CovR/S

The best-characterized TCS of S. pyogenes is control of virulence (CovR/S) or capsule synthesis regulator (CsrR/S). CovS senses external signals, including environmental Mg2+ and host antimicrobial peptides (Gryllos, Levin, & Wessels, 2003; Gryllos, et al., 2008a) and transduces them to CovR, which in turn regulates the expression of several virulence gene regulons (Table 2). Phosphorylation of CovR enhances oligomerization of the activated protein and subsequent DNA binding to long AT-rich sequences within the target promoter regions (Miller, Engleberg, & DiRita, 2001; Gusa, Gao, Stringer, Churchward, & Scott, 2006). CovR mechanisms of binding to DNA appear to vary across promoters and display a high affinity for single DNA binding sites (Churchward, Bates, Gusa, Stringer, & Scott, 2009) or cooperative binding along long stretches of promoter region DNA (Gao, Gusa, Scott, & Churchward, 2005). A consensus CovR-binding element (ATTARA) has been proposed on the basis of binding studies of the hasA promoter (Federle & Scott, 2002), but no single conserved binding box has been identified, even among the 18 most strongly CovR influenced genes (Graham, et al., 2002). For a thorough review of the molecular mechanisms of CovR transcriptional regulation, see (Churchward, 2007).

More recent research has further defined the molecular mechanisms that underlie CovRS-mediated changes in S. pyogenes global gene expression. CovS acts as both a kinase and phosphatase to modulate levels of CovR phosphorylation. Increased streptolysin O (Slo) and NADase (Nga/Spn) production from S. pyogenes exposure to subinhibitory concentrations of clindamycin was found to involve CovRS activity (Chiang-Ni, Tseng, Shi, & Chiu, 2019). Inactivation of CovS phosphatase activity, whether by mutation or growth medium supplementation with Mg²⁺, inhibited clindamycin-mediated upregulation of Slo production in a dose-dependent manner. Horstmann et al. established a key role for CovS phosphatase activity in S. pyogenes pathogenesis using iso-allelic M1 and M3 S. pyogenes strains that differed in mutations that abrogated either CovS kinase or phosphatase activity, as well as CovR phosphorylation, to define their respective contribution to S. pyogenes pathogenesis (Horstmann, et al., 2018). In both serotypes, mutants that lack CovS phosphatase activity (CovS^T284A) and that failed to respond to signaling by human cathelicidin (LL-37) were impaired in causing skin infection or colonizing the oropharynx in murine models and showed reduced survival in human blood. Impairment of CovS phosphatase activity also decreased transcription of multiple virulence genes, including emm and its regulator Mga (Horstmann, et al., 2018). Additionally, hypervirulent S. pyogenes generated by spontaneous CovRS-inactivating mutations emerged at a lower frequency in CovS^T284A strains than in the iso-allelic parents.

Alternately, CovS kinase activity is involved in repression of CovR-regulated genes (hasA, ska, slo) under acidic stress, as inactivation of CovS kinase activity or CovR protein phosphorylation derepresses transcription of these genes in an acidic environment (Chiang-Ni, Tseng, Hung, & Chiu, 2017). Under acidic conditions, activity of the CovRS promoter (Pcov) increased, and replacement of pPcov with a constitutively active promoter abrogated acid stress-mediated repression of CovR-regulated genes. A CovR phosphorylation site mutant strain (CovR^D53E), generated in an M3 background, displayed CovR-dependent differential expression of only a subset of the CovR regulon (Horstmann, Sahasrabhojane, Yao, Su, & Shelburne, 2017). The mutant CovR^D53E protein binds promoter DNA from both CovR^D53E-regulated and -nonregulated genes, which suggests that more than the ability to bind DNA is involved in CovR regulon modulation. Promoter sequences belonging to CovR^D53E-regulated and CovR^D53E-nonregulated genes exhibit distinct promoter structures, differing in the location and number of putative CovR-binding sites. Furthermore, the CovR^D53E protein cannot dimerize, a functional feature of OmpR/PhoB family regulators. Thus, differences in the CovR regulon associated with the CovR^D53E mutation suggest that dimerization-dependent and dimerization-independent modes of CovR-mediated gene repression exist (Horstmann, Sahasrabhojane, Yao, Su, & Shelburne, 2017). Examination of CovRS activation in response to external signals using strains mutagenized in CovS kinase and phosphatase activity reveals that extracellular Mg⁺² and the human antimicrobial peptide LL-37 have opposing effects on the phosphorylation of CovR (Finn, Ramsey, Dove, & Wessels, 2021). These mutants had similar but more pronounced effects on CovR phosphorylation relative to growth in magnesium or LL-37 and are correlated with repression or activation of CovR-regulated genes.

CovR/S TCS is considered to be a master regulator, given that microarray and quantitative RT–PCR-based analysis of S. pyogenes serotype M1 revealed that CovR influences the transcription of up to 15% (271 genes) of all chromosomal genes (Graham, et al., 2002), with its activity exerted during late exponential and stationary growth phases. CovR primarily acts as a transcriptional repressor, as it was observed that in a CovR mutant array analysis, only 26 and 4 of the 271 differentially expressed transcripts examined were down regulated during exponential and stationary phase growth, respectively (Graham, et al., 2002; Federle, McIver, & Scott, 1999). CovR both directly and indirectly regulates multiple virulence factors that repress the hyaluronic acid capsule operon (hasABC), and modulate the expression of streptolysin S (SagA), a cysteine protease (SpeB), an NAD glycohydrolase (Nga), streptolysin O (Slo), an IL-8 degrading protease (SpyCEP), streptokinase (Ska), streptodornase (Sda), an immunoglobulin modifying protein (EndoS), and a fibronectin binding protein (Fba) (Graham, et al., 2002; Heath, DiRita, Barg, & Engleberg, 1999; Darmstadt, Mentele, Podbielski, & Rubens, 2000; Levin & Wessels, 1998; Bernish & van de Rijn, 1999; Shelburne, et al., 2010). As a master regulator, CovR/S also controls the expression of other proven and putative transcriptional regulators, as observed in the up-regulation of 8 transcriptional regulators in the exponential growth phase, and that of 18 transcriptional regulators in the stationary phase of growth in a CovR-deficient mutant (Graham, et al., 2002). Chromatin immunoprecipitation and DNA sequencing (ChIPseq) has identified CovR binding sites at CovR/S-regulated promoters, as well as lower-affinity association at additional locations on the S. pyogenes chromosome (Finn, Ramsey, Dove, & Wessels, 2021). Intermediate transcriptional regulators were identified as directly affected by CovR that occupied a subset (22 out of 42) of CovR-regulated promoters with which no CovR was found to be associated by ChIPseq analysis (Finn, Ramsey, Dove, & Wessels, 2021).

One directly CovR-repressed regulator is RivR (Ralp4), a member of the RofA-like protein type (RALP) family of standalone virulence-related regulators that repress the expression of capsule (hasABC) and protein G-related α2-macroglobulin-binding proteins (Grab) involved in the inactivation of proteolytic blood plasma proteins (Roberts, Churchward, & Scott, 2007; Treviño, Liu, Cao, Ramirez-Peña, & Sumby, 2013). Significantly, CovR can regulate its target genes independently of CovS, as S. pyogenes strains with isogenic CovR or CovS mutations have different phenotypes (Treviño, et al., 2009) and the most studied invasive clinical isolate, the MGAS5005 (M1) strain, encodes a truncated and functionally inactive CovS protein (Treviño, et al., 2009; Sumby, Whitney, Graviss, DeLeo, & Musser, 2006; Shelburne, et al., 2008). The regulatory role of CovR/S in responding to the host environment and in metabolic control affecting virulence, whether directly or through the modulation of other virulence-related regulators (such as RALPs, Ihk/Irr, and LytR), is detailed in later sections.

RocA

The “Regulator of Cov” (RocA) was first identified by transposon mutagenesis of an M6 serotype strain, which isolated two independent insertions in the rocA open reading frame that resulted in enhanced CovR expression (Biswas & Scott, 2003). Disruption of rocA resulted in S. pyogenes mucoid colony appearance and increased transcription from the has promoter, both of which are CovR-related phenotypes. Biswas et al. also determined that while RocA negatively autoregulated its expression independently of CovR, RocA-associated phenotypes were dependent on the presence of a wild-type covR allele (Biswas & Scott, 2003).

The interaction of rocA with CovR/S has been validated in multiple S. pyogenes backgrounds by various experimental approaches. Levels of phosphorylated CovR increase in the presence of intact functional RocA, and RocA has no regulatory activity in the context of covR or covS mutation in an M3 S. pyogenes background (Miller, et al., 2015). Conversely, using isogenic mutant M89 S. pyogenes strains, deleting one or more nucleotide repeat units of a variable number tandem repeat upstream of the rocA gene abolished RocA production, reduced CovR phosphorylation, and altered levels of CovR-regulated transcripts (nga, slo, prtS, grab) (Zhu, et al., 2016). Reduction of RocA levels enhanced secretion of Spn NADase and streptolysin O (Slo) and enhanced virulence in a mouse model of necrotizing fasciitis. Further evidence of RocA and CovRS association was provided by the identification of RocA mutants isolated from S. pyogenes-infected mice exhibiting enhanced Sse expression, concomitant with normal levels of SpeB production (Feng, et al., 2016). These variants showed transcript levels of CovRS-controlled virulence genes comparable to those of a covS mutant, but had no covRS mutations.

These lines of evidence and sequence similarity with sensor protein kinases suggested that RocA acts in concert with CovRS phosphorylation to alter gene expression and influence virulence. Ectopic expression of a truncated RocA derivative, harboring the predicted membrane-spanning domains but not the dimerization or HATPase domain, was sufficient to complement an M1 serotype ∆rocA mutant strain (Jain, Miller, Danger, Pflughoeft, & Sumby, 2017). Complementation mutagenesis in an M3 background using intact M1 rocA, and naturally occurring truncated M3 and M18 rocA alleles indicated RocA is a pseudokinase that inhibits capsule expression and reduced M1 S. pyogenes resistance to killing in human blood (Jain, Miller, Danger, Pflughoeft, & Sumby, 2017). Varying the RocA concentration attenuates the regulatory activity of Mg²⁺ and the antimicrobial peptide LL-37, which positively and negatively regulate CovS function, respectively, which further suggests that RocA is an accessory protein to the CovRS system. Co-immunoprecipitation of CovS with tagged RocA transmembrane domain fragments demonstrated RocA interacts with CovS via its seven N-terminal transmembrane domains (Lynskey, Velarde, Finn, Dove, & Wessels, 2019). The effect of expressed RocA fragments on CovR phosphorylation in a rocA-null M18 S. pyogenes strain demonstrated that interaction of RocA with CovS is essential for RocA-mediated regulation of CovRS function. Lynskey et al. also showed that RocA forms homodimers via its cytoplasmic domain, and that RocA truncation observed in M3 isolates alters RocA homotypic interaction, resulting in protein aggregation and impairment of RocA-mediated regulation. Further research determined RocA is a membrane protein that directly interacts with itself (RocA) and CovS, but not CovR (Bernard, Duarte, Bogdanov, Musser, & Olsen, 2020). The seven transmembrane helices of RocA are oriented such that the C-terminus is cytoplasmic, whereas the N terminus is extracellular. Bernard et al. also showed that single amino acid polymorphisms along the entire length of RocA in an M28 strain background can disrupt homotypic and heterotypic (i.e. RocA-CovS) interactions (Bernard, Duarte, Bogdanov, Musser, & Olsen, 2020). This resulted in significant changes in S. pyogenes virulence phenotypes, including in vitro activity of secreted virulence factors (Spn, Slo, Ska), murine model tissue destruction and morbidity. These findings were confirmed in M1 and M3 S. pyogenes, with the exception that in these backgrounds the RocA regulon was found to include the secreted SpeB protease (Jain, Danger, Burgess, Uppal, & Sumby, 2020). Additional studies on the mechanism of RocA regulation determined that while CovR phosphorylation decreased in a rocA mutant during exponential growth, levels of phosphorylated of CovR increased during the stationary phase of growth and under acidic stress in a rocA mutant to levels similar to those in the wild-type strain (Chiang-Ni, Chiou, Tseng, Hsu, & Chiu, 2020). Similar CovR phosphorylation in a CovS phosphatase-inactivated mutant and a rocA mutant under acidic stress and exposure to Mg²⁺ suggest that CovS phosphatase activity is required for RocA to modulate CovR phosphorylation.

As is the case with other S. pyogenes virulence regulators, RocA heterogeneity correlates with variations in disease potential among and within S. pyogenes serotypes. Unlike M1 S. pyogenes that encode an intact RocA allele (Jain, Miller, Danger, Pflughoeft, & Sumby, 2017), naturally occurring truncation mutations of RocA conserved among serotype M18 (Lynskey, et al., 2013) and M3 serotype S. pyogenes (Lynskey, Turner, Heng, & Sriskandan, 2015; Miller, Pflughoeft, & Sumby, 2015) result in the increased production of hyaluronic acid capsule. In addition to capsule production, the premature stop codon in rocA, due to a single nucleotide polymorphism early in the open reading frame (T269A) in the M18 background enhances covR transcription as well as alters expression of genes involved in metabolism and virulence (spyCEP, cppA, salB) (Lynskey, et al., 2013). Allelic replacement with an intact rocA (from serotype M89) reverted the capsule production phenotype, reduced M18 S. pyogenes survival in human blood, and altered nasopharyngeal carriage longevity in a mouse model of infection. In contrast, the conserved truncated RocA allele in serotype M3 invasive disease isolates is missing 35 amino acids from its C-terminus. RocA truncation in the M3 background results in enhanced expression of several immunomodulatory virulence factor genes (nga/spn, slo, scpC, ska, sse, grab, spd), enhanced hemolysis and NAD(+) hydrolysis, as well as increased resistance to human phagocytic killing and virulence in a murine bacteremia model of infection (Miller, et al., 2015).

M1 RocA mutants exhibit intermediate skin invasion and virulence phenotypes relative to wild-type and CovS mutants in subcutaneous infection of mice (Feng, et al., 2016). RocA and CovS mutations enhanced hasA transcript levels more than those of spyCEP above wild-type M1 S. pyogenes levels, but mutation of RocA, unlike mutations of CovS, did not result in the downregulation of speB transcription at a stationary growth phase or in subcutaneous infection of mice. Additionally, an inverse correlation has been observed between the virulence of wild-type in M1 S. pyogenes, rocA mutant, covS mutant and covR mutant strains during invasive infection and their fitness in an ex vivo upper respiratory tract model (Jain, Danger, Burgess, Uppal, & Sumby, 2020).

M28 S. pyogenes strains collected from invasive infections in humans also exhibit missense and nonsense polymorphisms in rocA (Bernard, Kachroo et al., 2018). As in other serotypes, rocA-inactivating mutations enhanced M28 S. pyogenes virulence in a mouse model of necrotizing myositis. Genome-wide transcript analysis in M28 strains revealed thatRocA inactivation significantly alters transcript levels of 427 genes and 323 genes at mid-exponential and early stationary growth phases, respectively, including 21 virulence factors. Comparison to RocA transcriptomes in other S. pyogenes serotype backgrounds found them to differ in both size and the number of regulators affected (Bernard, et al., 2018). Further investigation of M28 rocA polymorphisms revealed each polymorphism had a unique effect on the global S. pyogenes transcriptome, including differential expression of virulence genes (nga, ifs, slo, spyCEP, sag operon, emm, enn, mrp, scpA, has operon), regulators (mga, spxA2) and metabolic genes (Bernard, et al., 2019). The varied rocA polymorphisms also distinctly altered production of Spn andSlo, as well as Ska and Sof activity. These variations in virulence factor production in turn were associated with an increase or decrease in virulence in mouse and nonhuman primate infection models. Conversely, unlike observations in M1, M3, and M18 S. pyogenes strains, rocA mutation reduced, rather than enhanced, survival of the M12 isolate in human blood ex vivo (Sarkar, et al., 2018).

LiaFSR/Spx

LiaFSR (formerly referred to as YvqFEC in S. pyogenes) is a three-component system (TCS), unique to Gram-positive bacteria, that consists of a predicted histidine kinase sensor (LiaS/YvqE), a response regulator (LiaR/YvqC), and an accessory membrane protein (LiaF/YvqF). In Firmicutes, the LiaFSR system is activated by exposure to antimicrobial peptides (AMPs) and antibiotics (e.g., bacitracin, daptomycin) that target the bacterial membrane, and is therefore critical to the cell envelope stress response (Mascher, 2006). Mutation of LiaFSR proteins or downstream effectors has been associated with changes in antimicrobial susceptibility in enterococci (Arias, et al., 2011; Khan, et al., 2019). The contribution of LiaFSR to S. pyogenes virulence was first described by the discovery of an arginine-to-glycine amino acid mutation in the LiaS histidine kinase (LiaS^R135G) that contributed to pharyngeal asymptomatic carriage of an M3 strain (Flores, Jewell, Yelamanchili, Olsen, & Musser, 2015). Mutagenesis of wild-type liaS to liaS^R135G and vice-versa altered adherence of M3 S. pyogenes to epithelial cells, S. pyogenes carriage in a mouse nasopharyngeal infection model, and invasiveness in a murine intramuscular infection model.

Recent research suggests that LiaFSR function connects the sensing of host antimicrobial peptide-mediated stress on the bacterial membrane with regulation of S. pyogenes virulence. Antimicrobial peptides (AMPs), which are produced in human skin and mucosal surfaces as well as immune cells, disrupt bacterial membranes through various mechanism of action to achieve killing and clearance of invading bacteria (Melo, Ferre, & Castanho, 2009; Cole & Nizet, Bacterial Evasion of Host Antimicrobial Peptide Defenses, 2016). One target of AMP activity are discrete bacterial membrane locations that carry out crucial cellular processes, which are referred to as functional membrane microdomains (FMM) (López & Kolter, 2010; García-Fernández, et al., 2017). In S. pyogenes, the “ExPortal” is a FMM that colocalizes protein secretion via the general secretory pathway and processing of virulence proteins (Rosch & Caparon, 2004), along with peptidoglycan precursor synthesis proteins (Vega, Port, & Caparon, 2013), in an anionic lipid-enriched microdomain. Polymyxin B and the alpha-defensin human neutrophil peptide 1 (hNP-1) produced in azurophilic granules of neutrophils (Ganz, et al., 1985) preferentially target and disrupt the ExPortal, which inhibits protein secretion and processing (Rosch & Caparon, 2005; Vega & Caparon, 2012). LiaF and LiaS have been found to colocalize to the ExPortal, which when disrupted following exposure to hNP-1, induces activation the LiaFSR system (Lin, Sanson et al., 2020). LiaS colocalization with the S. pyogenes ExPortal can also be disrupted by the elimination of membrane cardiolipin through deletion of cardiolipin synthase (cls), which leaves LiaF membrane localization unaffected. Conversely, production of LiaF appears to be required for maintaining ExPortal integrity (Lin, et al., 2020). Further characterization of LiaFSR-mediated gene regulation through RNAseq analysis of isogenic ∆liaF, ΔliaS and ΔliaR mutants in an M3 S. pyogenes background revealed a near perfect inverse correlation between ∆liaF and ∆liaR transcriptomes, suggesting LiaF acts as an inhibitor of LiaR-mediated transcriptional regulation (Sanson, Vega et al., 2021). The most highly differentially expressed gene in ∆liaF and ∆liaR mutants is that encoding the RNA polymerase-binding protein SpxA2, which has been shown to be directly regulated by activation of LiaR (Sanson, et al., 2021).

Spx regulatory proteins are highly conserved among Firmicutes, acting as anti-activators of expression via their interactions with RNA polymerase (Rojas-Tapias & Helmann, 2019). S. pyogenes encodes two highly similar SpxA paralogs (SpxA1 and SpxA2). An M14 serotype S. pyogenes mutant that lacks SpxA1 grows poorly under aerobic conditions, whereas an SpxA2-lacking strain is severely attenuated in its response to stressors, which can include thermal and oxidative stress (Port, Cusumano, Tumminello, & Caparon, 2017). Loss of SpxA1 reduced susceptibility to the cationic antimicrobial polymyxin B, whereas the absence of SpxA2 enhanced it. Virulence phenotypes in a murine model of soft tissue infection were also in direct opposition, as the ∆spxA1 strain showed highly attenuated virulence, while ∆spxA2 was hypervirulent and produced more extensive tissue damage and a greater bacterial burden than the wild-type isogenic parent (Port, Cusumano, Tumminello, & Caparon, 2017). SpxA1 loss correlated with reduced expression of several toxins (e.g., SpeB), in opposition to speB overexpression observed in the SpxA2-lacking background in which hypervirulence was reverted by deletion of speB.

Among the other genes differentially expressed as a result of LiaFSR mutagenesis were multiple virulence factors that are also regulated by CovRS, and high levels of spxA2 transcript resulting from LiaFSR activation correlate with increased CovR-regulated gene transcription (Sanson, et al., 2021). Moreover, changes in CovR phosphorylation inversely correlate with levels of SpxA2 expression that result from LiaFSR activity, which suggests that regulatory cross talk occurs between LiaFSR and CovRS systems.

Virulence phenotypes that result from LiaFSR mutagenesis suggest additional levels of regulatory complexity associated with the system, since a ΔliaF mutant exhibited decreased virulence in whole human blood and in a murine model of infection, despite the increased levels of CovR-regulated virulence gene expression associated with SpxA2 overexpression (Sanson, et al., 2021). Furthermore, higher than expected virulence was observed for a ∆liaR mutant, which was attributed to elevated SpxA2-associated speB transcription, which as mentioned previously is a phenotype observed in SpxA2 mutants in other S. pyogenes serotypes (Port, Cusumano, Tumminello, & Caparon, 2017). Other studies suggest LiaS function contributes to S. pyogenes biofilm formation (Isaka, et al., 2016) and environmental stress adaptation, including salt, acidic and oxidative stress (Roobthaisong, Aikawa, Nozawa, Maruyama, & Nakagawa, 2017), which potentially result from the substantial effect of LiaFSR activation on SpxA2 expression (Lin, et al., 2020; Sanson, et al., 2021) that in turn has been shown to contribute to S. pyogenes stress responses (Port, Cusumano, Tumminello, & Caparon, 2017). There is also evidence that LiaS mediates biofilm production and pilus expression in an environment acidified through glucose fermentation, as a ∆liaS isogenic mutant produced biofilms in acidified culture but not exposed to glucose (Isaka, et al., 2021).

RofA and the RofA-like protein type regulators (RALPs)

Four RALPs have been identified in S. pyogenes: RofA, Nra, Ralp3, and RivR (Ralp4). Among sequenced strains, they are 52% similar and 29% identical to each other on average (Granok, Parsonage, Ross, & Caparon, 2000), and are involved in the control of S. pyogenes–host-cell interactions, avoidance of host cell damage, and balanced virulence factor expression during stationary phase growth (Beckert, Kreikemeyer, & Podbielski, 2001; Podbielski, Woischnik, Leonard, & Schmidt, 1999; Molinari, et al., 2001). The most studied of the RALPs is the namesake of this family: RofA, a DNA-binding protein that was first identified in a serotype M6 strain as a transcriptional inducer under anaerobic growth conditions of prtF, the gene encoding fibronectin-binding protein F (sfbI) (Fogg & Caparon, 1997). RofA transcription occurs adjacent to and divergently from prtF and is positively autoregulated by RofA interaction with specific 17-bp sites within the intergenic region shared by the two genes (Granok, Parsonage, Ross, & Caparon, 2000; Fogg & Caparon, 1997). RofA directly modulates multiple virulence genes, up-regulating transcription of the FCT region genes (SfbI, the collagen binding protein Cpa, and T antigen) (Kreikemeyer, Beckert, Braun-Kiewnick, & Podbielski, 2002) and repressing transcription of the streptolysin S hemolysin (encoded by sagA), the SpeB protease, and the SpeA superantigen (Beckert, Kreikemeyer, & Podbielski, 2001). As a regulator of other virulence-related regulators, RofA down-regulates Mga in serotype M6 and thus can modulate Mga regulon virulence genes.

Nra is another well-studied RALP, which is only present in M serotypes that carry the FCT type 3 gene region (M3, 18 and 49), as all other M serotypes possess FCT gene regions that encode for RofA instead of Nra (Kratovac, Manoharan, Luo, Liziano, & Bessen, 2007). In the M49 strain, Nra was characterized as a repressor for the adjacent cpa, as well as for an unlinked gene that encodes a second fibronectin-binding protein, prtF2 (Podbielski, Woischnik, Leonard, & Schmidt, 1999). Transcriptome analysis of a serotype M49 strain and its isogenic nra mutant strain showed that Nra is active in the exponential, transition, and stationary growth phases (Kreikemeyer, et al., 2007). Maximum expression of Nra occurs in early stationary phase growth (Podbielski, Woischnik, Leonard, & Schmidt, 1999), and the highest number of differentially transcribed genes was observed in the transition phase of growth, with an Nra-knockout strain displaying 112 and 84 genes with increased and decreased transcript abundance, respectively (Kreikemeyer, et al., 2007). However, unlike some RofA-expressing strains, Nra does not appear to respond to changing atmospheric conditions (Podbielski, Woischnik, Leonard, & Schmidt, 1999). Nra has been shown to repress the transcription of FCT region genes, pilus protein-encoding genes, and the capsule biosynthesis operon (hasABC) (Kreikemeyer, et al., 2007). As a master regulator, Nra also represses virulence factors encoded by the Mga core regulon, and in the M49 serotype background, Nra has been observed to directly or indirectly repress expression of the global regulator Rgg, the Ihk/Irr TCS, additional RALPs (Ralp3 and RivR), and Mga itself (Podbielski, Woischnik, Leonard, & Schmidt, 1999; Kreikemeyer, et al., 2007). For other virulence genes, a negative regulatory effect of Nra has only been shown during one or two growth phases. These genes encode prtF2, a putative hemolysin (hylX), two hyaluronidases (hylP2 and hylP3), an extracellular matrix-binding protein (epf), the laminin-binding protein (lmb), speA, and ska.

Ralp3 is encoded in the so-called eno-ralp3-epf-sagA (ERES) pathogenicity region, which is identified in selected M serotypes (M1, 4, 12, 28 and 49) where it controls the expression of Epf and Streptolysin S (Kreikemeyer, et al., 2007). Inactivation of Ralp3 in an M49 strain resulted in a diminished binding capacity to human plasminogen, and SpeB activity was significantly decreased (Siemens, et al., 2012). Transcriptome analysis identified 16 genes as up regulated, and 43 genes as down regulated in the ralp3 mutant. Among those down regulated were the lac operon and the fru operon (encoding proteins involved in metabolism), the putative salivaricin operon (sal), and the whole Mga core regulon. The importance of the RALPs in metabolic control will be further discussed in the corresponding section. Every S. pyogenes strain tested to date encodes at least RofA or Nra, and some strains can express several RALP family members, which results in a widely variable combination of RALP-regulated genes and patterns of gene expression between different serotypes (Beckert, Kreikemeyer, & Podbielski, 2001; Podbielski, Woischnik, Leonard, & Schmidt, 1999; Kreikemeyer, Beckert, Braun-Kiewnick, & Podbielski, 2002; Kreikemeyer, et al., 2007).

Ralp4, or RivR (which stands for RALP, roman numeral “iv”) was identified and characterized as a RALP, based on its 29% identity with RofA and 32% identity with Nra at the amino acid level (Roberts, Churchward, & Scott, 2007). Since phosphorylated CovR represses expression of RivR (Roberts, Churchward, & Scott, 2007), analysis of the RivR-regulated transcriptome in a MGAS5005 (M1T1) strain required its deletion and complementation in a ∆covR background (Roberts & Scott, 2007). This study showed that the overexpression of RivR resulted in the altered transcription of 31 genes, among which there were multiple virulence related genes that were either induced, in the case of endoS and several genes of the Mga regulon (emm, scpA, fba, mga, sic, scl and grm), or repressed by RivR activity. Further characterization revealed that RivR enhances transcriptional activation by Mga in vitro, increases expression of the Mga regulon in vivo, and is necessary for virulence in a CovR-deficient strain (Roberts & Scott, 2007). Additionally, as previously indicated, RivR also acts as a repressor of hasABC and grab expression in a CovR-dependent manner in two distinct M1T1 serotype strains (MGAS2221 and MGAS5005) (Treviño, Liu, Cao, Ramirez-Peña, & Sumby, 2013), and RivR repression abrogates S. pyogenes survival in human blood (Ramalinga, Danger, Makthal, Kumaraswami, & Sumby, 2016). In contrast to the earlier report (Roberts & Scott, 2007), the Mga regulon was not activated by RivR in these M1T1 serotype strains (Treviño, Liu, Cao, Ramirez-Peña, & Sumby, 2013). Like Mga, RivR forms both dimers and a higher-molecular-mass multimer to exert its regulatory activity on hasA and grab transcripts (Ramalinga, Danger, Makthal, Kumaraswami, & Sumby, 2016). Using a library of expression plasmids in which each of the four cysteines in RivR was mutagenized to a serine, it was demonstrated that cysteine residues at positions 32, 377, and 470 are required for RivR dimerization.

Carbohydrate catabolite regulation (CcpA, LacD.1, Mga)

Bacteria of multiple species have been observed to coordinately alter the expression of carbohydrate utilization genes and virulence factor production in response to changes in carbohydrate availability (Moreno, Schneider, Maile, Weyler, & Saier, Jr., 2001; Deutscher, Francke, & Postma, 2006). Glucose is the primary carbohydrate source of energy for many bacteria, including S. pyogenes, which generates ATP from its conversion to pyruvate through the Embden-Meyerhof pathway. However, the utilization of alternative carbohydrates requires energy input to convert them to glucose in order to be metabolized. As a result, bacteria ensure that genes involved in alternative carbohydrate utilization are not expressed when glucose is available, through a process known as carbon catabolite repression (CCR) (Deutscher, Francke, & Postma, 2006). In S. pyogenes, CCR is not restricted to the regulation of carbohydrate catabolic enzymes and can affect the expression of virulence determinants via carbohydrate-sensitive regulators, such as CcpA, LacD.1, and Mga (Table 3, 4).

Phosphoenolpyruvate Phosphotransferase System (PTS) regulation

Regulatory pathways of carbohydrate metabolism contribute to S. pyogenes pathogenesis, including those involving phosphoenolpyruvate phosphotransferase system (PTS) components. PTS activity has been associated with S. pyogenes survival in whole human blood, and mutagenesis of PTS genes affects virulence factor expression (e.g., SLS) in a murine model of soft tissue infection (Gera, Le, Jamin, Eichenbaum, & McIver, 2014; Valdes, et al., 2016). Insertional inactivation of the 14 annotated PTS EIIC-encoding genes in an M1 serotype S. pyogenes determined that the mannose-specific EII locus (manLMN) acts to prevent the early onset of SLS-mediated hemolysis (Sundar, Islam, Gera, Le Breton, & McIver, 2017). Additional investigation of PTS permease (EIIC) genes identified the annotated β-glucoside PTS transporter (bglP) as a contributor to S. pyogenes growth and survival in human blood (Braza, et al., 2020).The operon encoding bglP, including antiterminator licT and phospho-β-glucosidase bglB, is repressed by glucose and induced by salicin. Mutants that lack bglP and bglB exhibit in vitro growth defects in distinct carbohydrate sources (fructose/mannose/salicin and salicin alone, respectively) as well as altered biofilm formation and SLS production affecting ulcerative lesion progression during subcutaneous infections in mice (Braza, et al., 2020).

CcpA and LacD.1

The catabolite control protein (CcpA) is a member of the LacI/GalR family of transcription factors that is common among Gram-positive pathogens, and a thorough review of its characterization can be found in (Deutscher, Francke, & Postma, 2006). Briefly, CcpA function centers on the activity of the phosphoenolpyruvate phosphotransferase system (PTS), which regulates carbohydrate uptake via a phosphorylation relay pathway that involves cytosolic (EI, HPr) and membrane bound (EII) enzymes (Deutscher, Francke, & Postma, 2006). Of these, HPr is tasked with determining the energy level of the cell by undergoing differential phosphorylation by the PTS and an HPr kinase at conserved histidine and serine residues, respectively. Phosphorylation of the serine residue enables the interaction of HPr with CcpA in order for CcpA to act as a carbohydrate-dependent transcriptional repressor by binding DNA at a palindromic consensus sequence in the promoter or coding regions of CCR-transcripts, known as a catabolite-responsive element or cre site (Deutscher, Francke, & Postma, 2006; Fujita, Miwa, Galinier, & Deutscher, 1995). Though primarily a repressor, CcpA is also known to function as an activator of transcripts that are required for glucose-dependent growth in other Gram-positive bacteria (Grundy, Waters, Allen, & Henkin, 1993). Transcriptome analysis of a MGAS5005 (M1T1) strain and its isogenic ccpA deletion mutant revealed that the transcription of 124 genes (~6% of the S. pyogenes genome) is affected by CcpA (Kinkel & McIver, 2008). In addition to the expected repression of genes relating to non-glucose sugar utilization, CcpA has influenced expression of virulence related genes, including the sag operon (repressing) and rivR (inducing). Under glucose starved conditions, the expression of additional virulence related genes (speB, mac, spd3) were affected in the same CcpA-deficient strain (Shelburne, et al., 2008).

A combination RNAseq and ChIPseq analysis determined that CcpA affects transcript levels of 514 M1 serotype S. pyogenes genes, of which 105 demonstrated direct DNA binding by CcpA, including those that encode the cytotoxin SLS, as well as PrtS and SpeB proteases (DebRoy, et al., 2021). Abolishing the interaction of CcpA with its co-factor HPr by mutation of a valine residue (V301A) affected the transcript levels of 205 genes (40%) of the CcpA regulon. ChIPseq analysis showed the mutagenized CcpA^V301A protein bound to 74% of gene targets bound by wild-type CcpA with lower affinity, thus elucidating HPr-dependent and independent virulence and metabolic gene regulation by CcpA in S. pyogenes (DebRoy, et al., 2021). Among the genes affected by Hpr-dependent CcpA regulation were prtS, lctO, arcA and malX. Analysis of CcpA interaction with a cre site in the promoter region of mga in the JRS4 (M6) strain revealed that CcpA is necessary for full expression of the virulence regulator Mga in the late logarithmic phase of growth (Almengor, Kinkel, Day, & McIver, 2007). CcpA regulatory activity appears to be exerted both directly and indirectly and varies among strains, as CcpA was observed to bind to the cre site of the mga promoter in JRS4 (Almengor, Kinkel, Day, & McIver, 2007)and speB in the HSC5 (M14) strain, but not sagA in the latter, despite still repressing the expression of the sag operon (Kietzman & Caparon, 2010).

Comparison in murine models of S. pyogenes virulence of a ∆ccpA strain with another mutant expressing CcpA locked into a high-affinity DNA-binding conformation (CcpA^T307Y) suggests that CcpA activity contributes to balancing bacterial growth with host tissue invasion (Paluscio, Watson, Jr., & Caparon, 2018). The ∆ccpA and CcpA^T307Y mutants displayed distinct patterns of virulence in models of acute soft tissue infection and of long-term mucosal colonization. The ∆ccpA strains exhibited reduced ability to grow in tissue, reduced tissue damage, and early clearance. The CcpA^T307Y strain produced high tissue burdens and extended time of carriage, accompanied with reduced tissue damage (Paluscio, Watson, Jr., & Caparon, 2018).

The CcpA regulon shares virulence gene targets with the CovR regulon, as demonstrated by the characterization of CcpA-deficient (2221∆ccpA), CovR-deficient (2221∆covR), and doubly-deficient (2221∆ccpA∆covR) mutant strains in an M1T1 background with a functional CovS (MGAS2221), as well as by comparison with similarly constructed mutations in a CovS-deficient strain (MGAS5005) (Shelburne, et al., 2010). According to this study, CcpA and CovR co-regulate transcription of the virulence factor encoding genes nga, slo, spyCEP, sagA, sdaD2, endoS, fba, and speB. CcpA directly regulates expression of these genes in the strains examined, as CcpA and its complex with phosphorylated HPr protein, CcpA-(HPr-Ser46-P), bind with high affinity to cre sites within their promoters. However, this shared regulation is dependent upon the status of CovS functionality; CcpA influences speB, slo, and spyCEP expression during in vitro growth only when CovS is functionally active (Shelburne, et al., 2010). Notably, CovR and CcpA do not regulate each other’s expression, at least not directly in the strains and conditions examined—despite the fact that all of the virulence genes affected by CcpA inactivation are also affected by CovR inactivation (Shelburne, et al., 2010).

The CCR of virulence-related genes by CcpA occurs in conjunction with the activity of another carbohydrate-dependent regulator, called LacD.1. Identified as a glucose responsive regulator of the SpeB protease (Loughman & Caparon, 2006a), LacD.1 is a member of the Lac.1 operon and has the characteristics of a tagatose 1,6-bisphosphate aldolase involved in lactose and galactose metabolism. Though LacD.1 possesses enzymatic activity, it does not function to metabolize galactose (Loughman & Caparon, 2006a; Loughman & Caparon, 2007). LacD.1 regulatory function is independent of its catalytic activity, but the protein must bind to its substrate to exert its regulation of SpeB, a function that appears to have evolved by conferring increased fitness to S. pyogenes, as observed using competitive assays under in vitro growth conditions that mimic deep tissue environments (Cusumano & Caparon, 2013). An in vivo analysis of the carbon catabolite-responsive regulatory pathways constituted by CcpA and LacD.1 revealed that they can regulate up to 15% of the genome in response to glucose, and that together, they contribute to regulation of 60% of this subset in various combinations, including CcpA and LacD.1-specific and independent regulation, co-regulation, and even regulation independent of glucose (Kietzman & Caparon, 2011). Among the affected genes were several secreted factors, putative virulence genes, and previously characterized genes that affect virulence, such as sagA and lctO (Kietzman & Caparon, 2010). The most important virulence gene affected by CcpA and LacD.1 co-regulation is speB, which was induced by CcpA and repressed by LacD.1 both in vitro and in vivo. In the latter condition, CcpA and

LacD.1 mutants evinced mis-regulation of SpeB at separate and distinct phases of infection, which highlights the spatial and temporal role that carbon catabolite sensing appears to play in virulence-related gene regulation during the course of infection (Kietzman & Caparon, 2011). Another important finding of this study was that multiple transcripts are responsive to glucose independent of CcpA and LacD.1 activity, which indicates that other carbohydrate responsive regulators act in S. pyogenes. One such regulator appears to be the virulence gene-related transcription factor Mga.

Mga

Mga is a large 500 amino acid DNA-binding protein with an approximate molecular size of 62 kDa. Orthologous proteins can be found in many pathogenic streptococci, including Streptococcus dysgalactiae, S. pneumoniae, S. equi, S. gordonii, S. mitis, S. sanguinis, and S. uberis (Geyer & Schmidt, 2000; Vasi, Frykberg, Carlsson, Lindberg, & Guss, 2000; Hava & Camilli, 2002; Vahling & McIver, 2006). Mga was first identified in S. pyogenes through spontaneous small deletions at a locus just upstream of the emm gene that resulted in M protein-negative strains (Spanier, Jones, & Cleary, 1984), and was later confirmed as a positive regulator of emm transcription and M protein production (Caparon & Scott, 1987). The identified loci immediately upstream of emm was predicted to produce a protein with homology to a DNA-binding transcriptional regulator (Perez-Casal, Caparon, & Scott, 1991; Chen, Bormann, & Cleary, 1993) and was eventually designated as mga for multiple gene regulator of group A streptococcus (Scott, et al., 1995). Mga is expressed in all S. pyogenes strains and its encoding gene is found in all strains examined to date (Podbielski, 1992; Bessen, Manoharan, Luo, Wertz, & Robinson, 2005). Its sequence varies up to 21% among S. pyogenes strains, and two divergent alleles of mga (mga-1, mga-2) have been identified (Haanes & Cleary, 1989; Hollingshead, Readdy, Yung, & Bessen, 1993), although the expressed Mga proteins maintain greater than 97% amino acid sequence identity. The mga-1 allele is linked to serum opacity factor negative (SOF-) class I strains isolated from throat infections, while the mga-2 allele is almost exclusively observed in SOF+ class II strains associated either with skin infections or with “generalist” strains (Bessen, Manoharan, Luo, Wertz, & Robinson, 2005), which suggests that the divergent Mga regulons likely evolved to adapt to distinct tissue environments (Bessen, Manoharan, Luo, Wertz, & Robinson, 2005).

Mga induces transcription of its encoding gene (mga) (Geist, Okada, & Caparon, 1993; Podbielski, Flosdorff, & Weber-Heynemann, 1995; McIver, Thurman, & Scott, 1999) and a core set of virulence genes during exponential growth in vitro. These include cell wall-associated surface molecules in addition to M protein involved in host tissue adherence, internalization into non-phagocytic cells and avoidance of the host innate and adaptive immune responses. The regulatory role of Mga during the course of infection in different tissue environments will be covered later in this chapter. Mga induces the transcription of multiple genes located downstream of mga and emm in a discrete region of the chromosome called the mga locus, identified in all S. pyogenes genomes thus far examined. This region includes, in varying organization and combinations depending on serotype, a cell wall-associated C5a peptidase (scpA), the M-like surface proteins Mrp (fcrA) and Enn (enn), secreted inhibitor of complement (sic), and a fibronectin-binding surface adhesin (fba) (Haanes, Heath, & Cleary, 1992; Podbielski, 1993; Hollingshead, Arnold, Readdy, & Bessen, 1994). The scpA gene product is involved in cleavage of the C5a chemotaxin, which inhibits recruitment of PMNs to infectious sites (Simpson, LaPenta, Chen, & Cleary, 1990). Mrp (fcrA) binds IgG and fibrinogen, and can provide resistance to phagocytosis; while Enn generally binds IgA, though antiphagocytic properties for it have not been shown (Bessen & Fischetti, 1992; Courtney & Li, 2013). The product of sic has the ability to block the membrane attack complex (MAC) of the complement, as well as inhibit the activity of innate immune effectors, such as lysozyme and host antimicrobial peptides (Akesson, Sjöholm, & Björck , 1996; Fernie-King, et al., 2001; Frick, Akesson, Rasmussen, Schmidtchen, & Björck, 2003). Fba constitutes a fibronectin-binding surface adhesin that is able to bind the complement regulators factor H and factor H-like protein 1 to block opsonophagocytosis (Terao, et al., 2001; Pandiripally, Gregory, & Cue, 2002). However, not all Mga-regulated genes are located within the mga locus. The fibronectin-binding surface proteins SfbX and SOF that enable S. pyogenes to interact with the host ECM (Courtney, et al., 1999; Jeng, et al., 2003) are encoded by sof and sfbX 12 kb away from mga in class II SOF+ strains, and their co-transcription is activated by Mga (Green, et al., 2005). Mga also influences the expression of sclA, a gene that is found over 30 kb upstream of the mga locus in a subset of serotypes and that encodes the streptococcal collagen-like protein A (sclA/scl1). This repeat-containing surface molecule found on all S. pyogenes strains absorbs human plasma LDL and binds to α2β1 integrins on host cells, which allows for internalization and subsequent re-emergence as a means of immune evasion (Caswell, Lukomska, Seo, Höök, & Lukomski, 2007). As previously indicated, Mga can also regulate the transcription of speB, which modulates the production of the secreted SpeB cysteine protease (Podbielski, Flosdorff, & Weber-Heynemann, 1995; Ribardo & McIver, 2006).

Of the carbohydrate-responsive virulence gene regulators, Mga is the best characterized in terms of its mechanism of action. A more thorough overview of Mga binding site and domain architecture can be found in (Hondorp & McIver, 2007; Rom, Hart, & McIver, 2021). The binding of Mga to upstream promoter regions is essential for high-level transcriptional activation of core regulon genes (McIver & Myles, 2002). Mga binding sites within these promoters vary widely in location with respect to the start of transcription and have thus far been classified into three categories based on biochemical studies ((Almengor & McIver, 2004), reviewed in (Hondorp & McIver, 2007)). Initial DNase I footprinting experiments indicate that Mga protects a large 45- to 59-base-pair non-palindromic region of DNA (McIver, Thurman, & Scott, 1999; McIver, Heath, Green, & Scott, 1995), and these studies suggest a nominal consensus sequence to guide the search for Mga binding sites. A mutagenesis-based analysis of the M1T1 emm promoter binding site (Pemm1) identified 34 nucleotides within Pemm1 that had an effect either on Mga binding, on Mga-dependent transcriptional activation, or on both (Hause & McIver, 2012). Among these critical nucleotides, guanines and cytosines within the major groove were disproportionately identified as clustered at the 5' and 3' ends of the binding site, along with runs of nonessential adenines between the critical nucleotides. On the basis of these results, a Pemm1 minimal binding site of 35 bp bound Mga was found at a level comparable to the level of binding of the larger 45-bp site. However, as more sites have been found, the sequence has become less defined, due to lack of conservation between the various promoters, and exhibits only 13.4% identity with no discernible symmetry (Hause & McIver, 2012). Nonetheless, in vitro transcription assays have established that Mga is sufficient to activate expression (Almengor, Walters, & McIver, 2006), so while additional factors might modify Mga regulation in vivo, they are not required. Despite the variability in promoter sites, Mga seems to employ the same protein domain(s) to bind locations with little sequence homology, as evidenced by competition of Pemm and PscpA for Mga binding to Pmga (McIver, Thurman, & Scott, 1999).

Mga has two conserved helix–turn–helix (HTH) domains, HTH-3 (residues 53–72) and HTH-4 (residues 107–126), that map near the N-terminus of the protein (Chen, Bormann, & Cleary, 1993; Podbielski, Flosdorff, & Weber-Heynemann, 1995; McIver & Myles, 2002). These domains have been shown to be required for DNA binding and the activation of transcription for all genes studied (Vahling & McIver, 2006; McIver & Myles, 2002). While HTH-4 is absolutely essential to these processes, HTH-3 appears to serve more of an accessory role during autoactivation. These HTH domains appear to be present in other virulence regulators that do not share homology throughout the entire length of the protein with Mga, such as the RALPs. This has led to the definition of the specific Pfam domains “HTH_Mga” and “Mga,” which encompass HTH-3 and HTH-4, respectively (Finn, et al., 2006). An in silico analysis comparing Mga to proteins of known structure found in the SCOP database found established Mga DNA-binding domains (Andreeva, Howorth, Brenner, Hubbard, Chothia, & Murzin, 2004; Vahling, 2006), as well as two regions within Mga that were predicted to have strong homology to the dual PTS regulatory domains (PRD) in the antiterminator LicT (Hondorp & McIver, 2007; Rom, Hart, & McIver, 2021). The overall size and domain structure of Mga, with N-terminal DNA-binding domains and two central PRD domains, closely resembles that of known PRD-containing transcriptional activators such as MtlR (Vahling, 2006). Subsequently, the Pfam database now designates these regions as a “PRD_Mga” domain, which is a member of the PRD superfamily (Finn, et al., 2006). Once the high-level purification of Mga was achieved, gel filtration analyses, analytical ultracentrifugation and co-immunoprecipitation experiments have demonstrated that Mga oligomerizes in solution, and that its ability to oligomerize correlates with transcriptional activation (Hondorp, et al., 2012). Examination of the previously uncharacterized C-terminus detected structural homology to EIIB domains used by the PTS. Truncation analyses of Mga from an M4 and an M1 strain revealed the C-terminal region of Mga is required for oligomerization and in vivo transcriptional activity, but not DNA binding (Hondorp, et al., 2012). Conversely, the DNA binding domains of Mga are necessary, but are insufficient to induce the expression of Mga-regulated transcripts.

As mentioned, Mga contains two central PRD domains that are similar to antiterminators (such as LicT), and activators (such as MtlR and LicR) that are involved in the regulation of sugar metabolism (reviewed in Deutscher, Francke, & Postma, 2006; Hondorp & McIver, 2007; Rom, Hart, & McIver, 2021). The activity of these regulators is modulated by phosphorylation of conserved histidine residues within their PRD domain(s) via the PTS phosphorelay in response to the utilization of different carbohydrate sources, as observed in the B. anthracis Mga homolog, AtxA (Tsvetanova, et al., 2007; Hammerstrom, et al., 2015). An M4 strain with a nonfunctional PTS that results from the deletion of the EI-encoding ptsI gene exhibited a decrease in Mga activity, and characterization of purified Mga showed that it could be phosphorylated at conserved PRD histidines by EI and HPr components of the PTS in vitro (Hondorp, et al., 2013). Investigation of glucose-dependent changes in the Mga regulon using in vitro RNAseq analysis revealed distinct sets of genes were differentially expressed in high glucose (THY medium) versus low glucose (C medium) conditions (Valdes, et al., 2018). Mga regulon genes activated in C medium correlated with those repressed by CcpA. Detected medium-associated changes in the phosphorylation status of Mga support a regulatory framework in which differences in the Mga regulon correspond to the effect of environmental glucose levels on PTS-mediated regulation of Mga (Valdes, et al., 2018). Confirmation that Mga activity is directly influenced by the PTS came from examination of Mga transcriptional activity using alanine (unphosphorylated) and aspartate (phosphomimetic) substitution mutations of conserved Mga PRD histidines, which established that a doubly-phosphorylated PRD1 phosphomimetic Mga (D/DMga4) is completely inactive in vivo and shuts down the expression of the Mga regulon (Hondorp, et al., 2013). Although the D/DMga4 protein is still able to bind DNA in vitro, homo-multimerization of Mga is disrupted and the protein is unable to activate transcription. As a result, carbohydrate availability can be linked to Mga-dependent expression of virulence-related genes. These studies also highlighted the role of a PRD-containing virulence regulator (PCVR) like Mga (Hondorp & McIver, 2007; Rom, Hart, & McIver, 2021) for pathogenesis, as both non-phosphorylated and phosphomimetic PRD1 Mga mutants were attenuated in a model of S. pyogenes invasive skin disease (Hondorp, et al., 2013). A similar study in an M59 serotype background that is highly polymorphic in Mga found that phosphomimetic substitutions at both PRD1 and PRD2 conserved histidine residues are inhibitory to Mga-dependent gene expression and attenuated S. pyogenes virulence by influencing Mga-dependent global gene regulation (Sanson, et al., 2015). Conversely, non-phosphorylation-mimicking alanine substitutions relieved inhibition and the mutants exhibited a wild-type phenotype in a mouse model of necrotizing fasciitis. These studies suggest that adequate regulation (positive and negative) of Mga activity as a PCVR is necessary for virulence and varies between genotypic backgrounds—an important finding, given that PCVRs appear to be widespread among Gram-positive pathogens (Rom, Hart, & McIver, 2021).

Mga expression is both autoregulated and influenced by other regulators. Transcription from Pmga appears not to be autoregulated in response to carbohydrate availability, as sensed by the PTS (Hondorp, et al., 2013). Nonetheless, Pmga is required for growth phase-regulated expression of the Mga regulon, which indicates that environmental regulation still occurs at the level of mga transcription (Okada, Geist, & Caparon, 1993; McIver & Scott, 1997). Such regulation of Pmga does not appear to occur through the direct influence of two-component signal transduction systems (Ribardo, Lambert, & McIver, 2004), although TCSs are involved in inducing transcription of Mga-regulated virulence genes in other serotypes, as was seen in the TrxRS-mediated activation of Pmga demonstrated by analysis of a luciferase reporter fusion in a MGAS5005 (M1) strain (Leday, et al., 2008). As previously indicated, CcpA binds to Pmga and maintains high-level mga expression during exponential growth (Almengor, Kinkel, Day, & McIver, 2007). Deletion analysis of Pmga identified a region overlapping the Mga binding site I that is involved in repression of mga transcription (McIver, Thurman, & Scott, 1999). Some RALPs (i.e., RofA, RALP3 and Nra) are known to repress mga expression (Beckert, Kreikemeyer, & Podbielski, 2001; Podbielski, Woischnik, Leonard, & Schmidt, 1999; Kwinn, et al., 2007). Another regulator, Rgg/RopB (Federle, 2012), has also been shown to repress Mga expression (Chaussee, et al., 2002); however, direct interaction of these at Pmga has not been demonstrated. In contrast, RivR (Ralp4) activity in an M1 strain is able to enhance the activation of mga and Mga-regulated genes both in vitro and in vivo, though this is controversial (Roberts, Churchward, & Scott, 2007; Treviño, Liu, Cao, Ramirez-Peña, & Sumby, 2013; Roberts & Scott, 2007). The inactivation of amrA, which produces a putative Wzx integral membrane sugar exporter, was found to be essential for exponential-phase expression of mga in a serotype M6 strain (Ribardo & McIver, 2003), possibly by altering sugar levels in the cell.

All of these studies suggest a general model for how changes in environmental carbohydrates influence expression of the Mga-regulated virulence repertoire of S. pyogenes (Figure 2). While glucose (the preferred carbohydrate source) is abundantly available, CcpA induction combined with reduced PTS activity and Mga autoregulation maintain the high-level expression of Mga and its regulon. As glucose becomes scarce, induction by CcpA decreases and mga transcription is reduced, but is maintained by autoregulation. As PTS activity increases to supply the bacterium with alternate carbohydrate sources, Mga activity is repressed by the PTS-dependent phosphorylation of its PRD region. Other factors like Rgg and RALPs, whose regulatory activity is mostly exerted under the stationary phase of growth when carbohydrate energy sources are exhausted, further maintain the repression of Mga until environmental carbohydrate supplies become available. Modulation of RivR by the CovR/S master regulator to influence Mga activity serves to further fine-tune the expression of the Mga regulon. Naturally, this is an oversimplified model that fails to account for the contributions made by different genomic backgrounds, the organization of regulatory regions, or the input of master regulators in controlling the Mga regulon. However, this model might be useful in guiding elucidation of the complex virulence gene regulatory network influencing S. pyogenes pathogenesis in response to changes in carbohydrate availability, which is an area of increasing research interest.

Figure 2.

Mga regulation exemplifies phase and metabolite-dependent regulation in S. pyogenes: Mga expression and activity is growth phase-dependent and is influenced by carbohydrate availability (indicated by decreasing glucose levels). The regulation of Mga involves (more...)

Rsh: stringent and relaxed responses to amino acid starvation

Originally cloned and sequenced (Mechold, Steiner, Vettermann, & Malke, 1993), as well as structurally and functionally characterized (Hogg, Mechold, Malke, Cashel, & Hilgenfeld, 2004; Mechold, Cashel, Steiner, Gentry, & Malke, 1996; Mechold & Malke, 1997), the stringent factor from S. dysgalactiae subsp. equisimilis (group C streptococci) has proved to be of seminal importance to the subsequent establishment of the RelA/SpoT homolog (Rsh) superfamily of bifunctional guanosine 5’-3’ polyphosphate ((p)ppGpp) synthetases and hydrolases and their distribution and function across all living organisms (Atkinson, Tenson, & Hauryliuk, 2011)). Given the amino acid sequence identities of the order of 95% (97% similarity) between the group C streptococci query sequence (739 residues) and the available S. pyogenes sequences, as well as identical linkage relationships of the respective genomic regions (Ferretti, et al., 2001; Mechold, Steiner, Vettermann, & Malke, 1993), all basic properties established for the group C streptococcus protein can also safely be taken to apply to the S. pyogenes homolog. This assumption was also confirmed in a recent review on S. dysgalactiae (Alves-Barroco, et al., 2021) and, henceforth, the GCS protein and its S. pyogenes homolog will be specified as RshStr. Like all full-length Rsh proteins, RshStr synthesizes the alarmone (p)ppGpp at the expense of ATP and GTP when cognate, but deacylated tRNAs occupy the aminoacyl acceptor site of the ribosomes (Hogg, Mechold, Malke, Cashel, & Hilgenfeld, 2004; Mechold, Cashel, Steiner, Gentry, & Malke, 1996; Mechold & Malke, 1997). Consequently, ribosomal complexes on mRNA templates are stalled, which prevents wasteful macromolecular syntheses by a rapid shutdown of futile RNA synthesis in stressful metabolic conditions, like amino acid starvation. Alarmone signaling pathways are primarily directed towards transcriptional inhibition of the stable RNAs, such as rRNA and tRNA (the so-called stringent response, reviewed in (Potrykus & Cashel, 2008). Accordingly, under specific nutritional stress conditions, S. pyogenes synthetase-deficient RshStr insertion mutants with rshStr truncated at codons 220 or 129 show survival rates about three orders of magnitude lower than wild types. The basic reason for this deficiency is the energetically wasteful continuation of RNA synthesis during amino acid deprivation (the so-called relaxed response (Steiner & Malke, 2000; Mechold & Malke, 1997; Potrykus & Cashel, 2008)).

As shown by functional RshStr analyses, this enzyme has also a strong (p)ppGpp hydrolytic activity that gives rise to GTP/GDP plus pyrophosphate (Mechold, Cashel, Steiner, Gentry, & Malke, 1996). The first-order rate constants of (p)ppGpp turnover reflect short half-lives of 20 and 60 sec for the penta- and tetraphosphates, respectively, of the alarmone, which enables the cells to recover quickly from the stasis-like survival state under improved nutritional conditions (Mechold, Cashel, Steiner, Gentry, & Malke, 1996). Defined RshStr fragments created by cloning and functionally analyzed by enzymatic activity assays showed that the two opposing activities reside in the 1-385 residue N-terminal half, bridged by an exposed hinge region to the C-terminal half, which is suggested to be involved in regulatory intramolecular interactions or intermolecular interactions with the ribosome (Atkinson, Tenson, & Hauryliuk, 2011; Mechold, Murphy, Brown, & Cashel, 2002). Dissecting the N-terminal half more precisely by a nested deletion analysis, combined with enzymatic activity assays of purified peptide fragments, revealed non-overlapping minimal fragments of residues 1-198 and 211-347 for full hydrolytic and synthetic activities, respectively (Mechold, 2009).

One challenging problem posed by the opposing enzymatic activities of RshStr concerned the prevention of futile cycling of the alarmone metabolism, which would waste ATP and hamper the rapid adjustment of (p)ppGpp levels as dictated by the prevailing niches of infection in vivo and indeed demonstrated in vitro (Mechold & Malke, 1997; Mechold, Murphy, Brown, & Cashel, 2002) and Figure 3). However, the resolved X-ray structure of the catalytic RshStr region (residues 1-385), which is the first to be established for any Rsh homolog, presented this fragment in distinct hydrolase-OFF/synthetase-ON and hydrolase-ON/synthetase-OFF conformations (Hogg, Mechold, Malke, Cashel, & Hilgenfeld, 2004). The structure localized the opposing enzymatic activities >30 A apart and rendered them mutually exclusive by a combination of induced fit principles and nucleotide ligand-induced intramolecular allosteric signaling between the opposing active sites (Hogg, Mechold, Malke, Cashel, & Hilgenfeld, 2004; Mechold, 2009). As a result, structural antagonism rules out the in vivo possibility of simultaneous (p)ppGpp synthesis and hydrolysis.

Figure 3.

Synthesis and hydrolysis of (p)ppGpp catalyzed by bifunctional RshStr.

Continuing this line of research, researchers (Mechold, Potrykus, Murphy, Murakami, & Cashel, 2013) have developed strategies that enable them to fine-tune the differential accumulation of pppGpp and ppGpp by activating different RshStr synthetic fragments in combination with E. coli pppGpp 5’-gamma phosphate hydrolase to get rid of the pentaphosphate and generate only ppGpp. They found that ppGpp is a more efficient regulator than pppGpp with respect to a plethora of five stress responses, with the most important one in the present context being rRNA synthesis regulation. Reducing the aura of the alarmone even further, multiple groups found that both nucleotides bind to a site at the interface between the beta’ and omega RNAP subunits (Ross, Vrentas, SanchezVasquez, Gaal, & Gourse, 2013; Zuo, Wang, & Steitz, 2013). This finding supports a model where ppGpp connects the core and the shelf of RNAP to form a mobile clamp around the DNA. By controlling the opening and closing of the clamp through an allosteric mechanism, ppGpp modulates RNAP activity. However, since the structural results were obtained with RNAP holoenzyme from E. coli, it remains to be seen if RNAP from S. pyogenes will behave in a similar fashion.

Apart from its primary function of stable RNA synthesis inhibition, (p)ppGpp also affects negatively the transcription of important groups of protein-encoding S. pyogenes genes, including regulators, virulence factors and genes for transport systems (Malke, Steiner, McShan, & Ferretti, 2006). Of pivotal importance is the strong negative stringent control of rshstr itself. Exposure to isoleucine plus valine starvation results in 40-fold changes in rshstr expression in an rshstr mutant, relative to the wild type. This high degree of negative autoregulation may be necessary for a gene with broad connectivity in order to sustain cellular homeostasis. Among 31 additional genes studied for their expression during the stringent response (and found to be strongly negatively controlled) were the virulence factors graB (see above) and speH encoding the superantigen pyrogenic exotoxin H. An approximately tenfold expression increase under relaxed conditions was also observed for the important branched-chain amino acid (BCAA, see below) transporter gene braB, which reflects a cost-effective means for the repression of a futile transport process under nutrient-rich conditions.

CodY: (p)ppGpp-independent response to amino acid starvation.

Experiments in which both S. pyogenes wild type and rshStr mutant strains were exposed to BCAA starvation led to the unexpected discovery of an rshStr-independent mode of global transcriptional regulation (Steiner & Malke, 2000; Steiner & Malke, 2001). This regulatory response to the deprivation of the essential amino acids isoleucine and valine is characterized by the up-regulation of a wide array of housekeeping genes, as well as accessory and dedicated virulence factors in both isogenic genotypes. Up-regulation under these conditions also applies, for example, to the CovRS regulation of virulence gene expression in a global fashion. Importantly, of the operons affected by the rshStr-independent response, the oligopeptide permease (opp), FAS regulatory system (fasBCAX) and streptolysin S operon (sag), exhibit internal transcriptional termination under nutritionally rich conditions. However, BCAA-starvation promotes full-length transcription of these operons, which supports the level of coordinate transcription of functionally related genes and contributes to the efficacy of these systems under stressful conditions. When all effects are considered, the rshStr-independent response to BCAA limitation appears to counteract the stringent response and enables S. pyogenes dynamically to exploit the protein-rich niches of their host to cope with stasis states under nutritional stress conditions.

The results summarized above were generated before anything was known about CodY action in S pyogenes. BCAAs had been shown to activate the pleiotropic repressor CodY in Lactococcus lactis (Guédon, Serror, Ehrlich, Renault, & Delorme, 2001), as well as in Bacillus subtilis (Shivers & Sonenshein, 2004). The codY of S. pyogenes was subsequently inactivated in strain NZ131 (M49) and experiments were performed to find possible instances of the codY mutants mimicking rshStr-independent stimulation of transcription upon BCAA deprivation under a variety of culture conditions, such as growth media and growth phases (Malke, Steiner, McShan, & Ferretti, 2006; Malke & Ferretti, 2007). Given the broad regulatory connectivity of codY among the Firmicutes (Sonenshein, 2007), CodY action could be thought to involve both direct and indirect effects. Direct action was suggested by finding the 15-base pair CodY binding sequence, as defined in Lactococcus lactis (den Hengst, et al., 2005; Guédon, Sperandio, Pons, Ehrlich, & Renault, 2005) upstream of several NZ131 genes, including the opp operon, the braB gene, and the negatively autoregulated codY gene itself (Malke & Ferretti, 2007). These findings obtained in silico were substantiated by electrophoretic mobility shift assays (EMSAs), which provided direct evidence for the function of the codY box in S. pyogenes (Kreth, Chen, Ferretti, & Malke, 2011). However, how much degeneracy of the consensus sequence is tolerated for function is still not known, given that a putative CodY-binding site with 4 mismatches (including loss of the inverted repeat of the box) was identified in the mga promoter (Flores, et al., 2013).

Concerning the fact that high BCAA levels render CodY a more active repressor, a unique feature of the S. pyogenes homolog deserves further consideration. Northern analysis showed for two strains, NZ131 (M49) and SF370 (M1), that codY is transcribed both monocistronically from its own promoter and dicistronically from the promoter of the co-oriented upstream aat gene that encodes aspartate aminotransferase (Malke, Steiner, McShan, & Ferretti, 2006). This implies that monocistronic transcript levels are down regulated at low BCAA levels (namely, under nutritional stress conditions) and are counterbalanced by the BCAA-independent dicistronic mode of codY transcription, with the caveat that nothing is known about the control of aat transcription.

Using quantitative RT-PCR, (qPCR), a selected set of about 50 NZ131 (M49) genes was studied for differential expression under various conditions. This included regulators with known targets, established virulence factors, physiologically important transporters, and metabolic enzyme genes (Malke, Steiner, McShan, & Ferretti, 2006; Malke & Ferretti, 2007; Malke, McShan, & Ferretti, 2009). The results revealed that CodY and RshStr did not significantly affect the expression of each other; instead, they appeared to act independently, as indicated by an rshStr-codY2 double mutant that showed a composite transcription pattern of the gene sets for the single mutants (Malke, Steiner, McShan, & Ferretti, 2006). Additional support for an independent action of the two regulatory systems was provided by their additive effects in the double mutant regarding the expression patterns of braB and graB, which are already strongly affected in both single mutants under specific conditions (Malke, Steiner, McShan, & Ferretti, 2006). Overall, specific transcription units from all four groups previously mentioned were under CodY control, often in a medium and growth-phase dependent manner. In particular, growth in human blood apparently involves environmental cues that are absent in laboratory media, which leads to partially discordant CodY-directed transcription profiles. The impact on transcription regulators (covRS, fasX, mga, sptRS) amplifies the connectivity of CodY and provides bona fide evidence for the indirect CodY regulation of diverse sets of genes. Virulence genes (cfa, emm49, graB, hasA, ideS, nga, prtS, sagA, scl, scpA, ska, slo, sof, speH), were shown to be under the control of CodY, presumably because their direct regulators (mainly covRS and mga) are in the CodY regulon. Therefore, CodY tends to upregulate the positive regulator Mga and to down-regulate the negative regulator CovRS, which indirectly enhances the expression of a broad range of virulence factors. Notably, a number of these up regulated virulence factors (such as Cfa, Ids, Nga, PrtS, ScpA, and Slo) also serve to release nutrients from the host (Malke & Ferretti, 2007). Transporter gene expression (braB, dppA, malX, opp, pbuX, ptsG, pyrP) turned out to be highly specific to the individual systems, with braB and opp being most strongly affected in a cost-effective manner, as accounted for by the culture conditions.

Regarding the metabolic enzyme genes, CodY-mediated control of the arcABC operon involved in the catabolism of arginine is particularly relevant, because it serves to integrate basic nitrogen metabolic and virulence pathways by yielding ammonia and ATP, as well as leading to host-generated nitric oxide production. Ammonia production supports survival of the cells in acidic conditions, such as those found within phagolysosomes (Degnan, et al., 2000), and nitric oxide mediates host innate immunity (Cusumano, Watson, Jr., & Caparon, 2014). Experiments carried out in near-natural conditions, namely with NZ131 (M49) and its isogenic codY1 mutant cultured in heparinized human blood and sampled at different points during the course of growth, found that arcABC transcript levels up regulated in the codY1 mutant decrease dynamically during growth (Malke, McShan, & Ferretti, 2009) and imply that strong CodY repressor activity occurs preferentially during the first hours of exposure to blood, when effective BCAA concentrations are still available. This activity does not appear to be directly or indirectly influenced by CodY action on the arc operon regulators argR and crp (Malke, McShan, & Ferretti, 2009).

Interaction of the global CodY and CovRS regulatory systems

Given that CodY and CovRS show a broad overlap regarding their target genes, their regulons were dissected using NZ131 and its codY1, covRS and codY1-covRS mutants grown in C medium (Loughman & Caparon, 2006b) to mid-exponential (ME) and early stationary (ST) phase. In contrast to the nonpolar codY1, covRS disrupting the operon at codon 174 is polar and inactivates both covR and the downstream covS, which prevents the possible cross talk of CovS with other regulators (Steiner & Malke, 2000). Transcriptome analysis using DNA microarrays (Kreth, Chen, Ferretti, & Malke, 2011) showed that CodY controls the transcription of about 17% of the core genome (1,605 genes, excluding prophage), which is a finding comparable to the 15% controlled by CovRS (Graham, et al., 2002). Regarding the action of the two systems on virulence gene control, CovR-directed repression generally appears to be stronger than CodY-mediated activation. This can be seen in Figure 4 by looking at the phenotypes of the four strains, with respect to capsule production (hasABC).

Figure 4.

Comparison of the colony morphology of NZ131 codY and covR mutants: colonies of wild-type NZ131 were compared to that of the indicated mutants grown on C-agar (Loughman & Caparon, 2006b). Figures reflect relative amounts of capsule formation; (more...)

The most intriguing results concerning the relationship between the actions of CodY and CovRS were observed upon robust correlation analysis of the DNA microarray results (Kreth, Chen, Ferretti, & Malke, 2011). This revealed a strong and highly significant negative correlation between the action of CodY and CovRS (r(1603) = -0.40; p<0.0001) in ME phase cells. The negative correlation is relieved in the double mutant, but stays slightly negative (r(1603) = -0.16; p<0.0001), which indicates that CovRS-directed repression overrides CodY-mediated gene activation. When ST phase cells were subjected to the same analysis, the negative correlations between the actions of the two systems ceased to operate. This was particularly evident under expression threshold parameters (Kreth, Chen, Ferretti, & Malke, 2011) where the low r values did not reach significance.

Therefore, CodY and CovRS appear to act in opposite directions, with the expression-balancing effect being largely restricted to actively growing cells. This leads to intermediate levels of not only virulence gene transcripts, but also lessens full transcriptional control of a substantial fraction of the whole genome. This could be due to CodY and Mga governing about 17% and 10%, of the genome, respectively (Hondorp & McIver, 2007; Kreth, Chen, Ferretti, & Malke, 2011). In light of the Red Queen principle, the interaction of CodY and CovRS may indicate that the host-pathogen association has coevolved to become less harmful to either partner.

VicR/S

VicR/S is one of the TCSs found in S. pyogenes and its orthologs in other Gram-positive pathogens appear to be essential (Fabret & Hoch, 1998; Lange, et al., 1999; Martin, Li, Sun, Biek, & Schmid, 1999). Work from several groups has indicated that vicR is essential to some S. pyogenes serotypes, including the 5448 (M1T1) strain (Le Breton, et al., 2015); however, the construction of an insertional inactivation mutant of vicR in an MGAS5005 (M1T1) strain was successful (Liu, et al., 2006). Characterization of this strain revealed that VicR induces expression of 13 genes, including those of virulence factors like the Mac/IgG endopeptidase (Mac/IdeS), a putative cell wall hydrolase gene (pcsB), and an operon that encodes a putative PTS mannose/fructose family EII (spy1058-1060) for carbohydrate transport. VicR also represses the expression of 5 genes, including ones that encode the osmoprotectant transporter OpuA (Liu, et al., 2006). The VicR-deficient strain showed no growth defects in rich culture media or any changes in susceptibility to phagocytic killing. However, the mutant grew poorly in non-immune human blood and serum and showed attenuated virulence in an intraperitoneal model of infection, which suggests that VicR/S influences virulence only in a specific host tissue environment. Admittedly, the insertional inactivation of vicR examined was unstable in vivo (Liu, et al., 2006). Nonetheless, further confirmation of the role of VicR/S as a transcriptional regulator influencing virulence through modulation of a metabolism-related gene came from deletion by allelic exchange of one of the genes in the PTS EII operon (spy1060) regulated by VicR, which recapitulates the attenuated virulence phenotype of the vicR insertional inactivation. Successful deletion of vicS partially replicates the growth defects and attenuation in vivo of the VicR-deficient strain (Liu, et al., 2006).

Metalloregulation and virulence (MtsR, PerR, CiaHR)

Transition metal ions are involved in a variety of biochemical processes in bacteria necessary for colonization, survival, and proliferation in their environmental niche, which present pathogens with the challenge of overcoming metal ion deprivation within host tissue environments. Metallo-regulatory, or “metal-sensing” proteins have evolved in bacteria to mediate metal ion homeostasis by modulating the expression of genes that encode metal ion transport systems upon binding their cognate metal ion, and emerging literature indicates that, in pathogens like S. pyogenes, such proteins have also been adapted to act as virulence regulators (Merchant & Spatafora, 2014).

Such is the case for MtsR, a transcriptional regulator of the DtxR family of metalloregulators, expressed from a gene proximal to the free iron and manganese cation specific transporter-encoding mtsABC operon (Hanks, et al., 2006). Examination of metallo-regulatory activity in S. pyogenes revealed that the deletion of mtsR abolishes the Mn2+ and Fe3+-induced repression of mtsA expression, as well as the Fe3+-dependent decrease in expression of the heme-specific transporter gene (htsA) (Hanks, et al., 2006). Additionally, MtsR represses the streptococcal iron acquisition (sia) operon during cell growth under conditions of high metal levels by specifically binding the promoter regions of these genes in an iron- and manganese-dependent manner (Hanks, et al., 2006; Bates, Toukoki, Neely, & Eichenbaum, 2005), which clearly establishes the function of MtsR as a metal-responsive transcriptional repressor. Mn occupancy at each of two Mn-sensing sites in MtsR influences its regulatory activity, which involves multimerization on DNA that is critical for virulence in a mouse model of invasive infection (Do, et al., 2019). The first observation that this metallo-regulatory protein is involved in virulence gene regulation in S. pyogenes was made in an M3 serotype strain subclone (MGAS9887), which was isolated from a patient with an invasive infection, and that showed a decreased ability to cause necrotizing fasciitis (Beres, et al., 2006). DNA–DNA microarray hybridization-based comparative genomic resequencing and polymorphism sequencing of the M3 isolates determined that MGAS9887 possesses a single base pair insertion in the mtsR gene just downstream of the start codon, which results in a truncated MtsR protein. The deletion of mtsR results in the deregulation of iron uptake, making S. pyogenes hypersensitive to both streptonigrin and hydrogen peroxide (Bates, Toukoki, Neely, & Eichenbaum, 2005) and attenuated for virulence in intramuscular and intraperitoneal models of infection (Bates, Toukoki, Neely, & Eichenbaum, 2005; Olsen, et al., 2010). Further characterization of the MtsR transcriptome in an mtsR-deficient MGAS315 (M3) strain revealed, in addition to the expected differential expression of genes involved in metal ion transport and oxidative stress, the repression of prsA, which encodes a peptidyl-prolyl isomerase (Olsen, et al., 2010). PrsA is required for proper maturation of the SpeB protease that plays an important role in the pathogenesis of necrotizing fasciitis. Isogenic mutants overexpressing PrsA or deficient in SpeB recapitulated the decreased necrotizing fasciitis phenotype of MtsR-deficient strains, which confirms the role of MtsR in regulating SpeB production.

The regulation of virulence genes by MtsR appears to vary among S. pyogenes serotypes and tissue sites of infection. Epidemiological studies ascribe a representative number of pneumonia, bacteremia, and synovitis cases to the naturally occurring mtsR mutant (MGAS9987), which indicates that the loss of MtsR regulation in an M3 serotype background only affects virulence in the context of necrotizing fasciitis (Merchant & Spatafora, 2014). Examination of an mtsR knockout strain in an NZ131 (M49) strain revealed differential expression of 64 genes involved in metal ion transport, nucleic acid synthesis, and regulation, many of which coincided with those affected in the M3 serotype strain (Toukoki, Gold, McIver, & Eichenbaum, 2010). However, there were a number of dysregulated transcripts exclusive to each strain. Among the MtsR-influenced virulence genes particular to the NZ131 strain were mga, emm, and ska. Further involvement of MtsR in S. pyogenes virulence relates to the management of oxidative stress, which, as detailed in another section of this chapter, must be overcome in order to colonize and persist in multiple host niches. Suffice it to say that the MtsR-dependent maintenance of iron homeostasis supports S. pyogenes virulence by aiding resistance to oxidative stress.

As previously indicated, the metalloregulator MtsR influences virulence gene expression in the context of necrotizing fasciitis in certain strains, in addition to directly repressing the transcription of metal transport operons (mtsABC, htsABC and siaABCD). However, virulence-related regulators besides MtsR also influence these operons. PerR is a transcriptional regulator primarily involved in oxidative stress resistance, a role described in another section of this chapter. PerR activity as a peroxide-responsive transcriptional regulator is inextricably linked to its function as a metalloregulator, as indicated by decreased iron uptake into S. pyogenes cells and reduced transcription of mtsA from the mtsABC operon in a PerR-deficient AP1 (M1) strain (Ricci, Janulczyk, & Björck, 2002). In an M3 isolate, PerR was shown to repress the three known iron and heme acquisition systems of S. pyogenes: the MtsABC iron transporter (Janulczyk, Pallon, & Björck, 1999; Sun, Ge, Chiu, Sun, & He, 2008), the heme/ferrichrome transport system (FhuGBDA or FtsDCBA) (Hanks, et al., 2006), and the Shr/Shp/SiaABCD (also dubbed HtsABC) transporter involved in hemoglobin acquisition (Bates, Montañez, Woods, Vincent, & Eichenbaum, 2003; Lei, et al., 2003; Lei, et al., 2002; Grifantini, Toukoki, Colaprico, & Gryllos, 2011). At the same time, PerR up regulated genes of the AdcRCB transport complex involved in Mn2+/Zn2+ import in streptococci (Grifantini, Toukoki, Colaprico, & Gryllos, 2011; Dintilhac & Claverys, 1997). Upregulation of the AdcR regulon in response to calprotectin-mediated Zn limitation is critical for S. pyogenes virulence in a murine necrotizing fasciitis infection model (Makthal, et al., 2020). The role that regulation of these metal cation transporters has in oxidative stress responses relating to virulence is specified later.

Oxidative and Acid Stress

In addition to the proteins that co-opt host processes for the pathogen’s benefit or subvert host immune responses, virulence factors also include proteins that function to protect the bacterium from environmental stressors in the host, especially those resulting from mechanisms of host immunity. S. pyogenes produces a variety of proteins to sense and respond to oxidative and acid stresses, the chemical stressors primarily encountered within its human host, and both dedicated and general regulators tightly control expression of these proteins.

Virulence-related biofilm regulation (Srv, Rgg2/3)

For an extensive review on current evidence of S. pyogenes biofilm formation, modelling S. pyogenes biofilms, and the contribution of S. pyogenes virulence factors to formation of biofilm, see (Vyas, Proctor, McArthur, Gorman, & Sanderson-Smith, 2019).

Growth phase/quorum sensing-mediated virulence gene control (Rgg/RopB, Rgg2/3, Sil, TrxSR, PepO)

A theme running through this chapter is that S. pyogenes continuously senses the conditions of its surrounding environment and simultaneously regulates the expression of its genomes to maintain and enhance its survival. Batch culture is the simplest method to investigate such bacterial adaptation to the depletion of sources of energy and resources for growth, as well as the accumulation of metabolic end products. S. pyogenes exhibits two primary growth phases in culture: exponential and stationary. The former is characterized by preferential use of glucose as the source of energy that is fermented to lactic acid, continuous cell division, expansion of the bacterial population and the expression of a regulatory program corresponding to the prevailing metabolic status of the cell (Chaussee, Dmitriev, Callegari, & Chaussee, 2008). The stationary phase of growth is evidenced by the depletion of glucose, acidification of the media, the arrest of cell division, and population growth and transition into a radically different regulatory program. Examination of the growth phase-dependent changes in gene expression in S. pyogenes has revealed that, unlike other bacteria that employ a variety of alternative sigma factors to modulate gene expression in response to changes in the environment (Helmann & Moran, Jr., 2001; Beyer-Sehlmeyer, Kreikemeyer, Hörster, & Podbielski, 2005), S. pyogenes alters the growth phase-associated patterns of gene expression through interactions among transcriptional regulators. Therefore, growth phase-dependent regulation of S. pyogenes virulence involves global regulators (such as CovR/S or RALPs), metabolite responsive regulators (namely, CodY and Mga) and dedicated regulators that have been observed to have genome-wide effects in response to changing growth phases.

One such dedicated virulence regulator, which was first identified by its influence on growth-phase dependent SpeB production, is the Rgg-family transcriptional regulator Rgg/RopB, which induces maximal expression of the extracellular SpeB protease (Chaussee, Ajdic, & Ferretti, 1999; Lyon, Gibson, & Caparon, 1998) during stationary phase growth (Unnikrishnan, Cohen, & Sriskandan, 1999). Rgg/RopB is thus a central component of the cell density-associated regulation of SpeB expression. The SpeB-inducing peptide (SIP) intercellular signaling system employs an eight-amino acid leaderless peptide to influence global gene expression through Rgg/RopB (Do, et al., 2017). S. pyogenes produce SIP, encoded in the intergenic region between ropB and speB, during growth at high cell density. The cell-to-cell SIP signaling pathway involves synthesis and secretion of SIP and reimportation of the peptide into the cytosol, followed by SIP interaction with Rgg/RopB, which in turn results in SIP-dependent modulation of RopB/DNA interaction and regulatory activity that terminates in RopB-mediated induction of speB expression (Do, et al., 2017). SIP signaling appears to be functional during S. pyogenes growth in human saliva and blood, and SIP-mediated speB expression contributes to S. pyogenes colonization of the mouse oropharynx, as well as survival in human blood (Makthal, et al., 2018).

Investigation of the mechanism by which SIP signaling influences RopB-mediated induction of SpeB expression revealed a pH-sensing mechanism involving a pH-sensitive histidine switch of the SIP-binding pocket of RopB (Do, et al., 2019). Environmental acidification induces protonation of the His¹⁴⁴ residue and reorganization of hydrogen bonding networks in RopB to enhance SIP recognition. Proteomic and qPCR approaches to analyze the influence of Rgg/RopB on the expression of other extracellular proteins in a SpeB-deficient NZ131 (M49) strain found that, during the stationary phase of growth, the loss of Rgg/RopB expression led to a decreased transcription of genes that encode lysozyme, autolysin and ClpB (a heat shock protein), but produced more streptodornase (DNase encoded by sdb/mf) and a DNA entry nuclease (Chaussee, Watson, Smoot, & Musser, 2001). As described in an earlier section, transcriptome analysis using DNA microarrays revealed that Rgg/RopB affects the stationary-phase expression of amino acid metabolic genes. Further characterization of rgg inactivation in the NZ131 (M49) strain found Rgg/RopB also controls amino acid and non-glucose carbohydrate metabolism genes (Chaussee, Somerville, Reitzer, & Musser, 2003; Chaussee, Callegari, & Chaussee, 2004; Dmitriev, et al., 2006). These studies also uncovered that transcripts that encode virulence factors involved in cytolysin-mediated translocation of NAD-glycohydrolase (spn), including the immunity factor (ifs) and streptolysin O (slo), were more abundant in the rgg-deficient strain, which correlated to increased levels of NADase (SPN) and SLO activity in both exponential and stationary growth phases (Dmitriev, et al., 2006). These data suggested that Rgg/RopB both directly and indirectly exerts growth-phase dependent control of virulence proteins and that it represses the transcription of spn, ifs, and slo during the exponential phase of growth, as well as controlling the degradation of the secreted proteins during the stationary phase by inducing the expression of the SpeB protease. More recent transcriptomic analyses report differential expression of ∼14% of the S. pyogenes genome, including upregulation of virulence factors (e.g. Spi, SagP, NdoS, and Sdn streptodornase) that promote pathogen dissemination and infection. Do et al. demonstrated that SIP system signaling and RopB-mediated induction of SpeB is required for S. pyogenes virulence in various murine infection models (Do, et al., 2017) and is conserved across various S. pyogenes serotypes (Makthal, et al., 2018).

Growth-phase–dependent changes in gene expression in S. pyogenes involve connections between Rgg and more globally acting regulatory networks. The inactivation of rgg in NZ131 (M49) increases the transcription of several virulence-associated genes, including speH (which encodes a superantigen), scl1/sclA (which encodes an adhesion protein), ska, hasABC, mf, mf3, grab, and mac, in addition to targets of the Mga regulon (emm, scpA, sagA, and slo) (Chaussee, et al., 2002). The altered expression of these virulence genes correlates with changes in the transcription of several regulatory genes, including mga, covR/S, and fasBCAX; indicating that Rgg/RopB influences other global regulators to exert its genome-wide modulation of virulence. Further evidence of this is in the increased transcription of the TCS genes lytR and lytS in an rgg-deficient strain during the exponential growth phase; in the stationary phase of growth, the transcripts of FasB, CpsY, and the RALP4 (RivR) regulator were less abundant in the mutant strain (Dmitriev, McDowell, Kappeler, Chaussee, Rieck, & Chaussee, 2006). However, inter-serotype and intra-serotype variations are a common feature of the Rgg/RopB regulon. The sequencing of rgg/ropB in 171 invasive serotype M3 strains identified 19 distinct alleles (Carroll, et al., 2011). The observed rgg/ropB polymorphisms alter regulator function, as inactivation of the gene in strains producing distinct Rgg/RopB variants had dramatically divergent effects on global gene expression. Iso-allelic S. pyogenes strains that differed by as little as a single amino acid in Rgg/RopB all exhibited differing transcript levels of speB. Comparison of parental, rgg/ropB-inactivated, and rgg/ropB iso-allelic strains in mouse infection models manifested differences in virulence and disease manifestations (Carroll, et al., 2011). Additionally, rgg-deficient mutants in three different strains, which represented M1 (SF370 and MGAS5005) and M49 (CS101) serotypes, all exhibited different changes in gene expression in the stationary phase of growth (Dmitriev, McDowell, & Chaussee, 2008). Specifically, the inactivation of rgg alters transcription of regulatory genes in SF370 and CS101 strains, but not in MGAS5005. These studies identified speB and an adjacent hypothetical gene (spy2040; later precisely defined as the SIP coding region (Do, et al., 2017) as the only transcripts similarly affected by rgg inactivation in all three strains. Nevertheless, a strain-specific Rgg/RopB subregulon was also detected, which could vary by as little as a single gene (nrdD) in the MGAS5005 strain, and by as much as 43 genes in SF370 (Dmitriev, McDowell, & Chaussee, 2008).

Given such variation among strains for Rgg/RopB-dependent gene regulation, it was important to define the mechanism of Rgg/RopB-mediated control of transcription in S. pyogenes. Chromatin immunoprecipitation followed by DNA microarray (ChIP-chip) analysis identified 65 DNA binding sites for Rgg in the NZ131 chromosome: 35 of them within noncoding DNA and 43% of which were adjacent to genes previously identified as regulated by Rgg (Anbalagan, McShan, Dunman, & Chaussee, 2011). EMSA demonstrated Rgg binding to noncoding DNA upstream of speB, and the genes that encode PulA, Spd-3, a predicted transcriptional regulator (Spy49_1113), prophage-associated genes that encode a putative integrase (Spy49_0746) and a surface antigen (Spy49_0396), both in vivo and in vitro. Rgg-mediated transcriptional regulation of these genes via an active promoter was confirmed using a luciferase reporter system (Anbalagan, McShan, Dunman, & Chaussee, 2011). Further characterization of Rgg-mediated promoter activation found that Rgg is unable to bind to the speB promoter to induce transcription when bound to LacD.1 during exponential growth, but as levels of glycolytic intermediates decrease upon entry into stationary phase, a change in LacD.1 conformation results in dissociation from Rgg and activation of SpeB expression (Loughman & Caparon, 2006a). This LacD.1-dependent effect on the DNA binding specificity of Rgg in response to changes in environmental carbohydrates was not limited to SpeB production, as demonstrated for emm49, sof, sfbX49, and speH genes in NZ131 (M49) using in vitro DNA-binding and in vivo transcriptional fusion assays (Anbalagan, Dmitriev, McShan, Dunman, & Chaussee, 2012). These results further support the paradigm that growth-phase–dependent gene modulation in S. pyogenes is based on the interactions of multiple transcriptional regulators, which vary greatly among strains, and which necessitates further examination of the manner in which regulatory networks are structured in different serotypes in order to understand strain differences and similarities when infecting a host.

Saliva

Multiple studies have examined virulence factor expression and transcriptional changes of S. pyogenes in body fluids, including saliva (Graham, et al., 2005; Shelburne, et al., 2005a; Shelburne, et al., 2005b). An MGAS5005 strain exposed to human saliva produces a variety of factors in a growth-phase dependent manner, including Sic, SpeB, streptococcal pyrogenic exotoxin A, Mac, streptococcal phospholipase A2, the lantibiotic salivaricin A (SalA), DNases, streptolysin S, and the 67-kDa myosin cross-reactive protein (spy0470) (Shelburne, et al., 2005a; Shelburne, et al., 2005b). Transcriptome profiling during exponential and stationary growth phases in blood identified a two component regulatory system, termed SptR/S, as essential to the persistence of S. pyogenes in human saliva (Shelburne, et al., 2005a). SptR/S acts as a transcriptional inducer of numerous virulence genes, including spd, spd3, sic, hasA, and speB, as well as genes that encode complex carbohydrate transporters and utilization enzymes. During exponential growth in saliva transcripts involved in pathogen-host interactions (such as emm and sic), oxidative stress response (e.g., ahpC and mtsA), ATP generation and pH balancing (such as the arginine deiminase operon) are up regulated, in accordance with the nutritional limitations and the low pH found in saliva; whereas in the stationary growth phase, starch-degrading and carbohydrate-metabolism genes are differentially regulated, either positively (malX, malE, amyA, agaD) or negatively (pulA, malE) (Shelburne, et al., 2005a). Additional virulence regulators observed to display activity in the saliva environment include RofA and several stress-response regulators (PerR, HrcA, Spy0583 and Ihk/Irr), which are all up regulated. Additional studies that examined the role of other virulence regulators found that CcpA and CovR influence gene expression in response to saliva, as it was observed that a ccpA-deficient MGAS2221 (M1) strain failed to induce the expression of speB, sagA, and arcA upon exposure to saliva and that a covR-deficient mutant in the same background exhibited reduced induction of these genes, as well as spyCEP, slo, and amyA, when grown in human saliva (Shelburne, et al., 2010).

Salivaricin A is one of the S. pyogenes factors induced in the presence of saliva. The salivaricin (sal) locus of S. pyogenes is ∼85% homologous to the sal locus of Streptococcus salivarius, the commensal bacterium in which SalA was originally identified. Paradoxically, only M4 serotype strains produce active SalA and are immune to the lantibiotic produced by S. salivarius, yet the sal locus is highly conserved (>92% homology) among sequenced S. pyogenes serotypes (Phelps & Neely, 2005; Upton, Tagg, Wescombe, & Jenkinson, 2001), suggesting an alternative function for the sal operon in S. pyogenes pathogenesis. In general, lantibiotic loci are transcribed as operons. In addition to the pre-lantibiotic peptide, these operons encode modifying enzymes to produce the mature lantibiotic, an immunity protein or specialized ABC transporter to provide resistance to the lantibiotic and a TCS to regulate expression of the operon (Willey & van der Donk, 2007). Transcription of the sal locus in S. pyogenes was determined to occur from two promoters, one of which is upstream of the operon. The other one is an internal promoter in the coding region of salY, directly upstream of the salKR genes that encode the putative TCS regulating expression of the sal locus (Namprachan-Frantz, Rowe, Runft, & Neely, 2014). Transposon insertions into salY and salK, as well as in frame deletion of salK, resulted in attenuated virulence of an HSC5 (M14) strain in the zebrafish infection model (Phelps & Neely, 2005). Additionally, the product of salR was found to repress activity of the salKR promoter, as did SalA, whereas human serum increased expression from the promoter (Namprachan-Frantz, Rowe, Runft, & Neely, 2014). Altogether, this indicates that regulation of the sal operon is complex and involves multiple inputs, while its activity influences S. pyogenes virulence through some as yet uncharacterized mechanisms.

Another regulator found to be involved in virulence in the context of human saliva is the maltose repressor, MalR, which, like CcpA, is a LacI/GalR family member. Insertional inactivation of MalR in the MGAS2221 strain reduced colonization of the mouse orophyarnx, but did not detrimentally affect invasive infection (Shelburne, et al., 2011), which suggests a specificity of MalR activity for the mucous membrane environment. The observed MalR transcriptome is limited to only 25 genes, and a highly conserved MalR DNA-binding sequence required for DNA interaction in vitro was identified. Inactivation of one of the transcriptional targets of MalR, the cell wall anchored carbohydrate binding and degrading enzyme encoded by pulA, significantly reduced S. pyogenes adhesion to human epithelial cells and mouse oropharyngeal colonization (Shelburne, et al., 2011).

Blood

Severe invasive infection by S. pyogenes, which involves its dissemination into blood and other sterile deep tissue sites, is most frequently caused by strains of serotypes M1T1, M3, and M12 (O'Loughlin, et al., 2007). Clinical isolates of these invasive strains exhibit hypervirulence and an enhanced capacity to invade soft tissues and evade neutrophil responses, in contrast with the more commonplace pharyngitis isolates (Kansal, et al., 2010; Li, et al., 2013). Thus, it has been of great importance to elucidate the virulence determinants required for S. pyogenes dissemination and survival in blood.

Characterization of the MGAS5005 (M1) transcript profile during growth in human blood revealed that the transcriptome of S. pyogenes undergoes extensive modulation in this environment (Graham, et al., 2005). Three-quarters (n = 1467) of encoded genes were differentially transcribed, with changes in expression that indicate the broad metabolic adaptation of S. pyogenes to the blood environment, shutting down glycolysis and activating amino acid-fermentation pathways. Induced virulence genes included Mga-dependent (such as emm and sic) and Mga-independent genes (hasABC, sagA, mac, slo, speA, speC, and smeZ), whereas speB and crgR transcripts were less abundant. In terms of regulation, exposure to blood resulted in the coordinated accumulation of mga, rofA, fasBCA and ihk/irr transcripts, as well as a temporal increase in covR/S expression. From these data, a regulatory model of S. pyogenes adaptation to the blood environment, composed of three phases, was proposed: persistence, adaptation, and dissemination. Persistence is mainly characterized by immune evasion through the up-regulation of mga and fasBCA, as well as down-regulation of crgR. Adaptation is typified by initiating cell contact, proteolysis, and peptide scavenging (along with up-regulation of covRS and ihk/ irr) and dissemination was defined as tissue destruction, cell necrosis, and apoptosis, accompanied by the up-regulation of RALPs and pyrogenic toxin antigens.

Further analysis of S. pyogenes virulence gene regulation in blood involved the examination of 51 gene transcripts in a codY-deficient NZ131 (M49) strain, which revealed differential responses for 26 genes that occasionally differed from responses seen in laboratory media (Malke & Ferretti, 2007). Differentially expressed regulators in this study included covR, fasX, mga, and sptR/S. Virulence genes with altered expression included cfa, emm49, grab, hasA, ideS, nga, prtS, scl, scpA, ska, slo, sof, and speH, as did genes encoding amino acid transporters and metabolic enzymes. Degenerate derivatives of the CodY binding box potentially serving as a cis-regulatory element for CodY regulation were identified in the upstream regions of 15 genes of the NZ131 genome, and these genes featured sequence motifs identical to the NZ131 CodY box in all completely sequenced S. pyogenes genomes. However, these genes consisted almost exclusively of metabolic and transporter encoding ORFs rather than virulence factors or regulators, which suggests that the observed differential transcription of the majority of virulence genes was caused by the indirect regulation by CodY of the S. pyogenes regulatory network.

Efforts have been recently made to identify the essential genes necessary for the survival of S. pyogenes in blood. Transposon-site hybridization (TraSH) analysis of a complex mariner-based transposon mutant library in a 5448 (M1T1) strain subjected to negative selection in human blood identified 81 genes that are important to S. pyogenes fitness in the blood environment (Le Breton, et al., 2013). This approach found regulatory genes already known to play a role in the survival of S. pyogenes in blood, such as mga, perR, and ralp3, as well as genes previously reported for their contribution to sepsis in other pathogens, such as genes involved in de novo nucleotide synthesis (purD, purA, pyrB, carA, carB, guaB), sugar metabolism (scrB, fruA), zinc uptake (adcC), and transcriptional regulators (cpsY). The LysR family transcriptional regulator CpsY is a transcriptional regulator found in several streptococci that regulate methionine transport and amino acid metabolism (MetR, MtaR). In S. pyogenes, CpsY is involved in resistance to human neutrophils and a ∆cpsY M1 strain does not exhibit the in vitro phenotypes of homologous mutants in other streptococcal species (Vega, et al., 2017). Furthermore, the S. pyogenes CpsY regulon is distinct from the regulons of its homologs and includes known virulence factors (mntE, speB, spd, nga/spn, prtS/spyCEP, and sse), as well as cell surface-associated factors of S. pyogenes (emm1, mur1.2, sibA/cdhA, and M5005_Spy0500). CpsY activity appears to be specific to the human host, as loss of CpsY does not result in S. pyogenes virulence defects in murine models of infection (Vega, et al., 2017). Further work characterizing genes that contribute to S. pyogenes behavior in blood will be important to identifying the major determinants of invasive infection.

The contribution of CovR/S regulation to S. pyogenes dissemination and survival in blood is also being actively investigated, due to this regulator’s association with invasive infection (reviewed in (Langshaw, Pandey, & Good, 2018)). Mutations in covS that attenuate the activity of its gene product toward CovR are a common cause of hypervirulence and enhancement of soft tissue invasion and innate immune evasion in invasive clinical isolates (Ikebe, et al., 2010; Shea, et al., 2011; Maamary, et al., 2012; Tatsuno, Okada, Zhang, Isaka, & Hasegawa, 2013; Garcia, et al., 2010; Masuno, et al., 2014) and animal passaged clones that arise from experimental invasive infection in mice (Sumby, Whitney, Graviss, DeLeo, & Musser, 2006; Walker, et al., 2007; Engleberg, Heath, Miller, Rivera, & DiRita, 2001). One outstanding feature of hypervirulent S. pyogenes strains with inactivated covS (CovR⁺S^-) is the lack of detectable SpeB in culture supernatants (Maamary, et al., 2010). CovR phosphorylation decreases in the absence of active CovS, resulting in de-repression of virulence factor expression. Although there is evidence the virulence factor SpeB is negatively regulated by CovR (Heath, DiRita et al. 1999), expression of SpeB is almost completely repressed in a ∆covS mutant (Treviño, Perez et al. 2009). In an M1 strain, non-phosphorylated CovR acts as a transcriptional repressor for the SpeB-inducing regulator Rgg (Chiang-Ni, Chu et al. 2016). In addition, the expression of the Rgg-repressing regulator LacD.1 is upregulated in the ∆covS mutant. Chiang-Ni et al showed that overexpression of rgg but not inactivation of lacD.1 in the ∆covS mutant partially restored speB expression, indicating that only rgg repression, but not lacD.1 upregulation, contributes to the speB repression in a ∆covS mutant (Chiang-Ni, Chu et al. 2016). Intact covS greatly enhances the speB expression that is necessary for establishing early infectivity, after which the downregulation of speB expression promotes virulence (Cole, Barnett, Nizet, & Walker, 2011). This switch in speB expression is further facilitated by covS gene mutations that allow CovR to repress speB expression, thereby permitting other proteins (such as the products of sda1, ska, and slo) to avoid degradation by SpeB and sustaining their activities as S. pyogenes virulence factors (Aziz, et al., 2004; Cole, et al., 2006; Liang, et al., 2013). The selection for CovR+S- strains suggests that only colonies maximally discharging their virulence factors are able to survive in host compartments that are particularly hostile to S. pyogenes (namely, blood), whereas at sites without host immune surveillance, CovS is important for alleviating the energy burden of producing virulence gene products and allows for long-term colonization.

The consistency of gene mutations in covS that engender hypervirulent strains was examined by comparing the invasive globally disseminated 5448 (CovR+S+, M1T1) strain and the non-virulent engineered strain AP53 (CovR+S+) in a number of mouse-passage studies, where the same wild type bacteria were used to infect different mice (Mayfield, et al., 2014). Infection and dissemination sites were screened for SpeB-deficient clones also exhibiting increased sda1 and ska expression and activity. Characterization of the isolated clones showed many types of mutations that occur in the covS gene (such as frame-shift insertions, deletions, and in-frame small and large deletions), which indicates that, when establishing infection, the entire covS gene is susceptible to mutations and that clones that contain CovS-inactivating mutations affect numerous phenotypes, which leads to serious invasive disease. Most interestingly, characterization of CovS function in clinical isolates with single amino acid substitutions demonstrated that point mutations partially, but not completely, impaired the function of the covS alleles (Tatsuno, Okada, Zhang, Isaka, & Hasegawa, 2013). Unlike covS-knockout (ΔcovS) mutants, S. pyogenes strains that exhibit partial loss of CovS function stemming from covS alleles with point mutations were not impaired for growth in culture. As a result, complete loss of CovS function may confer greater virulence at the expense of the ability to respond to environmental cues recognized by CovS, which explains the abundance of point mutations among hypervirulent clinical isolates. Both the consequences of different covS mutations on S. pyogenes adaptation to host environments like blood and the mechanism driving these mutations require further study, though recent evidence hints that the latter entails interaction with host innate immunity, as described in the next section.

Non-immune cells

The host cells infected by S. pyogenes are predetermined by the major entry ports the bacterium uses to enter the human body, thus making tonsillar epithelial cells and skin keratinocytes the primary targets in cases of infection of mucous membranes and the skin (Fiedler, Sugareva, Patenge, & Kreikemeyer, 2010). A prerequisite for colonization of these sites is the need to specifically adhere to epithelial cell surfaces, a task for which adhesion- and pilus-encoding genes found in a genotype-specific pattern within the FCT region are predominantly responsible. As previously indicated in this chapter, different members of the RALP family act as the primary regulators that influence the expression of FCT region genes in a serotype-dependent manner, but they are not the only ones to regulate them.

mRNA half-life of the S. pyogenes transcriptome influenced by RNase Y

Knowledge of global mRNA half-lives in bacterial species in general and pathogens in particular is limited, but can provide valuable insights on the adaptability of a species to changing environmental conditions. In Bacillus subtilis and Staphylococcus aureus, numerous transcription units are up or down regulated in mutants that carry a defective rny gene, which encodes the endoribonuclease RNase Y responsible for the initiation of mRNA degradation (Lehnik-Habrink, et al., 2011; Marincola, et al., 2012). A NZ131 rny mutant was assessed using a novel approach (steepest-slope method) for precise mRNA half-life determination on a transcriptome-wide scale (Chen, Itzek, Malke, Ferretti, & Kreth, 2013). The results encompassed 1,485 S. pyogenes genes (87.1% of the core genome) and revealed median and mean transcriptome half-lives of 0.89 min and 1.24 min, respectively, in the wild-type strain. The corresponding values for the rny mutant amount to 1.81 min and 2.79 min, respectively. In the wild type, the overwhelming majority of these genes (85%) show “unstable” mRNAs with half-lives shorter than 2.0 min, and only a small minority (2.3%) exhibit half-lives longer than 5.0 min (“stable” mRNAs). In contrast, the rny mutant gave corresponding percentage changes that changed to 56% and 12%, respectively. These data compare well with a 2-fold increase of overall mRNA stability, as affected by a non-functional rny gene. Compared to other bacterial species, which show longer transcriptome half-lives, the high mRNA turnover rate in S. pyogenes suggests that this species is particularly capable of adaptation to fast-changing environments during the course of the infection process (Figure 5).

Figure 5.

Independence and interaction of the regulatory systems important for amino acid starvation, mRNA stability, and global virulence in S. pyogenes.

RNase Y influences the expression of S. pyogenes virulence factors in a growth phase and nutrient-related manner (Kang, Caparon, & Cho, 2010). The influence of RNAse Y (previously dubbed CvfA) was most pronounced at the stationary phase of growth and in low carbohydrate media, conditions under which up to 30% of the transcriptome exhibited altered expression. Differentially expressed virulence genes included streptokinase ska, cfa, slo , emm, speB, sagA (Kang, Caparon, & Cho, 2010). While regulation of gene expression dependent on nutritional stress is commonly linked to the stringent response, loss of RNAse Y did not alter the levels of ppGpp; rather, RNAse Y interacted with enolase to control the transcript decay rates of virulence factors or their regulators (Kang, Caparon, & Cho, 2010). An RNAse Y-lacking mutant exhibited attenuated virulence in murine infection models. mRNA stability is also differentially affected in important functional groups of S. pyogenes genes (Chen, Itzek, Malke, Ferretti, & Kreth, 2013). For example, Clusters of Orthologous Groups (COGs (Tatusov, Koonin, & Lipman, 1997)) involved in energy production and translation are about 2- to 3-times more stable than the transcriptome average in the wild type S. pyogenes. The vast majority of mRNA in all 20 COGs studied showed a 1.7- 3.3-fold increase in the rny mutant. However, the stability profile of the different functional groups stays about the same in wild type and mutant strains, indicating that RNase Y does not preferentially affect certain functional groups. Resolving the general picture of rny-directed mRNA half-life control to the level of individual target genes leads to the identification of mRNAs, the half-lives of which are not significantly influenced by RNase Y (upp, ska, cfa; (Chen, Itzek, Malke, Ferretti, & Kreth, 2013)).

Further investigation of virulence gene regulation through mRNA stability in S. pyogenes revealed that RNase Y processing of the 5' untranslated (UTR) region of speB mRNA alters speB expression at the transcriptional level (Broglia, et al., 2018). Two RNase Y cleavage sites were identified downstream of a guanosine (G) residue, which in turn was found to be required for RNase Y processing of speB. Partial deletion of the speB 5' UTR resulted in accumulation of speB mRNA in stationary growth, due to retarded mRNA degradation (Chen, Mashburn-Warren, Merritt, Federle, & Kreth, 2017). In contrast to the observed role of RNAse Y in mRNA turnover during stationary growth (Kang, Caparon, & Cho, 2010; Broglia, et al., 2018), Chen et al. observed a weak interaction between the truncated speB 5' UTR and RNase Y compared with the wild-type, which suggests other unidentified RNA-degrading components are involved in the phenotypes produced by speB UTR truncation.

A study to define S. pyogenes mRNAs processed by RNase Y examined the transcriptomes of a S. pyogenes M49 strain and an isogenic RNase Y mutant (Δrny) using RNAseq and identified operons that display segmental stability in the wild type but not in the Δrny background (Chen, Raghavan, Qi, Merritt, & Kreth, 2019), which suggests RNAse Y processing. Of the 15 operons with mRNAs potentially processed by RNase Y, several associated with virulence were selected for confirmation (folC1, prtF, speG, ropB, and ypaA), of which only folC1 was confirmed to be processed, though whether solely RNAse Y was responsible is unclear (Chen, Raghavan, Qi, Merritt, & Kreth, 2019).

Type I Restriction Modification System and the S. pyogenes Methylome

It has become possible to quickly and reliably determine the complete methylation profile of bacterial genomes (Murray, et al., 2012) as part of the single molecule, real-time (SMRT) sequencing procedure (Flusberg, et al., 2010). The methylated bases, created by DNA methyltransferases and present practically in all bacterial genomes, not only serve functions in restriction-modification systems (Euler, Ryan, Martin, & Fischetti, 2007), but also regulate the transcription of virulence genes (Casadesús & Low, 2006). Since the previous edition of this chapter, genome-wide epigenetic data has been published for S. pyogenes, and SMRT sequencing analysis has identified 412 putative N6-methyladenosine (m6A) sites throughout the genome of an M28 serotype S. pyogenes strain (Nye, et al., 2019). Deletion of the Restriction, Specificity, and Methylation gene subunits (∆RSM) of a putative Type I restriction modification system resulted in abrogation of all detectable m6A at the recognition sites in the generated ΔRSM strain, which also failed to prevent transformation with foreign-methylated DNA. RNA-sequencing determined that 20 genes were significantly down-regulated in the ΔRSM strain relative to its isogenic parent (Nye, et al., 2019). Among these was mga, which suggested that the ablated Type I restriction modification system influenced virulence gene expression. This hypothesis was supported by the observations that the ΔRSM strain elicited an enhanced host immune response in a murine subcutaneous infection model (e.g.. greater inflammation and tissue damage, increased levels of pro-inflammatory cytokines), survived poorly within human neutrophils, and had reduced adherence to human epithelial cells (Nye, et al., 2019). A more comprehensive analysis of DNA methylation in S. pyogenes using a database of 224 S. pyogenes genomes encompassing 80 M serotypes determined that nearly all S. pyogenes strains encode a type I restriction modification (RM) system that lacks the hsdS' alleles that influence global gene expression in other streptococcal species (DebRoy, et al., 2021). This study confirmed the presence of the S. pyogenes Type I restriction modification system on the core chromosome, as well as identified the sporadic presence of Type II orphan methyltransferases on prophages. Furthermore, they assigned 13 methylation patterns to the S. pyogenes population and confirmed that inactivation of the Type I restriction modification system abrogated DNA methylation detectable via either SMRT or MinION sequencing, as well as significantly increasing plasmid transformation rates (DebRoy, et al., 2021). In contrast to the report from Nye et al, the inactivation of the Type I system in a different M28 serotype strain and an M87 strain did not alter either mga transcript levels nor Mga-regulated gene expression. Further research will potentially elucidate whether the methylome is environment-dependent, or if a given methylome determines the tissue specificity of infection. Likewise, it will be interesting to find out whether the methylome of carrier strains differs from that of their invasive counterparts, which might explain how CodY turns a gene activator. Clearly, we need to gain knowledge about the epigenetic regulation of transcription, and can foresee exciting new fields of research arising in the near future.