Biological Functions Of Sortase BCD Pilus Island In Highly Pathogenic Streptococcus Suis Serotype 2 Strain 05ZYH33

Streptococcus suis 2 (S. suis 2) is an emerging zoonotic pathogen responsible for a wide range of life-threatening diseases in pigs and humans. To date, thirty-five serotypes (types 1/2, and 1-34) of S. suis have been described, of which S. suis 2 is the most frequently isolated and associated with disease. Various cell-surface multisubunit protein polymers, known as pili or fimbriae, have a pivotal role in the colonization of specific host tissues by many pathogenic bacteria. In contrast to Gram-negative bacteria, Gram-positive bacteria assemble pili by a distinct mechanism involving a transpeptidase called sortase. Sortase cross-links individual pilin monomers and ultimately joins the resulting covalent polymer to the cell-wall peptidoglycan. Most importantly, pilins confer a significant protection, which indicated that it can serve as a novel vaccine candidate. Based on bioinformatics analysis of the whole genome of S. suis 2 Chinese high virulent strain 05ZYH33, we found two gene clusters similar to pilus-associated gene clusters of some Gram-positive bacteria in genetic organization phenomena, designated srtBCD and srtF pilus clusters, which srtBCD cluster exists only in the virulent strain. In this study,? we characterized a functional member of srtBCD clusters and SrtF from a Chinese S. suis 2 isolate, 05ZYH33, And the following experiments are conducted:1 Molecular cloning and immunological characterization of SSU2099 gene from srtBCD pilus island of Streptococcus suis 2An open reading fragment, SSU2099, encoding pilus gene from srtBCD pilus island was identified based on bioinformatics analysis of the genome sequence of 05ZYH33 and alignment with relative protein family. The target DNA fragment was amplified using a pair of primers specific to SSU2099 gene. SSU2099 gene was cloned into prokaryotic expression plasmid pET32a subsequently, and the recombinant protein was expressed and purified. The mice anti-SSU2099 serum was obtained by immunizing mice with recombinant SSU2099 protein, and the titer of the serum was analyzed by ELISA. Animal test showed that vaccinating mice with recombinant SSU2099 confer a significant protection, which indicated that SSU2099 can serve as a novel vaccine candidate.2 Construction the ?srtBCD mutantTo obtain the isogenic mutant srtBCD, the competent cells of 05ZYH33 were subjected to electrotrans formation with recombinant plasmid based on the principle of homologous recombination. The resulting mutant strains was further confirmed by a series of PCR and reverse transcription PCR.3 Biological functions of ?srtBCD mutantWe demonstrated that mutant srtBCD reduced capacity to adhere to human laryngeal epithelial cell line compared to the wild-type strain. However, disruption of srtBCD had less effect on the virulence of S.suis in a mouse intraperitoneal model of infection. These results indicated that surface proteins anchored by SrtBCD are required for a normal level of bacterial binding. However, other factors may also be important for S.suis 2 virulence and interactions with host tissues.4 Biological functions of ?srtF mutantThe resulting mutant strain exhibited growth kinetics equivalent to those of the WT parent strain upon cultivation in standard laboratory used in our in vitro assays. Abolishment of the expression of srtF did not result in impaired interactions of S.suis 2 with human laryngeal epithelial cell line. Furthermore, srtF mutant were as virulent as the wild type strain when evaluated in a murine model of S.suis . Taken together, these results suggest that srtF cluster in fact not critical for the full virulence of S. suis 2.