Identification and functional study of the truncated surface-associated subtilisin-like protease gene of Streptococcus suis serotype 2

Abstract:

[Objective] To clone and express the truncated surface-associated subtilisin-like protease gene 05SSU0811 of Streptococcus suis serotype 2 (SS2) highly virulent strain 05ZYH33 and measure the activity of the recombinant protease; Construct the 05SSU0811 gene knockout mutant strain and analyze its contribution to pathogenicity. [Methods] The 05SSU0811 gene encoding SspA was amplified and cloned into the expression plasmid pET28a and then transformed into Escherichia coli BL21 to overproduce the protein. The recombinant protease was purified by chromatography procedures. Its activity was measured by using the chromogenic substrate Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (pNa). The 05SSU0811 gene was replaced with spectinomycin resistance cassette through homologous recombination, then multiple-PCR and sequence analysis were adopted to identify the knockout strain ?05SSU0811. The virulence of SS2 wild type and ?05SSU0811 mutant strain were then evaluated by calculating the survival rate of the infected mice. [Results] The recombinant SspA was expressed and purified. Its activity was demonstrated by the subtilisin-like protease assay. The isogenic mutant ?05SSU0811 was successfully constructed and the virulence of the ?05SSU0811 mutant strain was attenuated remarkably compared to the wild type strain. [Conclusion] The truncated SspA encoded by 05SSU0811 gene in SS2 exhibits its activity in vitro. And it also contributes to the virulence of SS2.