Genetic variation in sialidase and linkage to N-acetylneuraminate catabolism in Mycoplasma synoviae

Abstract

We explored the genetic basis for intraspecific variation in mycoplasmal sialidase activity that correlates with virulence, and its potentially advantageous linkage to nutrient catabolism. Polymorphism in N-acetylneuraminate scavenging and degradation genes (sialidase, Nacetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate epimerase, N-acetylglucosamine-6-phosphate deacetylase, and glucosamine-6-phosphate deaminase) was evident among eight strains of the avian pathogen Mycoplasma synoviae. Most differences were single nucleotide polymorphisms, ranging from 0.34 ± 0.04 substitutions per 100 bp for N-acetylmannosamine kinase to 0.65 ± 0.03 for the single-copy sialidase gene nanI. Missense mutations were twice as common as silent mutations in nanI; 26% resulted in amino acids dissimilar to consensus; and there was a 12-base deletion near the nanI promoter in strain WVU1853 T , supporting a complex genetic basis for differences in sialidase activity. Two strains had identical frameshifts in the N-acetylneuraminate lyase gene nanA, resulting in nonsense mutations, and both had downstream deletions in nanA. Such genetic lesions uncouple extracellular liberation of sialic acid from generation of fructose-6-phosphate and pyruvate via intracellular N-acetylneuraminate degradation. Retention of nanI by such strains, but not others in the M. synoviae phylogenetic cluster, is evidence that sialidase has an important non-nutritional role in the ecology of M. synoviae and certain other mycoplasmas.

Diversity of Expressed vlhA Adhesin Sequences and Intermediate Hemagglutination Phenotypes in Mycoplasma synoviae

“…The conserved N-terminal region (consensus amino acids 1 to 240 of VlhA and MSPB [32]) includes a secretion signal peptide, signal peptidase recognition site, and a prolinerich repeat domain. This region, once thought to be invariably present and nearly identical among allelic variants generated by recombination (32,33), had a rate of substitutions in the sequences that we examined that was 10-fold higher than strain variation in highly conserved genes encoding the N-acetylneuraminate degradation pathway and 100-fold higher than the 16S rRNA gene heterogeneity in M. synoviae (24). Eleven of the 30 strains sequenced by Benčina et al (2) also were polymorphic in this region of MSPB, but it was particularly remarkable that our WVUCC specimen lacked MSPB entirely.…”

“…Although numerous residues inferred to experience strong diversifying selection ( Ͼ 3) occur on the exterior surface of the enzyme, strain variation in sialidase activity is not attributable to direct immune-mediated selection (24,25). We conclude that the VlhAs also experience diversifying selection, such that the species M. synoviae is at its fittest when substantial diversity in the MSPA region of the expressed VlhA is perpetuated among strains (27,45), and this does seem likely to be the outcome of direct immune-mediated selection.…”

“…The functional relationship between VlhA and sialidase in M. synoviae thus seems analogous to the receptor-binding hemagglutinin and receptor-destroying sialidase (neuraminidase) activities of influenza viruses. To test the prediction that sialidase activity correlates with adhesin affinity in this species (24,44), we examined the diversity in primary amino acid sequences and predicted secondary structures of the expressed VlhAs among 14 specimens of M. synoviae and the consequent hemagglutination phenotypes of specimens with differing sialidase activities.…”

Diversifying and Stabilizing Selection of Sialidase and N -Acetylneuraminate Catabolism in Mycoplasma synoviae

“…Genomic DNA was extracted from all strains of M. synoviae and M. gallisepticum using EasyDNA reagents according to the manufacturer's specifications (Invitrogen, Carlsbad, CA). Amplification and sequencing of the sialidase and N-acetylneuraminate catabolism locus of M. synoviae were described previously (25). Amplification of the sialidase gene from M. gallisepticum strains was performed by initial denaturation at 94°C followed by 30 cycles at 94°C (20 s), 50°C (20 s), and 72°C (3 min).…”

“…A nearly identical homolog (consensus 94.5% amino acid identity and 96.5% amino acid similarity) exists in M. gallisepticum, even though M. gallisepticum does not possess the enzymes that M. synoviae does for catabolizing liberated sialic acid (N-acetylneuraminate) for glycolysis. Because receptor desialylation reduces or abolishes cytadherence by both M. synoviae and M. gallisepticum, a balance between variable sialidase activity and avidity of adherence to sialylated receptors is predictable (25), but the selective pressures responsible for the observed correlation of sialidase activity with M. synoviae strain virulence remain unknown (3,4,26).…”