Abstract
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 WVU1853T. 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.
Highlights
The bacterial pathogen Mycoplasma synoviae is associated with a broad spectrum of clinical manifestations ranging from inapparent infection to systemic disease of poultry
Identification of the putative hemagglutination motifs (PHMs) When the full-length expressed VlhA protein MSPA sequences of nine strains of M. synoviae that vary in avidity of cytadherence were aligned with MSPA of the reference strain MS53 [8] and a 60-a.a. hemagglutinating peptide derived from the C-terminus of MSPA expressed by the Tunisian lineage of strain WVU1853T [15], an imperfectly conserved 12-a.a. motif was evident in all sequences (Figure 1)
It is known that the protein family VlhA is responsible for attachment by M. synoviae, but the functional motifs of the adhesin and the molecular basis for rheostasis in binding avidity have not been characterized
Summary
The bacterial pathogen Mycoplasma synoviae is associated with a broad spectrum of clinical manifestations ranging from inapparent infection to systemic disease of poultry. Attachment to sialylated receptors on host cells is mediated by the M. synoviae variable lipoprotein hemagglutinin VlhA [6,7]. Previous analyses indicated that the vlhA gene family has been laterally transferred between M. synoviae and Mycoplasma gallisepticum possibly during coinfection of a shared avian host [8,9]. The selective pressure of specific host immune responses to these antigens is thought to drive diversity in vlhA allele expression [10,11,12,13]. Despite the critical importance of cytadherence to the establishment and maintenance of infection, discrete VlhA types were demonstrated to have significantly different avidities for host cell binding, which can be quantified by agglutination of erythrocytes [14]. We sought to identify and characterize the specific motif that mediates adhesion of VlhA proteins to host cells
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