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溶血素以及對抗cationic antimicrobial peptides (CAP)的能力等。CAP屬innate immunity一部份,P. mirabilis天生對CAP例如polymyxin B (PB)具高抗性。之前由transposon mutagenesis我們找到rppA及cpxR兩個對PB感受性增加的突變株。cpxR及rppA的產物皆為雙組成系統之responseregulator。CpxAR是一個能感知envelope壓力的雙組成系統而RppAB藉由控制下游galU、ugd、 pmrI等負責lipopolysaccharide (LPS)合成及修飾的基因使P. mirabilis能對抗PB。本實驗室也發現galU、 ugd或pmrI突變會影響表面結構及其他致病因子的表現,包括表面移行等,也會誘導維護envelope完整性之σE的表現。在E. coli中,σE已知和致病因子的表現有關而且會透過small RNA(sRNA)來調控雙組成系統以影響下游基因的表現。最近本實驗室又挑到另一個對PB感受性增加的hfq突變株。Hfq在其他細菌中是一種RNA結合蛋白質,參與在sRNA調控基因表現的過程中,是一種global regulator,影響致病力及壓力反應。本實驗室的資料顯示hfq突變株外膜組成發生改變,而對抗H2O2及形成biofilm的能力皆下降,此外對gentamycin (GM)的感受性也提高。另一方面已有研究證實LPS會誘導巨嗜細胞產生腎上腺素此類賀爾蒙促進發炎反應, Salmonella 會感受腎上腺素的存在來釋放溶血素而此過程Hfq、sRNA及CpxAR皆參與其中。而本實驗室最近也發現腎上腺素會影響P. mirabilis PB感受性及對抗H2O2的能力。此外我們發現RppA、CpxR以及σE也會影響細菌對抗H2O2的能力。根據以上之背景,本計劃之目的為:一. 探討Hfq在P.mirabilis扮演的角色,著重在致病力、抗藥性以及和宿主細胞之交互作用; 二. 找尋Hfq的標的sRNAs並探討影響Hfq 表現之因子; 三. 探討Hfq影響膜恆定,對抗H2O2、biofilm形成以及抗藥性之機制; 四.探討Hfq和sRNA、CpxAR、σE及RppAB之關係以了解其訊息傳遞; 五.探討腎上腺素對P. mirabilis 的影響包括致病因子表現及PB感受性; 六. 探討腎上腺素影響P. mirabilis 的機制以及Hfq、sRNA及CpxAR在其中拌演的角色。故未來三年之計畫為: 第一年,I. 探討Hfq在P. mirabilis扮演的角色包括藥物感受性及致病因子表現等;II. 進行細胞素分析實驗、測定TLR4及chemokine receptor的表現、研究巨嗜細胞之吞嗜及毒殺、進行老鼠實驗來探討Hfq的角色; III.表現Hfq蛋白質利用RNomics配合sRNA array找尋Hfq的標的sRNAs並以gel mobility shift assay確認Hfq-sRNA的結合作用;IV.以qPCR探討影響Hfq 表現的因子,包括滲透壓、溫度等;第二年,I. 以qPCR探討σE、σH及Hfq的關係,並以immunoblotting觀察rpoE及hfq突變對σS的影響,接著以qPCR測定H2O2及superoxide相關基因之表現以了解Hfq蛋白質對抗氧化壓力的原因; II. 以qPCR研究σE和sRNA的關係,並在rpoE及sRNA突變株中觀察外膜蛋白質的變化以了解Hfq影響外膜蛋白質的原因; III. 測定纖毛基因表現,探討LPS、extrapolysaccharide及外膜蛋白質影響biofilm形成的原因; IV. 探討LPS的改變及RppA和Hfq的關係以了解Hfq影響PB感受性的原因; V. 以flow cytometry測定reactive oxygen species的產生以了解Hfq影響GM感受性的原因; VI. 以qPCR研究rpoE、cpxAR、rppA、sRNA及hfq的關係。第三年,I. 以2D-massspectrometry比較σE、CpxAR、RppA及Hfq/sRNA 之調控蛋白質來瞭解它們的關係及Hfq之訊息傳遞; II. 探討腎上腺素對P. mirabilis 的影響,包括致病因子表現及PB感受性; III.探討腎上腺素影響P. mirabilis 的過程中Hfq/sRNA及CpxAR扮演的角色並找尋其他腎上腺素作用的標的。本研究能揭開Hfq/sRNA 在 P. mirabilis 中調控致病因子表現、抗藥性及與宿主細胞交互作用之複雜網路,有利於hfq突變株減毒疫苗之研發,並瞭解Hfq/sRNA和RppAB、CpxAR及σE之關係。另一方面,可瞭解宿主腎上腺素如何和P.mirabilis作用以影響致病因子表現及抗藥性。
Abstract: Previously, we identified a P. mirabilis two-component response regulator gene “rppA” that isinvolved in virulence expression and polymyxin B(PB) susceptibility through regulating itsdownstream genes, ugd, galU and pmrI, which are responsible for synthesis and/or modification oflipopolysaccharide (LPS) in E. coli. The knockout mutants of rppA, ugd, galU and pmrI exhibitincreased PB susceptibility and altered virulence expression. Furthermore, we found mutation of P.mirabilis ugd or galU induces expression of σE and results in inhibition of hemolysin expression.Recently, σE has been shown to be required for resistance to PB, oxidative stress and virulence ofSalmonella. Besides protein misfolding, changes in LPS have been shown to induce the σEexpressionin E. coli. In the other way, several outer membrane protein (OMP)-regulating small RNAs (sRNAs)are under the control of σE and E. coli phoPQ is repressed by σE in an sRNA-dependent manner.Moreover, an sRNA regulated by PhoPQ fine-tunes σE-mediated LPS decoration and hence PBsusceptibility in E. coli. Both σE and CpxAR pathways are required to fully cope with envelopestresses. Therefore, it is likely that a complex network of PB susceptibility and virulence generegulation involving CpxAR, RppAB, sRNAs and σE exists in P. mirabilis. Recently, in searching PBsusceptible Tn5-mutagenized mutants of P. mirabilis, we found hfq mutant is more susceptible to PBand gentamicin (GM). Hfq has been shown to be required for the fitness and virulence of an increasingnumber of bacterial pathogens. We have successfully gotten hfq knockout mutant and found OMPprofile, biofilm formation and H2O2 susceptibiliy were altered in hfq mutant. Oxidative stress has beenimplicated as one of the mechanisms whereby GM kill bacteria and signaling through two-componentsystems is a key player in this process. Remarkably, bacterial LPS has been shown to induceproduction of epinephrine hormone by macrophages. Bacteria communicate by producing autoinducermolecules and sensing their concentration. In E. coli, autoinducer 3 functions synergistically withepinephrine to regulate motility and virulence. It has been shown that exposure of Salmonella Typhi toepinephrine results in haemolysis. This effect is attributed to increased expression of the sRNAmicA and Hfq. Deletion of CpxAR abolishes the phenotype. Our data demonstrated epinephrinemakes P. mirabilis more susceptible to PB and H2O2. Based on the above, the specific ends and thestrategies will be used are listed below. Aim I. investigate the roles of Hfq. We will analyze virulencefactor expression in wild-type and hfq mutant; analyze the cytokine production, expression of TLR4and chemokine receptor and macrophage function; use mouse model to confirm the Hfq roles. Aim II.find out target sRNAs of Hfq and identify factors affecting Hfq expression. We will performRNomics or sRNA array to find sRNA targets of Hfq and perform quantitative PCR(qPCR) to findfactors affecting hfq expression. Aim III. disclose the mechanisms for roles of Hfq. i. perform qPCRto test if σE affects σH and subsequent hfq transcription, test if σE and Hfq affect the level of σS byimmunoblotting and compare expression of H2O2 and superoxide responsive genes to know how hfqaffect H2O2 resistance. ii.use qPCR, reporter assay and SDS-PAGE to confirm the σEsRNAOMPsregulatory pathway. iii.investigate expression of the fimbriae gene by qPCR, measure amount of EPSand LPS, analyze LPS profiles and overexpress the OMP to see if they are involved in biofilmformation. iv. analyze LPS profiles by SDS-PAGE, perform PB binding assay and disclose the relationbetween hfq and rppA to know how hfq affect PB resistance. v. analyze ROS production by flowcytometry to know how hfq affect GM resistance. Aim IV. investigate the relationship amongHfq/sRNA, CpxAR, σE, and RppAB by qPCR and 2D-mass spectrometry analysis. Aim V.investigate the effects of epinephrine on P. mirabilis virulence and PB susceptibility. We willinvestigate the effects of epinephrine on phenotypic traits such as virulence expression in wild-type inthe presence of epinephrine. Aim VI. investigate the signaling pathway of epinephrine-sensing inP.mirabilis and disclose the roles of Hfq, sRNA and CpxAR in this process. i. analyze phenotypesmediated by epinephrine using wild-type, cpxAR and hfq mutants. ii. construct P. mirabilis qseBC,qseEF and basSR mutants and examine the epinephrine-related phenotypes. This will be the firstimportant study of Hfq/sRNA about virulence expression, drug susceptibility and bacterium-host cellinteraction in P. mirabilis and their relationship with CpxAR, RppAB, and σE. This will also be thefirst study about P. mirabilis-host endocrine interaction. Such a study will provide clues about thepotential of hfq mutant as a vaccine candidate for preventing urinary tract infections caused by P.mirabilis.