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

Ulrike Resch,Manfred Rohde,Philip Tinnefeld,Anaïs Le Rhun,Susanne Holzmeister,Emmanuelle Charpentier,Sergo Kasvandik,James Anthony Tsatsaronis,Sun Nyunt Wai,Gerald Stübiger,E Peter Greenberg

doi:10.1128/mbio.00207-16

Ulrike Resch, Manfred Rohde + Show 9 more

Open Access

https://doi.org/10.1128/mbio.00207-16

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Abstract

ABSTRACTExport 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.

Highlights

Export of macromolecules via extracellular membrane-derived vesicles (MVs) plays an important role in the biology of Gram-negative bacteria
Pellets obtained from ISS3348 cultures were examined by negativestaining transmission electron microscopy (TEM) and exhibited a heterogeneously sized population of circular structures suggestive of extracellular membrane-derived vesicles (MVs) (Fig. 1G; see Fig. S1A in the supplemental material)
To determine whether MV production is an active process, MVs from late-logarithmic-phase group A streptococcus (GAS) culture supernatants and the same cultures incubated in fresh media following heat inactivation were quantified by flow cytometry

Summary

Introduction

Export of macromolecules via extracellular membrane-derived vesicles (MVs) plays an important role in the biology of Gram-negative bacteria. No vaccines are currently licensed for commercial use against GAS infections [3] Both mild and severe forms of GAS diseases are mediated by a suite of secreted and membrane-associated virulence factors that interact with the human host [3, 4]. Previous studies illustrate the presence of multiple factors present in the GAS secretome that lack the N-terminal secretion signal peptide or C-terminal cell wall anchor motif required for Sec-mediated translocation [6] These factors, dubbed “anchorless surface proteins,” comprise many important GAS virulence determinants and vaccine candidates; the mechanisms responsible for transport. Mycobacterium tuberculosis and other mycobacterial species actively produce MVs, which are able to modulate host inflammatory responses [15] These studies hint at a more broadly conserved export strategy utilized by both Gram-negative and Gram-positive bacteria [16].

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