探討resveratrol及polymyxin B調控奇異變形桿菌表面移行及致病因子表現的機制

Abstract

奇異變形桿菌（Proteus mirabilis）屬於革蘭氏陰性菌，當它處在固態表面上時會從單細胞、少量鞭毛的swimmer cell變化成多細胞、多鞭毛且具有移行能力的swarmer cell。在之前研究中指出，奇異變形桿菌的表面移行能力與許多致病因子相關，例如溶血酶活性、鞭毛生成量等，也和此菌入侵泌尿道細胞能力有關，因此可知表面移行能力與奇異變形桿菌所造成泌尿道感染具有正相關。自80年代開始，Resveratrol（RV）在抗癌、抗氧化方面的研究很豐富，但對於細菌的影響則比較少被發表，主要是因為目前只有胃幽門桿菌以及腦膜炎雙球菌、淋病雙球菌對於RV具感受性。 Polymyxin B（cationic AMPs, 簡稱PB）為帶有正電荷之抗菌胜肽，可利用其正電與帶負電之細菌外膜結合，進一步造成細胞膜上的孔洞，而引發細菌的死亡。目前我們發現到RV60μg/ml以及PB 100μg/ml的濃度下，不會抑制P. mirabilis的生長，但卻會抑制表面移行。綜合以上各點，我將研究分成三個方向：臨床菌株對於RV抗藥情況的篩選、探討RV以及polymyxin B調控P. mirabilis表面移行於致病因子表現的機制。 首先利用純化的resveratrol進行臨床菌株抗藥性試驗，篩選台大醫院共572株臨床菌株及本實驗室原有菌株71隻，發現大部份菌株之MIC均大於256 μg/ml，其中S. maltophilia及A. baumannii有部分菌株之MIC只有64 μg/ml，但仍未能挑出對於RV有高度敏感性之菌株。本實驗室中P. mirabilis菌株亦對RV具高度抗藥性（MIC > 256 μglml）；但是我們發現到RV可以抑制P. mirabilis的表面移行，且對於一些致病因子如haemolysin、 urease等均有抑制現象。將實驗室之前建構的突變株S13（rsbA mutated；two-component system protein）及野生株N2做比較，可以發現到S13的移行以及致病因子的表現大多不受RV的抑制，表示RV對於P. mirabilis的作用可能是透過這個two-component的機制，而resveratrol抑制移行的現象亦普遍存在於其他P. mirabilis臨床菌株中。我們亦發現P. mirabilis雖然對於polymyxin B藥物具有高度抗藥性，但其表面移行現象、鞭毛表現以及溶血酶的活性均會受其抑制。因此，我們利用Tn5-mutagenesis的方式找出不受RV或是PB抑制移行能力的菌株，並加以分析可能參與調控的機制。 我希望透過這樣的研究，能對於P. mirabilis表面移行以及致病因子調控機制有更深入的暸解。

Proteus mirabilis is a pathogenic gram-negative bacterium that frequently causes kidney infections, typically established by ascending colonization of the urinary tract. The pathogen exhibits a cyclical differentiation process in which vegetative cells growing on solid media differentiate into long, hyperflagellated swarmer cells, which undergo rapid and coordinated population migration away from the initial colony. This migration behavior is coupled to expression of some virulence factors, such as haemolysin、urease in P. mirabilis. Resveratrol produced by several plants, berries and fruits, including grapes, is one of the best known natural food micro-components with potent chemopreventive properties towards the most severe contemporary human diseases: cardiovascular sickness, cancer and neurodegenerative pathologies. Polymyxin B, a cationic antimicrobial peptide, binds to outer-membrane of bacteria and leads to death. Before studying, we tried to screening clinical isolates for resveratrol susceptibility, but failed. We found that Proteus mirabilis, an important urinary tract pathogen, was highly resistant to RV (MIC > 256μg/ml). For the first time, we demonstrated that the anti-swarming and anti-virulence effects of RV on P. mirabilis and that such effects required the presence of rsbA, encoding a bacterial two-component sensor kinase in P. mirabilis. To investigate the inhibitory effects of RV on swarming in P. mirabilis, we performed haemolysin assay, flagellin assay, cell differentiation analysis, in wild-type P. mirabilis and its congenic rsbA mutant. We found RV inhibits swarming of wild-type P. mirabilis in a dose-dependent manner. Haemolysin activity, extent of cell differentiation, and flagellin level were reduced in wild-type P. mirabilis by RV. The above phenotypic traits of the RV treated wild-type P. mirabilis were lost in the rsbA mutant. Polymyxin B also inhibits swarming behavior、flagellin production and haemolysin activity of P. mirabilis. To determine the inhibitory mechanism of polymyxin B and resveratrol, we performed Tn5-mutagenesis to construct mutant that can swarm in the presence of PB or RV and analysed for these mechanisms. Through these studies, we can disclose the mechanisms of swarming behavior and virulence expression in P. mirabilis.