Molecular basis of attenuation of Mycoplasma synoviae

© 2013 Dr. Muhammad Akbar ShahidComparative genomic analysis of M. synoviae strains including temperature-sensitive (ts+) vaccine strain MS-H, non-temperature-sensitive (ts–) vaccine parent strain 86079/7NS and two of the ts– MS-H reisolates, revealed a large number of single nucleotide polymorphisms (SNPs) some of which were non-synonymous mutations in essential genes of MS-H. One such non-synonymous mutation causing Gly123Arg substitution in a highly conserved domain of GTP binding protein Obg was further investigated. In-silico analysis of M. synoviae Obg 3D structures, obtained by homology modeling, revealed that Gly123Arg substitution was likely to destablise the Obg structure whilst another substitution, Ala210Val, mapped in G3 motif of the conserved GTP binding domain, was likely to compensate the destablising effect of Gly123Arg change in the Obg domain. Plasmid shuttle vectors, based on the origin of replication (oriC) of M. synoviae, were developed and used to transform MS-H by electroporation. One plasmid vector, pMAS-LoriC, containing complete oriC region, was found to replicate autonomously in the MS-H and integrated at the chromosomal oriC locus. In order to complement the MS-H with the wild-type obg, an oriC-based vector, pKS-VOTL, was developed and used to transform MS-H. Variable lipoprotein haemagglutinine A (vlhA) promoter region was used to drive expression of the wild-type obg. The pKS-VOTL plasmid readily integrated at the chromosomal oriC with no homologous recombination event other than at oriC locus was observed. Over-expression of wild type obg was confirmed using Northern blot, SDS-PAGE, and Western blot analyses. The MS-H clones transformed with pKS-VOTL retained the ts+ phenotype of MS-H; however unlike MS-H, these MS-H transformants could grow, albeit to a very low titer, at 33°C after exposure to nonpermissive temperature of 39.5°C. It was postulated that overproduction of wild-type Obg exerted growth inhibitory effect at the nonpermissive temperature whilst promoted the growth at the permissive temperature. High-resolution melting curve analysis, targeting the informative SNPs in obg, was used to develop rapid genotyping technique which could reliably differentiate MS-H from its ts–reisolates and also from all field strains used in this study. The technique was also applied to clinical specimens taken from SPF chickens vaccinated with MS-H and commercial field chicken flocks infected with a field M. synoviae strain. Findings of this thesis indicated that, like many other mycoplasmas, genetic manipulations of M. synoviae are now technically feasible. Mutations in obg are most likely responsible for the temperature-sensitivity/attenuation of MS-H while mutations in other essential genes, especially those encoding aminoacyl-tRNA synthetase and tRNA (guanine-N1)-methyltransferease, might have a role to play in MS-H attenuation. Using oriC vectors, expression of foreign proteins is now also possible in MS-H. Such strategy can serve as foundations for production of diagnostic mycoplasma antigens and for the development of recombinant vaccines