RESUMO
OBJECTIVE: In order to study the role of SS2 Type IV Secretion System VirD4 in evasion of the host innate immune killing, we constructed a knockout mutant AVirD4. Then we studied its biological activity and virulence. METHODS: The two VirD4 flanking DNA sequences were amplified using genome of 05ZYH33 as template. We also amplified the Cm sequence of shuttle vector pSET1, and through overlap extension PCR we connected the three fragments together. Using suicide vector pSET4s, we constructed the recombinant gene knockout vector pSET4s::VirD4. The mutant AVirD4 was successfully constructed by allelic replacement. Virulence of mutant strain was compared with wild type strain 05ZYH33 through in vitro bactericidal assays, competitive infection and challenge experiment of CD1 mice. RESULTS: Mutant strain AVirD4 was constructed successfully, its virulence attenuated compared to the wild type strain. CONCLUSION: These findings indicated that Type IV Secretion System component VirD4 contributed to the virulence of S. suis with important functions in evading innate immunocyte killing.