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 ß(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.

Effects of immune serum on macrophage cell cultures infected with Mycoplasma mycoides subsp. mycoides small colony: morphological analysis by scanning electron microscopy.

Macrophages are pivotal cells of the immune system and play a key role in the host defence mechanism against pathogens. To date, the importance of macrophages and the role of humoral response in eliciting macrophage activity against Mycoplasma mycoides subsp. mycoides small colony (Mmm-SC), the causative agent of contagious bovine pleuropneumonia (CBPP), have only been marginally elucidated or are almost unknown. The present study was undertaken to investigate the changes in surface morphology of macrophages after in vitro infection with Mmm-SC in the presence of bovine immune serum. Morphological analysis was performed on macrophage cultures at 6 h post infection using the three-dimensional vision of scanning electron microscopy. Non-infected macrophages in the presence of negative or immune serum and macro phages infected with Mmm-SC in the absence of serum showed only minor cell surface changes. In contrast, clear surface modifications, broad veils, fine philopodia highlighting cell activation and small aggregates of mycoplasma closely attached to the macrophage membrane, were observed in infected macrophage cultures in the presence of immune serum. Our results suggest that specific humoral response to Mmm-SC may contribute and support phagocytic activity of macrophages.

Creation of a bacterial cell controlled by a chemically synthesized genome.

We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication.

Domain analysis of lipoprotein LppQ in Mycoplasma mycoides subsp. mycoides SC.

The lipoprotein LppQ is the most prominent antigen of Mycoplasma mycoides subsp. mycoides small colony type (SC) during infection of cattle. This pathogen causes contagious bovine pleuropneumonia (CBPP), a devastating disease of considerable socio-economic importance in many countries worldwide. The dominant antigenicity and high specificity for M. mycoides subsp. mycoides SC of lipoprotein LppQ have been exploited for serological diagnosis and for epidemiological investigations of CBPP. Scanning electron microscopy and immunogold labelling were used to provide ultrastructural evidence that LppQ is located to the cell membrane at the outer surface of M. mycoides subsp. mycoides SC. The selectivity and specificity of this method were demonstrated through discriminating localization of extracellular (i.e., in the zone of contact with host cells) vs. integral membrane domains of LppQ. Thus, our findings support the suggestion that the accessible N-terminal domain of LppQ is surface exposed and such surface localization may be implicated in the pathogenesis of CBPP.

[Tubular structures in Mycoplasma gallisepticum and the localization of a tubulin-like protein]. / Tubuliarnye struktury Mycoplasma gallisepticum i lokalizatsiia tubulinopodobnogo belka.

In all the strains of M. gallisepticum investigated, a protein with apparent molecular weight 40 kDa was revealed by immunoblotting with polyclonal anti-calf brain tubulin antibodies and monoclonal anti-chicken alpha-tubulin antibodies. In other 8 investigated Mycoplasma species no positive reactions with the same antibodies were found. The M. Gallisepticum cells were examined under electron microscope on fine serial sections and on some sections going at different angles to the long cell axis. Undermembrane system of tubules was revealed and the intracellular pattern of the tubular structures were reconstructed. The immunoelectron microscopic data suggest that tubulin-like protein may be included into the structures.

Adherence of Mycoplasma mycoides subspecies mycoides to cultured endothelial cells.

The LC- and SC-type strains of Mycoplasma mycoides subspecies mycoides were examined for adherence to guinea pig erythrocytes and bovine and caprine endothelial cells. The LC-type strains but not the SC-type strains adsorbed guinea pig erythrocytes and caprine endothelial cells. Difference in cytoadherence was observed between strains of the LC-type. The interaction between the most adherent LC-type strain and caprine endothelial cells was examined by transmission and scanning electron microscopy.

Effect of membrane cholesterol on potassium transport in Mycoplasma mycoides var. Capri (PG3).

Relationships between membrane lipid composition and physiological properties, particularly intracellular potassium levels, have been studied at 37 degrees C in Mycoplasma mycoides var. Capri (PG3). Native organisms grown on medium supplemented with either oleic acid plus palmitic acid or elaidic acid have identical growth characteristics, acidification properties and intracellular K content. On the other hand, when the cholesterol normally present in the membrane (20--25% of total lipids) is reduced to less than 2%, we observe: (1) the intracellular K content decreases (20 microgram K/mg cell protein instead of 40) and is independent of the phase of growth; (2) K passive permeability is drastically increased but K distribution remains in equilibrium with the transmembrane potential (delta psi); (3) organisms stop growing at pH 6.5 (instead of 5.2) and acidification is reduced by 40%, suggesting a large increase in H+ permeability, and (4) intracellular Na contents rise from 3 to 9 microgram Na/mg cell protein. Replenishing cholesterol in membranes of depleted cells results in a recovery of the high intracellular K level (35--40 microgram K/mg cell protein) and acidification properties. It is suggested that cholesterol affects the cation content via the increase in proton permeability which in turn controls the value of the delta psi responsible for the value of intracellular K equilibrium. Changes in K passive permeability, although related to the amount of cholesterol present in the plasma membrane, are probably not involved in the control of the intracellular K level.

Ultrastructural localization of tellurite reduction in Acholeplasma species.

Aerobic reduction of tellurite by five Acholeplasma species and two Mycoplasma species was investigated by light and electron microscopy. Among the Acholeplasma species, colonies of A. laidlawii and A. oculi exhibited a heavy, macroscopically visible reduction of tellurite, whereas the reaction of A. axanthum was weaker. A. granularum and A. modicum did not reduce the substrate under the experimental conditions employed. The two subspecies of M. mycoides also reacted with tellurite, as did also the investigated strain of M. bovigenitalium although to a lesser extent. Ultrastructurally, reduction sites were localized to the cytoplasmic membrane in the three tellurite positive Acholeplasma species and apparently to the cytoplasm of M. mycoides subsp. mycoides. Reduction sites could not be demonstrated in M. mycoides subsp. capri and in M. bovigenitalium. The results support previous evidence obtained by biochemical methods which indicates membrane localization of redox enzymes in Acholeplasmas.

Ultrastructural visualization of anionic sites on mycoplasma membranes by polycationic ferritin.

Anionic sites on mycoplasma membranes were visualized in the electron microscope by a polycationized ferritin derivative. The technique of thin sectioning was used. Staining prior to fixation led to clustering of ferritin granules on the mycoplasma cell surface. On glutaraldehyde-fixed Mycoplasma mycoides subsp. capri, M. gallisepticum, M. pneumoniae, and Acholeplasma laidlawii, the anionic sites were uniformly distributed over the entire membrane surface. M. hominis did not bind the polycationic ferritin label. Chemical and enzymatic treatments of the mycoplasmas indicated that the anionic sites may be lipid phosphate groups. Isolated M. mycoides subsp. capri membranes were labeled exclusively on only one membrane surface, presumably the outer one. Liposomes prepared from diphosphatidylglycerol and phosphatidylcholine were also labeled by the polycationic ferritin.

Ultrastructural visualization of surface carbohydrate structures on mycoplasma membranes by concanavalin A.

Surface carbohydrates of Mycoplasma mycoides var. capri were made visible by the cytochemical staining procedure with concanavalin A, horseradish peroxidase, and diaminobenzidine.

Morphological changes induced during fixation of Mycoplasma mycoides for electron microscopy.

Only spherical mycoplasmas 0.6-0.9 mum diam were observed by darkfield microscopy in single cell suspensions prepared from exponential broth cultures of Mycoplasma mycoides var mycoides and Mycoplasma mycoides var capri. Similar cells were seen in pseudoreplicas by electron microscopy and they are considered characteristic of the morphology of M. mycoides. When the mycoplasmas were fixed by the addition of 10% formalin to the suspensions or washed, centrifuged cells were fixed by glutaraldehyde, filamentous and ring forms were observed in electron micrographs. These are not considered typical of the morphology of M. mycoides but are artifacts produced during the preparation of the mycoplasmas for electron microscopy.

Studies with lectins on the surface carbohydrate structures of mycoplasma membranes.

The surface carbohydrate structures on the cell membranes of various mycoplasma species have been investigated by using lectins, which are sugar-specific proteins. Carbohydrate structures presumably bound to glycolipids, with both galactose and glucose units, were found to be exposed on the surface of Mycoplasma pneumoniae and its temperature-sensitive mutants, M. mycoides var. mycoides and capri, M. pulmonis, M. gallinarum, and M. gallisepticum. Lipid-bound glucose was found on M. neurolyticum. The possible relationship of the lipid-bound surface carbohydrate groups to the known serological cross-reactions and lipid compositions of the various mycoplasma species is discussed. Intact Acholeplasma laidlawii and M. fermentans have no lectin-binding sites exposed on their surfaces; galactose groups were discovered only after Pronase digestion of the organisms, suggesting that their glycolipids are hidden under a protein layer. Neither intact nor Pronase-digested M. hominis reacted with the lectins; this is fully consistent with the lipid composition of this organism, which contains glycolipids. The lectins from Vicia cracca and Phaseolus vulgaris, which react with N-acetyl-galactosamine groups, agglutinated M. gallinarum, M. gallisepticum, M. mycoides var. capri, and M. pulmonis. The agglutinability was lost after Pronase treatment, indicating that the corresponding carbohydrates are presumably protein bound. They may be correlated with the extracellular structures observed by electron microscopy of both sectioned and negatively stained mycoplasma species.