Ways And Functional Studies Of P. Aeruginosa Type Ⅲ Secretion System Gene Regulation TopA And PA0964 Regulation

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogens, and its pathogenicity is enabled by a variety of virulence factors. One of the important virulence factors is the type Ⅲ secretion system (T3SS). During acute infection, P. aeruginosa uses T3SS to inject the toxins called effectors into the host cell cytoplasm, promoting infection process. Continuing previous research carried out in our lab, the regulation the type Ⅲ secretion system by topA and the relationship between PA0964and type Ⅲ secretion sysem, as well as quorum sensing system (QS), have been investigated in this study.In order to analyze the effect of top A on T3SS in P. aeruginosa, a top A gene knockout mutant PAOl(△topA) was constructed. Expression reporters of T3SS genes, exoS, exoY, exoT based on the luminescence reporter gene cluster, luxCDABE, were introduced into the mutant construct The expression of the T3SS genes in PAOl(△topA)was compared with that in PAO1. The results show that the expression of T3SS genes decreased in PAOl(△topA), suggesting the T3SS genes were positively regulated by topA. The effect of topA on T3SS repression and pyocin synthesis was possibly through ptrB. In addition to decreased T3SS expression, decreased biofilm formation and motility were observed in the topA mutant. topA mutation also significantly reduced the bacterium’s tolerance to high temperature and osmotic stress, indicating an important role of topA plays in such tolerances.PA0964has been shown in vitro to directly bind the pqsR promoter and regulate the expression of QS system. In this study, a PA0964mutant PAOl(△0964) and PA0964/pqsR double mutant PAOl(△0964/△pqsR) were constructed. The expression of the T3SS genes and QS genes was measured in PAO1, PAOl(△0964) and PAOl(△0964/△pqsR)-The results showed that the expression of T3SS genes increased in PAOl(△0964) compared to wild type, suggesting the T3SS genes were also negatively regulated by PA0964. The expression of QS system increased in the two mutant strains to a similar level. Increased pyocyannin synthesis was observed in PAOl(△0964), but in PAOl(△0964/△pqsR) the opposite was observed.