研究兩種功能類似的蛋白酶Lon以及ClpYQ(HslUV)參與 Salmonella enterica Serovar Typhimurium致病力的關係

Abstract

Salmonellae are pathogenic bacteria to both human and animals. Salmonella invades intestinal epithelial cells by using a type III secretion system (TTSS) encoded by Salmonella pathogenicity island 1 (SPI1). The SPI1 TTSS forms a needle-like structure that injects effecter proteins directly into the cytosol of host cells. Salmonella pathogenicity is to maintain its survival and is able to replicate inside host cells. These virulence traits could cause systemic infections. SPI1 expression is influenced by a variety of environmental conditions and regulatory proteins, and the key regulator is the SPI1-encoded HilA, which activates their transcription directly or by increasing expression of another regulator, InvF. HilC and HilD accumulate in Lon-depleted cells and the enhancing effect on SPI1 expression. Lon and Clp family are ATP-dependent proteases shared of removing misfolded protein under stress conditions. In vivo proteolysis activities of Escherichia coli Lon and the ClpQY (HslUV) protease are also redundantly. In this study, we focused on the relationship between ClpQY (HslUV) protease and Salmonella SPI1 expression under different environmental conditions. The lon, clpQ and/or clpY single or double or triple mutation strains carrying hilA::Tn5lacZY, invF::Tn5lacZY, sipA::Tn5lacZY and sipC::Tn5lacZY fusions on the chromosome were constructed by bacteriophage P22 transductions. The resultant strains were incubated under different oxygen and osmolarity (0%-3.5% NaCl) environmental conditions. From β-galactosidase assay data, we found that bacteria with an optimal SPI1 gene expression were grown in oxygen limited and 1% NaCl condition. There is no significant difference in clpQ and clpY single or double mutants for SPI1 expression. However, the SPI1 gene expression in lon mutant is higher than wild type about 3 to 5 fold. The clpQ lon double mutant strain is slightly higher than lon single mutant in SPI1 gene expression. The influence of SPI1 gene expression in clpQ single mutant might be amplified in lon deletion background. ClpQY protease modulates Salmonella virulence; presumably that ClpQY shares redundant activities with Lon protease. Searching for the maximum difference in lon single mutant and clpQ lon double mutant, we found that the modulation by different environmental conditions are very diversely in invF and hilA gene expressions from wild type and all mutants. However, the SPI1 downstream gene expressions in clpQ lon double mutation strain are obvious higher than that in lon single mutant. We also notice that ClpQ and ClpY might play a different role in modulation the SPI1 gene expression. Afterwards, the SPI1 gene expression was repressed by incubation under high oxygen condition in wild type and all mutants.