O-linked protein glycosylation in Mycoplasma.

Although mycoplasmas have a paucity of glycosyltransferases and nucleotidyltransferases recognizable by bioinformatics, these bacteria are known to produce polysaccharides and glycolipids. We show here that mycoplasmas also produce glycoproteins and hence have glycomes more complex than previously realized. Proteins from several species of Mycoplasma reacted with a glycoprotein stain, and the murine pathogen Mycoplasma arthritidis was chosen for further study. The presence of M. arthritidis glycoproteins was confirmed by high-resolution mass spectrometry. O-linked glycosylation was clearly identified at both serine and threonine residues. No consensus amino acid sequence was evident for the glycosylation sites of the glycoproteins. A single hexose was identified as the O-linked modification, and glucose was inferred by (13) C-labelling to be the hexose at several of the glycosylation sites. This is the first study to conclusively identify sites of protein glycosylation in any of the mollicutes.

Rhamnose biosynthesis in mycoplasmas requires precursor glycans larger than monosaccharide.

Despite the apparent absence of genes coding for the known pathways for biosynthesis, the monosaccharide rhamnose was detected in the d configuration in Mycoplasma pneumoniae and Mycoplasma pulmonis, and in both the d and l configurations in Mycoplasma arthritidis. Surprisingly, the monosaccharide glucose was not a precursor for rhamnose biosynthesis and was not incorporated at detectable levels in glucose-containing polysaccharides or glycoconjugates. In contrast, carbon atoms from starch, a polymer of glucose, were incorporated into rhamnose in each of the three species examined. When grown in a serum-free medium supplemented with starch, M. arthritidis synthesized higher levels of rhamnose, with a shift in the relative amounts of the d and l configurations. Our findings suggest the presence of a novel pathway for rhamnose synthesis that is widespread in the genus Mycoplasma.

Novel interactions of a microbial superantigen with TLR2 and TLR4 differentially regulate IL-17 and Th17-associated cytokines.

Mycoplasma arthritidis, an inflammatory murine pathogen, secretes a potent superantigen, Mycoplasma arthritidis mitogen (MAM) that contributes to toxic shock, arthritis and skin necrosis. Previously we showed that MAM induced type 2 T-cell cytokines in mice that express functional TLR2 and TLR4, but type 1 cytokines in mice that lack TLR4 function. We show here that IL-17, pSTAT3 and retinoid-related orphan nuclear receptorγt are rapidly expressed in wild-type C3H/HeSnJ (TLR2+/4+) mice but are significantly delayed in mutant C3H/HeJ (TLR2+/4-) mice. This marked kinetic difference was associated with a high level of IL-6 in TLR2+/4+ mice versus high levels of IL-1ß and TNFα in TLR2+/4- mice. Also antibodies to IL-6 and IL-23, suppressed IL-17 responses to MAM in TLR2+/4+ mice whereas anti-IL-1ß, but not anti-TNFα, enhanced IL-17 in TLR2+/4- mice. Antibody blocking of TLR4 in TLR2+/4+ mice decreased IL-17 and IL-6 but not IL-23. In addition both IL-17 and IL-6 but not IL-23 were elevated in TLR2 KO mice versus wild-type TLR2+/4+ mice given MAM. We conclude that the MAM interaction with TLR2 versus TLR4 leads to distinct cytokine pathways mediated primarily by IL-1ß or IL-6/IL-17 signalling respectively. Our findings suggest that the differential interaction of MAM with different TLRs might play an important role in disease outcomes by M. arthritidis.

Crystal structure of the Mycoplasma arthritidis-derived mitogen in apo form reveals a 3D domain-swapped dimer.

Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing particular Vbeta elements of T cell receptor. Here, we report the crystal structure of a MAM mutant K201A in apo form (unliganded) at 2.8-A resolutions. We also partially refined the crystal structures of the MAM wild type and another MAM mutant L50A in apo forms at low resolutions. Unexpectedly, the structures of these apo MAM molecules display a three-dimensional domain-swapped dimer. The entire C-terminal domains of these MAM molecules are involved in the domain swapping. Functional analyses demonstrated that the K201A and L50A mutants do not show altered ability to bind to their host receptors and that they stimulate the activation of T cells as efficiently as does the wild type. Structural comparisons indicated that the "reconstituted" MAM monomer from the domain-swapped dimer displays large differences at the hinge regions from the MAM(wt) molecule in the receptor-bound form. Further comparison indicated that MAM has a flexible N-terminal loop, implying that conformational changes could occur upon receptor binding.

Isolation and partial purification of macrophage- and dendritic cell-activating components from Mycoplasma arthritidis: association with organism virulence and involvement with Toll-like receptor 2.

Mycoplasma arthritidis induces toxicity, arthritis, and dermal necrosis in mice. Virulence factors include a superantigen and membrane adhesins and possibly also a bacteriophage component. Here we compare the biological properties of Triton X-114 extracts derived from avirulent and virulent M. arthritidis strains. Macrophage cell lines and resident peritoneal macrophages were used to assess inflammatory potential as indicated by production of tumor necrosis factor alpha, interleukin-6, and/or nitric oxide. The activity resided exclusively within the hydrophobic detergent phase, was unaffected by heat treatment at 100 degrees C for 30 min, and was resistant to proteinase K digestion, suggesting involvement of a lipopeptide. Contamination of extracts with endotoxin or superantigen was excluded. Extracts of the more virulent strain had higher activity than did those of the avirulent strain. Using CHO cells expressing Toll-like receptor 2 (TLR2) or TLR4, both with transfected CD14, we showed that extracts activated these cells via TLR2 but not by TLR4. Also, macrophages from C57BL/6 TLR2(-/-) mice failed to respond to the extracts, whereas those from TLR2(+/+) cells did respond. The preparations from the virulent strain of M. arthritidis were also more potent in activating dendritic cells, as evidenced by up-regulation of major histocompatibility complex class II, CD40, B7-1, and B7-2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent elution of gel slices revealed the presence of three active moieties which corresponded to molecular masses of approximately 24, 28, and 40 kDa. Three active components were also found by reverse-phase chromatography. We suggest that macrophage activation by M. arthritidis could play a significant role in the inflammatory response induced in the host by this organism.

Mycoplasma arthritidis bacteriophage MAV1 prophage integration, deletions, and strain-related polymorphisms.

Temperate bacteriophage MAV1 is found in certain highly virulent strains of Mycoplasma arthritidis. Integration sites, portions of the right and left prophage ends, and flanking DNA from eight prophages in seven M. arthritidis strains were characterized in this study. attb and attp sites conformed for the most part to the consensus sequence TATTTTT, although minor polymorphisms were noted. Prophages were integrated into similar sites in four strains, suggesting that these strains may have had a common ancestor. Two strains had three prophage copies each, and integration sites were identical. Two strains had two copies each. One of these shared two of the integration sites occupied in the three-copy strains, while the other shared one of these sites and harbored a second prophage in a unique site. Integration sites in the two strains with one prophage each were unique. Four MAV1 copies contained extensive substitutions within a region encoding a putative structural protein and the putative repressor protein. A 3-kb fragment was deleted from the right side of two of these copies. It is proposed that polymorphisms within MAV1 prophage integration sites and within the prophages themselves may help to identify phylogenetic relationships among virulent M. arthritidis strains.

Crystal structure of Mycoplasma arthritidis mitogen complexed with HLA-DR1 reveals a novel superantigen fold and a dimerized superantigen-MHC complex.

Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing particular TCR Vbeta elements. Here we report the crystal structure of MAM complexed with a major histocompatibility complex (MHC) antigen, HLA-DR1, loaded with haemagglutinin peptide 306-318 (HA). The structure reveals that MAM has a novel fold composed of two alpha-helical domains. This fold is entirely different from that of the pyrogenic superantigens, consisting of a beta-grasped motif and a beta barrel. In the complex, the N-terminal domain of MAM binds orthogonally to the MHC alpha1 domain and the bound HA peptide, and to a lesser extent to the MHC beta1 domain. Two MAM molecules form an asymmetric dimer and cross-link two MHC antigens to form a plausible, dimerized MAM-MHC complex. These data provide the first crystallographic evidence that superantigens can dimerize MHC molecules. Based on our structure, a model of the TCR2MAM2MHC2 complex is proposed.

Crystallization and preliminary crystallographic analysis of Mycoplasma arthritidis-derived mitogen complexed with peptide/MHC class II antigen.

Mycoplasma arthritidis-derived mitogen (MAM), a bacterial superantigen, has been crystallized in complex with its human receptor, major histocompatibility complex (MHC) class II antigen, by the hanging-drop vapor-diffusion method. Crystals were obtained under three conditions, with ammonium sulfate, phosphate salt and PEG 8000 as the precipitant. The crystals grown under these conditions all belong to space group I222, with the same unit-cell parameters: a = 137.4, b = 178.2, c = 179.6 A. Diffraction data were collected to 3.3 and 3.4 A resolution from crystals of native and selenomethionylated MAM-MHC complexes, respectively. Self- and cross-rotation function calculations suggest the presence of two complex molecules in the asymmetric unit, resulting in a V(M) of 4.0 and a solvent content of 69%. An interpretable electron-density map was produced using a combination of molecular replacement and SAD phasing.