探討small RNA GlmY在奇異變形桿菌中所扮演的角色

Abstract

奇異變形桿菌(Proteus mirabilis)是革蘭氏陰性的兼性厭氧的桿菌，屬腸內菌科 (Enterobacteriaceae) 為健康人類腸道中的正常菌叢，在部分插尿導管的病患身上會造成伺機性感染，是尿道感染的重要病原菌，也會導致膀胱炎、腎結石，腎臟病等併發症。其中P. mirabilis的表面移行現象對該菌在宿主體內生存是非常重要的，可使細菌能於黏膜等固體表面移動，有利於入侵宿主細胞；協助細菌上行至泌尿道而引發感染，也與插尿管病人導尿管中的生物膜形成有關。在E. coli和某些腸內菌中發現small RNAs GlmY 和 GlmZ 會調控glmS (glucosamine-6-phosphate )的表現，影響合成細胞壁。另外GlmY 及 GlmZ會受到QseF及QseB調控，幫助細菌貼附並入侵腸道上皮細胞。本實驗室已建立qseEGF和qseF突變株，其表現型與運動性相關。近期研究發現E. coli和P. mirabilis的 glmY皆在qseEGF基因上游，因此本篇論文探討GlmY在P. mirabilis中所扮演的角色。我們建構glmY突變株(glmY knockout)，探討GlmY在P. mirabilis中所扮演的角色。我們比較∆qseF和野生株中GlmY表現量，證實 GlmY的確受到QseF的調控；並排除∆glmY影響qseEGF的可能性。觀察GlmY對於P. mirabilis的影響，發現∆glmY不會影響生長、抗藥性、通透性和生物膜的形成；而表面移行、泳動能力下降、鞭毛較少、細胞型態較短小和swarmer cell分化下降；利用pBAD33 plasmid 在野生株中過度表現GlmY，可發現表面移行、細胞長度與鞭毛都顯著的上升；而GlmY-complemented strain皆有補回。在基因調控方面，以生物資訊預測GlmY調控基因，發現與趨化因子相關的cheA。∆glmY中flhDC、cheA mRNA的表現量較野生株少；而rcsB mRNA則是上升。顯示GlmY可能間接影響flhDC與rcsB，直接調控cheA造成鞭毛合成、swarmer cell分化與運動性下降。目前我們正在進行找出GlmY在P. mirabilis中與cheA 的結合區域與探討GlmY與GlmZ之間的作用機制。目前GlmY尚未發現有相關功能；在P. mirabilis中，GlmY受到QseF的調控，進而影響cheA造成運動性相關的表現型。

Proteus mirabilis is a facultative anaerobic, Gram-negative bacterium and a member of the Enterobacteriaceae family. It is part of the normal flora of the human gastrointestinal tract, but an opportunistic pathogen that frequently causes complicated urinary tract infections in patients under long-term catheterization. P. mirabilis infections frequently develop into cystitis and pyelonephritis and can be further complicated by catheter encrustation and formation of urinary stones. The ability of P. mirabilis to invade host urothelial cells is closely coupled to swarming, a form of cyclical multicellular behavior in which vegetative bacteria differentiate into hyper flagellated. The flagellum of P. mirabilis is crucial to its motility, a characteristic that helps the organisms colonize. The flagellum has also been linked to the ability of P. mirabilis to form biofilms, aiding in the bacteria’s resistance to defenses of the host and select antibiotics. P. mirabilis also relies on its pili for adhesion to avoid being flushed out of the urinary tract system. The small RNAs (sRNAs) GlmY is present in Escherichia coli, Shigella flexneri, Yersinia pestis and Salmonella species, where it is found between the yfhk (qseE) and purL genes. The GlmY sRNA has been shown to activate the synthesis of glmS (glucosamine-6-phosphate, GlcN6P), which catalyses the rate limiting reaction in the biosynthesis of bacterial cell walls. GlmY's functions as an anti-adaptor, it binds to RapZ (RNase adaptor protein for sRNA GlmZ), this binding prevents RapZ from binding to GlmZ and targeting it for cleavage by RNase E. In enterohemorrhagic E. coli (EHEC) GlrR/GlrK (QseF/QseE) coordinately activate transcription of GlmY and GlmZ that post-transcriptionally further modulate and coordinate expression of the LEE and espFU, which is an EHEC-specific gene and encodes an effector protein translocated to the host cell. Our lab has qseEGF and qseF mutant, which relate to the ability of swarming in P. mirabilis. The sRNA between the qseE and purL genes in P. mirabilis has recently been identified as GlmY. Thus, we investigate the role of P. mirabilis sRNA GlmY in this study. We constructed glmY mutant by used homologous recombinant with kmr cassette and found the glmY mutant had reduced motility, fewer flagella and swarmer cell differentiation relative to wild-type and the GlmY-complemented strain. Then we ruled out the glmY deletion affect transcription of qseEGF by RT-PCR, to confirm that phenotypes are caused by glmY mutation. We also confirmed that transcription of GlmY and GlmZ is controlled by the two-component system QseF/QseE in P. mirabilis by glmY reporter assay and real-time PCR. We used bioinformatic tools to predict sRNA GlmY target, and found out the chemotaxis protein A (CheA) which had GlmY binding site and related to flagellar assembly. The real-time PCR data indicates GlmY likely modulates flhDC, rcsB and cheA mRNAs expression level and affects the production of flagellin, the subunit of flagella, and swarming differentiation in P. mirabilis. Alltogether, our data indicate that GlmY is important in P. mirabilis motility by modulating genes associated with swarming. This is the first report about the role of P. mirabilis GlmY.