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

Abstract

Mycoplasmas are minimal, wall-less bacteria but have retained the ability to secrete complex carbohydrate polymers that constitute a glycocalyx. In members of the Mycoplasma mycoides cluster, which are important ruminant pathogens, the glycocalyx includes both cell-attached and cell-free polysaccharides. This report explores the potential secretion of polysaccharides by M. agalactiae, another ruminant pathogen that belongs to a distant phylogenetic group. Comparative genomic analyses showed that M. agalactiae possesses all the genes required for polysaccharide secretion. Notably, a putative synthase gene (gsmA) was identified, by in silico reconstruction of the biosynthetic pathway, that could be involved in both polymerization and export of the carbohydrate polymers. M. agalactiae polysaccharides were then purified in vitro and found to be mainly cell attached, with a linear ␤-(1¡6)-glucopyranose structure [␤-(1¡6)-glucan]. Secretion of ␤-(1¡6)-glucan was further shown to rely on the presence of a functional gsmA gene, whose expression is subjected to high-frequency phase variation. This event is governed by the spontaneous intraclonal variation in length of a poly(G) tract located in the gsmA coding sequence and was shown to occur in most of the M. agalactiae clinical isolates tested in this study. M. agalactiae susceptibility to serum-killing activity appeared to be dictated by ON/OFF switching of ␤-(1¡6)-glucan secretion, suggesting a role of this phenomenon in survival of the pathogen when it invades the host bloodstream. Finally, ␤-(1¡6)-glucan secretion was not restricted to M. agalactiae but was detected also in M. mycoides subsp. capri PG3 T , another pathogen of small ruminants. IMPORTANCEMany if not all bacteria are able to secrete polysaccharides, either attached to the cell surface or exported unbound into the extracellular environment. Both types of polysaccharides can play a role in bacterium-host interactions. Mycoplasmas are no exception despite their poor overall metabolic capacity. We showed here that M. agalactiae secretes a capsular ␤-(1¡6)-glucopyranose thanks to a specific glycosyltransferase with synthase activity. This secretion is governed by high-frequency ON/OFF phase variation that might be crucial in mycoplasma host dissemination, as cell-attached ␤-(1¡6)-glucopyranose increases serum-killing susceptibility. Our results provide functional genetic data about mycoplasmal glycosyltransferases with dual functions, i.e., assembly and export of the sugar polymers across the cell membrane. Furthermore, we demonstrated that nonprotein epitopes can be subjected to surface antigenic variation in mycoplasmas. Finally, the present report contributes to unravel the role of secreted polysaccharides in the virulence and pathogenicity of these peculiar bacteria.

Metabolic Engineering of Saccharomyces cerevisiae to Improve Glucan Biosynthesis

“…In particular, overexpression of GsmA resulted in 43% increase of β-glucan content. The result here confirmed that GsmA from M. agalactiae is a functional β-1, 6-glucan synthase [18]. Moreover, the results demonstrate that introduction of heterologous β-1, 3-glucan synthases can also strengthen the biosynthesis of β-1, 3-glucan in S. cerevisiae.…”

“…This work [17]. In order to increase the overall polymerization capability, three heterologous β-glucan synthases, GsmA, Gtf and CrdS, from Mycoplasma agalactiae 5632 [18], Oenococcus oeni [19] and Agrobacterium sp. [20], were overexpressed in S. cerevisiae and the effects on the composition of cell wall polysaccharides were investigated.…”

Genome-wide detection of conservative site-specific recombination in bacteria

“…Additionally, DNA inversions are not confined to the bacterial realm, since they occur in bacteriophages, for example P1 and Mu, and plasmids such as R64 and p15B [ 32 – 34 ]. Finally, various non-surface components have been described to phase vary by mechanisms unrelated to DNA-inversion and ultimately influence multiple phenotypes related to immunoevasion, niche adaptation and virulence [ 35 – 42 ].…”

Comprehensive RNA-Seq Profiling to Evaluate the Sheep Mammary Gland Transcriptome in Response to Experimental Mycoplasma agalactiae Infection

“…agalactiae [16]. Although mycoplasma liposoluble proteins are demonstrated to induce NET release, the exact role of the recently identified phase variable β-(1→6)-Glucan is yet to be elucidated in its disease progression [17]. …”