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

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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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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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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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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Natural Variant of Collagen-Like Protein A in Serotype M3 Group A Streptococcus Increases Adherence and Decreases Invasive Potential

Infection and Immunity ◽

10.1128/iai.02860-14 ◽

2015 ◽

Vol 83 (3) ◽

pp. 1122-1129 ◽

Cited By ~ 12

Author(s):

Anthony R. Flores ◽

Brittany E. Jewell ◽

Erika M. Versalovic ◽

Randall J. Olsen ◽

Beth A. Bachert ◽

...

Keyword(s):

Molecular Mechanisms ◽

Group A Streptococcus ◽

Stop Codon ◽

Protein A ◽

Premature Stop Codon ◽

Invasive Disease ◽

Content Type ◽

Invasive Strain ◽

Group A ◽

Epithelial Barriers

Group AStreptococcus(GAS) predominantly exists as a colonizer of the human oropharynx that occasionally breaches epithelial barriers to cause invasive diseases. Despite the frequency of GAS carriage, few investigations into the contributory molecular mechanisms exist. To this end, we identified a naturally occurring polymorphism in the gene encoding the streptococcal collagen-like protein A (SclA) in GAS carrier strains. All previously sequenced invasive serotype M3 GAS possess a premature stop codon in thesclAgene truncating the protein. The carrier polymorphism is predicted to restore SclA function and was infrequently identified by targeted DNA sequencing in invasive strains of the same serotype. We demonstrate that a strain with the carriersclAallele expressed a full-length SclA protein, while the strain with the invasivesclAallele expressed a truncated variant. An isoallelic mutant invasive strain with the carriersclAallele exhibited decreased virulence in a mouse model of invasive disease and decreased multiplication in human blood. Further, the isoallelic invasive strain with the carriersclAallele persisted in the mouse nasopharynx and had increased adherence to cultured epithelial cells. Repair of the premature stop codon in the invasivesclAallele restored the ability to bind the extracellular matrix proteins laminin and cellular fibronectin. These data demonstrate that a mutation in GAS carrier strains increases adherence and decreases virulence and suggest selection against increased adherence in GAS invasive isolates.

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Novel Genes Required for the Fitness ofStreptococcus pyogenesin Human Saliva

mSphere ◽

10.1128/mspheredirect.00460-17 ◽

2017 ◽

Vol 2 (6) ◽

Cited By ~ 15

Author(s):

Luchang Zhu ◽

Amelia R. L. Charbonneau ◽

Andrew S. Waller ◽

Randall J. Olsen ◽

Stephen B. Beres ◽

...

Keyword(s):

Molecular Mechanisms ◽

Group A Streptococcus ◽

Human Saliva ◽

Mutant Library ◽

Acid Transport ◽

Wild Type ◽

Content Type ◽

Inorganic Ion ◽

New Information ◽

Group A

ABSTRACTStreptococcus pyogenes(group A streptococcus [GAS]) causes 600 million cases of pharyngitis each year. Despite this considerable disease burden, the molecular mechanisms used by GAS to infect, cause clinical pharyngitis, and persist in the human oropharynx are poorly understood. Saliva is ubiquitous in the human oropharynx and is the first material GAS encounters in the upper respiratory tract. Thus, a fuller understanding of how GAS survives and proliferates in saliva may provide valuable insights into the molecular mechanisms at work in the human oropharynx. We generated a highly saturated transposon insertion mutant library in serotype M1 strain MGAS2221, a strain genetically representative of a pandemic clone that arose in the 1980s and spread globally. The transposon mutant library was exposed to human saliva to screen for GAS genes required for wild-type fitness in this clinically relevant fluid. Using transposon-directed insertion site sequencing (TraDIS), we identified 92 genes required for GAS fitness in saliva. The more prevalent categories represented were genes involved in carbohydrate transport/metabolism, amino acid transport/metabolism, and inorganic ion transport/metabolism. Using six isogenic mutant strains, we confirmed that each of the mutants was significantly impaired for growth or persistence in human salivaex vivo. Mutants with an inactivated Spy0644 (sptA) or Spy0646 (sptC) gene had especially severe persistence defects. This study is the first to use of TraDIS to study bacterial fitness in human saliva. The new information we obtained will be valuable for future translational maneuvers designed to prevent or treat human GAS infections.IMPORTANCEThe human bacterial pathogenStreptococcus pyogenes(group A streptococcus [GAS]) causes more than 600 million cases of pharyngitis annually worldwide, 15 million of which occur in the United States. The human oropharynx is the primary anatomic site for GAS colonization and infection, and saliva is the first material encountered. Using a genome-wide transposon mutant screen, we identified 92 GAS genes required for wild-type fitness in human saliva. Many of the identified genes are involved in carbohydrate transport/metabolism, amino acid transport/metabolism, and inorganic ion transport/metabolism. The new information is potentially valuable for developing novel GAS therapeutics and vaccine research.Podcast: A podcast concerning this article is available.

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Neutrophils Select Hypervirulent CovRS Mutants of M1T1 Group A Streptococcus during Subcutaneous Infection of Mice

Infection and Immunity ◽

10.1128/iai.01458-13 ◽

2014 ◽

Vol 82 (4) ◽

pp. 1579-1590 ◽

Cited By ~ 31

Author(s):

Jinquan Li ◽

Guanghui Liu ◽

Wenchao Feng ◽

Yang Zhou ◽

Mengyao Liu ◽

...

Keyword(s):

Group A Streptococcus ◽

Regulatory System ◽

Lavage Fluid ◽

Content Type ◽

Enhanced Expression ◽

Group A ◽

Skin Invasion ◽

Selection For ◽

Innate Immune Evasion ◽

Selection Of

ABSTRACTPathogen mutants arise during infections. Mechanisms of selection for pathogen variants are poorly understood. We tested whether neutrophils select mutations in the two-component regulatory system CovRS of group AStreptococcus(GAS) during infection using the lack of production of the protease SpeB (SpeB activity negative [SpeBA−]) as a marker. Depletion of neutrophils by antibodies RB6-8C5 and 1A8 reduced the percentage of SpeBA−variants (SpeBA−%) recovered from mice infected with GAS strain MGAS2221 by >76%. Neutrophil recruitment and SpeBA−% among recovered GAS were reduced by 95% and 92%, respectively, in subcutaneous MGAS2221 infection of CXCR2−/−mice compared with control mice. In air sac infection with MGAS2221, levels of neutrophils and macrophages in lavage fluid were reduced by 49% and increased by 287%, respectively, in CXCR2−/−mice compared with control mice, implying that macrophages play an insignificant role in the reduction of selection for SpeBA−variants in CXCR2−/−mice. One randomly chosen SpeBA−mutant outcompeted MGAS2221 in normal mice but was outcompeted by MGAS2221 in neutropenic mice and had enhancements in expression of virulence factors, innate immune evasion, skin invasion, and virulence. This and nine other SpeBA−variants from a mouse all had nonsynonymouscovRSmutations that resulted in the SpeBA−phenotype and enhanced expression of the CovRS-controlled secreted streptococcal esterase (SsE). Our findings are consistent with a model that neutrophils select spontaneouscovRSmutations that maximize the potential of GAS to evade neutrophil responses, resulting in variants with enhanced survival and virulence. To our knowledge, this is the first report of the critical contribution of neutrophils to the selection of pathogen variants.

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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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Pathovar Transcriptomes

mSystems ◽

10.1128/msystems.00049-17 ◽

2017 ◽

Vol 2 (4) ◽

Cited By ~ 1

Author(s):

Amy Platenkamp ◽

Jay L. Mellies

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Regulatory Networks ◽

Virulence Gene ◽

Model System ◽

Genomic Sequences ◽

Bacterial Strains ◽

Content Type ◽

Virulence Gene Expression ◽

Link Type

ABSTRACT Archetypal pathogenic bacterial strains are often used to elucidate regulatory networks of an entire pathovar, which encompasses multiple lineages and phylogroups. With enteropathogenic Escherichia coli (EPEC) as a model system, Hazen and colleagues (mSystems 6:e00024-17, 2017, https://doi.org/10.1128/mSystems.00024-17 ) used 9 isolates representing 8 lineages and 3 phylogroups to find that isolates with similar genomic sequences exhibit similarities in global transcriptomes under conditions of growth in medium that induces virulence gene expression, and they found variation among individual isolates. Archetypal pathogenic bacterial strains are often used to elucidate regulatory networks of an entire pathovar, which encompasses multiple lineages and phylogroups. With enteropathogenic Escherichia coli (EPEC) as a model system, Hazen and colleagues (mSystems 6:e00024-17, 2017, https://doi.org/10.1128/mSystems.00024-17 ) used 9 isolates representing 8 lineages and 3 phylogroups to find that isolates with similar genomic sequences exhibit similarities in global transcriptomes under conditions of growth in medium that induces virulence gene expression. They also found variation among individual isolates. Their work illustrates the importance of moving beyond observing regulatory phenomena of a limited number of regulons in a few archetypal strains, with the possibility of correlating clinical symptoms to key transcriptional pathways across lineages and phylogroups.

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The Putative APSES Transcription Factor RgdA Governs Growth, Development, Toxigenesis, and Virulence in Aspergillus fumigatus

mSphere ◽

10.1128/msphere.00998-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Sang-Cheol Jun ◽

Yong-Ho Choi ◽

Min-Woo Lee ◽

Jae-Hyuk Yu ◽

Kwang-Soo Shin

Keyword(s):

Transcription Factor ◽

Aspergillus Fumigatus ◽

Molecular Mechanisms ◽

Pathogenic Fungus ◽

Mrna Levels ◽

Transcript Levels ◽

Content Type ◽

Asexual Sporulation ◽

Human Pathogenic Fungus ◽

Growth Development

ABSTRACT The APSES transcription factor (TF) in Aspergillus species is known to govern diverse cellular processes, including growth, development, and secondary metabolism. Here, we investigated functions of the rgdA gene (Afu3g13920) encoding a putative APSES TF in the opportunistic human-pathogenic fungus Aspergillus fumigatus. The rgdA deletion resulted in significantly decreased hyphal growth and asexual sporulation. Consistently, transcript levels of the key asexual developmental regulators abaA, brlA, and wetA were decreased in the ΔrgdA mutant compared to those in the wild type (WT). Moreover, ΔrgdA resulted in reduced spore germination rates and elevated transcript levels of genes associated with conidium dormancy. The conidial cell wall hydrophobicity and architecture were changed, and levels of the RodA protein were decreased in the ΔrgdA mutant. Comparative transcriptomic analyses revealed that the ΔrgdA mutant showed higher mRNA levels of gliotoxin (GT)-biosynthetic genes and GT production. While the ΔrgdA mutant exhibited elevated production of GT, ΔrgdA strains showed reduced virulence in the mouse model. In addition, mRNA levels of genes associated with the cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway and the SakA mitogen-activated protein (MAP) kinase pathway were increased in the ΔrgdA mutant. In summary, RgdA plays multiple roles in governing growth, development, GT production, and virulence which may involve attenuation of PKA and SakA signaling. IMPORTANCE Immunocompromised patients are susceptible to infections with the opportunistic human-pathogenic fungus Aspergillus fumigatus. This fungus causes systemic infections such as invasive aspergillosis (IA), which is one of the most life-threatening fungal diseases. To control this serious disease, it is critical to identify new antifungal drug targets. In fungi, the transcriptional regulatory proteins of the APSES family play crucial roles in controlling various biological processes, including mating, asexual sporulation and dimorphic growth, and virulence traits. This study found that a putative APSES transcription factor, RgdA, regulates normal growth, asexual development, conidium germination, spore wall architecture and hydrophobicity, toxin production, and virulence in A. fumigatus. Better understanding the molecular mechanisms of RgdA in human-pathogenic fungi may reveal a novel antifungal target for future drug development.

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A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus

mBio ◽

10.1128/mbio.00207-16 ◽

2016 ◽

Vol 7 (6) ◽

Cited By ~ 47

Author(s):

Ulrike Resch ◽

James Anthony Tsatsaronis ◽

Anaïs Le Rhun ◽

Gerald Stübiger ◽

Manfred Rohde ◽

...

Keyword(s):

Lipid Composition ◽

Secretory Pathway ◽

Group A Streptococcus ◽

Regulatory System ◽

Surface Proteins ◽

Streptococcal Toxic Shock Syndrome ◽

Gram Positive ◽

Gram Positive Bacteria ◽

Two Component ◽

Group A

ABSTRACT Export of macromolecules via extracellular membrane-derived vesicles (MVs) plays an important role in the biology of Gram-negative bacteria. Gram-positive bacteria have also recently been reported to produce MVs; however, the composition and mechanisms governing vesiculogenesis in Gram-positive bacteria remain undefined. Here, we describe MV production in the Gram-positive human pathogen group A streptococcus (GAS), the etiological agent of necrotizing fasciitis and streptococcal toxic shock syndrome. M1 serotype GAS isolates in culture exhibit MV structures both on the cell wall surface and in the near vicinity of bacterial cells. A comprehensive analysis of MV proteins identified both virulence-associated protein substrates of the general secretory pathway in addition to “anchorless surface proteins.” Characteristic differences in the contents, distributions, and fatty acid compositions of specific lipids between MVs and GAS cell membrane were also observed. Furthermore, deep RNA sequencing of vesicular RNAs revealed that GAS MVs contained differentially abundant RNA species relative to bacterial cellular RNA. MV production by GAS strains varied in a manner dependent on an intact two-component system, CovRS, with MV production negatively regulated by the system. Modulation of MV production through CovRS was found to be independent of both GAS cysteine protease SpeB and capsule biosynthesis. Our data provide an explanation for GAS secretion of macromolecules, including RNAs, lipids, and proteins, and illustrate a regulatory mechanism coordinating this secretory response. IMPORTANCE Group A streptococcus (GAS) is a Gram-positive bacterial pathogen responsible for more than 500,000 deaths annually. Establishment of GAS infection is dependent on a suite of proteins exported via the general secretory pathway. Here, we show that GAS naturally produces extracellular vesicles with a unique lipid composition that are laden with proteins and RNAs. Interestingly, both virulence-associated proteins and RNA species were found to be differentially abundant in vesicles relative to the bacteria. Furthermore, we show that genetic disruption of the virulence-associated two-component regulator CovRS leads to an increase in vesicle production. This study comprehensively describes the protein, RNA, and lipid composition of GAS-secreted MVs and alludes to a regulatory system impacting this process.

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