Identify Novel Small Regulatory RNAs Based On RNA-Seq In Shigella Flexneri And Determine Their Potential Pathogenic Mechanisms

The s RNAs(small regulatory RNAs) play an important role in all aspects of bacteria, such as bacterial resistance to environment, plasmid replication, and bacterial invasion regulation. So we tried to detect novel s RNAs and their regulation mechanism in Shigella flexneri using RNA-Seq technology.There were about 160 million people infected by Shigella annually, mostly in developing countries, and leading to the death of 1.1 million. Shigella infection is very serious in our country. The incidence rate of infection is fourth, and the mortality rate ranked third, and Shigella flexneri is most popular in our country. However, to date, only 2 s RNAs of shigella flexneri had been annotated. In Escherichia coli, more than 100 s RNAs had been found. So it is necessary to carry out the identification and functional analysis of s RNAs in Shigella flexneri, and the study will provide theoretical basis for further research and new drug targets.The RNA-Seq technology not only can detect RNA transcriptsbut also measure their expression levels. At the same time, the large number of unknown transcripts also can be identified. Due to its high accuracy, repeatability, high throughput, the RNA-Seq technology has been more and more applied to the s RNA identification in bacteria. According to the current research progress, the HFQ can regulate the environmental tolerance and virulence of bacteria through the combination with s RNA. Therefore, in our study, we carried out the transcriptional profile of Hfq gene deletion strains and wild strains based on RNA-Seq technology, and found the function and regulation mechanism of the HFQ. Meantimes, we found out the new s RNAs in Shigella flexneri combined with promoter and terminator prediction, and further were verified by experiments. The research mainly includes following five parts:Part A: we successfully constructed hfq gene mutant strains of Shigella flexneri using λ-RED method. The hfq gene deletion strains and wild strains were sequenced by RNA-Seq. We got firm foundationdata for clarifying function of the hfq gene in Shigella flexneri, and found new s RNAs and their possible regulatory network.Part B: We analyzed high-throughput sequencing data of wild strain 301 and the hfq gene deletion strains. The results show that HFQ regulates about 16.05% gene of Shigella flexneri, and that the expression of virulence genes in the hfq gene deletion strains demonstrated low expression. Further quantitative PCR showed the same results. So we concluded that HFQ direct or indirect regulate these virulence factors. At the same time, the T3 SS is essential for Shigella invasion, however, in the hfq gene deletion strains, the expression of T3 SS genes were significantly downregulated or not expressed, indicating that Hfq protein involved in the regulation of the T3 SS system. And some of the acid resistance genes in the deletion of the hfq gene also appeared to significantly downregulated, showing that the HFQ protein involved in acid resistance.Part C: We found the new transcriptsin intergenic regionsthrough in-depth analysis of the transcriptional profiling data, and the results showed that there were 717 new s RNAs in Shigella flexneri. The expression of 280 s RNAs has significant difference between wild strain 301 and the Hfq gene deletion strains. These s RNAs are likely to regulate gene expression associated with Hfq. In order to improve the accuracy of the new s RNA identification, we found a total of 243 s RNAs has a complete transcription unit based on promoter and terminator prediction. The length more than 80 bp were been used for experimental verification.Part D: The 92 s RNAs were used for experimental verification. These s RNAs were detected by RT-PCR in different growth stage(6 hours, 9 hours and 12 hours), and these s RNA are all detected. At the same time, we also detected s RNAs using Nothern hybridization. Finally, we found that 10 new s RNAs had hybridization signals, and the newly discovered 10 s RNAs were named as: sf29, sf46, sf47, SF63, sf69, sf80, sf83, sf86, sf87, sf90.Part E: In this part, we predicted the targets of the new 10 s RNAs to understand their possible mechanism and pathway. We found that sf46, sf69, sf80 may be involved in the regulation of virulence, because of their main targetsbelong to the type three secretion; sf83 is involved in the adaptation to environmental stress regulation, and the regulatory pathway is the sf83 s RNA combined with HFQ to regulate the expression of evg S, led to changes in environmental resistance protein evg A, thereby affecting the tolerance of Shigella; sf63, sf83, sf87, sf90 may be involved in both environmental and virulence, these s RNAs play a pathogenic regulation through the regulation of type three secretion system and by regulating the hde B and hde B protein to control acid tolerance.In summary, we first obtained transcriptional profiling of the Hfq gene deletion strains and wild strains in Shigella flexneri, and found the function and regulatory mechanism of HFQ protein in Shigella flexneri. We also found 10 new s RNAs and their possible pathwaysin Shigella flexneri. This study provides a more thorough theoretical basis and new potential drug targets for the infection of Shigella.