Characterization of Free Exopolysaccharides Secreted by Mycoplasma mycoides Subsp. mycoides

Clothilde Bertin,François Thiaucourt,Patrice Gaurivaud,Corinne Pau-Roblot,Dominique Le Grand,Lucía Manso-Silván,Florence Tardy,François Poumarat,Josiane Courtois

doi:10.1371/journal.pone.0068373

Abstract

Contagious bovine pleuropneumonia is a severe respiratory disease of cattle that is caused by a bacterium of the Mycoplasma genus, namely Mycoplasma mycoides subsp. mycoides (Mmm). In the absence of classical virulence determinants, the pathogenicity of Mmm is thought to rely on intrinsic metabolic functions and specific components of the outer cell surface. One of these latter, the capsular polysaccharide galactan has been notably demonstrated to play a role in Mmm persistence and dissemination. The free exopolysaccharides (EPS), also produced by Mmm and shown to circulate in the blood stream of infected cattle, have received little attention so far. Indeed, their characterization has been hindered by the presence of polysaccharide contaminants in the complex mycoplasma culture medium. In this study, we developed a method to produce large quantities of EPS by transfer of mycoplasma cells from their complex broth to a chemically defined medium and subsequent purification. NMR analyses revealed that the purified, free EPS had an identical β(1−>6)-galactofuranosyl structure to that of capsular galactan. We then analyzed intraclonal Mmm variants that produce opaque/translucent colonies on agar. First, we demonstrated that colony opacity was related to the production of a capsule, as observed by electron microscopy. We then compared the EPS extracts and showed that the non-capsulated, translucent colony variants produced higher amounts of free EPS than the capsulated, opaque colony variants. This phenotypic variation was associated with an antigenic variation of a specific glucose phosphotransferase permease. Finally, we conducted in silico analyses of candidate polysaccharide biosynthetic pathways in order to decipher the potential link between glucose phosphotransferase permease activity and attachment/release of galactan. The co-existence of variants producing alternative forms of galactan (capsular versus free extracellular galactan) and associated with an antigenic switch constitutes a finely tuned mechanism that may be involved in virulence.

Highlights

Bacteria of the Mycoplasma genus have evolved from their Gram-positive ancestors by massive reduction of their genome size
Following a 72 h-incubation in CMRL-1066, polysaccharide extracts were clearly detected in culture supernatants of Mycoplasma mycoides subsp. mycoides (Mmm) strain Afade, while no polysaccharides were detected in non-inoculated CMRL1066 used as negative control (Figure 1A)
Polysaccharide production by Mmm could not be assessed in PPLO-based medium because of detection of a strong background in the negative control corresponding to the saccharides contained in this growth medium (Figure 1A)

Summary

Introduction

Bacteria of the Mycoplasma genus have evolved from their Gram-positive ancestors by massive reduction of their genome size. They are considered the smallest and simplest self-replicating organisms, with many missing biosynthetic pathways and the notable absence of a cell wall [1]. Contrary to other mycoplasmas involved in cattle respiratory diseases, such as M. bovis [4], Mmm is not considered an agent of co-infection and is often isolated on its own. This clinical picture contrasts with that observed in infections by M. mycoides subsp. This clinical picture contrasts with that observed in infections by M. mycoides subsp. capri, its closest phylogenetic neighbor and one of the agents of contagious agalactia, a small-ruminant syndrome affecting diverse organs (udder, joints, eyes, lungs)

Methods

Results

Conclusion