Regulatory rewiring confers serotype‐specific hyper‐virulence in the human pathogen group A <scp><i>S</i></scp><i>treptococcus</i>

Miller, Eugene H.; Danger, Jessica L.; Ramalinga, Anupama; Horstmann, Nicola; Shelburne, Samuel A.; Sumby, Paul

doi:10.1111/mmi.13136

Cited by 42 publications

(116 citation statements)

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“…This could be tested by comparing our parental, rivR mutant, and complemented mutant strains in models of noninvasive infections, but such testing was beyond the scope of this study. Interestingly, serotype M3 GAS isolates, which are nonrandomly associated with particularly severe invasive infections and high mortality rates (32,33), are naturally rivR mutants, due to the presence of several inactivating mutations in rivR. Thus, our data are consistent with mutations of rivR contributing to the invasive disease hypervirulence of serotype M3 isolates, enhancing their ability to avoid the immune properties of human blood.…”

Section: Discussionsupporting

confidence: 77%

Multimerization of the Virulence-Enhancing Group A Streptococcus Transcription Factor RivR Is Required for Regulatory Activity

et al. 2017

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Group A Streptococcus (GAS) (Streptococcus pyogenes) causes more than 700 million human infections each year. The significant morbidity and mortality rates associated with GAS infections are in part a consequence of the ability of this pathogen to coordinately regulate virulence factor expression during infection. RofA-like protein IV (RivR) is a member of the Mga-like family of transcriptional regulators, and previously we reported that RivR negatively regulates transcription of the hasA and grab virulence factor-encoding genes. Here, we determined that RivR inhibits the ability of GAS to survive and to replicate in human blood. To begin to assess the biochemical basis of RivR activity, we investigated its ability to form multimers, which is a characteristic of Mga-like proteins. We found that RivR forms both dimers and a higher-molecular-mass multimer, which we hypothesize is a tetramer. As cysteine residues are known to contribute to the ability of proteins to dimerize, we created a library of expression plasmids in which each of the four cysteines in RivR was converted to serine. While the C68S RivR protein was essentially unaffected in its ability to dimerize, the C32S and C377S proteins were attenuated, while the C470S protein completely lacked the ability to dimerize. Consistent with dimerization being required for regulatory activity, the C470S RivR protein was unable to repress hasA and grab gene expression in a rivR mutant. Thus, multimer formation is a prerequisite for RivR activity, which supports recent data obtained for other Mga-like family members, suggesting a common regulatory mechanism.IMPORTANCE The modulation of gene transcription is key to the ability of bacterial pathogens to infect hosts to cause disease. Here, we discovered that the group A Streptococcus transcription factor RivR negatively regulates the ability of this pathogen to survive in human blood, and we also began biochemical characterization of this protein. We determined that, in order for RivR to function, it must self-associate, forming both dimers (consisting of two RivR proteins) and higher-order complexes (consisting of more than two RivR proteins). This functional requirement for RivR is shared by other regulators in the same family of proteins, suggesting a common regulatory mechanism. Insight into how these transcription factors function may facilitate the development of novel therapeutic agents targeting their activity.KEYWORDS multimer, transcription factor, virulence regulation, Streptococcus pyogenes T he human bacterial pathogen group A Streptococcus (GAS) causes a diverse range of infections, from the superficial, such as pharyngitis, to the severely invasive, such as necrotizing fasciitis (1-3). In addition, failure to adequately treat individuals with skin

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Section: Discussionsupporting

confidence: 77%

Multimerization of the Virulence-Enhancing Group A Streptococcus Transcription Factor RivR Is Required for Regulatory Activity

et al. 2017

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“…5). This result suggests that increased hyaluronic acid capsule production likely is not the only determining factor accounting for the hypervirulent phenotype of previously studied rocA mutants (16,18,19). That is, upregulation of other CovR-repressed virulence factors (e.g., SPN and SLO) is likely sufficient to significantly increase the virulence of S. pyogenes strains.…”

Section: Discussionmentioning

confidence: 69%

“…RocA is a known regulator of CovR activity (16,17). Hence, in addition to nga, slo, and speB, we measured the expression of two other CovR-regulated genes: prtS and grab.…”

Section: Resultsmentioning

confidence: 99%

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Intergenic Variable-Number Tandem-Repeat Polymorphism Upstream of rocA Alters Toxin Production and Enhances Virulence in Streptococcus pyogenes

et al. 2016

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dVariable-number tandem-repeat (VNTR) polymorphisms are ubiquitous in bacteria. However, only a small fraction of them has been functionally studied. Here, we report an intergenic VNTR polymorphism that confers an altered level of toxin production and increased virulence in Streptococcus pyogenes. The nature of the polymorphism is a one-unit deletion in a three-tandemrepeat locus upstream of the rocA gene encoding a sensor kinase. S. pyogenes strains with this type of polymorphism cause human infection and produce significantly larger amounts of the secreted cytotoxins S. pyogenes NADase (SPN) and streptolysin O (SLO). Using isogenic mutant strains, we demonstrate that deleting one or more units of the tandem repeats abolished RocA production, reduced CovR phosphorylation, derepressed multiple CovR-regulated virulence factors (such as SPN and SLO), and increased virulence in a mouse model of necrotizing fasciitis. The phenotypic effect of the VNTR polymorphism was nearly the same as that of inactivating the rocA gene. In summary, we identified and characterized an intergenic VNTR polymorphism in S. pyogenes that affects toxin production and virulence. These new findings enhance understanding of rocA biology and the function of VNTR polymorphisms in S. pyogenes. Streptococcus pyogenes (group A streptococcus, or GAS) is a human-specific bacterial pathogen causing ϳ10,000 invasive and 10 million noninvasive infections in the United States annually. Enhanced understanding of the molecular pathogenesis of S. pyogenes is necessary to develop novel therapeutic strategies. S. pyogenes expresses a large arsenal of virulence factors (1, 2), many of which are well studied. These include antiphagocytic hyaluronic acid capsule (3), the cytotoxins S. pyogenes NADase (SPN) and streptolysin O (SLO) (4, 5), and a potent extracellular cysteine protease, SpeB (6, 7). These virulence factors are regulated by the CovR/CovS two-component regulatory system (8-10). Polymorphisms in covR or covS have been reported to change the level of production of these virulence factors and thereby alter S. pyogenes virulence (11, 12).We recently sequenced 1,125 emm89 genomes (13, 14) and measured the levels of SPN and SLO production in some of these sequenced strains. Unexpectedly, two strains (MGAS28980 and MGAS27457) with functionally wild-type covR and covS genes produced increased amounts of SPN and SLO. We previously showed that polymorphisms in the nga upstream regulatory region can affect nga-slo transcription and production of SPN and SLO (13,14). However, all strains that we assayed for SPN and SLO production had the same variant-3 nga promoter (13). This suggests that other factors are contributing to overproduction of SPN and SLO in strains MGAS28980 and MGAS27457. Genetic interrogation revealed that both of these strains have a variablenumber tandem-repeat (VNTR) polymorphism upstream of rocA. The nature of the polymorphism is a single GAAGGA deletion in the GAACGA-GAAGGA-GAAGGA repeat locus three nucleotides upstream of the rocA trans...

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“…Mutation of the histidine kinase covS causes the downregulation of speB and enhancement of expression of many CovRS-controlled virulence genes, resulting in hypervirulence (19)(20)(21)(22). The rocA gene, an orphan kinase, regulates CovRS expression and capsule production (23) and functions through CovR (24). Natural mutations and deletion of rocA lead to considerable increases in the levels of transcription of multiple CovRScontrolled virulence genes, at least in M3 and M1 GAS isolates (24).…”

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confidence: 99%

Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of In Vitro and In Vivo Protease SpeB Expression and Virulence

et al. 2017

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Group A Streptococcus (GAS) acquires mutations of the virulence regulator CovRS in human and mouse infections, and these mutations result in the upregulation of virulence genes and the downregulation of the protease SpeB. To identify in vivo mutants with novel phenotypes, GAS isolates from infected mice were screened by enzymatic assays for SpeB and the platelet-activating factor acetylhydrolase Sse, and a new type of variant that had enhanced Sse expression and normal levels of SpeB production was identified (the variants had a phenotype referred to as enhanced Sse activity [Sse Aϩ ] and normal SpeB activity [SpeB Aϩ ]). Sse Aϩ SpeB Aϩ variants had transcript levels of CovRS-controlled virulence genes comparable to those of a covS mutant but had no covRS mutations. Genome resequencing of an Sse Aϩ SpeB Aϩ isolate identified a C605A nonsense mutation in orphan kinase gene rocA, and 6 other Sse Aϩ SpeB Aϩ isolates also had nonsense mutations or small indels in rocA. RocA and CovS mutants had similar levels of enhancement of the expression of CovRS-controlled virulence genes at the exponential growth phase; however, mutations of RocA but not mutations of CovS did not result in the downregulation of speB transcription at stationary growth phase or in subcutaneous infection of mice. GAS with RocA and CovS mutations caused greater enhancement of the expression of hasA than spyCEP in mouse skin infection than wild-type GAS did. RocA mutants ranked between wild-type GAS and CovS mutants in skin invasion, inhibition of neutrophil recruitment, and virulence in subcutaneous infection of mice. Thus, GAS RocA mutants can be selected in subcutaneous infections in mice and exhibit gene expression patterns and virulences distinct from those of CovS mutants. The findings provide novel information for understanding GAS fitness mutations in vivo, virulence gene regulation, in vivo gene expression, and virulence.KEYWORDS CovRS, PAF acetylhydrolase, RocA, SpeB, Streptococcus pyogenes, group A Streptococcus, in vivo expression, mutation, virulence, virulence regulation T he human pathogen group A Streptococcus (GAS) commonly causes pharyngitis and superficial skin infections. GAS also causes severe invasive infections, such as necrotizing fasciitis and sepsis. The most recently available data indicate that from 2005 to 2012 severe invasive infections in the United States were most frequently associated with GAS of the M protein serotypes M1, M12, M28, M89, and M3 (1), and the currently circulating M1 GAS belongs to the pandemic M1T1 clone (2). GAS produces an

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Regulatory rewiring confers serotype‐specific hyper‐virulence in the human pathogen group A Streptococcus

Cited by 42 publications

References 73 publications

Multimerization of the Virulence-Enhancing Group A Streptococcus Transcription Factor RivR Is Required for Regulatory Activity

Multimerization of the Virulence-Enhancing Group A Streptococcus Transcription Factor RivR Is Required for Regulatory Activity

Intergenic Variable-Number Tandem-Repeat Polymorphism Upstream of rocA Alters Toxin Production and Enhances Virulence in Streptococcus pyogenes

Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of In Vitro and In Vivo Protease SpeB Expression and Virulence

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