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



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.
The Laboratory for Molecular Infection Medicine Sweden (MIMS), Department of Molecular Biology, Umeå University, Umeå, Sweden.
Ulrike
U
Resch
Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.|Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
James Anthony
JA
Tsatsaronis
The Laboratory for Molecular Infection Medicine Sweden (MIMS), Department of Molecular Biology, Umeå University, Umeå, Sweden.|Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.|Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
Anaïs
A
Le Rhun
Department of Biomedical Imaging and Image-Guided Therapy, Medical University, Vienna, Vienna, Austria.
Gerald
G
Stübiger
Central Facility for Microscopy, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Manfred
M
Rohde
University of Tartu, Institute of Technology, Tartu, Estonia.
Sergo
S
Kasvandik
NanoBioScience Group, Institute for Physical and Theoretical Chemistry, TU, Braunschweig, Braunschweig, Germany.
Susanne
S
Holzmeister
NanoBioScience Group, Institute for Physical and Theoretical Chemistry, TU, Braunschweig, Braunschweig, Germany.
Philip
P
Tinnefeld
The Laboratory for Molecular Infection Medicine Sweden (MIMS), Department of Molecular Biology, Umeå University, Umeå, Sweden.
Sun Nyunt
SN
Wai
The Laboratory for Molecular Infection Medicine Sweden (MIMS), Department of Molecular Biology, Umeå University, Umeå, Sweden charpentier@mpiib-berlin.mpg.de.|Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.|Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.|Hannover Medical School, Hannover, Germany.
Emmanuelle
E
Charpentier
D001426:Bacterial Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D059014:High-Throughput Nucleotide Sequencing; D000071677:Histidine Kinase; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008055:Lipids / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis; D012313:RNA / Q000032:analysis / Q000235:genetics; D012097:Repressor Proteins / Q000378:metabolism*; D022142:Secretory Vesicles / Q000737:chemistry / Q000378:metabolism*; D013297:Streptococcus pyogenes / Q000378:metabolism*; D037521:Virulence Factors / Q000032:analysis
D001426:Bacterial Proteins; C432143:CsrR protein, Streptococcus pyogenes; D047908:Intracellular Signaling Peptides and Proteins; D008055:Lipids; D008565:Membrane Proteins; D012097:Repressor Proteins; D037521:Virulence Factors; D012313:RNA; C534547:CovS protein, Streptococcus pyogenes; D000071677:Histidine Kinase