Highly Dynamic Genomic Loci Drive the Synthesis of Two Types of Capsular or Secreted Polysaccharides within the Mycoplasma mycoides Cluster

Abstract

iMycoplasmas of the Mycoplasma mycoides cluster are all ruminant pathogens. Mycoplasma mycoides subsp. mycoides is responsible for contagious bovine pleuropneumonia and is known to produce capsular polysaccharide (CPS) and exopolysaccharide (EPS). Previous studies have strongly suggested a role for Mycoplasma mycoides subsp. mycoides polysaccharides in pathogenicity. Mycoplasma mycoides subsp. mycoides-secreted EPS was recently characterized as a ␤(1¡6)-galactofuranose homopolymer (galactan) identical to the capsular product. Here, we extended the characterization of secreted polysaccharides to all other members of the M. mycoides cluster: M. capricolum subsp. capripneumoniae, M. capricolum subsp. capricolum, M. leachii, and M. mycoides subsp. capri (including the LC and Capri serovars). Extracted EPS was characterized by nuclear magnetic resonance, resulting in the identification of a homopolymer of ␤(1¡2)-glucopyranose (glucan) in M. capricolum subsp. capripneumoniae and M. leachii. Monoclonal antibodies specific for this glucan and for the Mycoplasma mycoides subsp. mycoides-secreted galactan were used to detect the two polysaccharides. While M. mycoides subsp. capri strains of serovar LC produced only capsular galactan, no polysaccharide could be detected in strains of serovar Capri. All strains of M. capricolum subsp. capripneumoniae and M. leachii produced glucan CPS and EPS, whereas glucan production and localization varied among M. capricolum subsp. capricolum strains. Genes associated with polysaccharide synthesis and forming a biosynthetic pathway were predicted in all cluster members. These genes were organized in clusters within two loci representing genetic variability hot spots. Phylogenetic analysis showed that some of these genes, notably galE and glf, were acquired via horizontal gene transfer. These findings call for a reassessment of the specificity of the serological tests based on mycoplasma polysaccharides. Within the class Mollicutes, the so-called Mycoplasma mycoides cluster (MMC) (1) is unique: all the members of the MMC are pathogenic for ruminants, yet from a phylogenetic point of view, this cluster belongs to the Spiroplasma clade, which includes a number of species isolated from plants or insects (2). The taxonomy of the MMC was modified recently to reflect more precisely the phylogeny of this group. Because all the cluster members are closely related, their 16S rRNA gene sequences did not provide sufficient resolution to discriminate them accurately (3). A multilocus sequence typing approach was used to obtain a more precise phylogeny (4), which led to a simplification of the taxonomy (5) ( Table 1). The cluster currently comprises five species or subspecies: Mycoplasma mycoides subsp. mycoides, which was known formerly as the "small colony" (SC) biotype (1), M. mycoides subsp. capri, M. capricolum subsp. capricolum, M. capricolum subsp. capripneumoniae, and M. leachii. In addition, M. mycoides subsp. capri is subdivided in two serovars: the "large colony" serovar (M. mycoides subsp...

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

“…Antibodies raised against the ␤-(1¡2)-glucopyranose and ␤-(1¡6)-galactofuranose polysaccharides produced by Bertin et al (12) failed to react with purified CPS, suggesting a different structure for the polysaccharide secreted by M. agalactiae 14628 (data not shown). The chemical composition and structure of the CPS in M. agalactiae were therefore elucidated by HPAEC analysis and NMR spectra, as described in Materials and Methods.…”

“…BLASTP analyses were conducted in the Molligen database (29) to search for enzymes potentially involved in glucose uptake and phosphorylation and for the synthesis of UDP-sugar in M. agalactiae genomes, using the proteins described for members of the M. mycoides cluster (12,13). Homologs of enzymes described in Fig.…”

“…Interestingly, comparative genomic data have shown that several genes involved in polysaccharide biosynthesis have been horizontally transferred between members of the M. mycoides cluster and other mycoplasma species that are also ruminant pathogens but belong to a distant and distinct phylogenetic group (12,18,19). These include M. agalactiae, a major pathogen responsible for contagious agalactia in small ruminants, which is listed by the World Organization of Animal Health (OIE) due to its economic impact in countries with important dairy industries.…”

“…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).…”