• Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin.

      Boehm, Manja; Hoy, Benjamin; Rohde, Manfred; Tegtmeyer, Nicole; Bæk, Kristoffer T; Oyarzabal, Omar A; Brøndsted, Lone; Wessler, Silja; Backert, Steffen; From the School for Medicine and Medical Science, University College Dublin, Belfield Campus, Dublin-4, Ireland. Steffen.Backert@ucd.ie. (2012)
    • Reclassification and emended description of Caulobacter leidyi as Sphingomonas leidyi comb. nov., and emendation of the genus Sphingomonas.

      Chen, Hong; Jogler, Mareike; Rohde, Manfred; Klenk, Hans-Peter; Busse, Hans-Jürgen; Tindall, Brian J; Spröer, Cathrin; Overmann, Jörg; Bereich Mikrobiologie, Department Biologie I, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany. (2012-12)
      'Caulobacter leidyi' DSM 4733(T) has been shown to be affiliated with the family Sphingomonadaceae instead of the Caulobacteraceae, and due to its poor characterization has been omitted from the current edition of Bergey's Manual of Systematic Bacteriology and removed to limbo. We isolated a novel sphingoglycolipid-containing dimorphic prosthecate bacterium, designated strain 247, from a pre-alpine freshwater lake. Strain 247 and 'Caulobacter leidyi' DSM 4733(T) were characterized in detail. The rod-shaped cells were Gram-stain-negative, aerobic, catalase- and oxidase-positive, and formed a stalk or polar flagellum. Both strains grew optimally at 28-30 °C, and pH 6.0-8.0. The major fatty acids were C(18 : 1)ω7c, C(16 : 0) and 11-methyl C(18 : 1)ω7c. C(14 : 0) 2-OH represents the major 2-hydroxy fatty acid. Q-10 was the major respiratory quinone and the major polar lipids were diphosphatidylglycerol, phosphatidyldimethylethanolamine, phosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, phosphatidylcholine, three glycolipids, two phosphoaminolipids and two unidentified sphingoglycolipids. The major polyamine was sym-homospermidine. The G+C content of genomic DNA of strains 247 and DSM 4733(T) was 67.6 mol% and 67.0 mol%, respectively. According to 16S rRNA gene sequence analysis and DNA-DNA hybridization, strains DSM 4733(T) and 247 were phylogenetically closely related (99.6 % 16S rRNA gene sequence similarity, 82.9 % DNA-DNA hybridization value) and affiliated to the genus Sphingomonas. The closest recognized species was Sphingomonas aquatilis DSM 15581(T) (98.1 % sequence similarity). In addition, the presence of cystine arylamidase, absence of β-galactosidase, and the inability to utilize l-arabinose, galactose and sucrose distinguished strains DSM 4733(T) and 247 from most other members of the family Sphingomonadaceae. So far, the dimorphic life cycle that involves a prosthecate and a flagellated stage is unique for strains DSM 4733(T) and 247 among all members of the family Sphingomonadaceae. Therefore, Caulobacter leidyi is reclassified as Sphingomonas leidyi, with the type strain DSM 4733(T) ( = ATCC 15260(T) = CIP 106443(T) = VKM B-1368(T)) and strain 247 (DSM 25078 = LMG 26658) as an additional strain of this species.
    • Region specific and worldwide distribution of collagen-binding M proteins with PARF motifs among human pathogenic streptococcal isolates.

      Reissmann, Silvana; Gillen, Christine M; Fulde, Marcus; Bergmann, René; Nerlich, Andreas; Rajkumari, Reena; Brahmadathan, Kootallur N; Chhatwal, Gursharan S; Nitsche-Schmitz, D Patric; Department of Microbial Pathogenesis, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2012)
      Some of the variety of Streptococcus pyogenes and Streptococcus dysgalactiae ssp. equisimilis (SDSE) M proteins act as collagen-binding adhesins that facilitate acute infection. Moreover, their potential to trigger collagen autoimmunity has been implicated in the pathogenesis of acute rheumatic fever and attributed to a collagen-binding motif called PARF (peptide associated with rheumatic fever). For the first time we determine the rate of clinical isolates with collagen-binding M proteins that use a PARF motif (A/T/E)XYLXX(L/F)N in a defined geographic region, Vellore in South India. In this region both, incidence of streptococcal infections and prevalence of acute rheumatic fever are high. M proteins with PARF motif conferred collagen-binding activity to 3.9% of 153 S. pyogenes and 10.6% of 255 SDSE clinical isolates from Vellore. The PARF motif occurred in three S. pyogenes and 22 SDSE M protein types. In one of the S. pyogenes and five of the SDSE M proteins that contained the motif, collagen-binding was impaired, due to influences of other parts of the M protein molecule. The accumulated data on the collagen binding activity of certain M protein types allowed a reanalysis of published worldwide emm-typing data with the aim to estimate the rates of isolates that bind collagen via PARF. The results indicate that M proteins, which bind collagen via a PARF motif, are epidemiologically relevant in human infections, not only in Vellore. It is imperative to include the most relevant collagen-binding M types in vaccines. But when designing M protein based vaccines it should be considered that collagen binding motifs within the vaccine antigen remain potential risk factors.
    • Rheumatic fever–associated Streptococcus pyogenes isolates aggregate collagen

      Dinkla, Katrin; Rohde, Manfred; Jansen, Wouter T.M.; Kaplan, Edward L.; Chhatwal, Gursharan S.; Talay, Susanne R. (American Society for Clinical Investigation, 2003-06-15)
    • RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes.

      Le Rhun, Anaïs; Beer, Yan Yan; Reimegård, Johan; Chylinski, Krzysztof; Charpentier, Emmanuelle; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-02)
      Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.
    • Role of glucose and CcpA in capsule expression and virulence of Streptococcus suis.

      Willenborg, J; Fulde, M; de Greeff, A; Rohde, Manfred; Smith, H E; Valentin-Weigand, P; Goethe, R; Institute for Microbiology, University of Veterinary Medicine, Hannover, Germany. (2011-06)
      Streptococcus suis is one of the most important pathogens in pigs and is also an emerging zoonotic agent. After crossing the epithelial barrier, S. suis causes bacteraemia, resulting in meningitis, endocarditis and bronchopneumonia. Since the host environment seems to be an important regulatory component for virulence, we related expression of virulence determinants of S. suis to glucose availability during growth and to the sugar metabolism regulator catabolite control protein A (CcpA). We found that expression of the virulence-associated genes arcB, representing arcABC operon expression, cps2A, representing capsular locus expression, as well as sly, ofs, sao and epf, differed significantly between exponential and early stationary growth of a highly virulent serotype 2 strain. Deletion of ccpA altered the expression of the surface-associated virulence factors arcB, sao and eno, as well as the two currently proven virulence factors in pigs, ofs and cps2A, in early exponential growth. Global expression analysis using a cDNA expression array revealed 259 differentially expressed genes in early exponential growth, of which 141 were more highly expressed in the CcpA mutant strain 10ΔccpA and 118 were expressed to a lower extent. Interestingly, among the latter genes, 18 could be related to capsule and cell wall synthesis. Correspondingly, electron microscopy characterization of strain 10ΔccpA revealed a markedly reduced thickness of the capsule. This phenotype correlated with enhanced binding to porcine plasma proteins and a reduced resistance to killing by porcine neutrophils. Taken together, our data demonstrate that CcpA has a significant effect on the capsule synthesis and virulence properties of S. suis.
    • Role of Macrophages in Host Resistance to Group A Streptococci

      Goldmann, Oliver; Rohde, Manfred; Chhatwal, Gursharan Singh; Medina, Eva (American Society for Microbiology, 2004-05)
    • SCM, a novel M-like protein from Streptococcus canis, binds (mini)-plasminogen with high affinity and facilitates bacterial transmigration.

      Fulde, Marcus; Rohde, Manfred; Hitzmann, Angela; Preissner, Klaus T; Nitsche-Schmitz, D Patric; Nerlich, Andreas; Chhatwal, Gursharan Singh; Bergmann, Simone; Department of Medical Microbiology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany. Marcus.Fulde@helmholtz-hzi.de (2011-02-24)
      Streptococcus canis is an important zoonotic pathogen capable of causing serious invasive diseases in domestic animals and humans. In the present paper we report the binding of human plasminogen to S. canis and the recruitment of proteolytically active plasmin on its surface. The binding receptor for plasminogen was identified as a novel M-like protein designated SCM (S. canis M-like protein). SPR (surface plasmon resonance) analyses, radioactive dot-blot analyses and heterologous expression on the surface of Streptococcus gordonii confirmed the plasminogen-binding capability of SCM. The binding domain was located within the N-terminus of SCM, which specifically bound to the C-terminal part of plasminogen (mini-plasminogen) comprising kringle domain 5 and the catalytic domain. In the presence of urokinase, SCM mediated plasminogen activation on the bacterial surface that was inhibited by serine protease inhibitors and lysine amino acid analogues. Surface-bound plasmin effectively degraded purified fibrinogen as well as fibrin clots, resulting in the dissolution of fibrin thrombi. Electron microscopic illustration and time-lapse imaging demonstrated bacterial transmigration through fibrinous thrombi. The present study has led, for the first time, to the identification of SCM as a novel receptor for (mini)-plasminogen mediating the fibrinolytic activity of S. canis.
    • Sequencing and characterization of Pseudomonas aeruginosa phage JG004.

      Garbe, Julia; Bunk, Boyke; Rohde, Manfred; Schobert, Max; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2011)
      Phages could be an important alternative to antibiotics, especially for treatment of multiresistant bacteria as e.g. Pseudomonas aeruginosa. For an effective use of bacteriophages as antimicrobial agents, it is important to understand phage biology but also genes of the bacterial host essential for phage infection.
    • Simultaneous Deficiency of both MurA and p60 Proteins Generates a Rough Phenotype in Listeria monocytogenes

      Machata, Silke; Hain, Torsten; Rohde, Manfred; Chakraborty, Trinad (American Society for Microbiology, 2005-12)
    • Sphingomonas starnbergensis sp. nov., isolated from a prealpine freshwater lake.

      Chen, Hong; Jogler, Mareike; Tindall, Brian J; Klenk, Hans-Peter; Rohde, Manfred; Busse, Hans-Jürgen; Overmann, Jörg; Bereich Mikrobiologie, Department Biologie I, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany. (2013-03)
      A novel type of freshwater bacterium was isolated from the prealpine mesotrophic Starnberger See (Bavaria, southern Germany). Cells of strain 382(T) were Gram-negative and rod-shaped, motile and creamy-white. The isolate was strictly aerobic, catalase- and oxidase-positive, and grew at pH values of 6-9 (optimum, pH 7) and temperatures of 10-37 °C (optimum, 28 °C). The genomic G+C content of strain 382(T) was 64.1 mol%. Based on 16S rRNA gene sequence analyses, strain 382(T) belongs to the family Sphingomonadaceae and clusters within the genus Sphingomonas. Sphingomonas histidinilytica UM 2(T) and Sphingomonas wittichii DSM 6014(T) were the closest relatives, as indicated by the highest 16S rRNA gene sequence similarities (97.1 % and 96.8 %, respectively). Sphingomonas paucimobilis DSM 1098(T) (the type species of the genus Sphingomonas) exhibited 95.3 % sequence similarity. This affiliation of strain 382(T) to the genus Sphingomonas is confirmed by the presence of Q-10 as the major respiratory quinone, two sphingoglycolipids, C14 : 0 2-OH as the major 2-hydroxy fatty acid and sym-homospermidine as the major polyamine. The main cellular fatty acids of strain 382(T) were C18 : 1ω7c (39 %), C16 : 1ω7c (21 %), C16 : 0 (10 %) and C14 : 0 2-OH (10 %). Based on the phylogenetic distance from other species of the genus Sphingomonas and its unusually high C16 : 1ω7c content, strain 382(T) represents a novel species of the genus Sphingomonas, for which the name Sphingomonas starnbergensis is proposed. The type strain is 382(T) ( = DSM 25077(T)  = LMG 26763(T)).
    • Streptococcal protein FOG, a novel matrix adhesin interacting with collagen I in vivo.

      Nitsche, D Patric; Johansson, Helena M; Frick, Inga-Maria; Mörgelin, Matthias (2006-01-20)
      Group G streptococcus (GGS) is a human pathogen of emerging clinical significance. It causes skin and soft tissue infections, occasionally resulting in life-threatening conditions such as sepsis and necrotizing fasciitis. We recently identified FOG, a novel surface protein of GGS with fibrinogen binding and immune evasion properties. Here we investigated the role of FOG in streptococcal primary adhesion to host tissue. A FOG-expressing clinical isolate adhered more efficiently to human skin biopsies ex vivo and to the murine dermis in vivo than a FOG-deficient strain. Scanning and transmission electron microscopy of skin specimens exhibited that this property was assigned to the ability of FOG to interact with collagen I, a major interstitial component of the dermis. Overlay experiments with human skin extracts and radiolabeled FOG followed by matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis identified both the alpha1- and alpha2-chains of collagen I as targets for FOG. Transmission electron microscopy of the molecular complexes revealed thread-like FOG molecules binding via their NH2 termini to distinct sites on collagen I monomers and fibrils. The results demonstrate that FOG is important for GGS adhesion in vivo, implying a pathogenic role for this surface protein.
    • Streptococcal surface proteins activate the contact system and control its antibacterial activity.

      Wollein Waldetoft, Kristofer; Svensson, Lisbeth; Mörgelin, Matthias; Olin, Anders I; Nitsche-Schmitz, D Patric; Björck, Lars; Frick, Inga-Maria; Division of Infection Medicine, Department of Clinical Sciences, Lund University, SE-221 84 Lund, Sweden. kristofer.wollein_waldetoft@med.lu.se (2012-07-20)
      Group G streptococci (GGS) are important bacterial pathogens in humans. Here, we investigated the interactions between GGS and the contact system, a procoagulant and proinflammatory proteolytic cascade that, upon activation, also generates antibacterial peptides. Two surface proteins of GGS, protein FOG and protein G (PG), were found to bind contact system proteins. Experiments utilizing contact protein-deficient human plasma and isogenic GGS mutant strains lacking FOG or PG showed that FOG and PG both activate the procoagulant branch of the contact system. In contrast, only FOG induced cleavage of high molecular weight kininogen, generating the proinflammatory bradykinin peptide and additional high molecular weight kininogen fragments containing the antimicrobial peptide NAT-26. On the other hand, PG protected the bacteria against the antibacterial effect of NAT-26. These findings underline the significance of the contact system in innate immunity and demonstrate that GGS have evolved surface proteins to exploit and modulate its effects.
    • Structure-informed design of an enzymatically inactive vaccine component for group A Streptococcus.

      Henningham, Anna; Ericsson, Daniel J; Langer, Karla; Casey, Lachlan W; Jovcevski, Blagojce; Chhatwal, G Singh; Aquilina, J Andrew; Batzloff, Michael R; Kobe, Bostjan; Walker, Mark J; et al. (2013)
      Streptococcus pyogenes (group A Streptococcus [GAS]) causes ~700 million human infections/year, resulting in >500,000 deaths. There is no commercial GAS vaccine available. The GAS surface protein arginine deiminase (ADI) protects mice against a lethal challenge. ADI is an enzyme that converts arginine to citrulline and ammonia. Administration of a GAS vaccine preparation containing wild-type ADI, a protein with inherent enzymatic activity, may present a safety risk. In an approach intended to maximize the vaccine safety of GAS ADI, X-ray crystallography and structural immunogenic epitope mapping were used to inform vaccine design. This study aimed to knock out ADI enzyme activity without disrupting the three-dimensional structure or the recognition of immunogenic epitopes. We determined the crystal structure of ADI at 2.5 Å resolution and used it to select a number of amino acid residues for mutagenesis to alanine (D166, E220, H275, D277, and C401). Each mutant protein displayed abrogated activity, and three of the mutant proteins (those with the D166A, H275A, and D277A mutations) possessed a secondary structure and oligomerization state equivalent to those of the wild type, produced high-titer antisera, and avoided disruption of B-cell epitopes of ADI. In addition, antisera raised against the D166A and D277A mutant proteins bound to the GAS cell surface. The inactivated D166A and D277A mutant ADIs are ideal for inclusion in a GAS vaccine preparation. There is no human ortholog of ADI, and we confirm that despite limited structural similarity in the active-site region to human peptidyl ADI 4 (PAD4), ADI does not functionally mimic PAD4 and antiserum raised against GAS ADI does not recognize human PAD4.
    • Study of the in vivo role of Mce2R, the transcriptional regulator of mce2 operon in Mycobacterium tuberculosis

      Forrellad, Marina A; Bianco, María V; Blanco, Federico C; Nuñez, Javier; Klepp, Laura I; Vazquez, Cristina L; Santangelo, María d l P; Rocha, Rosana V; Soria, Marcelo; Golby, Paul; et al. (2013-09-05)
      Abstract Background Tuberculosis is one of the leading causes of mortality throughout the world. Mycobacterium tuberculosis, the agent of human tuberculosis, has developed strategies involving proteins and other compounds called virulence factors to subvert human host defences and damage and invade the human host. Among these virulence-related proteins are the Mce proteins, which are encoded in the mce1, mce2, mce3 and mce4 operons of M. tuberculosis. The expression of the mce2 operon is negatively regulated by the Mce2R transcriptional repressor. Here we evaluated the role of Mce2R during the infection of M. tuberculosis in mice and macrophages and defined the genes whose expression is in vitro regulated by this transcriptional repressor. Results We used a specialized transduction method for generating a mce2R mutant of M. tuberculosis H37Rv. Although we found equivalent replication of the MtΔmce2R mutant and the wild type strains in mouse lungs, overexpression of Mce2R in the complemented strain (MtΔmce2RComp) significantly impaired its replication. During in vitro infection of macrophages, we observed a significantly increased association of the late endosomal marker LAMP-2 to MtΔmce2RComp-containing phagosomes as compared to MtΔmce2R and the wild type strains. Whole transcriptional analysis showed that Mce2R regulates mainly the expression of the mce2 operon, in the in vitro conditions studied. Conclusions The findings of the current study indicate that Mce2R weakly represses the in vivo expression of the mce2 operon in the studied conditions and argue for a role of the proteins encoded in Mce2R regulon in the arrest of phagosome maturation induced by M. tuberculosis.
    • Subunit stoichiometry and juxtaposition of the photosynthetic coupling factor 1: Immunoelectron microscopy using monoclonal antibodies

      Tiedge, Henri; Lünsdorf, Heinrich; Schäfer, Günter; Schairer, Hans Ulrich (1985-12)
    • Toward Whole-Transcriptome Editing with CRISPR-Cas9.

      Heckl, Dirk; Charpentier, Emmanuelle; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015-05-21)
      Targeted regulation of gene expression holds huge promise for biomedical research. In a series of recent publications (Gilbert et al., 2014; Konermann et al., 2015; Zalatan et al., 2015), sophisticated, multiplex-compatible transcriptional activator systems based on the CRISPR-Cas9 technology and genome-scale libraries advance the field toward whole-transcriptome control.
    • The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems.

      Chylinski, Krzysztof; Le Rhun, Anaïs; Charpentier, Emmanuelle; The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Department of Molecular Biology, Umeå University, Umeå, Sweden. (2013-05)
      CRISPR-Cas is a rapidly evolving RNA-mediated adaptive immune system that protects bacteria and archaea against mobile genetic elements. The system relies on the activity of short mature CRISPR RNAs (crRNAs) that guide Cas protein(s) to silence invading nucleic acids. A set of CRISPR-Cas, type II, requires a trans-activating small RNA, tracrRNA, for maturation of precursor crRNA (pre-crRNA) and interference with invading sequences. Following co-processing of tracrRNA and pre-crRNA by RNase III, dual-tracrRNA:crRNA guides the CRISPR-associated endonuclease Cas9 (Csn1) to cleave site-specifically cognate target DNA. Here, we screened available genomes for type II CRISPR-Cas loci by searching for Cas9 orthologs. We analyzed 75 representative loci, and for 56 of them we predicted novel tracrRNA orthologs. Our analysis demonstrates a high diversity in cas operon architecture and position of the tracrRNA gene within CRISPR-Cas loci. We observed a correlation between locus heterogeneity and Cas9 sequence diversity, resulting in the identification of various type II CRISPR-Cas subgroups. We validated the expression and co-processing of predicted tracrRNAs and pre-crRNAs by RNA sequencing in five bacterial species. This study reveals tracrRNA family as an atypical, small RNA family with no obvious conservation of structure, sequence or localization within type II CRISPR-Cas loci. The tracrRNA family is however characterized by the conserved feature to base-pair to cognate pre-crRNA repeats, an essential function for crRNA maturation and DNA silencing by dual-RNA:Cas9. The large panel of tracrRNA and Cas9 ortholog sequences should constitute a useful database to improve the design of RNA-programmable Cas9 as genome editing tool.
    • Tumor necrosis factor alpha modulates the dynamics of the plasminogen-mediated early interaction between Bifidobacterium animalis subsp. lactis and human enterocytes.

      Centanni, Manuela; Bergmann, Simone; Turroni, Silvia; Hammerschmidt, Sven; Chhatwal, Gursharan Singh; Brigidi, Patrizia; Candela, Marco; Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy. (2012-04)
      The capacity to intervene with the host plasminogen system has recently been considered an important component in the interaction process between Bifidobacterium animalis subsp. lactis and the human host. However, its significance in the bifidobacterial microecology within the human gastrointestinal tract is still an open question. Here we demonstrate that human plasminogen favors the B. animalis subsp. lactis BI07 adhesion to HT29 cells. Prompting the HT29 cell capacity to activate plasminogen, tumor necrosis factor alpha (TNF-α) modulated the plasminogen-mediated bacterium-enterocyte interaction, reducing the bacterial adhesion to the enterocytes and enhancing migration to the luminal compartment.
    • A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus.

      Resch, Ulrike; Tsatsaronis, James Anthony; Le Rhun, Anaïs; Stübiger, Gerald; Rohde, M; Kasvandik, Sergo; Holzmeister, Susanne; Tinnefeld, Philip; Wai, Sun Nyunt; Charpentier, Emmanuelle; et al. (2016)
      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.