May Meghan, Dunne Dylan W, Brown Daniel R
Department of Biomedical Sciences, College of Osteopathic Medicine, University of New England, Biddeford, Maine, United States of America.
Department of Biological Sciences, Jess and Mildred Fisher College of Science and Mathematics, Towson University, Towson, Maryland, United States of America.
PLoS One. 2014 Oct 22;9(10):e110360. doi: 10.1371/journal.pone.0110360. eCollection 2014.
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.
滑膜支原体依靠其黏附素VlhA介导与唾液酸化宿主细胞受体的细胞黏附。VlhA的等位基因变体通过一组无启动子的vlhA假基因与单个转录启动子位点之间的重组产生，从而形成滑膜支原体谱系，每个谱系表达不同的vlhA等位基因。将与滑膜支原体九个谱系启动子相邻的预测全长VlhA序列（这些谱系在细胞黏附亲和力上有所不同）与参考菌株MS53的序列以及来自菌株WVU1853(T)突尼斯谱系的60个氨基酸的血凝VlhA C末端片段进行比对。在所检查的11个总序列的侧翼可变残基中，明显存在一个不完全保守的、单拷贝的、12个氨基酸的候选细胞黏附基序的七种不同序列变体。预测该基序在VlhA C末端附近的低复杂性区域采用短发夹结构。代表12个氨基酸基序的四个选定变体的生物素化合成寡肽，以整个合成的60个氨基酸片段作为阳性对照，它们与鸡红细胞膜结合的程度不同（P<0.01）。所有这些寡肽的结合程度均比随机排列或不相关的VlhA结构域阴性对照肽更高（P<0.01）。实验性引入的支链氨基酸（BCAA）取代Val3Ile和Leu7Ile并未显著改变结合，而使结构不稳定的取代Thr4Gly和Ala9Gly则倾向于降低结合（P<0.05）。当肽暴露于去唾液酸化膜时，或在暴露于未处理膜之前用游离唾液酸预饱和时，结合也降低至背景水平（P<0.01）。根据这些证据，我们得出结论，基序P-X-(BCAA)-X-F-X-(BCAA)-X-A-K-X-G结合唾液酸，并可能介导VlhA依赖性滑膜支原体与宿主细胞的附着。这种保守机制保留了结合亲和力精细调节的潜力，这可能是依赖于类似抗原可变黏附素系统的病原体的一个普遍特征。该基序可能有助于识别以前未被认识的黏附素。
