摘要:Proteus mirabilis 為尿道感染的重要病原菌,特別是由導管引起的。具有許多致病因子例如運動能力,溶血素以及pili 的黏附作用等,目前詳細的致病機制仍未明。Small regulatory RNAs (sRNAs)已被證明參與在許多細菌體致病力及抗藥性等運作之中,之前我們發表sRNAs 之伴侶蛋白質Hfq, 影響P.mirabilis 外膜組成,致病因子表現,壓力反應及藥物感受性, 也找到Hfq 伴侶sRNAs, GlmY, GlmZ, Spf,GcvB 等。近來本實驗室分離一無運動性之轉位子突變株分析得知其crp 基因遭破壞, Crp 為cyclicAMP 受體蛋白質其活性受葡萄醣影響,乃global regulator 影響代謝,致病力等,廣泛存在於革蘭陰性細菌中也調控參與壓力反應之RpoS 及sRNA Spf。將其剔除發現它不易在腎臟形成菌落。我們也發現Crp 受Hfq 正向調控。另一運動性減弱之突變株為qseF 基因遭破壞, QseF 乃QseEF 雙組成系統之response regulator,其剔除菌株MR/P pili 表現下降。在P. mirabilis qseEF 上游比對到glmY (轉錄GlmY),而reporter assay 證明QseEF 調控GlmY 之表現。E. coli glmY promoter 具RpoN 結合位, GlmY 保護GlmZ 免於被降解以活化GlmS (glucosamine-6-P synthase)表現,進行細胞壁及外膜合成。而QseEF 已被報導能感受宿主腎上腺素而調節致病力及代謝。此外RpoN 已被報導參與氮代謝,運動性,致病力及壓力反應, 也會調控sRNAs 例如和胺基酸代謝,致病相關之GcvB。我們也在P. mirabilis 發現尿道致病E. coli 中影響感染力的D-serine deaminase (DsdA)之調節蛋白質基因dsdC 類似物, 也發現其受RpoN 調控, 也證明GcvB 調控ureC (urease 次單元)及gdhA (尿道感染之因子glutamate dehydrogenase)表現。此外,已有報導合成表面移行訊號因子之GlnA (glutamine synthetase)同時受Crp 及RpoN 調控。為了進一步了解P. mirabilis 尿道感染之機制,擬進行三年計劃,目的為探討Hfq/sRNAs, Crp,QseEF 及RpoN 在尿道感染及藥物感受性的角色,其作用機制及調控網路並找尋可能之訊號因子。第一年: 探討Hfq, Crp, Spf 及RpoS 對P. mirabilis 致病力,藥物感受性,運動性及對抗壓力的影響並釐清P. mirabilis Crp 在腎臟形成菌落的角色。第二年: 探討QseEF, GlmY, GlmZ 及GlmS 相關表現型及機制,探討它們的關係並測試QseEF 是否能感知urea, glucosamine 及腎上腺素以啟動下游基因之表現,同時也研究QseEF 如何影響MR/P pili 的表現。第三年:探討RpoN, GcvB, DsdC 及DsdA 對運動性,藥物感受性,致病力的影響並釐清它們的關係。同時找尋RpoN 所調控之sRNAs 並分析Hfq 伴侶sRNAs 對藥物感受性的影響。本計畫將採用尿道細胞,巨噬細胞培養及小鼠尿道感染模型並測試尿液對相關基因表現之影響來探討相關問題。本研究能揭開P. mirabilis Hfq/sRNA, Crp, QseEF 及RpoN調控致病力,抗藥性,運動性,壓力反應及與宿主細胞交互作用之複雜網路,也能瞭解影響致病因子表現之訊號因子之作用機制。本研究將有利於P. mirabilis 治療藥物及減毒疫苗之研發。
Abstract: Proteus mirabilis is a common cause of urinary tract infection (UTI). The detailed underlyingmechanism to cause UTI by P. mirabilis still needs to be explored. The pleiotropic nature displayed by theP. mirabilis hfq mutant highlights the importance of the Hfq/sRNAs complex regulatory network inpathogenesis. We first identified Hfq-dependent sRNAs, Spf, CyaR, GlmY, GlmZ, and GcvB in P.mirabilis. Recently, we isolated two swarming-defective mutants found to have the Tn-5 inserted into crpand qseF, respectively. We found deletion of crp and qseF affects swarming and virulence and hfq mutanthad a lower crp mRNA level. Crp is a global regulator for bacteria to optimize their metabolism andenhance their fitness. E. coli can sense the epinephrine hormone by QseEF TCS to modulate virulence andthe epinephrine-mediated phenotype was associated with Hfq. Highly similar sRNAs GlmY and GlmZcontrol synthesis of GlmS, a key enzyme for synthesis of cell walls and LPS. We identified glmY, glmZand glmS homologues in P. mirabilis. We found an RpoN binding site exists in the glmY promoter regionand QseF regulates expression of GlmY and GlmZ in P. mirabilis. GcvB regulates expression of manygenes involved in amino acid metabolism and virulence. We disclosed GcvB overexpression increasedureC (urease subunit) and gdhA (glutamate dehydrogenase, a potential virulence factor) mRNA levels.Several lines of evidence support RpoN controls expression of sRNAs and genes associated with motilityand virulence. Seeing that the 5’-untranslated region of GlmY possesses QseF binding site and σN/σDoverlapping promoters both in E. coli and P. mirabilis and E. coli glnA (glutamine synthesizing glutaminesynthetase, a swarming signal) was regulated by Crp and σN, it indicates that σN together with QseEF andCrp may work in concert to modulate motility and virulence in P. mirabilis. For P. mirabilis to establish aUTI, multiple regulatory pathways of Hfq/sRNAs may synergize to induce gene sets at the appropriatetime and/or location. Therefore, a 3-year plan will be performed to investigate the roles of Hfq/sRNAs, Crp,QseEF, and RpoN in the uropathogenic P. mirabilis using in vitro and in vivo approaches. The specificaims of the plan are as follows, I. investigate the P. mirabilis Hfq-Crp regulatory circuit involving sRNAs(Spf, CyaR and others) and RpoS in motility, stress resistance, and virulence. II. investigate the P.mirabilis QseEF signaling pathway invoving GlmY, GlmZ and GlmS in motility, MR/P pili expression andvirulence. III. investigate the P. mirabilis RpoN-mediated phenotypes invoving GcvB and DsdC innitrogen utilization, motility, stress resistance and virulence. IV. investigate the role of Hfq-dependent sRNAsin drug susceptibility. This will be the first important study to unveil an Hfq/sRNAs complex regulatorynetwork involving catabolite repression protein Crp, QseEF TCS and alternative sigma factors RpoN/RpoS,which modulates carbon/nitrogen metabolism, motility, stress responses, virulence, drug susceptibility andother cellular functions. Such a study will provide clues for developing anti-virulence drugs and assessingthe mutant strains of reduced virulence as potential vaccine candidates for treating and preventing UTIscaused by P. mirabilis.