一、奇異變形桿菌中Cpx雙組成系統之研究 二、探討Compound X在奇異變形桿菌中的作用

Abstract

Proteus mirabilis為革蘭氏陰性的腸內菌，在健康人類腸道屬於正常菌叢，但在長期使用導尿管之病患身上，會造成伺機性感染，嚴重甚至可能導致腎臟病、肺炎等併發症。 Polymyxin B (PB) 屬cationic antimicrobial peptides(CAMPs)的一種，其利用與格蘭陰性菌的lipopolysaccharide (LPS)結合，進而造成細胞膜的破壞。P. mirabilis天生就對PB具有很高的抗性，其中一機制可能是透過LPS 上lipid A之修飾，使細胞表面負電性降低，造成PB與細胞結合減少而產生抗性。先前研究指出在Salmonella中lipid A修飾是透過雙組成系統 two-component system (TCS) 來調控。本實驗室先前發現P. mirabilis中有RppA-RppB雙組成系統，會透過調控表面特性之基因來影響PB的感受性及致病因子表現，為了探討P. mirabilis中是否還有其他雙組成系統可以感知細胞外來的訊息以調控對抗生素的抗性及致病力，我們選擇與表面特性調控相關之Cpx雙組成系統來研究其在P. mirabilis中所扮演的角色。Cpx pathway在E. coli中是一個廣泛被研究的TCS，它是由CpxA (histidine kinase)接收外界訊息後，藉由磷酸根活化將訊息傳給CpxR (response regulator)，磷酸化的CpxR-P則會結合其下游基因之啟動子進而調控基因表現。E. coli Cpx TCS扮演維持細胞外膜穩定的角色，Cpx pathway活化後也會影響細菌的swarming、biofilm生成能力、抗藥性及細胞入侵能力等毒力因子的表現。而在Salmonella中Cpx pathway和致病力有密切的關係。 在本論文中，首先將Cpx雙組成系統中的cpxR knockout，觀察相關表現型是否有改變。結果發現cpxR突變株有以下特徵：1.對抗PB及gentamycin的感受性分別上升為四倍及兩倍，且CpxR有助於此菌形成biofilm後之對抗gentamycin及PB的能力2.表面移行能力稍顯上升 3.鞭毛蛋白合成在第5~8小時大量增加 4.隔夜培養後長細胞數變多5.入侵細胞能力下降 6.生物膜生成量降低 7.外膜蛋白及LPS的改變。而將cpxAR operon補回突變株中，可觀察到PB MIC、biofilm、LPS pattern及swarming能力的恢復。 在E. coli中已發現cpxA* (缺 phosphatase 活性) 突變株cpxR會過度表現，進而造成細菌envelope的改變，使得生物膜形成力受到破壞、swarming能力下降，而且對aminoglycosides類的藥物抗性增加。我們利用transposon mutagenesis在P. mirabilis中篩選到一株cpxA突變株，其插入位點為phosphatase 活性所在。利用cpxR reporter assay發現Proteus的cpxA突變株cpxR會大量表現，因此想了解其表現型，結果發現表現型態如: swarming、swimming、生物膜生成能力、細菌外膜的改變、對抗gentamycin及PB的MIC與cpxR突變株相較皆呈現相反的情形。 先前文獻指出，E. coli中滲透壓的改變會刺激Cpx的活化，在本論文中，利用高滲透壓來觀察是否會刺激Proteus的Cpx pathway，由cpxR reporter assay證實高滲透壓為其訊號分子。之前研究室發現，cpxR突變株對PB的感受性上升，推測PB也有可能為其訊號分子，以cpxR reporter assay得知PB為其訊號分子。而在高滲透壓或PB存在下也觀察到swarming能力的下降、ampicillin及tetracycline的MIC上升、biofilm生成力上升。 利用mrpJ及flhDC reporter assay探究Cpx與表面移行能力的關係，發現Cpx會促進與生物膜生成相關的mrpJ基因表現進而使鞭毛合成相關的flhDC基因表現減少，另外mrpJ reporter assay結果顯示，在高滲透壓及PB的存在下會促進mrpJ基因的表現進而抑制表面移行能力。此外，我們發現Proteus cpxA突變株OmpF的表現量大輻減少，而對ampicillin及tetracycline呈現抗性較高的現象，也發現高滲透壓會藉由活化Cpx而造成ampicillin及tetracycline的MIC上升，在E. Coli中也是相同的情形。 由於TCS會造成細菌抗藥性和致病性，因此近年來有關用以抑制這些TCS之小分子藥物的研究相當多。我們意外發現小分子藥物Compound X可以上升Proteus PB的感受性，降低細胞入侵能力，此兩項表現型態與rppA突變株相當。而由rppA reporter assay證實Compound X會抑制rppA的表現，表示Compound X會藉由抑制rppA造成PB的感受性上升，因此推測Compound X可和PB合併使用以降低PB的治療使用量。 本研究讓我們了解到Cpx 雙組成系統在P. mirabilis中所扮演的角色，以及Compound X對rppA的相關調控，並發現Compound X和PB可合併使用以治療對CAMPs有高度抗性的細菌。

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.