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

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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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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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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Phosphorylation Events in the Multiple Gene Regulator of Group A Streptococcus Significantly Influence Global Gene Expression and Virulence

Infection and Immunity ◽

10.1128/iai.03023-14 ◽

2015 ◽

Vol 83 (6) ◽

pp. 2382-2395 ◽

Cited By ~ 13

Author(s):

Misu Sanson ◽

Nishanth Makthal ◽

Maire Gavagan ◽

Concepcion Cantu ◽

Randall J. Olsen ◽

...

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Mutant Strain ◽

Group A Streptococcus ◽

Global Gene Expression ◽

Sequencing Analysis ◽

Wild Type ◽

Content Type ◽

Group A

Whole-genome sequencing analysis of ∼800 strains of group AStreptococcus(GAS) found that the gene encoding themultiple virulencegene regulator of GAS (mga) is highly polymorphic in serotype M59 strains but not in strains of other serotypes. To help understand the molecular mechanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS, we constructed an isogenicmgamutant strain. Transcriptome studies indicated a significant regulatory influence of Mga and altered metabolic capabilities conferred by Mga-regulated genes. We assessed the phosphorylation status of Mga in GAS cell lysates with Phos-tag gels. The results revealed that Mga is phosphorylated at histidinesin vivo. Using phosphomimetic and nonphosphomimetic substitutions at conserved phosphoenolpyruvate:carbohydrate phosphotransferase regulation domain (PRD) histidines of Mga, we demonstrated that phosphorylation-mimicking aspartate replacements at H207 and H273 of PRD-1 and at H327 of PRD-2 are inhibitory to Mga-dependent gene expression. Conversely, non-phosphorylation-mimicking alanine substitutions at H273 and H327 relieved inhibition, and the mutant strains exhibited a wild-type phenotype. The opposing regulatory profiles observed for phosphorylation- and non-phosphorylation-mimicking substitutions at H273 extended to global gene regulation by Mga. Consistent with these observations, the H273D mutant strain attenuated GAS virulence, whereas the H273A strain exhibited a wild-type virulence phenotype in a mouse model of necrotizing fasciitis. Together, our results demonstrate phosphoregulation of Mga and its direct link to virulence in M59 GAS strains. These data also lay a foundation toward understanding how naturally occurring gain-of-function variations inmga, such as H201R, may confer an advantage to the pathogen and contribute to M59 GAS pathogenesis.

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Regulation of virulence factor expression in group A Streptococcus

Trends in Microbiology ◽

10.1016/s0966-842x(99)01604-2 ◽

1999 ◽

Vol 7 (11) ◽

pp. 428-430 ◽

Cited By ~ 8

Author(s):

Michael R Wessels

Keyword(s):

Virulence Factor ◽

Group A Streptococcus ◽

Factor Expression ◽

Group A

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SpyAD, a Moonlighting Protein of Group A Streptococcus Contributing to Bacterial Division and Host Cell Adhesion

Infection and Immunity ◽

10.1128/iai.00064-14 ◽

2014 ◽

Vol 82 (7) ◽

pp. 2890-2901 ◽

Cited By ~ 11

Author(s):

Marilena Gallotta ◽

Giovanni Gancitano ◽

Giampiero Pietrocola ◽

Marirosa Mora ◽

Alfredo Pezzicoli ◽

...

Keyword(s):

Cell Division ◽

Mammalian Cells ◽

Group A Streptococcus ◽

Host Cells ◽

Knockout Mutant ◽

Content Type ◽

Moonlighting Protein ◽

Group A

ABSTRACTGroup A streptococcus (GAS) is a human pathogen causing a wide repertoire of mild and severe diseases for which no vaccine is yet available. We recently reported the identification of three protein antigens that in combination conferred wide protection against GAS infection in mice. Here we focused our attention on the characterization of one of these three antigens, Spy0269, a highly conserved, surface-exposed, and immunogenic protein of unknown function. Deletion of thespy0269gene in a GAS M1 isolate resulted in very long bacterial chains, which is indicative of an impaired capacity of the knockout mutant to properly divide. Confocal microscopy and immunoprecipitation experiments demonstrated that the protein was mainly localized at the cell septum and could interactin vitrowith the cell division protein FtsZ, leading us to hypothesize that Spy0269 is a member of the GAS divisome machinery. Predicted structural domains and sequence homologies with known streptococcal adhesins suggested that this antigen could also play a role in mediating GAS interaction with host cells. This hypothesis was confirmed by showing that recombinant Spy0269 could bind to mammalian epithelial cellsin vitroand thatLactococcus lactisexpressing Spy0269 on its cell surface could adhere to mammalian cellsin vitroand to mice nasal mucosain vivo. On the basis of these data, we believe that Spy0269 is involved both in bacterial cell division and in adhesion to host cells and we propose to rename this multifunctional moonlighting protein as SpyAD (StreptococcuspyogenesAdhesion andDivision protein).

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RocA Regulates Phosphatase Activity of Virulence Sensor CovS of Group A Streptococcus in Growth Phase- and pH-Dependent Manners

mSphere ◽

10.1128/msphere.00361-20 ◽

2020 ◽

Vol 5 (3) ◽

Author(s):

Chuan Chiang-Ni ◽

He-Jing Chiou ◽

Huei-Chuan Tseng ◽

Chih-Yun Hsu ◽

Cheng-Hsun Chiu

Keyword(s):

Stationary Phase ◽

Phosphatase Activity ◽

Virulence Factor ◽

Growth Phase ◽

Exponential Phase ◽

Acidic Stress ◽

Phosphorylation Level ◽

Factor Expression ◽

Group A ◽

Ph Dependent

ABSTRACT The control of the virulence response regulator and sensor (CovR-CovS) two-component regulatory system in group A Streptococcus (GAS) strains regulates more than 15% of gene expression and has critical roles in invasive GAS infection. The membrane-embedded CovS has kinase and phosphatase activities, and both are required for modulating the phosphorylation level of CovR. Regulator of Cov (RocA) is a positive regulator of covR and also been shown to be a pseudokinase that interacts with CovS to enhance the phosphorylation level of CovR; however, how RocA modulates the activity of CovS has not been determined conclusively. Although the phosphorylation level of CovR was decreased in the rocA mutant in the exponential phase, the present study shows that phosphorylated CovR in the rocA mutant increased to levels similar to those in the wild-type strain in the stationary phase of growth. In addition, acidic stress, which is generally present in the stationary phase, enhanced the phosphorylation level of CovR in the rocA mutant. The phosphorylation levels of CovR in the CovS phosphatase-inactivated mutant and its rocA mutant were similar under acidic stress and Mg2+ (the signal that inhibits CovS phosphatase activity) treatments, suggesting that the phosphatase activity, but not the kinase activity, of CovS is required for RocA to modulate CovR phosphorylation. The phosphorylation level of CovR is crucial for GAS strains to regulate virulence factor expression; therefore, the growth phase- and pH-dependent RocA activity would contribute significantly to GAS pathogenesis. IMPORTANCE The emergence of invasive group A streptococcal infections has been reported worldwide. Clinical isolates that have spontaneous mutations or a truncated allele of the rocA gene (e.g., emm3-type isolates) are considered to be more virulent than isolates with the intact rocA gene (e.g., emm1-type isolates). RocA is a positive regulator of covR and has been shown to enhance the phosphorylation level of intracellular CovR regulator through the functional CovS protein. CovS is the membrane-embedded sensor and modulates the phosphorylation level of CovR by its kinase and phosphatase activities. The present study shows that the enhancement of CovR phosphorylation is mediated via the repression of CovS’s phosphatase activity by RocA. In addition, we found that RocA acts dominantly on modulating CovR phosphorylation under neutral pH conditions and in the exponential phase of growth. The phosphorylation level of CovR is crucial for group A Streptococcus species to regulate virulence factor expression and is highly related to bacterial invasiveness; therefore, growth phase- and pH-dependent RocA activity and the sequence polymorphisms of rocA gene would contribute significantly to bacterial phenotype variations and pathogenesis.

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The Mga Regulon but Not Deoxyribonuclease Sda1 of Invasive M1T1 Group A Streptococcus Contributes toIn VivoSelection of CovRS Mutations and Resistance to Innate Immune Killing Mechanisms

Infection and Immunity ◽

10.1128/iai.00857-15 ◽

2015 ◽

Vol 83 (11) ◽

pp. 4293-4303 ◽

Cited By ~ 9

Author(s):

Guanghui Liu ◽

Wenchao Feng ◽

Dengfeng Li ◽

Mengyao Liu ◽

Daniel C. Nelson ◽

...

Keyword(s):

Group A Streptococcus ◽

Regulatory System ◽

Innate Immune ◽

M Protein ◽

Content Type ◽

Group A ◽

Innate Immune Evasion ◽

Selection Of

ABSTRACTInvasive M1T1 group AStreptococcus(GAS) can have a mutation in the regulatory system CovRS, and this mutation can render strains hypervirulent. Interestingly, via mechanisms that are not well understood, the host innate immune system's neutrophils select spontaneous M1T1 GAS CovRS hypervirulent mutants, thereby enhancing the pathogen's ability to evade immune killing. It has been reported that the DNase Sda1 is critical for the resistance of M1T1 strain 5448 to killing in human blood and provides pressure forin vivoselection of CovRS mutations. We reexamined the role of Sda1 in the selection of CovRS mutations and in GAS innate immune evasion. Deletion ofsda1or all DNase genes in M1T1 strain MGAS2221 did not alter emergence of CovRS mutants during murine infection. Deletion ofsda1in strain 5448 resulted in Δsda1mutants with (5448 Δsda1M+strain) and without (5448 Δsda1M−strain) M protein production. The 5448 Δsda1M+strain accumulated CovRS mutationsin vivoand resisted killing in the bloodstream, whereas the 5448 Δsda1M−strain lostin vivoselection of CovRS mutations and was sensitive to killing. The deletion ofemmand a spontaneous Mga mutation in MGAS2221 reduced and preventedin vivoselection for CovRS mutants, respectively. Thus, in contrast to previous reports, Sda1 is not critical forin vivoselection of invasive M1T1 CovRS mutants and GAS resistance to innate immune killing mechanisms. In contrast, M protein and other Mga-regulated proteins contribute to thein vivoselection of M1T1 GAS CovRS mutants. These findings advance the understanding of the progression of invasive M1T1 GAS infections.

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Expression Microarray and Mouse Virulence Analysis of Four Conserved Two-Component Gene Regulatory Systems in Group A Streptococcus

Infection and Immunity ◽

10.1128/iai.74.2.1339-1351.2006 ◽

2006 ◽

Vol 74 (2) ◽

pp. 1339-1351 ◽

Cited By ~ 39

Author(s):

Izabela Sitkiewicz ◽

James M. Musser

Keyword(s):

Mutant Strain ◽

Group A Streptococcus ◽

Expression Microarray ◽

Regulatory Systems ◽

Significant Difference ◽

Two Component ◽

Mutant Strains ◽

Group A ◽

Gene Regulatory ◽

Component Gene

ABSTRACT Group A streptococcus (GAS) is a gram-positive human bacterial pathogen that causes diseases ranging from relatively mild epithelial cell surface infections to life-threatening invasive episodes. Much is known about the extracellular molecules that contribute to host-pathogen interactions, but in contrast, far less information is available about regulatory genes that control the expression of individual or multiple GAS virulence factors. The eight GAS genomes that have been sequenced have 12 conserved two-component gene regulatory systems (TCSs), but only 3 of these 12 have been studied in detail. Using an allelic replacement strategy with a nonpolar cassette, we inactivated the response regulator of four TCSs that have only weak homology with TCS genes of known or inferred function in other bacteria. The mutant strains were analyzed by expression microarray analysis at four time points and tested in two mouse infection models. Each TCS influenced expression (directly or indirectly) of 12 to 41% of all chromosomal genes, as assessed by growth in Todd-Hewitt broth and a custom Affymetrix GeneChip. None of the isogenic mutant strains was significantly altered for mouse virulence based on intraperitoneal inoculation. Similarly, compared to the wild-type strain, there was no significant difference in skin lesion size for three of the four mutants. In contrast, the ΔM5005_Spy_0680 mutant strain produced significantly larger abscesses after subcutaneous inoculation into mice, consistent with a hypervirulence phenotype. The mutant strain had significantly higher in vitro expression of several proven and putative virulence genes, including scpA, encoding a peptidase that inactivates complement protein C5a. Together, the data provide new information about previously uncharacterized GAS TCSs.

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Capsule Growth in Cryptococcus neoformans Is Coordinated with Cell Cycle Progression

mBio ◽

10.1128/mbio.00945-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 42

Author(s):

Rocío García-Rodas ◽

Radames J. B. Cordero ◽

Nuria Trevijano-Contador ◽

Guilhem Janbon ◽

Frédérique Moyrand ◽

...

Keyword(s):

Cell Cycle ◽

Cryptococcus Neoformans ◽

Mutant Strain ◽

Virulence Factor ◽

Cell Cycle Progression ◽

Regulatory Elements ◽

Pathogenic Yeast ◽

Cycle Progression ◽

Content Type ◽

Polysaccharide Capsule

ABSTRACT The fungal pathogen Cryptococcus neoformans has several virulence factors, among which the most important is a polysaccharide capsule. The size of the capsule is variable and can increase significantly during infection. In this work, we investigated the relationship between capsular enlargement and the cell cycle. Capsule growth occurred primarily during the G1 phase. Real-time visualization of capsule growth demonstrated that this process occurred before the appearance of the bud and that capsule growth arrested during budding. Benomyl, which arrests the cells in G2/M, inhibited capsule growth, while sirolimus (rapamycin) addition, which induces G1 arrest, resulted in cells with larger capsule. Furthermore, we have characterized a mutant strain that lacks a putative G1/S cyclin. This mutant showed an increased capacity to enlarge the capsule, both in vivo (using Galleria mellonella as the host model) and in vitro. In the absence of Cln1, there was a significant increase in the production of extracellular vesicles. Proteomic assays suggest that in the cln1 mutant strain, there is an upregulation of the glyoxylate acid cycle. Besides, this cyclin mutant is avirulent at 37°C, which correlates with growth defects at this temperature in rich medium. In addition, the cln1 mutant showed lower intracellular replication rates in murine macrophages. We conclude that cell cycle regulatory elements are involved in the modulation of the expression of the main virulence factor in C. neoformans. IMPORTANCE Cryptococcus neoformans is a pathogenic fungus that has significant incidence worldwide. Its main virulence factor is a polysaccharide capsule that can increase in size during infection. In this work, we demonstrate that this process occurs in a specific phase of the cell cycle, in particular, in G1. In agreement, mutants that have an abnormal longer G1 phase show larger capsule sizes. We believe that our findings are relevant because they provide a link between capsule growth, cell cycle progression, and virulence in C. neoformans that reveals new aspects about the pathogenicity of this fungus. Moreover, our findings indicate that cell cycle elements could be used as antifungal targets in C. neoformans by affecting both the growth of the cells and the expression of the main virulence factor of this pathogenic yeast.

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