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

Resch, Ulrike; Tsatsaronis, James A.; Rhun, Anaïs Le; Stübiger, Gerald; Rohde, Manfred; Kasvandik, Sergo; Holzmeister, Susanne; Tinnefeld, Philip; Wai, Sun Nyunt; Charpentier, Emmanuelle

doi:10.1128/mbio.00207-16

Cited by 142 publications

(178 citation statements)

References 51 publications

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“…Thus, conserved Spo0A may be important for the regulation of vesiculogenesis in sporulating Gram-positive bacteria. Recently, it was reported that in the nonsporulating Gram-positive bacterium Streptococcus pyogenes, the two-component system CovRS downregulates MV formation (49). Given the results of our study, which indicates the phosphorylated/activated response regulator is required for MV production, signal transduction from sensor kinase to response regulator or two-component system could be a key regulatory system of vesiculogenesis in both sporulating and nonsporulating Gram-positive bacteria.…”

Section: Discussionmentioning

confidence: 80%

Immunoactive Clostridial Membrane Vesicle Production Is Regulated by a Sporulation Factor

et al. 2017

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Recently, many Gram-positive bacteria as well as Gram-negative bacteria have been reported to produce membrane vesicles (MVs), but little is known regarding the regulators involved in MV formation. We found that a Gram-positive anaerobic pathogen, Clostridium perfringens, produces MVs predominantly containing membrane proteins and cell wall components. These MVs stimulated proinflammatory cytokine production in mouse macrophage-like cells. We suggested that MVs induced interleukin-6 production through the Toll-like receptor 2 (TLR2) signaling pathway. Thus, the MV could have a role in the bacterium-host interaction and bacterial infection pathogenesis. Moreover, we found that the sporulation master regulator gene spo0A was required for vesiculogenesis. A conserved, phosphorylated aspartate residue of Spo0A was indispensable for MV production, suggesting that the phosphorylation of Spo0A triggers MV production. Multiple orphan sensor kinases necessary for sporulation were also required to maximize MV production. These findings imply that C. perfringens actively produces immunoactive MVs in response to the environment changing, as recognized by membrane-spanning sensor kinases and by modulating the phosphorylation level of Spo0A.

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Section: Discussionmentioning

confidence: 80%

Immunoactive Clostridial Membrane Vesicle Production Is Regulated by a Sporulation Factor

et al. 2017

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“…EVs are enriched in sphingolipids ,phosphatidylethanolamine, with a higher component of unsaturated fatty acids. Remarkably their compositions were distinct from other bacterial EVs: EVs from Group A Streptococcus (42) displayed an enrichment in phosphatidylcholine and monoacylglycerol. Lipid composition of EVs may vary with the bacterial species and possibly is finely adapted for each environmental niche.…”

Section: Discussionmentioning

confidence: 91%

“…For Gram-positive bacteria these questions are further complicated by concerns as to how EV traverse the bacterial cell wall (23). Raising additional suspicion for their existence was the absence of EV-null mutants, although recent work have identified genes modulating EV synthesis in Gram-positive bacteria (42) and mycobacteria (43), and gene deletions that alter morphology of EVs (17). In this study we establish that EVs are associated with LLO via biochemical purification approaches, functional detection, immunoEM and fluorescence microscopy.…”

Section: Discussionmentioning

confidence: 99%

Listeria monocytogenesvirulence factors are secreted in biologically active Extracellular Vesicles

Coelho

Brown

Maryam

et al. 2017

Preprint

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Outer membrane vesicles produced by Gramnegative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secreted extracellular vesicles (EVs) was not pursued due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that is the etiological agent of listeriosis. Here we report that L. monocytogenes produces EVs with diameter ranging from 20-200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multi-omics we characterized protein, lipid and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Cell-free EV preparations were toxic to the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC can restrain LLO activity. Using immunogold electron microscopy we detect LLO localization at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes that colocalize with LLO during infection. Our findings demonstrate that L. monocytogenes utilize EVs for toxin release and implicate these structures in mammalian cytotoxicity.

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“…As predicted in this study, the majority of EVs proteins were cytoplasmic in both G. adiacens and G. elegans proteomes (71% and 67% respectively). Cytoplasmic proteins located in other bacterial vesicles have been reported in several earlier studies [36, 38]. Existing evidence suggests that the enormous location of cytoplasmic proteins into vesicles is due to specific sorting mechanisms, and not due to lysis of dead cells [39].…”

Section: Resultsmentioning

confidence: 98%

Proteomics of Extracellular Vesicles Produced byGranulicatellaSpecies that cause Infective Endocarditis

Ellepola

et al. 2019

Preprint

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30When oral bacteria accidentally enter the bloodstream due to transient tissue 31 damage during dental procedures, they have the potential to attach to the endocardium 32 or an equivalent surface of an indwelling prosthesis and cause infection. Many bacterial 33 species produce extracellular vesicles (EVs) as part of normal physiology, but also use 34 it as a virulence strategy. In this study, it was hypothesized that Granulicatella species 35 produce EVs that possibly help them in virulence. Therefore, the objectives were to 36 isolate and characterize EVs produced by these species and to investigate their immune-37 stimulatory effects. The reference strains G. adiacens CCUG 27809 and G. elegans 38 CCUG 38949 were cultured on chocolate blood agar for 2 days. From subsequent broth 39 cultures, the EVs were isolated using differential centrifugation and filtration protocol 40 and then observed using scanning electron microscopy. Proteins in the vesicle 41 preparations were identified by nano LC-ESI-MS/MS. The EVs proteomes were 42 analyzed and characterized using different bioinformatics tools. The immune-43 stimulatory effect of the EVs was studied via ELISA quantification of IL-8, IL-1β and 44 CCL5, major proinflammatory cytokines, produced from stimulated human PBMCs. It 45 was revealed that both G. adiacens and G. elegans produced EVs, ranging in diameter 46 from 30 to 250 nm. Overall, G. adiacens EVs contained 160 proteins, and G. elegans 47 EVs contained 107 proteins. Both proteomes consist of several ribosomal proteins, 48DNA associated proteins, binding proteins, and metabolic enzymes. It was also shown 49 that these EVs carry putative virulence factors including moonlighting proteins. These 50 EVs were able to induce the production of IL-8, IL-1β and CCL5 from human PBMCs. 51The diversity in EVs content indicates that these vesicles could have possible roles in 3 52 bacterial survival, invasion, host immune modulation as well as infection. Further 53 functional characterization of the Granulicatella EVs may provide new insights into 54 virulence mechanisms of these important but less studied oral bacterial species. 55 56 57 Granulicatella species, formerly known as nutritionally variant streptococci 58 based on their characteristic dependence on pyridoxal or cysteine supplementation for 59 their growth in standard media [1], are catalase and oxidase negative, non-motile, non-60 spore-forming, facultatively anaerobic Gram-positive cocci [2, 3]. They are part of the 61 normal oral flora [4], but cause serious infections such as infective endocarditis. The 62 genus Granulicatella consists of 3 species: Granulicatella adiacens, Granulicatella 63 elegans and Granulicatella balaenopterae [3]. The species G. balaenopterae has not 64 been isolated from human samples, whereas both G. adiacens and G. elegans have been 65 reported from IE cases [5, 6]. In addition, these oral commensal cocci have been 66 associated with endodontic infections [7, 8], dental caries [9], and periodontitis [8, 10] 67 via DNA-based s...

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

Cited by 142 publications

References 51 publications

Immunoactive Clostridial Membrane Vesicle Production Is Regulated by a Sporulation Factor

Immunoactive Clostridial Membrane Vesicle Production Is Regulated by a Sporulation Factor

Listeria monocytogenesvirulence factors are secreted in biologically active Extracellular Vesicles

Proteomics of Extracellular Vesicles Produced byGranulicatellaSpecies that cause Infective Endocarditis

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