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Diversifying and stabilizing selection of sialidase and N-acetylneuraminate catabolism in Mycoplasma synoviae.

ABSTRACT: Sialidase activity varies widely among strains and tends to correlate with strain virulence in the avian pathogen Mycoplasma synoviae. To characterize the forms of selection acting on enzymes required for sialic acid scavenging and catabolism, the ratios of nonsynonymous (K(a)) to synonymous (K(s)) mutation frequency were calculated for codons in the sialidase gene of 16 strains of M. synoviae and for its nearly identical homolog in four strains of Mycoplasma gallisepticum. The K(a)/K(s) (omega) values for the linked genes required for nutritive N-acetylneuraminate catabolism (nanA, nagC, nanE, nagA, and nagB) from nine strains of M. synoviae were also determined. To provide context, omega was determined for all corresponding genes of 26 strains of Clostridium perfringens and Streptococcus pneumoniae. Bayesian models of sequence evolution showed that only the sialidase of M. synoviae was under significant (P < 0.001) diversifying selection, while the M. synoviae genes for N-acetylneuraminate catabolism and all genes examined from M. gallisepticum, C. perfringens, and S. pneumoniae were under neutral to stabilizing selection. Diversifying selection acting on the sialidase of M. synoviae, but not on the sialidase of M. gallisepticum or the sialidases or other enzymes essential for sialic acid scavenging in other Firmicutes, is evidence that variation in specific activity of the enzyme is perpetuated by a nonnutritive function in M. synoviae that is influenced by the genomic context of the organism.

SUBMITTER: May M

PROVIDER: S-EPMC2681885 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Diversifying and stabilizing selection of sialidase and N-acetylneuraminate catabolism in Mycoplasma synoviae.

May Meghan M Brown Daniel R DR

Journal of bacteriology 20090327 11

Sialidase activity varies widely among strains and tends to correlate with strain virulence in the avian pathogen Mycoplasma synoviae. To characterize the forms of selection acting on enzymes required for sialic acid scavenging and catabolism, the ratios of nonsynonymous (K(a)) to synonymous (K(s)) mutation frequency were calculated for codons in the sialidase gene of 16 strains of M. synoviae and for its nearly identical homolog in four strains of Mycoplasma gallisepticum. The K(a)/K(s) (omega) ...[more]

PMID: 19329630

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Project description:BACKGROUND: Mycoplasma synoviae is an avian pathogen that can lead to respiratory tract infections and arthritis in chickens and turkeys, resulting in serious economic losses to the poultry industry. Enolase reportedly plays important roles in several bacterial pathogens, but its role in M. synoviae has not been established. Therefore, in this study, the enolase encoding gene (eno) of M. synoviae was amplified from strain WVU1853 and expressed in E. coli BL21 cells. Then the enzymatic activity, immunogenicity and binding activity with chicken plasminogen (Plg) and human fibronectin (Fn) was evaluated. RESULTS: We demonstrated that the recombinant M. synoviae enolase protein (rMsEno) can catalyze the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP), the Km and Vmax values of rMsEno were 1.1 × 10(-3) M and 0.739 ?mol/L/min, respectively. Western blot and immuno-electron microscopy analyses confirmed that enolase was distributed on the surface and within the cytoplasm of M. synoviae cells. The binding assays demonstrated that rMsEno was able to bind to chicken Plg and human Fn proteins. A complement-dependent mycoplasmacidal assay demonstrated that rabbit anti-rMsEno serum had distinct mycoplasmacidal efficacy in the presence of complement, which also confirmed that enolase was distributed on the surface of M. synoviae. An inhibition assay showed that the adherence of M. synoviae to DF-1 cells pre-treated with Plg could be effectively inhibited by treatment with rabbit anti-rMsEno serum. CONCLUSION: These results reveal that M. synoviae enolase has good catalytic activity for conversion of 2-PGA to PEP, and binding activity with chicken Plg and human Fn. Rabbit anti-rMsEno serum displayed an obvious complement-dependent mycoplasmacidal effect and adherent inhibition effect. These results suggested that the M. synoviae enolase plays an important role in M. synoviae metabolism, and could potentially impact M. synoviae infection and immunity.

Project description:Mycoplasma synoviae depends on its adhesin VlhA to mediate cytadherence to sialylated host cell receptors. Allelic variants of VlhA arise through recombination between an assemblage of promoterless vlhA pseudogenes and a single transcription promoter site, creating lineages of M. synoviae that each express a different vlhA allele. The predicted full-length VlhA sequences adjacent to the promoter of nine lineages of M. synoviae varying in avidity of cytadherence were aligned with that of the reference strain MS53 and with a 60-a.a. hemagglutinating VlhA C-terminal fragment from a Tunisian lineage of strain WVU1853(T). Seven different sequence variants of an imperfectly conserved, single-copy, 12-a.a. candidate cytadherence motif were evident amid the flanking variable residues of the 11 total sequences examined. The motif was predicted to adopt a short hairpin structure in a low-complexity region near the C-terminus of VlhA. Biotinylated synthetic oligopeptides representing four selected variants of the 12-a.a. motif, with the whole synthesized 60-a.a. fragment as a positive control, differed (P<0.01) in the extent they bound to chicken erythrocyte membranes. All bound to a greater extent (P<0.01) than scrambled or irrelevant VlhA domain negative control peptides did. Experimentally introduced branched-chain amino acid (BCAA) substitutions Val3Ile and Leu7Ile did not significantly alter binding, whereas fold-destabilizing substitutions Thr4Gly and Ala9Gly tended to reduce it (P<0.05). Binding was also reduced to background levels (P<0.01) when the peptides were exposed to desialylated membranes, or were pre-saturated with free sialic acid before exposure to untreated membranes. From this evidence we conclude that the motif P-X-(BCAA)-X-F-X-(BCAA)-X-A-K-X-G binds sialic acid and likely mediates VlhA-dependent M. synoviae attachment to host cells. This conserved mechanism retains the potential for fine-scale rheostasis in binding avidity, which could be a general characteristic of pathogens that depend on analogous systems of antigenically variable adhesins. The motif may be useful to identify previously unrecognized adhesins.

Dataset Information

Diversifying and stabilizing selection of sialidase and N-acetylneuraminate catabolism in Mycoplasma synoviae.

Publications

Diversifying and stabilizing selection of sialidase and N-acetylneuraminate catabolism in Mycoplasma synoviae.

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