Roles of the Cpx two-component system in Proteus mirabilisThe effects of Compound X on Proteus mirabilis

• Main Authors: Yu-Han Yuan, 袁于涵
• Other Authors: Shwu-Jen Liaw
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/38099654431726801055

碩士 === 臺灣大學 === 醫學檢驗暨生物技術學研究所 === 98 === Proteus mirabilis is a facultative Gram-negative bacterium and a member of the Enterobacteriaceae family. It’s a normal flora in intestines of healthy human. However, it commonly causes urinary tract infection (UTI), even kidney disease, pneumonia and septicemia in individuals with long-term catheterization or with structural or functional abnormalities in the urinary tract. Polymyxin B (PB) is a kind of cationic antimicrobial peptides. While the positive-charge structure of PB combine with the negative-charge bacterium membrane, the bacterium membrane is disrupted and leak the cytoplasm contents out. The well known mechanism about the resistance of PB is the modification of the lipid A of the lipopolysaccharides (LPSs) in Gram-negative bacteria by 4-amino-4-deoxy-L-arabinose (L-Ara4N), which decreases negative charge of the membrane. In this way, the binding ability decreases between PB and membrane, leading to the the resistance of PB. P. mirabilis is naturally resistant to PB, previous studies showed that the modification of L-Ara4N is modulated by PhoP-PhoQ and PmrA-PmrB two-component system (TCS), causing poor binding between LPS and PB in Salmonella. Our lab also found that there was a TCS, RppA-RppB, which can regulate surface gene expression to affect PB sensitivity and virulence in P. mirabilis. In order to know if there is any other TCS which can also regulate surface gene expression in P. mirabilis, we knockout cpxR gene to study the Cpx TCS, previous studies showed that perturbations on cell envelope are postulated to be the triggers of this pathway, the Cpx regulon has been implicated in the virulence of a number of bacterial pathogens, and more-recent work on the CpxAR regulon has identified a greater range of targets for phosphorylated CpxR in E. coli. The cpxR mutant exhibited inceasing swarming and swimming ability, increased flagellin synthesis, increased number of swarmer cells, increased haemolysin activity, decreased cell invasion ability, decreased biofilm formation, and altered membrane integrity in P. mirabilis. In E. coli, all of the phenotypes of cpxA* (lacks phosphatase activity) mutants can be accounted for by elevated levels of phosphorylated CpxR (CpxR-P), in this study, we found a cpxA mutant which phosphatase activity was also disrupted by transposon-mutagenesis, by using cpxR reporter assay, we also observed that cpxA mutant significantly increase cpxR expression in P. mirabilis. Complementation of cpxAR operon restored swarming, biofilm, LPS, and PB MIC in the cpxR mutant. Previous studies showed that, in E. coli, high osmolarity is one of the signals of Cpx pathway. We used cpxR reporter assay, and found that after treating high osmolarity, Cpx pathway could be activated in Proteus. Furthermore, we observed that cpxR mutant cause PB sensitivity in Proteus, so we think that PB could be the signal of Cpx, by using cpxR reporter assay, we also found that PB is one of the Cpx signals. In addition, under such signals like PB and high osmolarity, Cpx could be activated to cause decreased swarming ability in Proteus. We used mrpJ and flhDC reporter assay to investigate the mechanism of swarming of the cpxR mutant in Proteus. We found that Cpx can up-regulate mrpJ gene, which increased biofilm formation and down-regulate flhDC expression, therefore leading decreased swarming ability. In addition, previous studies showed that Cpx activation decreased the extracytoplasmic function (ECF) sigma factor σE (RpoE) expression in E. coli, by using rpoE reporter assay, we found that cpxR gene mutation led to decreased expression of rpoE in P. mirabilis. Furthermore, previous studies showed that cpxA* highly decreased OmpF expression, and high osmolarity induce Cpx to cause decreased expression of OmpF, therefore leading increased ampicillin and tetracycline MIC in E. coli, we also observed that an outer membrane prorin OmpF is highly reduced in cpxA mutant in Proteus, and under high osmolarity, Cpx can be induced to increase ampicillin and tetracyclin resistance. In this report, we also investigate if there is any small molecular drug can inhibit TCS activation, we found that Compound X can inhibit P. mirabilis swarming, increased PB sensitivity and decreased invasion ability, by using rppA reporter assay, it showed that Compound X can decrease rppA expression to cause polymyxin B sensitivity, thus, it is possible to combine Compound X and PB to reduce the dosage of PB during therapy. In this study, we investigated the roles of Cpx in antibiotics resistance, swarming ability and virulence factor expression in P. mirabilis. In addition, we also found that Compound X and PB can combine to treat on pathogens which are highly resistant to CAMPs.