摘要:尿道感染是世界上最常見的感染症之一,而Proteus mirabilis (P. mirabilis)為重要病原菌,特別是導管引起的尿道感染。P. mirabilis具有許多致病因子例如纖毛(pili)、鞭毛、溶血素以及對抗antimicrobial peptides (AP)的能力等。AP屬innate immunity一部份,P. mirabilis天生對AP例如polymyxin B (PB)具高抗性。之前我們發現一雙組成系統RppAB調控對PB的抗性,進一步研究得知RppAB藉由控制下游galU、 ugd、 pmrI等負責LPS合成及修飾的基因使P. mirabilis能對抗PB。galU、 ugd或pmrI突變並會影響表面結構及其他致病因子的表現,包括表面移行等,其中galU及ugd突變也會誘導負責維護envelope完整性之σE的表現。在E. coli中,σE已知和LPS修飾有關且會透過small RNA (sRNA)來調控雙組成系統的表現而影響PB抗性及致病因子的表現。此外經由transposonmutagenesis(TM)篩選對PB具感受性之突變株我們找到P. mirabilis一個能感知envelope壓力的雙組成系統CpxAR會影響gentamicin(GM)的感受性。由於臨床上抗GM的P. mirabilis有增多的趨勢故擬研究Cpx如何影響GM感受性。於是我們進行TM實驗找到比野生株對GM更具抗性的ispA(電子傳遞鏈相關的基因)和cpxA突變株及較具感受性的speA(抗氧化壓力相關)突變株,進一步以流式細胞儀分析發現speA突變株在GM存在下產生的reactive oxygen species (ROS)較野生株多而cpxA突變株則較少,這和Collins等人所提GM引發細菌產生ROS的多少會決定殺菌的效果相呼應,也許GM搭配speA抑制劑可用於高GM抗性菌之治療。在E. coli已被發現某些具調節膜蛋白作用的基因改變會提高GM感受性而Lee等人也報告破壞一雙組成系統可以提高GM對Pseudomonasaeruginosa的療效,這些發現啟發對GM高抗性菌株治療之思維。最近我們找到一天然物(CX)會透過RppAB使P. mirabilis PB 感受性提高,也發現TiO2奈米分子會使PB感受性上升。另外由2D電泳發現RppA會調控superoxide dismutase (SOD)、σE也被報告會調控抗氧化基因而氧化壓力和鐵衡定有關。根據以上之背景,本計劃之目的為:一. 探討CpxAR、σE、sRNA及RppAB在P. mirabilis致病因子表現及PB感受性上扮演的角色; 二. 探討TiO2 如何影響PB感受性,CX如何經由RppAB影響PB感受性並測試CX對P. mirabilis致病力的影響以評估PB搭配CX作為治療高PB抗性菌之可行性; 三. 探討Cpx影響GM感受性之機制及GM非核醣體標的基因(包括speA,ispA)在GM殺菌之角色以提供未來合併治療之用; 四. 由於CpxAR、σE及RppAB皆調控envelope相關基因且和抗氧化壓力有關,故擬探討這些基因突變是否會導致envelope壓力並探討會引起envelope壓力之突變株之氧化壓力及鐵衡定之狀態以瞭解envelope壓力之訊息傳遞; 五. 研究envelope及抗氧化壓力相關之基因是否會影響P.mirabilis和尿上皮細胞和巨嗜細胞之作用。故未來三年之計畫為: 第一年,I. 探討CpxAR、σE、sRNA及RNAchaperone Hfq在P. mirabilis PB感受性、致病因子表現扮演的角色;II. 探討RppAB、CpxAR、σE及sRNA之關係;III. 分析CX如何影響RppAB之訊息傳遞; IV. 測試CX對於臨床上其他具高PB抗性之革蘭陰性菌是否有作用;V. 研究TiO2使P. mirabilis PB感受性上升之機制。第二年,I. 研究cpxAR、 ispA及speA影響GM感受性的原因並探討氧化壓力和GM殺菌之關係;II. 探討抗氧化相關之ArcAB在GM感受性的角色; III.分析GM存在下抗氧化相關基因oxyR、 soxR之表現; IV. 分析GM存在下cpxAR及arcAB regulon之表現; V. 分析具不同GM感受性之P. mirabilis 臨床菌株處理GM後ROS產生的情形並探討其ispA、 speA、cpxA及相關抗氧化基因表現之差異以了解其感受性不同之機制以利於未來GM合併治療之應用;VI. 繼續篩選影響GM感受性的突變株以探討GM毒殺細菌之全貌以利於臨床上GM高抗性菌株感染之治療; VII. 探討envelope相關基因突變是否會導致envelope壓力。第三年,I.分析導致envelope壓力之突變株對抗氧化壓力之能力;II. 測定導致envelope壓力之突變株ROS的產生量並分析Cpx及Arc是否參與其中; III. 分析導致envelope壓力之突變株抗氧化及鐵衡定相關基因之表現;IV.觀察 envelope相關基因突變對CpxAR、ArcAB、RppAB、及σE 表現之影響來瞭解envelope壓力之訊息傳遞; V.進行尿上皮細胞和巨嗜細胞細胞素分析實驗以了解野生株及抗氧化壓力及envelope相關突變株和宿主之交互作用; VI. 探討抗氧化壓力及envelope相關基因突變是否會影響巨嗜細胞之吞嗜及毒殺。本研究能揭開RppAB、CpxAR、σE 及 sRNA 在P. mirabilis 中調控PB及GM抗藥性、對抗氧化壓力及致病因子表現之複雜網路並瞭解這些相關基因如何影響宿主之反應,另一方面,可利於PB及GM combination therapy藥物之發展,以用於未來對高PB或高GM抗性細菌之治療。
Abstract: Proteus mirabilis is a common cause of urinary tract infections. The ability of P. mirabilis to survive thekilling action of polymyxin B(PB), a kind of APs which are key effectors of the host innate immunity, isclearly important in the pathogenesis. Recently, σE has been shown to be required for resistance to AP andvirulence and changes in LPS has been shown to induce the σE. Moreover, a two-component system isregulated by small RNAs (sRNA) upon activation of σE and a two-component system regulated sRNA canfine-tune σE- mediated LPS decoration. Previously, we found a two-component response regulator gene (rppA)is involved in virulence expression and PB resistance via regulating the expression of its downstream ugd,galU and pmrI genes which are all responsible for synthesis and/or modification of LPS. Moreover, our ugdand galU mutants displayed induction of σE expression. Our preliminary data also showed P. mirabilisCpxAR , a two-component system, was involved in PB resistance. Therefore, it is likely that a complexnetwork of PB susceptibility and virulence gene regulation involving CpxAR, RppAB, sRNAs and σE exists inP. mirabilis. PB has now been used in the treatment of infections with highly drug-resistant Gram-negativeorganisms. However, elevated PB MICs are being reported with increasing frequency. Accidentally, we foundCX, a compound from the lacquer tree, can increase PB susceptibility of P. mirabilis through anRppAB-dependent pathway and sublethal concentration of TiO2 also increases PB susceptibility. By accident,in searching PB susceptible Tn5-mutagenized mutants of P. mirabilis, we found Cpx is involved ingentamicin (GM) susceptibility. We got several mutants with altered GM susceptibility. Among them are speA(more sensitive) and cpxA (more resistant) mutants. By flow cytometry, we demonstrated that speA mutantproduced more ROS than the wild-type, in contrast to the cpxA mutant. This finding supports the view thatGM utilizes a killing mechanism by stimulating the production of ROS. Envelope perturbation has beenshown to induce oxidative stress and changes in iron homeostasis. Because CpxAR,σE, sRNA and RppABregulate envelope-related genes, we wonder if mutation of envelope-related genes does create membranestress and consequently results in oxidative stress and changes in iron homeostasis. During UTI, E. colistimulates mucosal cells to secrete cytokines such as IL-8 and IL-6. It has been known urinary tract infectioncan augment expression of chemokine receptor (CXCR) by human neutrophils and Toll-like receptor 4 (TLR4)is well expressed on uroepithelial cells. The degree of cytokine secretion and the activation of mucosal TLR4are influenced by the properties of the infecting strain. Pathogens should avoid phagocytosis and survivephagocytic killing in order to establish an infection. Capsules and other surface structures are all associatedwith antiphagocytosis. Because CpxAR, σE, sRNA and RppAB regulate surface structures and are correlatedwith bacterial susceptibility to oxidative stress, we plan to investigate the effect of envelope or oxidative stressassociated genes on phagocytosis and phagocytic killing and assess their effects on expression of TLR4 onuroepithelial cells and expression of CXCR1 and CXCR2 on the macrophages. In summary, to disclose thecomplex regulatory network involving CpxAR, σE, sRNA and RppAB in PB susceptibility, virulenceexpression, envelope stress responses and bacteria-host cell interaction; the mechanisms of CX and TiO2affecting PB susceptibility and the GM killing mechanism in P. mirabilis, there are five specific aims of ourplan : I. investigate the roles of CpxAR, σE, sRNA and RppAB in PB susceptibility and virulence expression.II. study how CX and TiO2 decrease PB susceptibility to provide information for PB combination therapy. III.disclose the roles of CpxAR and other genetic determinants of GM susceptibility in GM killing to provideinformation for GM combination therapy. IV. investigate the effect of mutation of envelope-related genes onoxidative stress status, stress response and iron homeostasis to know the signaling pathways of envelopestresses. V. investigate the roles of envelope and antioxidation-related genes in the bacteria-host interaction.This will be the first important study about CpxAR, σE, Hfq, sRNA and RppAB in PB susceptibility, virulenceexpression and bacteria-host cell interaction in P. mirabilis. This will also be the first important study aboutbacterial two-component system controlling GM killing in P. mirabilis. This study also will disclose themechanisms how CX and TiO2 affect PB susceptibility. Such a study will facilitate the development ofcombination therapies for treating infections caused by highly PB or GM resistant bacteria.