Diversity of expressed vlhA adhesin sequences and intermediate hemagglutination phenotypes in Mycoplasma synoviae

Abstract

A reservoir of pseudogene alleles encoding the primary adhesin VlhA occurs in the avian pathogen Mycoplasma synoviae. Recombination between this reservoir and its single expression site was predicted to result in lineages of M. synoviae that each express a different vlhA allele as a consequence of host immune responses to those antigens. Such interstrain diversity at the vlhA expression site, including major differences in the predicted secondary structures of their expressed adhesins, was confirmed in 14 specimens of M. synoviae. Corresponding functional differences in the extent to which they agglutinated erythrocytes, a quantitative proxy for VlhA-mediated cytadherence, were also evident. There was a >20-fold difference between the highest- and lowest-agglutinating strains and a rheostatic distribution of intermediate phenotypes among the others (Tukey-Kramer honestly significant difference [HSD], P < 0.001). Coincubation with the sialic acid analog 2-deoxy-2,3-didehydro-N-acetylneuraminate inhibited hemagglutination in a pattern correlated with endogenous sialidase activity (r = 0.91, P < 0.001), although not consistently to the same extent that erythrocyte pretreatment with sialidase purified from Clostridium perfringens did (P < 0.05). The striking correlation between the ranked hemagglutination and endogenous sialidase activities of these strains (Spearman's r = 0.874, P < 0.001) is evidence that host-induced vlhA allele switching indirectly drives sequence diversity in the passenger sialidase gene of M. synoviae.

Fig. 1.

Sequence diversity of expressed VlhA adhesins in Mycoplasma synoviae. (A) Schematic of the vlhA translation product and its posttranslational cleavage products MSPA and MSPB. (B) Single-nucleotide substitution frequencies in the conserved and semivariable regions of M. synoviae vlhA with respect to the consensus of specimens aligned using ClustalW2. (C) Dissimilarity by blocks of 20 amino acids in the VlhA hypervariable region of the aligned specimens. This region, while having a low overall degree of similarity, includes short stretches of 70 to 100% identity. The sites and lengths of insertions (+) or deletions (−) are indicated for strains that were unique with respect to the consensus sequence.

Fig. 2.

Predicted secondary structures of expressed full-length Mycoplasma synoviae VlhA adhesins. Long vertical bars represent α-helices, short vertical bars represent β-sheets, and horizontal bars represent coiled coils. Shading indicates the MSPB region. WVUAU, low-passage-number Australian lineage; WVUCC, unpassaged lineage; WVUFL, >30-passage lineage; and WVUTU, ca.-12-passage lineage, all of the type strain WVU1853^T. Sequences were aligned with respect to the predicted MSPB-MSPA posttranslational cleavage site of the VlhA expressed by WVUFL and are shown, beginning with WVUCC, in descending order of hemagglutination activity.

Fig. 3.

Rank correlation between hemagglutination and endogenous sialidase activity in Mycoplasma synoviae. Shading indicates the 90% confidence interval.