奇異變形桿菌中PmrI及GalU調控多黏菌素B的感受性、表面移行能力、毒力因子表現及其受RppA調控之研究

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Proteus mirabilis is a gram-negative bacterium and a member of the family Enterobacteriaceae. It is an important pathogen of the urinary tract, especially in patients with indwelling urinary catheters. Antimicrobial peptides (APs) are important components of the innate defenses of animals and plants, which can be found in neutrophils and macrophages and are produced by epithelial cells at mucosal surfaces. Most APs are cationic, amphipathic molecules of small molecule weight peptides and have an activity against infection of pathogens. Polymyxin B (PB), a kind of cationic antimicrobial peptides (CAMPs), is composed of a polycationic peptide ring and a hydrophobic tail. In gram-negative bacteria, CAMPs which have positive charge, can bind to negative charged portion of LPS such as lipid A and core and then alter the membrane integrity by solubilization or pore formation. P. mirabilis is naturally resistant to PB, and one of the possible mechanisms is through the modification of lipid A by 4-amino-4-deoxy-L-arabinose (L-Ara4N) which is also found in Escherichia.coli and Salmonella enteric serovar Typhimurium. In Salmonella Typhimurium, L-Ara4N modification of lipid A is regulated by two-component systems, PhoP-PhoQ and PmrA-PmrB. The modification reduces the negative charge of LPS and consequently decreases the binding of PB. In our previous study, we found a PhoP-PhoQ homologue and designated RppA-RppB. The rppA knockout strain is highly sensitive to PB and the expression of rppA is up-regulated by a low concentration of PB. To better understand the underlying mechanisms of P. mirabilis resistance to PB, mini-Tn5 transposon mutagenesis is used to identify more genes involved in PB susceptibility and to characterize the function of these genes. Three unique PB-sensitive P. mirabilis mutants were identified and these mutants were over 10000-fold more sensitive than the wild-type. DNA sequence analysis of the transposon insertion gene reveals similarities to a galU, pmrI and PMI 1781 loci of E. coli and Salmonella. Though the galU and PM3 mutants have lower concentration of LPS and altered LPS profile than the wild-type, the pmrI mutant has normal LPS concentration and profile. Further, we found that the LPS of the pmrI mutant can bind more PB than the wild-type. The atomic force microscopy also showed that the surface structure of galU mutant is more roughness than wild-type. Moreover, in the presence of PB, LPS ladder occurs shifting in wild-type LPS profile but not in pmrI and rppA mutants, and the expression of galU and pmrI mRNA has four-fold increase. These data imply that galU and pmrI expression maybe regulated by response regulator RppA and pmrI is involved in LPS modification. Furthermore, the pmrI belongs to a pmrHFIJKLM operon, and we find that the promoter of this operon is enhanced by low concentration of PB, and we demonstrate that RppA protein can directly bind to the promoter to enhance this operon expression by EMSA experiment. When the mutants are streaked on 1.5% LB agar plate, the swarming motility of galU and PM3 mutants (LPS-defective mutants) are completely inhibited due to the defect in swarmer cell differentiation and decreased mRNA expression of flagellin biosynthesis genes (flhDC, fliA and flaA) in galU mutant. The other phenotypes such as haemolysin, urease activity and biofilm production are also decreased in galU and PM3 mutants. These data support the roles of galU and PMI 1781 in LPS synthesis, and LPS integrity plays an essential role in PB susceptibility, swarming and virulence factor expression in P. mirabilis. LPS modification, which is mediated by pmrI, also demonstrated to be the mechanism in the resistance of PB. Finally, our data also highlight the important role of RppA-RppB two-component system in the presence of PB to regulate downstream regulon, such as galU and pmrI and confer the ability against PB.