奇異變型桿菌Hfq對於壓力抵抗能力、毒力因子表現及外膜蛋白之調控機制探討

Abstract

奇異變型桿菌(Proteus mirabilis)為兼性厭氧之革蘭氏陰性腸內菌，屬於健康人類腸道的正常菌叢，但在長期使用導尿管之病患身上容易造成伺機性感染，嚴重甚至可能導致腎臟病、肺炎等併發症。 Hfq是細菌體內的RNA chaperone protein，其功能主要是藉由協助sRNAs的作用來進行基因的轉錄後調控(post-transcriptional regulation)進而促進或抑制目標蛋白質的轉譯，Hfq序列在很多革蘭氏陰性菌中是非常保守的，在P. mirabilis也不例外，且其基因的排列都是miaA-hfq-hflX；目前越來越多的研究顯示Hfq在許多革蘭氏陰性的細菌體內扮演著重要角色，影響細菌的生理及致病力。 本篇論文利用knockout方式建構P. mirabilis hfq突變株，研究其表現型後發現hfq突變會使得P. mirabilis生長些微緩慢、泳動能力下降、表面移行能力上升、鞭毛蛋白表現量下降、生物膜生成能力下降、溶血酶活性降低並延後、細胞入侵能力(NTUB1 )降低、巨噬細胞(Raw)清除能力上升、外膜蛋白表現量及通透性上升、抗生素感受性上升、熱、高滲透壓、氧化壓力抵抗能力降低、大鼠皮膚感染能力降低等，綜合以上結果顯示Hfq在P. mirabilis中扮演重要角色，對其生理及致病因子表現均有影響。由於hfq突變株對PB感受性有十倍上升，利用LPS定量、PB結合能力測試、rppA reporter assay分析結果hfq突變株與野生株均無明顯差異，所以目前Hfq參與之PB藥物感受性改變機制還是未知的。 先前研究指出，Hfq可做為RNA chaperone protein穩定rpoS mRNA，並同時藉由DsrA, RprA, ArcZ,RyhA等小RNAs協助促進轉譯，本論文利用real-time PCR發現當hfq突變時rpoS mRNA表現量就會降低，顯示在P. mirabilis中Hfq可扮演穩定rpoS mRNA的角色，同時利用建構rpoS knockout突變株並分析其表現型後發現其細胞侵入能力降低、對氧化壓力及高滲透壓環境抵抗能力降低，顯示hfq突變株可能因為失去穩定及幫助rpoS mRNA轉譯的角色，使rpoS表現量降低而造成這些現象。 研究顯示細菌的Hfq會與小RNA協同作用在外膜上，使得外膜蛋白(OMP)表現量改變，進而產生外膜壓力(envelop stress)，促使RpoE活化；本論文利用先前實驗室學長建構之rpoE reporter plasmid測試野生株與hfq突變株的rpoE活性，發現在hfq突變株中rpoE活性明顯高於野生株，顯示在P. mirabilis中hfq突變同樣會產生外膜壓力而促使rpoE活化；進一步利用SDS-PAGE分析其外膜蛋白組成，並用real-time PCR驗證，發現hfq突變導致PMI0044、PMI0540、PMI1017、OmpA、OmpF大量上升，尤以PMI0540最為明顯，利用PMI0540過度表現株也證實其過度表現足以產生外膜壓力，使rpoE活性上升，並使其對SDS, Cm藥物感受性上升及高滲透壓環境抵抗能力、生物膜生成能力下降。PMI0540經比對後發現其胺基酸序列與YbfM (ChiP)具有高度相似性，同樣屬於OprD family，YbfM的功能在Salmonella及E. coli中已有相關研究，而參與YbfM調控的基因或蛋白包括Hfq, MicM, chbBC IGR，這三者在P. mirabilis中均有相對應之基因，而在PMI0540 5’UTR、chbBC IGR及MicM中也都具有相對應之結合位序列，本篇論文發現在P. mirabilis中三者均有參與PMI0540的調控，不管是在hfq突變株或MicM突變株或者是在野生株接受chitobiose刺激的情況下，PMI0540的蛋白量均會明顯上升，而chb操作組在接受chitobiose的刺激下也會大量表現，證實PMI0540應該就是P. mirabilis中YbfM的homologue，同時也發現此外膜蛋白會影響生物膜生成能力，當其大量表現時會使得P. mirabilis生物膜生成能力降低；而在SDS, Cm藥物感受性方面MicM突變株較野生株僅有兩倍之上升。 總結，Hfq在P. mirabilis中扮演重要角色；影響其生長、運動性、毒力因子、壓力抵抗能力、藥物感受性、致病力；同時在基因調控方面，Hfq可能藉由穩定rpoS mRNA進而影響到部分表現型，而對外膜蛋白部分，Hfq與MicM小RNA均可調控PMI0540表現，使其在野生株中維持在一個很低的量，而當環境中出現chitobiose時chb操作組可被活化，同時PMI0540會大量表現，以幫助細菌利用chitosugars。

Proteus mirabilis is a facultative anaerobic, Gram-negative bacterium and a member of the Enterobacteriaceae family. It is a normal flora in human intestine. It usually causes, however, urinary tract infection (UTI), and leads to kidney disease, pneumonia and septicemia in individuals with long-term catheterization or with structural or functional abnormalities in the urinary tract. Hfq is a bacterial RNA chaperone protein. Its regulation is mediated through small RNAs (sRNAs), which belong to post-transcriptional regulation. Hfq can help sRNA function, as to increase or decrease target proteins translation. The sequence of Hfq is highly conserved in several Gram negative pathogens, including Proteus mirabilis. In almost all of the Gram negative bacteria, hfq gene is located in a miaA-hfq-hflX cascade, and there’s no exception for P. mirabilis. Recently, ever-increasing studies of Hfq have been reported, focusing on its impact on bacterial physiology and virulence. To understand the role of Hfq in P. mirabilis, in our study we constructed an hfq mutant by knockout assay. In the following experiments we found that hfq mutation caused massive effects on P. mirabilis, leading to growth defect, changes in motility, decreased flagellin expression, impaired biofilm formation, delayed haemolysin cycle and decreased activity, decreased NTUB1 cell invasion ability, increased macrophage clearance, changes in outer membrane protein profile, increased outer membrane permeability, increased sensitivity to antibiotics, lowered heat, oxidative and hyperosmotic stress resistance, and attenuated rat burn wound skin infection ability. According to the aboved-mentaioned results we concluded that Hfq played a vital role in P. mirabilis, affecting its physiology and virulence. Since there’s a ten-fold increase in polymyxin B sensentivity in hfq mutant, we used LPS quantification, PB binding assay, and rppA reporter assay to study the underlying mechanisms involved. So far, none of the results have been found desirable to explain our findings. Thus, the effect of Hfq on polymyxin B sensentivity in P. mirabilis remained unclear. It has been proved that Hfq can function as an RNA chaperone protein to stabilize rpoS mRNA. Furthermore, it can up-regulate RpoS translation via DsrA, RprA, ArcZ, RyhA-sRNAs. In our study, using real-time PCR we found that rpoS mRNA was significantly decreased in hfq mutant. This indicated that Hfq has a similar effect on rpoS mRNA stability in P. mirabilis. Also, we constructed rpoS mutant and found that it showed some coomon phenotypes with the hfq mutant, including decreased NTUB1 cell invasion ability as well as lowered resistance to oxidative and hyperosmotic stresses. Studies have revealed that Hfq can act in concert with sRNAs to influence targets on bacterial outer membrane. When hfq is mutated, loss of these actions can change the outer membrane protein composition, and thus dirupt the outer membrane intergrity. Changes in outer membrane intergrity can then activate the rpoE-envelop stress sigma factor. In our data, we showed that hfq mutant possessed stronger rpoE activity than wild type, indicating that there’s a similar role of Hfq in P. mirabilis. Using SDS-PAGE outer membrane protein profiling we further iderntified five outer membrane proteins that accumulated in the hfq mutant. These are PMI0044, PMI0540, PMI1017, OmpA, and OmpF. Subsequent real-time PCR quantification revealed that PMI0540 is the most significantly increased one. After constructing the PMI0540 overexpression strain and assaying its phenotypes, we observed that rpoE reporter acitivity was increased. The result indicated that PMI0540 overexpression alone can activiate rpoE response. In other phenotypic assays we found that SDS and Cm sensitivity was increased and PMI0540 overexpression strain showed lowered hyperosmotic stress resistance and biofilm formation ability. After searching for PMI0540 homologue in other bacteria, we found that PMI0540 was closely related to YbfM. They both belong to OprD family outer membrane protein. The specific function of YbfM have been published in E. coli and Salmonella. There are three important genes involved in YbfM regulation, hfq, MicM, and chbBC intergenic region. In P. mirabilis, all of the three genes do exist and their functionally important binding sites are also conserved. In this study we found that similar roles of these regulators applied to P. mirabilis. When Proteus encountered chitobiose, PMI0540 was then induced and synthesized for transportation of these sugars. And chb operon, MicM, Hfq were all involved in this kind of regulation, which was called an mRNA-mediated antisense regulation of the antisense RNA. To sum up, we found that Hfq played an important role in P. mirabilis, affecting its growth, motility, stress resistance, antibiotics sensitivity, and virulence factors. We also observed that RpoS did attribute to some of the hfq mutant phenotypes, but not all. In the regulation of outer membrane proteins part we found that MicM and Hfq can repress PMI0540 translation unless the signal molecule, chitobiose, occurred.