Mechanisms Of Gene Expressional Regulation By RpoE Of Salmonella Enterica Serovar Typhi In Response To Environmental Hyperosmotic Stress

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.