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

2016 ◽  
Vol 199 (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.

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

2014 ◽  
Vol 82 (5) ◽  
pp. 1744-1754 ◽  
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.

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

2012 ◽  
Vol 81 (1) ◽  
pp. 364-372 ◽  
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.

scholarly journals Identification and Characterization of Serotype-Specific Variation in Group A Streptococcus Pilus Expression

2017 ◽  
Vol 86 (2) ◽  
Author(s):  
Gregory Calfee ◽  
Jessica L. Danger ◽  
Ira Jain ◽  
Eric W. Miller ◽  
Poulomee Sarkar ◽  
...  
Keyword(s):  
Human Blood ◽  
Vaccine Development ◽  
Group A Streptococcus ◽  
Bacterial Pathogen ◽  
Host Cells ◽  
Dependent Manner ◽  
Content Type ◽  
Low Level ◽  
Invasive Infections ◽  
Group A

ABSTRACTIsolates of a given bacterial pathogen often display phenotypic variation, and this can negatively impact public health, for example, by reducing the efficacy of preventative measures. Here, we identify that the human pathogen group AStreptococcus(GAS;Streptococcus pyogenes) expresses pili on its cell surface in a serotype-specific manner. Specifically, we show that serotype M3 GAS isolates, which are nonrandomly associated with causing particularly severe and lethal invasive infections, produce negligible amounts of pili relative to serotype M1 and M49 isolates. Performance of an interserotype transcriptome comparison (serotype M1 versus serotype M3) was instrumental in this discovery. We also identified that the transcriptional regulator Nra positively regulates pilus expression in M3 GAS isolates and that the low level of pilus expression of these isolates correlates with a low level ofnratranscription. Finally, we discovered that the phenotypic consequences of low levels of pilus expression by M3 GAS isolates are a reduced ability to adhere to host cells and an increased ability to survive and proliferate in human blood. We propose that an enhanced ability to survive in human blood, in part due to reduced pilus expression, is a contributing factor in the association of serotype M3 isolates with highly invasive infections. In conclusion, our data show that GAS isolates express pili in a serotype-dependent manner and may inform vaccine development, given that pilus proteins are being discussed as possible GAS vaccine antigens.

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

2014 ◽  
Vol 82 (7) ◽  
pp. 2890-2901 ◽  
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).

scholarly journals Conversion of an M- group A streptococcus to M+ by transfer of a plasmid containing an M6 gene.

1986 ◽  
Vol 164 (5) ◽  
pp. 1641-1651 ◽  
Author(s):  
J R Scott ◽  
P C Guenthner ◽  
L M Malone ◽  
V A Fischetti
Keyword(s):  
Structural Gene ◽  
Human Blood ◽  
Group A Streptococcus ◽  
M Protein ◽  
Gene Encoding ◽  
Hyperimmune Serum ◽  
Amino Terminal ◽  
Group A ◽  
Hyperimmune Rabbit ◽  
Streptococcal Strain

An M28-derived group A streptococcal strain deleted for the gene encoding M protein was converted to M+ by introduction of a plasmid carrying emm6, the structural gene for type 6 M protein from strain D471. The reconstituted M+ strain, JRS2, resists phagocytosis in human blood and is opsonized by anti-M6 hyperimmune serum, but not by anti-M28 serum. Immunofluorescent microscopy and ELISA demonstrate the presence of M protein on its surface. In addition, JRS2 removes opsonic antibodies from hyperimmune rabbit sera generated by immunization with purified ColiM6 protein and with a synthetic amino-terminal peptide derived from M6. Immunization of rabbits with JRS2 generates opsonic anti-M6 antibodies. These results indicate that the cloned emm6 gene contains the information necessary to convert a phagocytosis-sensitive streptococcus to phagocytosis resistance. Furthermore, it also contains the determinants for M type specificity and those required to elicit opsonic antibodies. It thus appears to determine all the traits associated with M protein.

scholarly journals Malleilactone Is a Burkholderia pseudomallei Virulence Factor Regulated by Antibiotics and Quorum Sensing

2018 ◽  
Vol 200 (14) ◽  
Author(s):  
Jennifer R. Klaus ◽  
Jacqueline Deay ◽  
Benjamin Neuenswander ◽  
Wyatt Hursh ◽  
Zhe Gao ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Caenorhabditis Elegans ◽  
Quorum Sensing ◽  
Virulence Factor ◽  
Mammalian Cells ◽  
Burkholderia Pseudomallei ◽  
Gene Clusters ◽  
Close Relative ◽  
Burkholderia Thailandensis ◽  
Content Type

ABSTRACT Burkholderia pseudomallei , the causative agent of melioidosis, encodes almost a dozen predicted polyketide (PK) biosynthetic gene clusters. Many of these are regulated by LuxR-I-type acyl-homoserine (AHL) quorum-sensing systems. One of the PK gene clusters, the mal gene cluster, is conserved in the close relative Burkholderia thailandensis . The B. thailandensis mal genes code for the cytotoxin malleilactone and are regulated by a genetically linked LuxR-type transcription factor, MalR. Although AHLs typically interact with LuxR-type proteins to modulate gene transcription, the B. thailandensis MalR does not appear to be an AHL receptor. Here, we characterize the mal genes and MalR in B. pseudomallei . We use chemical analyses to demonstrate that the B. pseudomallei mal genes code for malleilactone. Our results show that MalR and the mal genes contribute to the ability of B. pseudomallei to kill Caenorhabditis elegans . In B. thailandensis , antibiotics like trimethoprim can activate MalR by driving transcription of the mal genes, and we demonstrate that some of the same antibiotics induce expression of B. pseudomallei malR . We also demonstrate that B. pseudomallei MalR does not respond directly to AHLs. Our results suggest that MalR is indirectly repressed by AHLs, possibly through a repressor, ScmR. We further show that malleilactone is a B. pseudomallei virulence factor and provide the foundation for understanding how malleilactone contributes to the pathology of melioidosis infections. IMPORTANCE Many bacterially produced polyketides are cytotoxic to mammalian cells and are potentially important contributors to pathogenesis during infection. We are interested in the polyketide gene clusters present in Burkholderia pseudomallei , which causes the often-fatal human disease melioidosis. Using knowledge gained by studies in the close relative Burkholderia thailandensis , we show that one of the B. pseudomallei polyketide biosynthetic clusters produces a cytotoxic polyketide, malleilactone. Malleilactone contributes to B. pseudomallei virulence in a Caenorhabditis elegans infection model and is regulated by an orphan LuxR family quorum-sensing transcription factor, MalR. Our studies demonstrate that malleilactone biosynthesis or MalR could be new targets for developing therapeutics to treat melioidosis.

scholarly journals 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

2015 ◽  
Vol 83 (11) ◽  
pp. 4293-4303 ◽  
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.

scholarly journals Genome-Wide Identification of Genes Required for Fitness of Group A Streptococcus in Human Blood

2013 ◽  
Vol 81 (3) ◽  
pp. 862-875 ◽  
Author(s):  
Yoann Le Breton ◽  
Pragnesh Mistry ◽  
Kayla M. Valdes ◽  
Jeffrey Quigley ◽  
Nikhil Kumar ◽  
...  
Keyword(s):  
Human Blood ◽  
De Novo ◽  
Group A Streptococcus ◽  
Response Regulator ◽  
Wide Spectrum ◽  
Sugar Metabolism ◽  
Nucleotide Synthesis ◽  
Rich Media ◽  
Group A ◽  
Virulence Regulator

ABSTRACTThe group A streptococcus (GAS) is a strict human pathogen responsible for a wide spectrum of diseases. Although GAS genome sequences are available, functional genomic analyses have been limited. We developed amariner-based transposon,osKaR, designed to perform Transposon-Site Hybridization (TraSH) in GAS and successfully tested its use in several invasive serotypes. A complexosKaRmutant library in M1T1 GAS strain 5448 was subjected to negative selection in human blood to identify genes important for GAS fitness in this clinically relevant environment. Mutants underrepresented after growth in blood (output pool) compared to growth in rich media (input pool) were identified using DNA microarray hybridization of transposon-specific tagsen masse. Using blood from three different donors, we identified 81 genes that met our criteria for reduced fitness in blood from at least two individuals. Genes known to play a role in survival of GAS in blood were found, including those encoding the virulence regulator Mga (mga), the peroxide response regulator PerR (perR), and the RofA-like regulator Ralp-3 (ralp3). We also identified genes previously reported for their contribution to sepsis in other pathogens, such asde novonucleotide synthesis (purD,purA,pyrB,carA,carB,guaB), sugar metabolism (scrB,fruA), zinc uptake (adcC), and transcriptional regulation (cpsY). To validate our findings, independent mutants with mutations in 10 different genes identified in our screen were confirmed to be defective for survival in blood bactericidal assays. Overall, this work represents the first use of TraSH in GAS to identify potential virulence genes.

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

2000 ◽  
Vol 97 (5) ◽  
pp. 2235-2240 ◽  
Author(s):  
T. F. Kagawa ◽  
J. C. Cooney ◽  
H. M. Baker ◽  
S. McSweeney ◽  
M. Liu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Virulence Factor ◽  
Cysteine Protease ◽  
Group A Streptococcus ◽  
Group A

scholarly journals The scfCDE Operon Encodes a Predicted ABC Importer Required for Fitness and Virulence during Group A Streptococcus Invasive Infection

2019 ◽  
Vol 87 (12) ◽  
Author(s):  
Rezia Era Braza ◽  
Yoann Le Breton ◽  
Kevin S. McIver
Keyword(s):  
Soft Tissue ◽  
Group A Streptococcus ◽  
Defined Medium ◽  
Growth Defect ◽  
Invasive Infection ◽  
Competitive Growth ◽  
Content Type ◽  
Wide Range ◽  
Integral Role ◽  
Group A

ABSTRACT As a strict human pathogen, Streptococcus pyogenes (group A Streptococcus, or GAS) causes a wide range of infections, from superficial to life-threatening diseases, upon dissemination. Thus, it is necessary to gain a better understanding of how GAS successfully overcomes host-mediated challenges and infects various host niches. We previously identified subcutaneous fitness (scf) genes in the clinically relevant wild-type (WT) GAS M1T1 5448 strain that are critical for fitness during murine soft-tissue infection at both 24 h and 48 h postinfection. The uncharacterized locus scfCDE was transcribed as an operon and is predicted to encode an ABC importer for nutrient uptake (e.g., amino acids). Individual scfCDE deletion mutants grew comparably to WT 5448 in rich medium but exhibited reduced fitness during competitive growth in murine soft tissue and in nutrient-limiting chemically defined medium (CDM). A deletion of the permease gene scfD resulted in a monoculture growth defect in CDM that could be rescued by addition of excess peptides, suggesting a role as an amino acid importer. Interestingly, the ΔscfC substrate-binding and ΔscfD permease mutants, but not the ΔscfE ATPase mutant, were highly attenuated in murine soft tissue. Moreover, all three genes were required for GAS survival in human blood, indicating their impact is not limited to superficial infections. As such, scfCDE plays an integral role in enhancing GAS adaptation during localized infection as well as dissemination to deeper host environments. Since scfCDE is conserved throughout Firmicutes, this work may contribute to the development of therapeutic strategies against GAS and other Gram-positive pathogens.

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