A novel signature-tagged mutagenesis system for Streptococcus suis serotype 2

Streptococcus suis is an economically important, zoonotic pathogen causing death and disease in swine. The objectives of this study were to develop a signature-tagged mutagenesis (STM) system for S. suis serotype 2 and to identify genes required for in vivo virulence. Identification of such candidate genes may lead to a better understanding of the pathogenesis of S. suis and may provide substrate for the discovery of new vaccines. A novel STM approach was designed to allow for a higher throughput assay of mutants using the Luminex xMAP system. Additionally, to speed the identification process, a direct genomic DNA sequencing method was developed that overcomes the problems associated with the presence of repetitive insertion sequences. Approximately 2600 mutants were screened through both mouse and caesarian-derived, colostrum-deprived (CDCD) pig models. The disrupted ORF was identified for each potential attenuated mutant, and mutants with distinct and unique mutated ORFs were analyzed individually for attenuation in mouse and CDCD pig models. A variety of genes were identified, including previously known genes essential to the virulence of other organisms, genes involved in capsule biosynthesis, a regulator of suilysin expression, and several conserved or predicted genes. Of the 22 mutants identified as attenuated in either animal model, eight insertion mutants caused no mortality in both mouse and pig models.