RpoE在伤寒沙门菌克服环境高渗应激中的基因表达调节机制研究
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摘要
伤寒沙门菌(Salmonella enterica serovar Typhi, S. Typhi)是一种重要的人类肠道致病菌,是目前研究最为广泛和深入的原核生物之一。伤寒沙门菌常通过污染食物经消化道入体内,在空肠远端入侵肠上皮细胞,并进一步在局部肠系膜淋巴组织中存活和增殖,再经淋巴液和血液进入肝、脾等全身组织,造成全身性感染并能引发肠穿孔等严重的并发症从而危及患者生命。
作为人类食源性致病菌,伤寒沙门菌在从环境到使宿主致病的过程中,需要遇到一系列剧烈的环境应激,包括人的肠道高渗应激环境。在高渗应激条件下,细菌需要及时有效地调节某些基因的表达和蛋白的活性来适应环境。原核生物基因表达均是由sigma因子介导转录起始,且多数呈操纵子模式,并受各种调节因子的影响。不同的sigma因子(σ因子)特异性识别并引导RNA聚合酶结合到目的基因启动子区域,启动相应基因表达,无疑是原核生物对其生命活动进行调控的最基本的方式。已知不同的sigma因子的作用不同,不同的sigma因子对某些基因或有共调节作用,但缺乏直接的研究证据。RpoE是响应环境应激的sigma因子。已有研究发现RpoE是肠杆菌科细菌中一个重要的sigma因子,在响应多种环境应激时发挥着重要作用,对细菌生存和致病性至关重要,但其在伤寒沙门菌中作用的研究报道尚少。
目的：
本研究旨在了解与RpoE相关的参与伤寒沙门菌克服高渗应激的效应蛋白基因,深入分析RpoE对重要致病因子表达调控的机制,研究RpoE在伤寒沙门菌克服高渗应激过程中与另一sigma因子RpoS对基因表达的共调节作用,以加深对伤寒沙门菌基因表达调控网络和致病分子机制的认识。
方法：
(1)基因缺陷变异株的制备采用自杀质粒pGMB151介导的同源重组方法,制备伤寒沙门菌rpoS缺陷变异株及rpoE/rpoS双缺陷变异株。
(2) rpoE基因缺陷回补株的构建PCR扩增rpoE基因,酶切后与表达载体pBAD/gⅢ定向连接,热击法转入大肠杆菌DH5a中筛选阳性质粒,经序列分析鉴定后,电击法转入rpoE基因缺陷变异株。
(3)高渗应激下生长情况分析以生长时间为横坐标,细菌OD600值为纵坐标绘制生长曲线,对比缺陷变异株、野生株及基因缺陷回补株在高渗应激条件下的生长情况。
(4)动力试验分析缺陷变异株和野生株37℃培养过夜,分别取4μ1菌液接种于0.3%高渗半固体LB培养基,37℃培养8小时后测量动力圈直径,观察缺陷株及野生株的动力。
(5)伤寒沙门菌基因表达谱分析伤寒沙门菌全基因组芯片分析目的基因在转录水平上对其他基因的表达调控。体外模拟高渗环境应激后分别提取伤寒沙门菌野生株和基因缺陷变异株的总RNA,反转录成cDNA并标记荧光(cy3或cy5),与基因组芯片杂交,扫描后根据荧光信号分析各基因转录表达水平,比较伤寒沙门菌野生株和基因缺陷变异株在高渗应激早期的基因表达谱差异。
(6)实时荧光定量PCR(qRT-PCR)分析选择基因芯片结果中部分差异表达基因,设计特异性引物,进行反转录和实时定量PCR,以验证DNA芯片分析结果。
(7)蛋白质二维电泳和质谱分析高渗应激后分别提取rpoE缺陷变异株和野生株的菌体蛋白和分泌蛋白。取适量蛋白进行等点聚焦和SDS-PAGE电泳分离后,以考马斯亮蓝G-250染色后扫描成像,分析蛋白表达差异,选取明显表达差异蛋白进行质谱分析；质谱分析的肽指纹图在www.mascot进行比对。
(8) Western免疫印迹分析分泌蛋白高渗应激条件培养rpoE缺陷株、野生株,利用三氯醋酸-丙酮法提取培养上清中的分泌蛋白,以抗H：z66多克隆抗体进行Western免疫印迹分析rpoE缺陷株、野生株分泌Z66蛋白的差异。
(9)凝胶阻滞试验根据伤寒沙门菌rpoE基因序列设计引物,扩增rpoE基因片段,与表达载体pET-28a连接经热击法转化入大肠杆菌JM109,用IPTG诱导表达RpoE蛋白,用镍柱纯化RpoE蛋白。根据伤寒沙门菌fliA基因序列设计特异性引物,扩增fliA基因启动子区域,取2μg PCR产物与不同浓度的RpoE蛋白混合,加入相应体积的GSM缓冲液,反应总体积为20μ1,30℃孵育15 min。选取真核生物DNA片段作为阴性对照,8%的丙烯酰胺胶电泳分离条带,溴化乙锭染色。
(10) HeLa细胞侵袭试验细菌培养至OD600为1.0,加入培养有HeLa细胞的24孔板中(MOI=20),90min后收集一部分细胞,加入裂解液,涂LB平板过夜培养,计细菌克隆数,代表细菌粘附细胞水平T0；另一部分加入庆大霉素杀死胞外细菌,继续培养90 min,裂解细胞后涂板过夜培养,计克隆数代表细菌的侵袭水平T90,用T90/T0的值代表细菌的侵袭力。
结果：
(1)研究用菌株制备经PCR及序列分析证实成功构建伤寒沙门菌rpoS基因缺陷变异株、rpoE/rpoS双缺陷变异株；成功构建pBAD-rpoE阳性重组载体,并成功将重组载体导入相应的缺陷株中,制备成rpoE基因缺陷回补株。
(2)生长曲线分析结果显示rpoE基因缺陷变异株在高渗应激条件下生长能力明显弱于野生株,在回补rpoE基因后得到明显恢复；rpoS基因缺陷株在高渗应激条件下生长能力亦明显弱于野生株,rpoE/rpoS双缺陷变异株在高渗应激条件下生长能力均明显弱于野生株和rpoE、rpoS单基因缺陷株。
(3)高渗应激下RpoE影响基因表达分析基因组芯片分析显示,高渗应激30分钟后,rpoE基因缺陷株相比野生株有74个基因表达下调,56个基因表达上调,选取其中部份差异表达基因利用RT-PCR进行验证,结果显示所选基因表达变化与基因芯片分析结果基本一致,且在回补rpoE基因后表达得到明显恢复。
(4)高渗应激下RpoE对蛋白表达影响的分析二维电泳、质谱分析显示,rpoE缺陷株与野生株相比18个菌体蛋白明显有差异,质谱分析鉴定包括有氨酰组氨酸二肽酶PepD、氨酰组氨酸二肽酶AspA等；分泌蛋白二维电泳后发现rpoE缺陷株与野生株相比有16个明显差异点,质谱分析鉴定包括NAD合成酶NadE,外膜蛋白OmpC等。
(5) RpoE对鞭毛及动力影响机制研究动力试验结果显示,在高渗半固体LB培养平板上,rpoE基因缺陷变异株动力比野生株明显下降,回补rpoE基因后动力明显恢复,提示RpoE在高渗条件下参与调控鞭毛动力；结合高渗应激早期基因芯片发现rpoE缺陷变异株中二级鞭毛基因fliA和三级鞭毛基因表达明显下调,利用qRT-PCR分析一级鞭毛基因flhD、二级鞭毛基因fliA和三级鞭毛基因fljB:z66的表达情况发现,rpoE缺陷变异株中flhD相比野生株没有明显差异,而fliA与fljB:z66的表达在rpoE缺陷变异株中明显下调；Western-bolt发现鞭毛蛋白FljB:z66在rpoE缺陷变异株的表达明显低于野生株；凝胶阻滞试验证实RpoE蛋白可以直接结合fliA基因启动子。
(6) RpoE对伤寒沙门菌侵袭力影响分析rpoE基因缺陷变异株的侵袭力比野生株明显下降,而rpoE回补株侵袭能力明显恢复；结合基因芯片结果,挑选部分侵袭毒力相关基因进行qRT-PCR,发现这些基因在rpoE缺陷株中表达相对野生株明显下调,在回补rpoE基因后表达明显恢复。
(7)高渗应激下RpoE和RpoS对基因表达的共调节分析利用基因组芯片,进一步分析rpoE基因缺陷株表达谱、rpoS基因缺陷株表达谱和rpoE/rpoS双缺陷株表达谱,发现有38个基因,包括osmC、osmY、otsBA、narUZY、dpS等,在rpoE、rpoS缺陷株中相比野生株表达变化不明显,但在rpoE/rpoS双缺陷株表达明显下调,提示这些基因受RpoE与RpoS的双重调控；选取不同操纵子的6个基因CosmC, osmY, otsB, narU, ugpB和dps)进行qRT-PCR进行验证,结果显示,所选基因表达与基因芯片分析结果一致。
结论：
(1) RpoE在伤寒沙门菌高渗应激时参与fliA、flgK、cheW、glpX、ompC、phoP等致病因子基因的表达调控。
(2)高渗应激下伤寒沙门菌RpoE能直接启动FliA表达,进而调控鞭毛基因表达和影响细菌动力。
(3) RpoE能促进orgA、prgK、sipA、invF、sopE、spaS等侵袭毒力基因表达,进而帮助伤寒沙门菌侵袭上皮细胞。
(4)高渗应激早期,RpoE因子与RpoS因子对osmC、osmY、otsBA、narUZY、dpS等38个基因存在共调控作用。
Salmonella enterica serovar Typhi (S. Typhi) is an important human intestinal bacteria and one of the most extensive and in-depth investigation of Prokaryotes. S. Typhi often enters the human digestive tract by contaminated food and invades into intestinal epithelial cells of the distal jejunum. After numerously proliferation, the bacteria of S. Typhi survive in the local mesenteric lymph tissue and shift to the liver, spleen and other tissues through the lymph and blood system. The systemic infection of S. Typhi leads to serious complications such as intestinal perforation and even death.
As a kind of human food-borne pathogen, S. Typhi needs a series of self-regulation to overcome some different kinds of severe environmental stresses during infection, including intestinal hyperosmotic stress. In the hyperosmotic condition,S. Typhi should promptly regulate the expression of many genes to adapt the new environment. Gene transcription in Prokaryotes is initiated by sigma factors (σfactor) in a manner of operon mode and controlled by various regulatory factors, including activators and inhibitors. Sigma factors specifically recognize the promoter region of target genes and helps RNA polymerase to assemble and promote the expression of relative genes, which is the most basic and important way for Prokaryotes to perform life activities undoubtedly. Different Sigma factors are responsible for different activity. Some researchers guessed that different sigma factors could co-regulate some gene expression, but there is no direct evidence yet. There was reported that RpoE was one of the important sigma factors in Enterobacteriaceae to adapt to some severe conditions. However, the role of RpoE involved in the hyperosmotic stress has been rarely reported in S. Typhi.
Objective:
The study will demonstrate the response genes of S. Typhi regulated by RpoE at the hyperosmotic condition, illustrate the mechanism of the expression of some important pathogenic factor genes regulated by RpoE, and investigate the co-regulation of gene expression by RpoE with RpoS, another important sigma factor of S. Typhi in the hyperosmotic condition. This study may be helpful to understand the regulatory networks of gene expression and the molecular mechanism of pathogenesis of S. Typhi.
Method:
(1) Preparation of gene mutant strains. The rpoS mutant and rpoE / rpoS double mutant S. Typhi was constructed by homologous recombinant with suicide plasmid pGMB151.
(2) Construction of the rpoE rescue strain. The rpoE gene was amplified by PCR and connected to the expression vector pBAD/gⅢ. The recombinant plasmid was transferred into Escherichia coli DH5a by heat shock. The positive plasmids were screened by sequence analysis and then transferred into rpoE mutant strain by electroporation.
(3) Analysis of bacterial growth under hyperosmotic conditions. To compare the survival ability of wild-type strain and gene mutant strain under the hyperosmotic stress conditions, growth curves were drawn by using growth time as the abscissa and value of OD60o as the ordinate.
(4) Motility assay. The wild-type strain and gene mutant strain were cultured overnight at 37℃without agitation on LB broth. Each 4μl of culture was inoculated into the centre of a 0.3% LB agar plate, which was containing 300 mM of NaCl. The plates were incubated at 37℃for 8 h and motility was assessed qualitatively by examining the diameter of circular swimming which was formed by the growing motile bacterial cells.
(5) Gene expression profile analysis of S. Typhi. The genome-wide gene expression at transcriptional level was analyzed by genomic microarray assay. After culturing in hyperosmotic conditions, the total RNAs from wild-type strain and mutant strain was extracted. cDNAs would be obtained by reverse transcription and labeled with fluorescent (cy3 or cy5). Cy3-cDNAs or Cy5-cDNAs hybridized with genomic microarray were captured by scanning analysis of fluorescent intensity. The gene expression difference between wild-type strain and mutant strain was reflected by fluorescent intensity.
(6) Quantitative real time PCR (qRT-PCR) assay. Primers specific to some selected genes were designed and used for qRT-PCR to validate the results of microarray assay.
(7) Protein two-dimensional electrophoresis and mass spectrometry assay. After culturing in hyperosmotic stress conditions, the bacterial proteins and secreted proteins were extracted from the rpoE mutant strain and the wild-type strain. The proteins were separated by isoelectrofocusing and SDS-PAGE electrophoresis. After staining with Coomassie Brilliant Blue G-250, the expressional levels of proteins were showed. Mass spectrometry was used to verify the selected proteins and the existing peptide fingerprinting was blasted with data at www. mascot.
(8) Western-blot. After culturing in hyperosmotic conditions, the secreted proteins from rpoE mutant strain and the wild-type strain were extracted by trichloroacetic acid-acetone. The different levels of secreted protein expression between rpoE mutant strain and the wild-type strain were analyzed by Western-blot with rabbit anti-H:z66 antiserum.
(9) Gel-shifting assay. One pair of primers was designed to amplify DNA fragments of the rpoE gene of S. Typhi, which was then inserted into the expression plasmid pET-28a. Escherichia coli JM109 were then transformed by the expression plasmid by electroporation. Expression of the RpoE protein was induced by IPTG. RpoE protein was purified with Ni-colum. Two pairs of primers were designed to amplify the promoter region of fliA of S. Typhi. Each 2μg amplicon and different amount of RpoE were mixed with GSM solution in final 20μl, incubated at 30 for 15℃min before electrophoresis. The PCR product from the eukaryotic cell was used as the control. The electrophoresis in 8% acrylamide gel was performed for separating DNA fragments. Gel was stained with Ethidium Bromide.
(10) Invasion ability assay with Hela cells. The bacteria were cultured until the OD600 was approximately 1.0, and then added to 24-well culture plate which was planted with Hela cells (MOI=20). After incubating for 90 min, a portion of Hela cells was lysed and then incubated on LB plate for calculating bacterial clones. The clones represented the ability of bacterial adhesion (To). The remaining cells were incubated for another 90 min, treating with gentamicin to kill extra cellular bacteria. The cells were then lysed and then incubated on LB plate for calculating bacterial clones. The clones represented the ability of bacterial invasion (T90). The ratio of T9o/To was used to evaluate the invasion ability of bacteria.
Results:
(1) Preparation of bacterial strains. The rpoS mutant strain and rpoE/rpoS double-defective mutant strain were constructed successfully, which was verified by PCR and DNA sequencing. The recombinant pBAD-rpoE plasmids were constructed and then transferred into gene mutant strain to generate the rpoE rescue strain.
(2) Analysis of the growth curve. The results showed that the survival ability of rpoE mutant strain was significantly weaker than the wild-type strain under hyperosmotic conditions. However, the survival ability was obviously restored in the rpoE rescue strain. The rpoS mutant strain also grew more slowly than the wild-type strain in the hyperosmotic condition. Moreover, the growth rate of the double rpoE/rpoS mutant strain was significantly reduced compared to rpoE mutant and rpoS mutant strain.
(3) Analysis of gene expression regulation by RpoE under hyperosmotic condition. Microarray analysis showed that 74 genes were down-regulated and 56 genes were up-regulated in the rpoE mutant strains after culturing in hypertonic conditions for 30 min. The results of some selected genes were verified by qRT-PCR. The expression of genes was significantly restored in rpoE rescue strain.
(4) Analysis of the expression of proteins affected by RpoE under hyperosmotic conditions. Two-dimensional electrophoresis revealed that eighteen bacterial proteins were expressed differently between rpoE mutant strain and wild-type strain. Among them aminoacyl-histidine, dipeptidase PepD and aminoacyl-histidine acid peptidase AspA were confirmed by mass spectrometry. Sixteen different points of secreted protein between rpoE mutant and wild-type strains were found by two-dimensional electrophoresis analysis. Among these secreted protein points, the NAD synthetase NadE and the outer membrane proteins OmpC were verified by mass spectrometry.
(5) Mechanism of flagellar and motility regulation by RpoE. The motility of rpoE mutant strain was apparently decreased compared to that of the wild-type strain in semi-solid LB medium hypertosmotic plate. On the contrary, the motility of the rpoE rescue strain was similar to that of the wild-type strain. It indicated RpoE participated in the regulation of flagella genes under hypertosmotic conditions. Microarray analysis showed expression of classⅡflagellar gene fliA and most classⅢflagellar gene were significantly reduced in rpoE mutant strains. qRT-PCR was used to analysis expression of classⅠflagellar gene flhD, classⅡflagellar gene fliA and classⅢflagellar gene fljB:z66. The results showed that the flhD expression was not significantly changed in rpoE mutant stain compared to the wild-type strain, whereas the fliA and fljB:z66 expressions were significantly reduced in rpoE mutant strain. Western-bolt showed that flagellin protein FljB:z66 was significantly reduced in rpoE mutant stain compared to the wild-type strain. The result of gel-shifting experiments showed that RpoE could bind the promoter region of fliA.
(6) Analysis of invasion ability affected by RpoE. The ability of invasion was significantly decreased in rpoE deleted mutant strain compared to that of the wild-type strain. Correspondingly, the invasion ability was rescued in the rpoE complementary strain. Considering the results from microarray, some invasion or virulence genes were detected by qRT-PCR. The results showed that those gene expressions were significantly reduced in the rpoE mutant stain compared to wild-type strain. However, those gene expressions would restore when rpoE gene was compensated.
(7) Analysis of co-regulating gene expression by RpoE and RpoS under hyperosmotic condition. Results of DNA microarray analysis showed that 38 gene expressions including osmC, osmY, otsB, narU, ugpB and dps were markedly reduced in the rpoE/rpoS double mutant strain after exposure in hyperosmotic conditions for 30 min. Those gene expressions were only weakly affected by either the rpoE or rpoS single mutation. The data suggested that those genes were co-regulated by RpoE and RpoS. qRT-PCR was also used to analyzed the expression levels of six genes from different operons (osmC, osmY, otsB, narU, ugpB and dps), which were highly consistent with the microarray results.
Conclusion:
(1) RpoE could regulate a large number of gene expressions in S. Typhi in hypertosmotic conditions, including fliA,flgK, chew, glpX, ompC, phop, dnaK and dnaJ.
(2) RpoE could directly promote flagellar gene expression through FliA in S. Typhi and manipulate flagellar motility in hyperosmotic conditions.
(3) RpoE could promote expression of invasion virulence genes (such as orgA, prgK, sipA, invF, sopE and spaS) and thereby help S. Typhi invading into epithelial cells.
(4) 38 genes were co-regulated by RpoE and RpoS in S. Typhi under hyperosmotic conditions including osmC, osmY, otsB, narU, ugpB and dps.
作为人类食源性致病菌,伤寒沙门菌在从环境到使宿主致病的过程中,需要遇到一系列剧烈的环境应激,包括人的肠道高渗应激环境。在高渗应激条件下,细菌需要及时有效地调节某些基因的表达和蛋白的活性来适应环境。原核生物基因表达均是由sigma因子介导转录起始,且多数呈操纵子模式,并受各种调节因子的影响。不同的sigma因子(σ因子)特异性识别并引导RNA聚合酶结合到目的基因启动子区域,启动相应基因表达,无疑是原核生物对其生命活动进行调控的最基本的方式。已知不同的sigma因子的作用不同,不同的sigma因子对某些基因或有共调节作用,但缺乏直接的研究证据。RpoE是响应环境应激的sigma因子。已有研究发现RpoE是肠杆菌科细菌中一个重要的sigma因子,在响应多种环境应激时发挥着重要作用,对细菌生存和致病性至关重要,但其在伤寒沙门菌中作用的研究报道尚少。
目的：
本研究旨在了解与RpoE相关的参与伤寒沙门菌克服高渗应激的效应蛋白基因,深入分析RpoE对重要致病因子表达调控的机制,研究RpoE在伤寒沙门菌克服高渗应激过程中与另一sigma因子RpoS对基因表达的共调节作用,以加深对伤寒沙门菌基因表达调控网络和致病分子机制的认识。
方法：
(1)基因缺陷变异株的制备采用自杀质粒pGMB151介导的同源重组方法,制备伤寒沙门菌rpoS缺陷变异株及rpoE/rpoS双缺陷变异株。
(2) rpoE基因缺陷回补株的构建PCR扩增rpoE基因,酶切后与表达载体pBAD/gⅢ定向连接,热击法转入大肠杆菌DH5a中筛选阳性质粒,经序列分析鉴定后,电击法转入rpoE基因缺陷变异株。
(3)高渗应激下生长情况分析以生长时间为横坐标,细菌OD600值为纵坐标绘制生长曲线,对比缺陷变异株、野生株及基因缺陷回补株在高渗应激条件下的生长情况。
(4)动力试验分析缺陷变异株和野生株37℃培养过夜,分别取4μ1菌液接种于0.3%高渗半固体LB培养基,37℃培养8小时后测量动力圈直径,观察缺陷株及野生株的动力。
(5)伤寒沙门菌基因表达谱分析伤寒沙门菌全基因组芯片分析目的基因在转录水平上对其他基因的表达调控。体外模拟高渗环境应激后分别提取伤寒沙门菌野生株和基因缺陷变异株的总RNA,反转录成cDNA并标记荧光(cy3或cy5),与基因组芯片杂交,扫描后根据荧光信号分析各基因转录表达水平,比较伤寒沙门菌野生株和基因缺陷变异株在高渗应激早期的基因表达谱差异。
(6)实时荧光定量PCR(qRT-PCR)分析选择基因芯片结果中部分差异表达基因,设计特异性引物,进行反转录和实时定量PCR,以验证DNA芯片分析结果。
(7)蛋白质二维电泳和质谱分析高渗应激后分别提取rpoE缺陷变异株和野生株的菌体蛋白和分泌蛋白。取适量蛋白进行等点聚焦和SDS-PAGE电泳分离后,以考马斯亮蓝G-250染色后扫描成像,分析蛋白表达差异,选取明显表达差异蛋白进行质谱分析；质谱分析的肽指纹图在www.mascot进行比对。
(8) Western免疫印迹分析分泌蛋白高渗应激条件培养rpoE缺陷株、野生株,利用三氯醋酸-丙酮法提取培养上清中的分泌蛋白,以抗H：z66多克隆抗体进行Western免疫印迹分析rpoE缺陷株、野生株分泌Z66蛋白的差异。
(9)凝胶阻滞试验根据伤寒沙门菌rpoE基因序列设计引物,扩增rpoE基因片段,与表达载体pET-28a连接经热击法转化入大肠杆菌JM109,用IPTG诱导表达RpoE蛋白,用镍柱纯化RpoE蛋白。根据伤寒沙门菌fliA基因序列设计特异性引物,扩增fliA基因启动子区域,取2μg PCR产物与不同浓度的RpoE蛋白混合,加入相应体积的GSM缓冲液,反应总体积为20μ1,30℃孵育15 min。选取真核生物DNA片段作为阴性对照,8%的丙烯酰胺胶电泳分离条带,溴化乙锭染色。
(10) HeLa细胞侵袭试验细菌培养至OD600为1.0,加入培养有HeLa细胞的24孔板中(MOI=20),90min后收集一部分细胞,加入裂解液,涂LB平板过夜培养,计细菌克隆数,代表细菌粘附细胞水平T0；另一部分加入庆大霉素杀死胞外细菌,继续培养90 min,裂解细胞后涂板过夜培养,计克隆数代表细菌的侵袭水平T90,用T90/T0的值代表细菌的侵袭力。
结果：
(1)研究用菌株制备经PCR及序列分析证实成功构建伤寒沙门菌rpoS基因缺陷变异株、rpoE/rpoS双缺陷变异株；成功构建pBAD-rpoE阳性重组载体,并成功将重组载体导入相应的缺陷株中,制备成rpoE基因缺陷回补株。
(2)生长曲线分析结果显示rpoE基因缺陷变异株在高渗应激条件下生长能力明显弱于野生株,在回补rpoE基因后得到明显恢复；rpoS基因缺陷株在高渗应激条件下生长能力亦明显弱于野生株,rpoE/rpoS双缺陷变异株在高渗应激条件下生长能力均明显弱于野生株和rpoE、rpoS单基因缺陷株。
(3)高渗应激下RpoE影响基因表达分析基因组芯片分析显示,高渗应激30分钟后,rpoE基因缺陷株相比野生株有74个基因表达下调,56个基因表达上调,选取其中部份差异表达基因利用RT-PCR进行验证,结果显示所选基因表达变化与基因芯片分析结果基本一致,且在回补rpoE基因后表达得到明显恢复。
(4)高渗应激下RpoE对蛋白表达影响的分析二维电泳、质谱分析显示,rpoE缺陷株与野生株相比18个菌体蛋白明显有差异,质谱分析鉴定包括有氨酰组氨酸二肽酶PepD、氨酰组氨酸二肽酶AspA等；分泌蛋白二维电泳后发现rpoE缺陷株与野生株相比有16个明显差异点,质谱分析鉴定包括NAD合成酶NadE,外膜蛋白OmpC等。
(5) RpoE对鞭毛及动力影响机制研究动力试验结果显示,在高渗半固体LB培养平板上,rpoE基因缺陷变异株动力比野生株明显下降,回补rpoE基因后动力明显恢复,提示RpoE在高渗条件下参与调控鞭毛动力；结合高渗应激早期基因芯片发现rpoE缺陷变异株中二级鞭毛基因fliA和三级鞭毛基因表达明显下调,利用qRT-PCR分析一级鞭毛基因flhD、二级鞭毛基因fliA和三级鞭毛基因fljB:z66的表达情况发现,rpoE缺陷变异株中flhD相比野生株没有明显差异,而fliA与fljB:z66的表达在rpoE缺陷变异株中明显下调；Western-bolt发现鞭毛蛋白FljB:z66在rpoE缺陷变异株的表达明显低于野生株；凝胶阻滞试验证实RpoE蛋白可以直接结合fliA基因启动子。
(6) RpoE对伤寒沙门菌侵袭力影响分析rpoE基因缺陷变异株的侵袭力比野生株明显下降,而rpoE回补株侵袭能力明显恢复；结合基因芯片结果,挑选部分侵袭毒力相关基因进行qRT-PCR,发现这些基因在rpoE缺陷株中表达相对野生株明显下调,在回补rpoE基因后表达明显恢复。
(7)高渗应激下RpoE和RpoS对基因表达的共调节分析利用基因组芯片,进一步分析rpoE基因缺陷株表达谱、rpoS基因缺陷株表达谱和rpoE/rpoS双缺陷株表达谱,发现有38个基因,包括osmC、osmY、otsBA、narUZY、dpS等,在rpoE、rpoS缺陷株中相比野生株表达变化不明显,但在rpoE/rpoS双缺陷株表达明显下调,提示这些基因受RpoE与RpoS的双重调控；选取不同操纵子的6个基因CosmC, osmY, otsB, narU, ugpB和dps)进行qRT-PCR进行验证,结果显示,所选基因表达与基因芯片分析结果一致。
结论：
(1) RpoE在伤寒沙门菌高渗应激时参与fliA、flgK、cheW、glpX、ompC、phoP等致病因子基因的表达调控。
(2)高渗应激下伤寒沙门菌RpoE能直接启动FliA表达,进而调控鞭毛基因表达和影响细菌动力。
(3) RpoE能促进orgA、prgK、sipA、invF、sopE、spaS等侵袭毒力基因表达,进而帮助伤寒沙门菌侵袭上皮细胞。
(4)高渗应激早期,RpoE因子与RpoS因子对osmC、osmY、otsBA、narUZY、dpS等38个基因存在共调控作用。
Salmonella enterica serovar Typhi (S. Typhi) is an important human intestinal bacteria and one of the most extensive and in-depth investigation of Prokaryotes. S. Typhi often enters the human digestive tract by contaminated food and invades into intestinal epithelial cells of the distal jejunum. After numerously proliferation, the bacteria of S. Typhi survive in the local mesenteric lymph tissue and shift to the liver, spleen and other tissues through the lymph and blood system. The systemic infection of S. Typhi leads to serious complications such as intestinal perforation and even death.
As a kind of human food-borne pathogen, S. Typhi needs a series of self-regulation to overcome some different kinds of severe environmental stresses during infection, including intestinal hyperosmotic stress. In the hyperosmotic condition,S. Typhi should promptly regulate the expression of many genes to adapt the new environment. Gene transcription in Prokaryotes is initiated by sigma factors (σfactor) in a manner of operon mode and controlled by various regulatory factors, including activators and inhibitors. Sigma factors specifically recognize the promoter region of target genes and helps RNA polymerase to assemble and promote the expression of relative genes, which is the most basic and important way for Prokaryotes to perform life activities undoubtedly. Different Sigma factors are responsible for different activity. Some researchers guessed that different sigma factors could co-regulate some gene expression, but there is no direct evidence yet. There was reported that RpoE was one of the important sigma factors in Enterobacteriaceae to adapt to some severe conditions. However, the role of RpoE involved in the hyperosmotic stress has been rarely reported in S. Typhi.
Objective:
The study will demonstrate the response genes of S. Typhi regulated by RpoE at the hyperosmotic condition, illustrate the mechanism of the expression of some important pathogenic factor genes regulated by RpoE, and investigate the co-regulation of gene expression by RpoE with RpoS, another important sigma factor of S. Typhi in the hyperosmotic condition. This study may be helpful to understand the regulatory networks of gene expression and the molecular mechanism of pathogenesis of S. Typhi.
Method:
(1) Preparation of gene mutant strains. The rpoS mutant and rpoE / rpoS double mutant S. Typhi was constructed by homologous recombinant with suicide plasmid pGMB151.
(2) Construction of the rpoE rescue strain. The rpoE gene was amplified by PCR and connected to the expression vector pBAD/gⅢ. The recombinant plasmid was transferred into Escherichia coli DH5a by heat shock. The positive plasmids were screened by sequence analysis and then transferred into rpoE mutant strain by electroporation.
(3) Analysis of bacterial growth under hyperosmotic conditions. To compare the survival ability of wild-type strain and gene mutant strain under the hyperosmotic stress conditions, growth curves were drawn by using growth time as the abscissa and value of OD60o as the ordinate.
(4) Motility assay. The wild-type strain and gene mutant strain were cultured overnight at 37℃without agitation on LB broth. Each 4μl of culture was inoculated into the centre of a 0.3% LB agar plate, which was containing 300 mM of NaCl. The plates were incubated at 37℃for 8 h and motility was assessed qualitatively by examining the diameter of circular swimming which was formed by the growing motile bacterial cells.
(5) Gene expression profile analysis of S. Typhi. The genome-wide gene expression at transcriptional level was analyzed by genomic microarray assay. After culturing in hyperosmotic conditions, the total RNAs from wild-type strain and mutant strain was extracted. cDNAs would be obtained by reverse transcription and labeled with fluorescent (cy3 or cy5). Cy3-cDNAs or Cy5-cDNAs hybridized with genomic microarray were captured by scanning analysis of fluorescent intensity. The gene expression difference between wild-type strain and mutant strain was reflected by fluorescent intensity.
(6) Quantitative real time PCR (qRT-PCR) assay. Primers specific to some selected genes were designed and used for qRT-PCR to validate the results of microarray assay.
(7) Protein two-dimensional electrophoresis and mass spectrometry assay. After culturing in hyperosmotic stress conditions, the bacterial proteins and secreted proteins were extracted from the rpoE mutant strain and the wild-type strain. The proteins were separated by isoelectrofocusing and SDS-PAGE electrophoresis. After staining with Coomassie Brilliant Blue G-250, the expressional levels of proteins were showed. Mass spectrometry was used to verify the selected proteins and the existing peptide fingerprinting was blasted with data at www. mascot.
(8) Western-blot. After culturing in hyperosmotic conditions, the secreted proteins from rpoE mutant strain and the wild-type strain were extracted by trichloroacetic acid-acetone. The different levels of secreted protein expression between rpoE mutant strain and the wild-type strain were analyzed by Western-blot with rabbit anti-H:z66 antiserum.
(9) Gel-shifting assay. One pair of primers was designed to amplify DNA fragments of the rpoE gene of S. Typhi, which was then inserted into the expression plasmid pET-28a. Escherichia coli JM109 were then transformed by the expression plasmid by electroporation. Expression of the RpoE protein was induced by IPTG. RpoE protein was purified with Ni-colum. Two pairs of primers were designed to amplify the promoter region of fliA of S. Typhi. Each 2μg amplicon and different amount of RpoE were mixed with GSM solution in final 20μl, incubated at 30 for 15℃min before electrophoresis. The PCR product from the eukaryotic cell was used as the control. The electrophoresis in 8% acrylamide gel was performed for separating DNA fragments. Gel was stained with Ethidium Bromide.
(10) Invasion ability assay with Hela cells. The bacteria were cultured until the OD600 was approximately 1.0, and then added to 24-well culture plate which was planted with Hela cells (MOI=20). After incubating for 90 min, a portion of Hela cells was lysed and then incubated on LB plate for calculating bacterial clones. The clones represented the ability of bacterial adhesion (To). The remaining cells were incubated for another 90 min, treating with gentamicin to kill extra cellular bacteria. The cells were then lysed and then incubated on LB plate for calculating bacterial clones. The clones represented the ability of bacterial invasion (T90). The ratio of T9o/To was used to evaluate the invasion ability of bacteria.
Results:
(1) Preparation of bacterial strains. The rpoS mutant strain and rpoE/rpoS double-defective mutant strain were constructed successfully, which was verified by PCR and DNA sequencing. The recombinant pBAD-rpoE plasmids were constructed and then transferred into gene mutant strain to generate the rpoE rescue strain.
(2) Analysis of the growth curve. The results showed that the survival ability of rpoE mutant strain was significantly weaker than the wild-type strain under hyperosmotic conditions. However, the survival ability was obviously restored in the rpoE rescue strain. The rpoS mutant strain also grew more slowly than the wild-type strain in the hyperosmotic condition. Moreover, the growth rate of the double rpoE/rpoS mutant strain was significantly reduced compared to rpoE mutant and rpoS mutant strain.
(3) Analysis of gene expression regulation by RpoE under hyperosmotic condition. Microarray analysis showed that 74 genes were down-regulated and 56 genes were up-regulated in the rpoE mutant strains after culturing in hypertonic conditions for 30 min. The results of some selected genes were verified by qRT-PCR. The expression of genes was significantly restored in rpoE rescue strain.
(4) Analysis of the expression of proteins affected by RpoE under hyperosmotic conditions. Two-dimensional electrophoresis revealed that eighteen bacterial proteins were expressed differently between rpoE mutant strain and wild-type strain. Among them aminoacyl-histidine, dipeptidase PepD and aminoacyl-histidine acid peptidase AspA were confirmed by mass spectrometry. Sixteen different points of secreted protein between rpoE mutant and wild-type strains were found by two-dimensional electrophoresis analysis. Among these secreted protein points, the NAD synthetase NadE and the outer membrane proteins OmpC were verified by mass spectrometry.
(5) Mechanism of flagellar and motility regulation by RpoE. The motility of rpoE mutant strain was apparently decreased compared to that of the wild-type strain in semi-solid LB medium hypertosmotic plate. On the contrary, the motility of the rpoE rescue strain was similar to that of the wild-type strain. It indicated RpoE participated in the regulation of flagella genes under hypertosmotic conditions. Microarray analysis showed expression of classⅡflagellar gene fliA and most classⅢflagellar gene were significantly reduced in rpoE mutant strains. qRT-PCR was used to analysis expression of classⅠflagellar gene flhD, classⅡflagellar gene fliA and classⅢflagellar gene fljB:z66. The results showed that the flhD expression was not significantly changed in rpoE mutant stain compared to the wild-type strain, whereas the fliA and fljB:z66 expressions were significantly reduced in rpoE mutant strain. Western-bolt showed that flagellin protein FljB:z66 was significantly reduced in rpoE mutant stain compared to the wild-type strain. The result of gel-shifting experiments showed that RpoE could bind the promoter region of fliA.
(6) Analysis of invasion ability affected by RpoE. The ability of invasion was significantly decreased in rpoE deleted mutant strain compared to that of the wild-type strain. Correspondingly, the invasion ability was rescued in the rpoE complementary strain. Considering the results from microarray, some invasion or virulence genes were detected by qRT-PCR. The results showed that those gene expressions were significantly reduced in the rpoE mutant stain compared to wild-type strain. However, those gene expressions would restore when rpoE gene was compensated.
(7) Analysis of co-regulating gene expression by RpoE and RpoS under hyperosmotic condition. Results of DNA microarray analysis showed that 38 gene expressions including osmC, osmY, otsB, narU, ugpB and dps were markedly reduced in the rpoE/rpoS double mutant strain after exposure in hyperosmotic conditions for 30 min. Those gene expressions were only weakly affected by either the rpoE or rpoS single mutation. The data suggested that those genes were co-regulated by RpoE and RpoS. qRT-PCR was also used to analyzed the expression levels of six genes from different operons (osmC, osmY, otsB, narU, ugpB and dps), which were highly consistent with the microarray results.
Conclusion:
(1) RpoE could regulate a large number of gene expressions in S. Typhi in hypertosmotic conditions, including fliA,flgK, chew, glpX, ompC, phop, dnaK and dnaJ.
(2) RpoE could directly promote flagellar gene expression through FliA in S. Typhi and manipulate flagellar motility in hyperosmotic conditions.
(3) RpoE could promote expression of invasion virulence genes (such as orgA, prgK, sipA, invF, sopE and spaS) and thereby help S. Typhi invading into epithelial cells.
(4) 38 genes were co-regulated by RpoE and RpoS in S. Typhi under hyperosmotic conditions including osmC, osmY, otsB, narU, ugpB and dps.
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