Characterization of Free Exopolysaccharides Secreted by Mycoplasma mycoides Subsp. mycoides

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.

Mycoplasma agalactiae Secretion of β-(1→6)-Glucan, a Rare Polysaccharide in Prokaryotes, Is Governed by High-Frequency Phase Variation

“…After counting the cells (h 72 [T72] in Table 3), the cultures were centrifuged at 14,000 ϫ g for 1 h at 4°C to separate the cells from the supernatant. EPS were extracted from the cell-free supernatant as described previously (13). CPS were purified from the cell pellet recovered after centrifugation using the method described by Shi et al (22) with some modifications.…”

“…13 C NMR experiments were conducted using the same spectrometer operating at 125.48 MHz with 2 s as the relaxation delay. The two-dimensional (2D) 1 Detection of polysaccharide secretion by colony immunostaining.…”

“…Our group recently contributed to identification of the polysaccharides secreted by several species that are pathogenic for ruminants and classified in the so-called M. mycoides cluster (12,13). These are (i) galactan, a ␤-(1¡6)-galactofuranose polymer, secreted by M. mycoides subsp.…”

“…Polysaccharide biosynthesis in members of the M. mycoides cluster is predicted to occur as a two-step process (12,13) in which sugars are first activated by the addition of a nucleoside-diphosphate group and then linked to an appropriate acceptor molecule by a variety of glycosyltransferases, resulting in the synthesis of different glycoconjugates (16). Among the glycosyltransferases involved in EPS/CPS secretion, one specific subfamily of membrane-embedded synthases has been described in several Gramnegative bacteria whose members are able both to assemble and to export the sugar polymers across the cell membrane (17).…”

“…S1 in the supplemental material). The homology of the MSC_0108 product with the BcsA subunit of Rhodobacter sphaeroides cellulose synthase, an enzyme responsible for cellulose synthesis and translocation across the cytoplasmic membrane, prompted us to propose that the MSC_0108 product is the galactan synthase catalyzing both UDP-galactofuranose polymerization and galactan secretion across the mycoplasma cytoplasmic membrane (13).…”