Mycoplasmas are no exception to extracellular vesicles release: Revisiting old concepts.

Patrice Gaurivaud,Véronique Monnet,Lucia Manso-Silvan,Sarah Ganter,Alexandre Villard,Florence Tardy,Didier Chevret,Christelle Boulé,Catherine A Brissette

doi:10.1371/journal.pone.0208160

Abstract

Release of extracellular vesicles (EV) by Gram-negative and positive bacteria is being frequently reported. EV are nano-sized, membrane-derived, non-self-replicating, spherical structures shed into the extracellular environment that could play a role in bacteria-host interactions. Evidence of EV production in bacteria belonging to the class Mollicutes, which are wall-less, is mainly restricted to the genus Acholeplasma and is scanty for the Mycoplasma genus that comprises major human and animal pathogens. Here EV release by six Mycoplasma (sub)species of clinical importance was investigated. EV were obtained under nutritional stress conditions, purified by ultracentrifugation and observed by electron microscopy. The membrane proteins of EV from three different species were further identified by mass spectrometry as a preliminary approach to determining their potential role in host-pathogen interactions. EV were shown to be released by all six (sub)species although their quantities and sizes (30–220 nm) were very variable. EV purification was complicated by the minute size of viable mycoplasmal cells. The proteins of EV-membranes from three (sub)species included major components of host-pathogen interactions, suggesting that EV could contribute to make the host-pathogen interplay more complex. The process behind EV release has yet to be deciphered, although several observations demonstrated their active release from the plasma membrane of living cells. This work shed new light on old concepts of “elementary bodies” and “not-cell bound antigens”.

Highlights

A. laidlawii strain PG8T was used as a control to validate our capacity to induce Extracellular vesicles (EV) formation and perform purification
The same experimental conditions were tested on M. mycoides subsp. mycoides (Mmm) strain Afadeused as a representative of the Mycoplasma genus
Our in-house experimental procedure confirmed the capacity of A. laidlawii PG8T to produce EV and demonstrated for the first time and in a reproducible manner the production of EV by Mmm strain Afade

Summary

Introduction

Extracellular vesicles (EV) are nano-sized, membrane-derived, non-self-replicating, spherical structures shed into the extracellular environment by both eukaryotic and prokaryotic cells. Within the Gram-positive phylum, bacteria belonging to the class Mollicutes hold a special place as they are wall-less, minute-sized cells limited only by a cytoplasmic membrane. The genetic relatedness with the EV-producing Clostridium genus [7] and the lack of a cell wall physical barrier are two reasons why we could expect EV production from Mollicutes This was demonstrated by Chernov and collaborators in 2011 using Acholeplasma (A.) laidlawii as a model [14]. The same group observed vesicles from Mycoplasma (M.) gallisepticum by atomic force microscopy but did not characterize them further [14] They belong to the same Mollicutes class, bacteria from the genus Mycoplasma differ from those of the genus Acholeplasma. Proteomic analyses further revealed that EV-membranes contained proteins potentially involved in hostpathogen interactions

Material and methods

Results & discussion

Conclusions