Crystal structure of the zymogen form of the group A Streptococcus virulence factor SpeB: An integrin-binding cysteine protease

ABSTRACT Streptococcus pyogenes expresses a highly conserved extracellular cysteine protease that is a virulence factor for invasive disease, including soft tissue infection. Site-directed mutagenesis was used to generate a His340Ala recombinant mutant protein that was made as a stable 40-kDa zymogen by Escherichia coli. Purified His340Ala protein was proteolytically inactive when bovine casein and human fibronectin were used as substrates. Wild-type 28-kDa streptococcal protease purified from S. pyogenes processed the 40-kDa mutant zymogen to a 28-kDa mature form, a result suggesting that the derivative protein retained structural integrity. The data are consistent with the hypothesis that His340 is an enzyme active site residue, an idea confirmed by recent solution of the zymogen crystal structure (T. F. Kagawa, J. C. Cooney, H. M. Baker, S. McSweeney, M. Liu, S. Gubba, J. M. Musser, and E. N. Baker, Proc. Natl. Acad. Sci. USA 97:2235–2240, 2000). The data provide additional insight into structure-function relationships in thisS. pyogenes virulence factor.

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A natural variant of the cysteine protease virulence factor of group A Streptococcus with an arginine-glycine-aspartic acid (RGD) motif preferentially binds human integrins v 3 and IIb 3

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.96.1.242 ◽

1999 ◽

Vol 96 (1) ◽

pp. 242-247 ◽

Cited By ~ 72

Author(s):

K. E. Stockbauer ◽

L. Magoun ◽

M. Liu ◽

E. H. Burns ◽

S. Gubba ◽

...

Keyword(s):

Aspartic Acid ◽

Virulence Factor ◽

Cysteine Protease ◽

Group A Streptococcus ◽

Rgd Motif ◽

Natural Variant ◽

Group A ◽

Arginine Glycine Aspartic Acid

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Natural Disruption of Two Regulatory Networks in Serotype M3 Group A Streptococcus Isolates Contributes to the Virulence Factor Profile of This Hypervirulent Serotype

Infection and Immunity ◽

10.1128/iai.01639-13 ◽

2014 ◽

Vol 82 (5) ◽

pp. 1744-1754 ◽

Cited By ~ 24

Author(s):

Tram N. Cao ◽

Zhuyun Liu ◽

Tran H. Cao ◽

Kathryn J. Pflughoeft ◽

Jeanette Treviño ◽

...

Keyword(s):

Virulence Factor ◽

Regulatory Networks ◽

Molecular Mechanisms ◽

Group A Streptococcus ◽

Regulatory System ◽

Mrna Levels ◽

Bacterial Strains ◽

Content Type ◽

Small Regulatory Rna ◽

Group A

ABSTRACTDespite the public health challenges associated with the emergence of new pathogenic bacterial strains and/or serotypes, there is a dearth of information regarding the molecular mechanisms that drive this variation. Here, we began to address the mechanisms behind serotype-specific variation between serotype M1 and M3 strains of the human pathogenStreptococcus pyogenes(the group AStreptococcus[GAS]). Spatially diverse contemporary clinical serotype M3 isolates were discovered to contain identical inactivating mutations within genes encoding two regulatory systems that control the expression of important virulence factors, including the thrombolytic agent streptokinase, the protease inhibitor-binding protein-G-related α2-macroglobulin-binding (GRAB) protein, and the antiphagocytic hyaluronic acid capsule. Subsequent analysis of a larger collection of isolates determined that M3 GAS, since at least the 1920s, has harbored a 4-bp deletion in thefasCgene of thefasBCAXregulatory system and an inactivating polymorphism in therivRregulator-encoding gene. ThefasCandrivRmutations in M3 isolates directly affect the virulence factor profile of M3 GAS, as evident by a reduction in streptokinase expression and an enhancement of GRAB expression. Complementation of thefasCmutation in M3 GAS significantly enhanced levels of the small regulatory RNA FasX, which in turn enhanced streptokinase expression. Complementation of therivRmutation in M3 GAS restored the regulation ofgrabmRNA abundance but did not alter capsule mRNA levels. While important, thefasCandrivRmutations do not provide a full explanation for why serotype M3 strains are associated with unusually severe invasive infections; thus, further investigation is warranted.

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Biofilm formation by group A Streptococcus: a role for the streptococcal regulator of virulence (Srv) and streptococcal cysteine protease (SpeB)

Microbiology ◽

10.1099/mic.0.021048-0 ◽

2009 ◽

Vol 155 (1) ◽

pp. 46-52 ◽

Cited By ~ 41

Author(s):

Christopher D. Doern ◽

Amity L. Roberts ◽

Wenzhou Hong ◽

Jessica Nelson ◽

Slawomir Lukomski ◽

...

Keyword(s):

Cysteine Protease ◽

Biofilm Formation ◽

Group A Streptococcus ◽

Immunoblot Analysis ◽

Wild Type ◽

Flow Conditions ◽

Group A ◽

Streptococcus A ◽

Insight Into ◽

Western Immunoblot Analysis

Recently, biofilms have become a topic of interest in the study of the human pathogen group A Streptococcus (GAS). In this study, we sought to learn more about the make-up of these structures and gain insight into biofilm regulation. Enzymic studies indicated that biofilm formation by GAS strain MGAS5005 required an extracellular protein and DNA component(s). Previous results indicated that inactivation of the transcriptional regulator Srv in MGAS5005 resulted in a significant decrease in virulence. Here, inactivation of Srv also resulted in a significant decrease in biofilm formation under both static and flow conditions. Given that production of the extracellular cysteine protease SpeB is increased in the srv mutant, we tested the hypothesis that increased levels of active SpeB may be responsible for the reduction in biofilm formation. Western immunoblot analysis indicated that SpeB was absent from MGAS5005 biofilms. Complementation of MGAS5005Δsrv restored the biofilm phenotype and eliminated the overproduction of active SpeB. Inhibition of SpeB with E64 also restored the MGAS5005Δsrv biofilm to wild-type levels.

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The Majority of 9,729 Group A Streptococcus Strains Causing Disease Secrete SpeB Cysteine Protease: Pathogenesis Implications

Infection and Immunity ◽

10.1128/iai.00989-15 ◽

2015 ◽

Vol 83 (12) ◽

pp. 4750-4758 ◽

Cited By ~ 24

Author(s):

Randall J. Olsen ◽

Anjali Raghuram ◽

Concepcion Cantu ◽

Meredith H. Hartman ◽

Francisco E. Jimenez ◽

...

Keyword(s):

Necrotizing Fasciitis ◽

Cysteine Protease ◽

Protease Activity ◽

Sequence Data ◽

Group A Streptococcus ◽

Whole Genome Sequence ◽

Wild Type ◽

Tissue Destruction ◽

Lethal Challenge ◽

Group A

Group A streptococcus (GAS), the causative agent of pharyngitis and necrotizing fasciitis, secretes the potent cysteine protease SpeB. Several lines of evidence suggest that SpeB is an important virulence factor. SpeB is expressed in human infections, protects mice from lethal challenge when used as a vaccine, and contributes significantly to tissue destruction and dissemination in animal models. However, recent descriptions of mutations in genes implicated in SpeB production have led to the idea that GAS may be under selective pressure to decrease secreted SpeB protease activity during infection. Thus, two divergent hypotheses have been proposed. One postulates that SpeB is a key contributor to pathogenesis; the other, that GAS is under selection to decrease SpeB during infection. In order to distinguish between these alternative hypotheses, we performed casein hydrolysis assays to measure the SpeB protease activity secreted by 6,775 GAS strains recovered from infected humans. The results demonstrated that 84.3% of the strains have a wild-type SpeB protease phenotype. The availability of whole-genome sequence data allowed us to determine the relative frequencies of mutations in genes implicated in SpeB production. The most abundantly mutated genes were direct transcription regulators. We also sequenced the genomes of 2,954 GAS isolates recovered from nonhuman primates with experimental necrotizing fasciitis. No mutations that would result in a SpeB-deficient phenotype were identified. Taken together, these data unambiguously demonstrate that the great majority of GAS strains recovered from infected humans secrete wild-type levels of SpeB protease activity. Our data confirm the important role of SpeB in GAS pathogenesis and help end a long-standing controversy.

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RivR Is a Negative Regulator of Virulence Factor Expression in Group A Streptococcus

Infection and Immunity ◽

10.1128/iai.00703-12 ◽

2012 ◽

Vol 81 (1) ◽

pp. 364-372 ◽

Cited By ~ 19

Author(s):

Jeanette Treviño ◽

Zhuyun Liu ◽

Tram N. Cao ◽

Esmeralda Ramirez-Peña ◽

Paul Sumby

Keyword(s):

Mutant Strain ◽

Virulence Factor ◽

Regulatory Networks ◽

Group A Streptococcus ◽

Human Keratinocytes ◽

Negative Regulator ◽

Upstream Region ◽

Content Type ◽

Factor Expression ◽

Group A

The bacterial pathogen group AStreptococcus(GAS) causes human diseases ranging from self-limiting pharyngitis (also known as strep throat) to severely invasive necrotizing fasciitis (also known as the flesh-eating syndrome). To control virulence factor expression, GAS utilizes both protein- and RNA-based mechanisms of regulation. Here we report that the transcription factor RivR (RofA-like protein IV) negatively regulates the abundance of mRNAs encoding the hyaluronic acid capsule biosynthesis proteins (hasABC; ∼7-fold) and the protein G-related α2-macroglobulin-binding protein (grab; ∼29-fold). Our data differ significantly from those of a previous study of the RivR regulon. Given thatgrabandhasABCare also negatively regulated by the two-component system CovR/S (controlofvirulence), we tested whether RivR functions through CovR/S. A comparison ofrivandcovsingle and double mutant strains showed that RivR requires CovR activity forgrabandhasABCregulation. Analysis of the upstream region ofrivRidentified a novel promoter the deletion of which reducedrivRmRNA abundance by 70%. ArivRmutant strain had a reduced ability to adhere to human keratinocytes relative to that of the parental and complemented strains, a phenotype that was abolished upon GAS pretreatment with hyaluronidase, highlighting the importance of capsule regulation by RivR during colonization. TherivRmutant strain was also attenuated for virulence in a murine model of bacteremia infection. Thus, we identify RivR as an important regulator of GAS virulence and provide new insight into the regulatory networks controlling virulence factor production in this pathogen.

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Screening Of Zinc Database Against Streptococcal Cysteine Protease Enzyme For Identification Of Novel Group A Streptococcus Inhibitors

10.33774/chemrxiv-2021-jwlbz ◽

2021 ◽

Author(s):

Ratul Bhowmik ◽

Ranajit Nath ◽

Ratna Roy

Keyword(s):

Cysteine Protease ◽

Group A Streptococcus ◽

Protease Enzyme ◽

Zinc Database ◽

Group A

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

Journal of Bacteriology ◽

10.1128/jb.00452-16 ◽

2016 ◽

Vol 199 (1) ◽

Cited By ~ 1

Author(s):

Anupama Ramalinga ◽

Jessica L. Danger ◽

Nishanth Makthal ◽

Muthiah Kumaraswami ◽

Paul Sumby

Keyword(s):

Transcription Factor ◽

Virulence Factor ◽

Human Blood ◽

Biochemical Characterization ◽

Regulatory Mechanism ◽

Group A Streptococcus ◽

Regulatory Activity ◽

Biochemical Basis ◽

Content Type ◽

Group A

ABSTRACT 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.

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Human Disease Isolates of Serotype M4 and M22 Group A Streptococcus Lack Genes Required for Hyaluronic Acid Capsule Biosynthesis

mBio ◽

10.1128/mbio.00413-12 ◽

2012 ◽

Vol 3 (6) ◽

Cited By ~ 37

Author(s):

Anthony R. Flores ◽

Brittany E. Jewell ◽

Nahuel Fittipaldi ◽

Stephen B. Beres ◽

James M. Musser

Keyword(s):

Hyaluronic Acid ◽

Virulence Factor ◽

Group A Streptococcus ◽

Ex Vivo ◽

Upper Respiratory Tract ◽

Major Virulence Factor ◽

Invasive Infections ◽

Group A ◽

Human Infections ◽

Hyaluronic Acid Capsule

ABSTRACTGroup A streptococcus (GAS) causes human pharyngitis and invasive infections and frequently colonizes individuals asymptomatically. Many lines of evidence generated over decades have shown that the hyaluronic acid capsule is a major virulence factor contributing to these infections. While conducting a whole-genome analysis of thein vivomolecular genetic changes that occur in GAS during longitudinal human pharyngeal interaction, we discovered that serotypes M4 and M22 GAS strains lack thehasABCgenes necessary for hyaluronic acid capsule biosynthesis. Using targeted PCR, we found that all 491 temporally and geographically diverse disease isolates of these two serotypes studied lack thehasABCgenes. Consistent with the lack of capsule synthesis genes, none of the strains produced detectable hyaluronic acid. Despite the lack of a hyaluronic acid capsule, all strains tested multiplied extensivelyex vivoin human blood. Thus, counter to the prevailing concept in GAS pathogenesis research, strains of these two serotypes do not require hyaluronic acid to colonize the upper respiratory tract or cause abundant mucosal or invasive human infections. We speculate that serotype M4 and M22 GAS have alternative, compensatory mechanisms that promote virulence.IMPORTANCEA century of study of the antiphagocytic hyaluronic acid capsule made by group A streptococcus has led to the concept that it is a major virulence factor contributing to human pharyngeal and invasive infections. However, the discovery that some strains that cause abundant human infections lack hyaluronic acid biosynthetic genes and fail to produce this capsule provides a new stimulus for research designed to understand the group A streptococcus factors contributing to pharyngeal infection and invasive disease episodes.

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