Free exopolysaccharide from Mycoplasma mycoides subsp. mycoides possesses anti-inflammatory properties

In this study we explored the immunomodulatory properties of highly purified free galactan, the soluble exopolysaccharide secreted by Mycoplasma mycoides subsp. mycoides (Mmm). Galactan was shown to bind to TLR2 but not TLR4 using HEK293 reporter cells and to induce the production of the anti-inflammatory cytokine IL-10 in bovine macrophages, whereas low IL-12p40 and no TNF-α, both pro-inflammatory cytokines, were induced in these cells. In addition, pre-treatment of macrophages with galactan substantially reduced lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines TNF- and IL-12p40 while increasing LPS-induced secretion of immunosuppressive IL-10. Also, galactan did not activate naïve lymphocytes and induced only low production of the Th1 cytokine IFN-γ in Mmm-experienced lymphocytes. Finally, galactan triggered weak recall proliferation of CD4+ T lymphocytes from contagious bovine pleuropneumonia-infected animals despite having a positive effect on the expression of co-stimulatory molecules on macrophages. All together, these results suggest that galactan possesses anti-inflammatory properties and potentially provides Mmm with a mechanism to evade host innate and adaptive cell-mediated immune responses. (Résumé d'auteur

Study Of Secreted Exopolysaccharides Synthesis By Mycoplasmas From The Mycoplasma mycoides Cluster And Notabily By Mycoplasma mycoides Subsp. mycoides, The Contagious Bovine Pleuropneumonia Agent

Notre modèle d'étude, Mycoplasma mycoides subsp. mycoides (Mmm), est l'agent de la péripneumonie contagieuse bovine (PPCB). Cette maladie fut un des plus grands fléaux de l'élevage bovin au XIXème siècle et sévit encore largement en Afrique. A cause de son impact sur l'économie, elle a bénéficié de grandes attentions dans les pays industrialisés, notamment lors des épisodes de résurgence européens dans les années 1980-90. Inscrite sur les listes de l'OIE, la PPCB est à déclaration obligatoire, ce qui entraine de sévères mesures de restriction sur le commerce des animaux vivants. Plusieurs espèces de mycoplasmes sont phylogénétiquement proches de Mmm. Pour mieux comprendre la pathogénie de cette maladie, il est nécessaire d'analyser les facteurs qui peuvent concourir à la virulence de l'agent pathogène incriminé. Dans ce contexte, nous nous sommes intéressés aux exopolysaccharides (EPS) de Mmm, candidats clés dans la virulence de cette bactérie. Ils ont été caractérisés chimiquement par HPLC et RMN, puis comparés aux polysaccharides capsulaires (CPS) dans le cadre d'une étude sur des variants intraclonaux de Mmm. Ces expériences ont été rendues possibles grâce à l'élaboration d'un protocole offrant une production optimale des EPS pour les analyses et un affranchissement des contaminants du milieu lors de leur isolement à partir des surnageants de culture. Ce protocole a permis, par ailleurs, d'élargir l'étude de caractérisation chimique des EPS au groupe « mycoides » auquel appartient Mmm. La production d'anticorps monoclonaux anti-polysaccharides a offert la possibilité d'étudier les communautés antigéniques au sein du groupe et de caractériser la localisation du polymère sécrété (CPS et/ou EPS). Les génomes séquencés et annotés disponibles ont fait l'objet d'études in silico sur les voies de biosynthèse potentiellement impliquées dans la production des polysaccharides. Les résultats montrent que dans le cas de Mmm, les variants d'un même clone présentent des différences de production de polysaccharides se traduisant par un phénotype opaque/translucide en culture sur milieu solide pouvant être associé à un « switch ON/OFF » du gène qui code un transporteur de glucose. Mmm sécréte du galactane (polymère de β-D-(1-6) galactofuranose) identique au composé associé à la membrane. Au sein du groupe « mycoides », deux espèces sécrètent un β-D-(1-2) glucane dont la structure linéaire est originale. L'analyse des voies de biosynthèse des génomes concernés concorde avec les deux produits mis en évidence. De nombreux gènes impliqués semblent résulter de transferts latéraux venant de mycoplasmes éloignés phylogénétiquement mais qui partagent le même habitatOur model, Mycoplasma mycoides subsp. mycoides (Mmm), is the agent of contagious bovine pleuropneumonia (CBPP). CBPP is a severe contagious disease currently present in Africa. CBPP is classed as a notifiable disease by the OIE, which implies severe restrictive measures in in the trade of live animals. Because of its economic importance, CBPP has received much attention in indusdrialized country, notabily when the disease had reemerged in Europe in 1980’s and 90’s. To better understand the pathogenesis of this disease, it is necessary to analyze the factors that may contribute to the virulence of this agent. In this context, we were interested in Mmm exopolysaccharides (EPS), potential key molecules in the virulence of this bacterium. First, Mmm EPS were chemically characterized by HPLC and NMR and compared to the capsular polysaccharides (CPS) in a study on intraclonal Mmm variants. To conduct these experiments, a suitable methodology was developed to produce and to purify EPS from culture supernatants by avoiding technical difficulties due to the complexity of the mycoplasma growth medium. Then, this protocol allowed extending the study to the chemical characterization of EPS from mycoplasmas belonging to the Mycoplasma mycoides cluster (MMC) as Mmm. The production of monoclonal antibodies recognizing Mmm and Mccp polysaccharides was used to study the antigenic communities within this group of mycoplasmas and to characterize the location of the secreted polymer (CPS and/or EPS). The sequenced and annotated genomes were used to manage in silico studies of biosynthetic pathways potentially involved in the production of polysaccharides. The results showed that Mmm intraclonal variants resulting in an opaque / translucent phenotype on solid culture may be associated with an “ON/OFF switch" of the gene encoding a glucose transporter and, in turn associated to the production of either CPS or EPS. Mmm secretes a β-(1-6) galactofuranose polymer identical to that of the membrane associated compound, excepted it has no lipid anchor. Within MMC, there are two species that secrete a β -(1-2) glucan, the linear structure of which is original. The analyses of the biosynthetic pathways of the different genomes were consistent with the structure of the related secreted products. Many genes could appear to originate from phylogenetically distant mycoplasmas that share the same habita

Characterization of free exopolysaccharides secreted by Mycoplasma mycoides subsp. mycoides.

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

Highly dynamic genomic loci drive the synthesis of two types of capsular or secreted polysaccharides within the "Mycoplasma mycoides" cluster.

Mycoplasmas of the "Mycoplasma mycoides" cluster are all ruminant pathogens. Mycoplasma mycoides subsp. mycoides (MmmSC) 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 MmmSC polysaccharides in pathogenicity. MmmSC-secreted EPS was recently characterized as a β-(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 (Mccp) M. capricolum subsp capricolum (Mcc), M. leachii and M. mycoides subsp capri (Mmc, including the "LC" and "capri" serovars). Extracted EPS were characterized by nuclear magnetic resonance, resulting in the identification of a homopolymer of β-(1-2)-glucopyranose (glucan) in Mccp and M. leachii. Monoclonal antibodies specific for this glucan and for the MmmSC-secreted galactan, were used to detect the two polysaccharides. While Mmc strains of the "LC" serovar produced only capsular galactan, no polysaccharide could be detected in strains of the "capri" serovar. All strains of Mccp and M. leachii produced glucan CPS and EPS, whereas glucan production and localization varied among Mcc 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 hotspots. 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

Proteolytic Post-Translational Processing of Adhesins in a Pathogenic Bacterium.

Mollicutes, including mycoplasmas and spiroplasmas, have been considered as good representatives of the « minimal cell » concept: these wall-less bacteria are small in size and possess a minimal genome and restricted metabolic capacities. However, the recent discovery of the presence of post-translational modifications unknown so far, such as the targeted processing of membrane proteins of mycoplasma pathogens for human and swine, revealed a part of the hidden complexity of these microorganisms. In this study, we show that in the phytopathogen, insect-vectored Spiroplasma citri GII-3 adhesion-related protein (ScARP) adhesins are post-translationally processed through an ATP-dependent targeted cleavage. The cleavage efficiency could be enhanced in vitro when decreasing the extracellular pH or upon the addition of polyclonal antibodies directed against ScARP repeated units, suggesting that modification of the surface charge and/or ScARP conformational changes could initiate the cleavage. The two major sites for primary cleavage are localized within predicted disordered regions and do not fit any previously reported cleavage motif; in addition, the inhibition profile and the metal ion requirements indicate that this post-translational modification involves at least one non-conventional protease. Such a proteolytic process may play a role in S. citri colonization of cells of the host insect. Furthermore, our work indicates that post-translational cleavage of adhesins represents a common feature to mollicutes colonizing distinct hosts and that processing of surface antigens could represent a way to make the most out of a minimal genome

Kinetics of exopolysaccharides secretion and viability of <b><i>Mmm</i></b><b> strain Afadé following transfer into CMRL-1066 medium with (red) or without (blue) glucose supplementation.</b>

<p>Glucose (1 g/L) was added at 24, 48 and 72 h (arrows).</p