen RJ, Nasser W, et al. Trading capsule for increased cytotoxin production: contribution to virulence of a newly emerged clade of emm89 Streptococcus pyogenes. MBio 2015; 6: e01378-01315.
The study describes the genetic steps leading to the emergence of a new, highly prevalent M89 strain, involving loss of hyaluronic acid capsule and increased production of the toxins NADase and SLO.
81▪▪. Zhu L, Olsen RJ, Nasser W, et al. A molecular trigger for intercontinental epidemics of group A streptococcus. J Clin Invest 2015; 125:3545–3559.
The study demonstrates a role for the increased production of NADase and SLO toxins in the emergence of highly prevalent M1strains.
82. Nasser W, Beres SB, Olsen RJ, et al. Evolutionary pathway to increased virulence and epidemic group A streptococcus disease derived from 3,615 genome sequences. Proc Natl Acad Sci U S A 2014; 111:E1768–E1776.
83. Friaes A, Machado MP, Pato C, et al. Emergence of the same successful clade among distinct populations of emm89 Streptococcus pyogenes in multiple geographic regions. MBio 2015; 6:e01780-15.
84. Turner CE, Lamagni T, Holden MT, et al. Turner et al.
Reply to ‘Emergence of the Same Successful Clade among Distinct Populations of emm89 Streptococcus pyogenes in Multiple Geographic Regions’. MBio 2015; 6:e01883-15.
85. Musser JM, Zhu L, Olsen RJ, Nasser W. Musser et al.
Reply to ‘Emergence of the Same Successful Clade among Distinct Populations of emm89 Streptococcus pyogenes in Multiple Geographic Regions’. MBio 2015; 6:e01838-15.
86. Shulman ST, Tanz RR, Dale JB, et al. Seven-year surveillance of north american pediatric group a streptococcal pharyngitis isolates. Clin Infect Dis 2009; 49:78–84.
87. Riddle DJ, Bessen DE, Caparon MG. Variation in Streptococcus pyogenes NAD + glycohydrolase is associated with tissue tropism. J Bacteriol 2010; 192:3735–3746.
88. Stevens DL, Tanner MH, Winship J, et al. Severe group A streptococcal infections associated with a toxic shock-like syndrome and scarlet fever toxin A. N Engl J Med 1989; 321:1–7.
89. Kotb M, Norrby-Teglund A, McGeer A, et al. An immunogenetic and molecular basis for differences in outcomes of invasive group A streptococcal infections. Nat Med 2002; 8:1398–1404.
90▪▪. Kasper KJ, Zeppa JJ, Wakabayashi AT, et al. Bacterial superantigens promote acute nasopharyngeal infection by Streptococcus pyogenes in a human MHC Class II-dependent manner. PLoS Pathog 2014; 10:e1004155.
The study uses a humanized mouse expressing human HLA class II molecules and demonstrates a critical role for GAS SAgs in the enhancement of URT infection.
91▪. Davies MR, Holden MT, Coupland P, et al. Emergence of scarlet fever Streptococcus pyogenes emm12 clones in Hong Kong is associated with toxin acquisition and multidrug resistance. Nat Genet 2015; 47:84–87.
The study delineates genetic changes involving acquisition of SAg genes and determinants of antibiotic resistance in the emergence of scarlet fever caused by M12 clones.
92. Ben Zakour NL, Davies MR, You Y, et al. Transfer of scarlet fever-associated elements into the group A streptococcus M1T1 clone. Sci Rep 2015; 5:15877.
93▪. Commons RJ, Smeesters PR, Proft T, et al. Streptococcal superantigens: categorization and clinical associations. Trends Mol Med 2014; 20:48–62.
This is a comprehensive review on SAg genes and proposes new guidelines for their categorization.
94. Bessen DE, Izzo MW, Fiorentino TR, et al. Genetic linkage of exotoxin alleles and emm gene markers for tissue tropism in group A streptococci. J Infect Dis 1999; 179:627–636.
95. Wang B, Dileepan T, Briscoe S, et al. Induction of TGF-beta1 and TGF-beta1-dependent predominant Th17 differentiation by group A streptococcal infection. Proc Natl Acad Sci U S A 2010; 107:5937–5942.
96. Park HS, Francis KP, Yu J, Cleary PP. Membranous cells in nasal-associated lymphoid tissue: a portal of entry for the respiratory mucosal pathogen group A Streptococcus
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97. Dileepan T, Linehan JL, Moon JJ, et al. Robust antigen specific th17 T cell response to group A streptococcus is dependent on IL-6 and intranasal route of infection. PLoS Pathog 2011; 7:e1002252.
98▪▪. Dileepan T, Smith ED, Knowland D, et al. Group A streptococcus intranasal infection promotes CNS infiltration by streptococcal-specific Th17 cells. J Clin Invest 2016; 126:303–317.
The study shows that human tonsils contain GAS-specific effector memory Th17 cells, and uses a mouse model to demonstrate that GAS exposure leads to migration of GAS-specific Th17 cells from NALT into the brain, with important implications for autoimmune processes.
99. Kirvan CA, Swedo SE, Heuser JS, Cunningham MW. Mimicry and autoantibody-mediated neuronal cell signaling in Sydenham chorea. Nat Med 2003; 9:914–920.
100. Shulman ST, Stollerman G, Beall B, et al. Temporal changes in streptococcal M protein types and the near-disappearance of acute rheumatic fever in the United States. Clin Infect Dis 2006; 42:441–447.
101. Stollerman GH. Rheumatogenic and nephritogenic Streptococci. Circulation 1971; 43:915–921.