Identification And Functional Research Of MalS-5’utr In Salmonella Enterica Serovar Typhi

BackgroundSalmonella enterica serovar Typhi (S. Typhi) is an important human intestinal bacteria. The regulation of gene expression play an important role in the process of entry to the host from environment. In recent years, with the application of bioinformatics, DNA microarrays and high-throughput sequencing, research on the gene expression regulation of biological system has been developed rapidly. Besides proteins, a great number of the noncoding RNAs (ncRNAs) were found involving in the gene expressional regulation network in various organisms. Researchers have found that ncRNAs mainly regulate the expression of target genes at the posttranscriptional level. These target genes play crucial regulatory roles in various physiological responses in bacterial, including the regulation of metabolism, quorum sensing, response to oxidative stress, iron homeostasis and virulence regulation of pathogens. Nowadays, the identification, function and regulatory mechanism of ncRNAs has already been the focus in biomedical research.ObjectiveThe expression indication of many new ncRNAs was discovered in S. Typhi by high-throughput sequencing and bioinformatics analysis in our group. The purpose of this work was to identify the molecular characteristics of a new ncRNA, malS-5’UTR, in S. Typhi, and further investigate its expressional pattern, the effect on the gene expression regulation, mechanisms of action.Methods1. Identification of the molecular characteristics of malS-5’UTR:According to the information obtained from high-throughput sequencing, specific probe and primers were designed, and the expression of malS-5’UTR in S. Typhi was confirmed by RT-PCR and Northern hybridization.5’-and3’-rapid amplification of cDNA ends (RACE) analysis were used to find the transcription initiation site and transcription termination site of malS-5’UTR respectively. The results of Northern blot and RACE were used to determine the full length of malS-5’UTR.2. Analysis of malS-5’UTR expression characteristic under different growth conditions:Northern blot and qRT-PCR assays were used to investigate the expression levels under different growth phases. The same analysis was also used to investigate the expression pattern of malS-5’UTR under different environmental stimuli, namely, acidic, oxidative, and hyperosmotic stresses, and the influence of rpoE and rpoS on the expression of the malS-5’UTR.3. Phenotype analysis of gene deletion mutants(1) the assay of growth curve of S. Typhi under different conditions:The growth of wild type S. Typhi and malS-5’UTR deletion mutant strains were assessed. Strains were subjected to growth under normal as well as stress conditions and the absorbance of the culture media was taken hourly over a time period to determine the growth curve.(2) motility assays:Motility agar was used to characterize the motility phenotypes of malS-5’UTR deletion mutant, box deletion mutant and wild type strain of S.Typhi. These strains were inoculated onto semi-solid LB plates (0.3%agar) and incubated at37℃for several hours. The diameter of motility circle was measured in order to assess the motility of bacteria.(3) Invasion capacity assay with HeLa cells:malS-5’UTR deletion mutant, box deletion mutant and wild type strain were incubated in LB medium to OD600of0.4. HeLa cells were seeded into24-well plates and infected by bacteria as a MOI of20:1. After infection90min, they were then either lysed and incubated on LB plate, and the number of clone was regarded as the level of adherence (To), or incubated in medium containing gentamicin for a further90min to kill extracellular bacteria and then lysed to assess the level of invasion (T90). The value of T90/T0represents the bacterial capacity invading into HeLa cells.4. Gene expression profile analysis of S. Typhi regulated by malS-5’UTR and Bax:The genome-wide gene expression profile was analyzed by genomic microarray assay. malS-5’UTR deletion mutant, box deletion mutant and wild type strain were incubated in LB medium for4h. Total RNAs of mutant and wild type were extracted, transcribed into cDNAs and reciprocally labeled with cy3-and cy5-dCTP one time. After Hybridization with Salmonella genomic DNA microarray, the slides were scanned, and the signals were digitalized. The gene expression difference between the wild type strain and mutant strain was analyzed. To confirm the findings of the microarray experiments, some genes with expressional change in the microarray assay were selected for qRT-PCR analysis.5. Analysis of effect malS-5’UTR on the expression of the target bax gene:The recombinant plasmid pBAD-malS-5’UTR was constructed. The wild type strain was transformed with the recombinant plasmid pBAD-malS-5’UTR, which expressed a240nt transcript of the malS-5’UTR in the strain after induction with L-arabinose. After the overexpression of the malS-5’UTR, the level of bax mRNA was investigated with Northern blot and qRT-PCR. To further investigate the effect of the malS-5’UTR on the expression of Bax protein, a Bax-3xFLAG fusion strain was constructed by homologous recombinant and transformed with the expressional plasmid of malS-5’UTR (pBAD-5’UTR). L-Arabinose (0.2%w/v) was added to the cultures to induce the overexpression of the malS-5’UTR. The effect of malS-5’UTR overexpression on the expression of Bax protein was assessed by immunoblotting with anti-FLAG-tag antibody.6. Analysis of the mechanism of malS-5’UTR regulating invasiveness:To gain further insight into the mechanism of the malS-5’UTR in affecting bacterial invasion, pBAD-5’UTR and pBAD empty plasmid were transferred into the bax mutant of S. Typhi. The mRNA level of invasion-associated gene invF was measured with qRT-PCR, and bacterial invasiveness to HeLa cells was also examined.Results1. Identification of the molecular characteristics of malS-5’UTR:The analysis with RNAseq of S. Typhi showed a236-nt transcript encoded by the minus strand of bax, extending from137nt downstream to99nt upstream from the start codon of bax. The transcript was confirmed by Northern blot and RT-PCR assay and its length was about2000nt.5’-RACE analysis showed the transcription start site was located453bp upstream from the malS start codon.3’-RACE detected different ends of the transcript, and the longest located1112bp downstream from the start codon of malS. However, no relative fragments was seen in the result of Northern hybridization assay. Considering the same transcriptional direction of the transcript with the malS, combining the results of RACE, Northern blot, and bioinformatics analysis of the promoter, the transcript encoding region overlaping malS is the5’-untranslated region of the malS gene, and therefore named as matS-5’UTR.2. Expression characteristics of malS-5’UTR in S. Typhi:The expression characteristics of the malS-5’UTR were investigated with Northern blot and qRT-PCR. Results reflect the fact that the malS-5’UTR is expressed in all growth phases of S. Typhi, with an highest expression during the lag phase (OD6000.4). The expression of the malS-5’UTR in S. Typhi was decreased under various stresses. The relative expression levels of the malS-5’UTR decreased in the rpoE and rpoS mutant strains compared with the wild type strains under stresses. The result suggests that the transcription of malS-5’UTR is related with sigma factors RpoE and RpoS.3. Phenotype analysis of the malS-5’UTR mutant:(1) Growth assay:Under normal condition, the growth rate of the AmalS-5’UTR mutant was slightly lower than the wild type, which indicates that the malS-5’UTR can accelerate the growth of bacteria. The growth curves under acidic and high osmotic stress conditions showed that there is no significant difference between wild type strain and AmalS-5’UTR mutant. Under oxidative stress condition, the growth rate of the△malS-5’UTR mutant was significantly lower than that of the wild type, which shows that the malS-5’UTR can accelerate the growth of bacteria under oxidative stress condition.(2) Motility assay:The motility experiment displayed a significant increase in migration from the spot of inoculation in the mutant AmalS-5’UTR compared with the wild type. This result suggests that the malS-5’UTR is involved in the regulation of motility in S. Typhi.(3) Invasiveness assay:The invasive capacity with HeLa cells of the malS-5’UTR deletion mutant was stronger than that of wild type strain. It indicates that malS-5’UTR is participated in the regulation of bacteria invasiveness. 4. Effect of malS-5’UTR on gene expression profiling:A comparison of the global transcription profiles revealed that334genes were differentially expressed in the malS-5’UTR mutant and the wild-type strain. Of these genes,254were up-regulated in the malS-5’UTR mutant compared with the wild type, including genes involved in flagellum, motility and invasion.80were down-regulated in the malS-5’UTR mutant, including ribosome-associated and the metabolism-associated genes. To confirm the findings of the microarray experiments, some genes with expression change in the microarray assay were selected for qRT-PCR analysis. Results indicated that the expression levels of selective genes from qRT-PCR analysis were consistent with the levels from microarray assay. It shows that the malS-5’UTR is participated in the systemic gene expression regulation in S. Typhi.5. Effect of malS-5’UTR on the expression of target box gene:The box mRNA level decreased gradually with increasing induction time by Northern blot and qRT-PCR analysis. These results indicate that the malS-5’UTR can decrease the stability of box mRNA. Compared with the control strain containing empty plasmid, the box gene protein level reduced gradually with increasing induction time by Western blot analysis. Together, these results indicate that the expression of Bax is negatively affected by the malS-5’VTR.6. Analysis of the mechanism of the malS-5’UTR action on invasion of S. Typhi:(1) Effect of Bax on the invasiveness of S. Typhi:The invasive capacity to HeLa cells of the bax deletion mutant strain was stronger than that of the wild type strain. It indicates that bax may be participated in the regulation of bacteria invasiveness. A comparison of the global transcription profiles revealed that67genes were differentially expressed in the bax mutant and the wild-type strain. Of these genes,55were up-regulated in the bax mutant, including SPI-1, virulence and invasion genes.12were down-regulated in the bax mutant, involving in the metabolism-associated genes. To confirm the findings of the microarray experiments, some genes with expressional change in the microarray assay were selected for qRT-PCR analysis. Results of qRT-PCR analysis was consistent with the the results of microarray analysis. It shows that the Bax is a invasion negative factor, and the malS-5’UTR may increase invasive capacity of S. Typhi by inhibiting the expression of Bax.(2) malS-5’UTR regulating the invasiveness of S. Typhi associated with the Bax. After over-expressing the malS-5’UTR in the deletion mutant of bax, the mRNA level of invF and invasiveness to HeLa cells were increased compared with the control strain, the bax mutant containing an empty plasmid. These finding suggest that the effect of malS-5’UTR on invasion may be partially dependent on its target bax gene. malS-5’UTR may affect the expression of invasion-associated gene and invasiveness by other ways.Conclusions1. A non-coding RNA, malS-5’UTR expressed in S. Typhi was first identified.2. The deletion of the malS5’-UTR can increase the bacterial motility and the invasiveness to the HeLa cells.3. The deletion of the malS5’-UTR led to the change of the expression of system genes, including flagellar structure and function, invasion, regulatory-protein, and some metabolism-related genes.4. The overexpression of the malS5’-UTR can reduce the expression of bax.5. Bax may be a negative factor of invasion in S. Typhi.6. ma/S-5’UTR increases bacterial invasiveness by inhibiting the expression of Bax, which is not a exclusive pathway.