探討σ因子 RpoN的 兩個增強子結合蛋白FhlA以及QseF在奇異變形桿菌中所扮演的角色

The roles of two enhancer binding proteins FhlA and QseF of sigma factor RpoN in uropathogenic Proteus mirabilis

林文淵(Wen-Yuan Lin)

指導教授 ： 廖淑貞

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/博士(2021年)

https://doi.org/10.6342/NTU202104245

摘要

奇異變型桿菌 (Proteus mirabilis)是主要造成泌尿道感染 (urinary tract infection, UTI)的病原菌之一，在長期使用尿導管的患者，容易造成伺機性感染。細菌為了適應環境，必須快速改變基因組表現， alternative sigma factors的功能便是因應外來環境變化，將轉錄組重新定向 (redirection)。RpoN為alternative sigma factors的一員，常與移動性、生物膜和尿素酶等細菌毒性因子相關，需要特定的enhancer binding proteins (EBPs)進行活化後，始能啟動轉錄。透過BLAST分析P. mirabilis N2 genome找出4個可能EBPs FhlA, QseF, PspF與NtrC，並挑選fhlA與qseF突變株分析重要毒性因子如尿素酶活性、移動性以及生物膜形成能力等。在尿素酶活性的部分，rpoN與fhlA突變株尿素酶活性顯著低於野生株；移動性的部分，rpoN及qseF突變株表面移行能力較野生株差；而生物膜形成能力上相較於野生株，qseF突變株則顯著低於野生株，而rpoN突變株則沒有明顯差異。
FhlA為formate hydrogenlyase (FHL)activator，擁有GAF domain來感受外界因子，可以活化的下游基因fdhF及hyf operon表現並形成FHL complex，FHL complex可以將生長環境去酸化。我們發現P. mirabilis N2尿素酶活性隨著pH值增加而增加，在pH9的時候達到最佳活性，因此我們假設FHL會協同尿素酶將環境去酸化，而使得尿素酶活性增加，實驗顯示隨著環境pH值增加到pH 9，hyfG及fhlA失去調控尿素酶活性的能力，而將野生株及hyfG突變株培養在pH 5人工尿中，發現hyf突變株去酸化的速度顯著低於野生株。我們利用人工尿模擬尿液環境，測試尿結石形成能力，可以看到hyfG及fhlA突變株尿結石形成能力顯著低於野生株。利用ICR母鼠泌尿道感染模型進行試驗，不論在膀胱或腎臟hyfG及fhlA突變株定殖能力皆顯著下降。
QseF為雙組成系統QseEF的response regulator，我們進行小鼠泌尿道感染試驗，發現qseF突變株定殖能力不論在膀胱或腎臟皆顯著低於野生株。
QseF正向調控glmY (sRNA)、flhDC (鞭毛調控轉錄因子)、cheA (化學趨化蛋白)以及負向調控rcsB (轉錄因子)等基因表現，其中QseF正向調控glmY基因轉錄，而GlmY可以直接與cheA mRNA作用，調控cheA的轉譯，進一步影響細菌移動性。
我們看到QseF正向調控glmZ及rpoS表現，而先前實驗室發現GlmZ透過正向調控rpoS表現可以幫助細菌抵抗酸性與氧化壓力環境，增加在巨噬細胞存活率，此外相對於野生株，qseEGF以及glmZ突變株對膀胱細胞侵入能力顯著下降。在本文我們也發現溶血素基因hpmAB表現量在qseF突變株中降低，同時qseF突變株對於膀胱細胞毒性也顯著低於野生株。
除了移動性、壓力抗性以及細胞毒性之外，QseF同時調控了生物膜的形成，利用螢光顯微鏡可以觀察到24小時qseF突變株生物膜呈網狀分布，而野生株則能聚集發展。我們將時間細分為2小時一個單位，監測生物膜形成的量，可以發現在8小時之前(含)，qseF突變株生物膜形成量大於野生株，而10小時開始qseF突變株生物膜量維持不變而野生株持續增加，以至於24小時顯著低於野生株。RNA-seq資料及qPCR確認，可以看到zapABCD蛋白酶相關基因在qseF突變株中較野生株下降，並利用qPCR發現QseF在24h時正向調控sRNA RyhB3、RprA以及alternative sigma factor RpoE，先前文獻顯示P. mirabilis生物膜相關纖毛MR/P受RpoE調控，在qPCR中發現RyhB3正向調控zapA, rpoE以及mrpA；RprA則正向調控mrpA。我們測試生物膜相關因子發現qseF突變株自聚集及細胞貼附能力較野生株上升，而僅有蛋白酶活性下降，因此我們認為蛋白酶為生物膜發展所必需，影響生物膜繼續發展，而受RpoE調控的MR/P纖毛在排除自聚集以及細胞貼附能力之外，仍然為生物膜生成之重要因子。
本研究我們發現了RpoN相關EBPs FhlA及QseF能調控移動性、尿素酶活性、生物膜形成能力等重要致病因子，我們推論一、在開始形成extracellular cluster時RpoN-FhlA調控FHL表現並與尿素酶協同作用，加速環境去酸化使礦物質結晶沉積，進一步形成感染性尿結石及生物膜。二、在細菌懸浮時QseF受尿素及油酸(oleic acid)抑制，使得纖毛表現上升，鞭毛表現下降，一方面增加細菌貼附細胞能力，另一方面減少鞭毛抗原躲避宿主免疫反應；三、在遇到固態表面時，油酸則從負向調控轉為正向調控QseF，QseF正向調控細菌移動性，使P. mirabilis可以上行感染、促進細菌侵入宿主細胞，躲避免疫反應、增加細胞毒性，以獲取更多養分、增加細菌壓力抗性，以提高被巨噬細胞吞噬後的存活率。綜合上述，P. mirabilis利用FhlA及QseF進行整體轉錄組的調控，使細菌可以迅速改變其轉錄組，以利在環境多變的泌尿道感染中生存。

關鍵字

尿道致病性奇異變形桿菌 ； FhlA ； HyfG ； 尿素酶活性 ； QseF ； RpoN ； 毒力因子

並列摘要

Introduction: Proteus mirabilis is one of the leading causes of urinary tract infections, especially in patients with indwelling catheters. Since catheter-associated urinary tract infection is a major health concern due to the complications and recurrence, researches directed at understanding the pathogenesis are warranted. P. mirabilis pathogenesis is closely coupled to urease, swarming and biofilm. Many studies showed that alternative sigma factor RpoN contributed to urease activity, swarming motility and biofilm formation. Previously, we found the RpoN and its cognate enhancer binding protein (EBP) QseF contributed to swarming in P. mirabilis. Next, we found another three EBPs FhlA, PspF and NtrC in P. mirabilis N2. Specific aims: we investigated the roles of two EBPs FhlA and QseF in uropathogenic Proteus mirabilis. Results: we constructed rpoN, fhlA and qseF mutant and did the urease, swarming and biofilm assay. Urease activity decreased in rpoN or fhlA mutant; swarming motility decreased in rpoN or qseF mutant; biofilm formation ability decreased in qseF mutant only. Formate hydrogenlyase (FHL), consisting of formate dehydrogenase H and hydrogenase for converting proton to hydrogen, has been reported to regulate by FhlA. We constructed hyfG (encoding hydrogenase large subunit) mutant and fhlAcSD (FhlA with mutation in the conserved RpoN-interacting motif). The acid resistance, urease activity, medium deacidification and urinary stone formation decreased in fhlA, hyfG and fhlAcSD. In addition, the loss of fhlA or hyfG exhibited attenuated colonization in mice. For QseF, we found the loss of qseF decreased mouse colonization, acid resistance, oxidative stress resistance and cytotoxicity. qPCR data revealed rpoS expression decreased in qseF mutant. Besides, qseF gene locating in glmYqseEGF operon. We found QseF positively regulated glmY expression and the loss of glmY or qseF exhibited reduced swarming motility, swimming motility, cell length, hemolysin activity and flagellin production. We found GlmY and QseF regulated flhDC (encoding a flagellum master regulator) and cheA (encoding a central regulator of chemotaxis) positively and rcsB (encoding a negative transcription regulator of swarming) negatively by reporter assay and qPCR. Furthermore, site-direct mutagenesis analysis confirmed the direct interaction between sRNA GlmY and cheA mRNA to improve cheA translation. Finally, we investigated how QseF regulated biofilm formation. By fluorescence observation of biofilm-forming during a 24 h-period, we found a better ability of qseF mutant to form biofilm at the early phase. The biofilm of qseF mutant after 10 h did not obviously grow, different from the biofilm formation in the wild-type strain which had a steady increase of biofilm production during the 24-h period. We denoted the biofilm formation into two phases: initial adhesion (4h) and mature biofilm (24h). Expression of biofilm-related genes in wt and qseF mutant by qPCR indicated QseF negatively regulated fimbrial genes (mrpA, pmfA, cfaB) and sRNA (rprA and ryhB3) and positively regulated protease genes (zapA, zapD) at initial adhesion stage, while QseF positively regulated protease genes (zapA, zapD), mrpA and sRNA (rprA and ryhB3) at mature biofilm stage. zapA or zapD mutation only decreased biofilm formation at mature biofilm stage. In addition, overexpression of rprA of ryhB3 increased biofilm formation at both stage. Conclusion: in this study, we found expression of fhlA and hyf confers medium deacidification together with urease activity, thereby facilitating urinary stone formation and mouse colonization. QseF modulated stress resistance, cytotoxicity, motility, biofilm formation and mouse colonization. These regulation network may provide a perspective to conquer urinary tract infections by P. mirabilis.

並列關鍵字

uropathogenic Proteus mirabilis ； FhlA ； HyfG ； urease activity ； QseF ； RpoN ； virulence factor

參考文獻

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