The Discovery Of Novel Small RNA And Their Biological Function In Shigella Flexneri

Shigella spp. is a group of Gram-negative and one of the main pathogens causing bacillus dysentery. The bacteria invade the intestinal epithelial cells and caused the typical symptoms of bacillus dysentery in patients, such as tenesmus, fever, abdominal pain and diarrhea and so on. Four species involved in Shigella based on the biochemical reactions and the structure of O antigen. The Shigella flexneri is the main group in the developing countries and the main serotype is 2a. According to the World Health Organization reported that Shigella caused 160 million cases each year with 1.1 million deaths and more than two-thirds of cases occurring in the children under 5 years old in the worldwide. Due to the low infection dose(10-100 bacteria) and the unclear of the pathogenic mechanism and the host immune protection mechanism, the outbreak of bacillus dysentery serious harm to human health. Thus it is important to perform basic research Shigella flexneri for implications for health and epidemic prevention work.In addition to the three classic RNA(r RNA, t RNA and m RNA), there also exists a new class of RNA, called non-coding RNA(nc RNA). s RNAs which exsit in bacterial genomes are a kind of newly discovered small RNA, which range from 50 to 500 nt in length and characterized by base pairing with target m RNAs; this affects the activity of the target m RNA or its translated protein product at the transcriptional level. s RNAs regulate various physiological processes in bacteria, such as metabolism, virulence, and stress responses. In prokaryotes, the study of s RNA mainly focused on E.coli, which hundreds of s RNAs have been discovered and their functions have also been elucidated. For example, in E.coli, Mic F length of 93 nt s RNA, which binding with omp F m RNA to inhibite the expression of outmember omp F; Oxys could paired with fhl A to inhibit the translation. Although the whole genome sequencing of Shigella have been completed, the system identification and function of s RNA have not been performed. Therefore the research of the s RNA on accommodate extreme environment and pathogenic mechanism will be better for understanding the drug and vaccine development and of great significance.Firstly, we use the bioinformatic method to predict s RNA candidate in Shigella. We used a transcription unit-based method to predict s RNAs in Shigella and found 57 candidate s RNAs. The RT-PCR and Northern blot was perfomed to verify the predicted s RNAs, 22 s RNAs were then validated by RT-PCR and 4 s RNAs were validated by Northern blot. Finally, the transcriptional start site and end site of Ssr1、Ssr9、Ssr44 and Ssr54 were determined by RACE assays. To study the function of these two s RNAs on accommodated environment stress and virulence, we used the λ-Red-mediated recombination method to construct a S. flexneri 301 Ssr1 and Ssr54 deletion mutant. To further study the effect of growth status under various environment stress, we perform a growth curve on wild type, s RNAs mutant and complemented strains. The results showed that Ssr1 mutants grown in medium adjusted to p H 5.0 exhibited a significantly prolonged lag in the exponential phase, and Ssr54 mutants grown in medium contain 640 m M Na Cl exhibited a increased in the stationary phase compared with the wild-type. To study the response to environment stress on s RNAs, we used q RT-PCR to exposed wild type and s RNAs mutant strain to different stress medium. q RT-PCR revealed that Ssr1 was most highly expressed in the p H 2.0–4.0 range compared to the p H7.0 condition, and Ssr54 were highly expressed in the p H 5.5–6.5, oxidative stress,limited nutrition and low osmotic. These results indicated that these two s RNAs play essential functions in response to environment stress signals. To investigate the functions of two s RNAs survive in different stress, we perform the survival rate assays in wild type and s RNAs mutant strains. The results showed that the survival rate of the Ssr1 deletion mutant decreased by 22% under low p H stress, while the survival rate of the Ssr54 deletion mutant increased by 30% under oxidative stress. It indicated that these two novel s RNAs in S. flexneri plays an important role in the resistance to various stress. These results suggested that Ssr1 and Ssr54 enhance the ability to adapt to the environment by changing their expression levels and responding to the external environment. To determine ascertain these two s RNAs are essential for virulence, guinea pig conjunctival sacs and lung invasion assays were performed with the s RNAs mutant and wild-type S. flexneri strains. The results showed that the Ssr1 deletion results the serious keratoconjunctivitis, and the capacity of invasion was also increased; while in the Ssr54 mutant, it results the non keratoconjunctivitis, and the capacity of invasion was decreased. Together, these results suggest that the expressions of these two s RNAs may be related to the virulence of S. flexneri. To further explore the functions of Ssr1 and Ssr54, we analyzed the cytokine in wild type strain and the Ssr1 and Ssr54 mutant strains. The cytokines contain TNF-α、IL-1β、IL-6、IFN-γ. The results showed that IL-1β was significantly increased in Ssr1 mutant at 24 hour infection. It indicated that at the early infection stage Ssr1 mutant could induce the elevated expression of TNF-α and IL-1β, which may lead to increasing virulence in Shigella flexneri. Ssr54 mutant was no significant different compared to the wild type.To further explored the specific mechanism of Ssr1 and Ssr54, the two-dimensional gel electrophoresis(2-DE) assay was used to compare the s RNAs mutant to the wild-type to confirm the potential targets of s RNAs. 51 differentially proteins were successfully found in the Ssr1 mutant strain, among them 27 proteins were down-regulated and 24 proteins were up-regulated. 40 differentially proteins were successfully found in he the Ssr54 mutant strain, among them 25 proteins were down-regulated and 15 proteins were up-regulated. Using q RT-PCR, we analyzed the expression of the stress resistance genes and virulence genes, including dnak,ipa A,mxi C,ipg C and ipa D genes in Ssr1 mutant strain, and tol C,hns,tre F and omp A genes in Ssr54 mutant strain. Ssr1 positively regulated dnak to resistance to acidic stress, and negatively regulated the ipa and mxi genes of the T3 SS system to reduce the virulence; Ssr54 positively regulated the tol C and hns to increase the virulence, and paired with hns and tre F to adapt to the changes in environmental stresses. These results were consistent with those of the 2-DE analysis. Our studies provide a new insight for future s RNA function mechanism analysis and understanding of the process of pathogenic and control of infection.The second part of this paper is to study the function of Hfq in Shigella flenxeri. Hfq protein is host factor for RNA phage Qβ replicase and RNA binding protein. Hfq was initially identified in E. coli and is a member of the Sm/Sm-like protein family. It can form ring-shaped hexameric structures that are known to bind single RNA molecules. It has been reported that the functions of Hfq focus mainly on growth-dependent metabolism, resistance to environmental stress, modulation of virulence and so on. However, the size of Hfq is different in bacteria, such as 102 amino acids in E.coli, and 77 amino acids in Staphylococcus. Also the function and mechanism of Hfq is different in bacteria, such as Hfq affect the toxin expression in Brucellosis and Pseudomonas aeruginosa. In E.coli, the Hfq has the ATP enzyme activity and then accelerating the process of transcription and translation. Therefore, the role of Shigella hfq will greatly facilitates the study of Shigella pathogenic mechanisms.To analyze the function of hfq, we used λ-Red-mediated recombination method to construct a Δhfq strain. The resulting plasmid p ACYC184-hfq was used to transform the Δhfq strain, and the recombinant DNA products were verified by sequencing. For stress growth curve experiments, the Δhfq mutant had a significant lengthening of its lag phase and a significant reduction of its entire growth phase compared with the wild-type strain under the p H 5.0 environment, the Δhfq strain entered the exponential phase in a similar manner as the wild-type strain; however, the mutant was unable to maintain its survival in the stationary phase in normal condition. To determine whether the Hfq protein is important in the stress response of S. flexneri, the stress tolerance of the wild-type, Δhfq, and Δhfq+phfq strains was measured using survival assays. The results showed that the survival rate of hfq mutant was reduced under acid environment, high osmotic and oxidative stress. These results indicated that hfq play a key role in environmental stress resistance. To discover the role of the S. flexneri hfq gene in response to various stress, the expression of hfq was examined using q RT-PCR assays. hfq was higher expression in the acid and oxidative stress. Interestingly, a significant linear correlation between the hfq and the T3 SS related genes, as well as between the expression of hfq and acid resistance genes was identified. These observations suggest that the hfq gene product is important for the S. flexneri response to environment stress. To examine the function of hfq on virulence, the He La cell invasion assays and lung invasion assays was performed in wild type and hfq mutant strain. The low invasion suggesting that hfq deletion has an impact on the ability of S. flexneri to effectively invade cultured cells in vitro. We detected the cytokines of alveolar lavage fluid in the mice which infected by the wild type and hfq muant strain. The results showed that the TNF-α were significantly decreased in hfq mutant. To assess whether the hfq product regulates virulence through the T3 SS, gene expression was analyzed in the wild-type, Δhfq, and Δhfq+phfq strains by q RT-PCR to identify the genes coding for the T3 SS and acid resistance genes. The expression levels of these T3 SS genes were almost undetectable in the Δhfq strain being weakly expressed relative to the wild-type strain. The q RT-PCR results indicated that the expression levels of acid resistance genes were significantly decreased in the Δhfq mutant relative to the wild-type strain. In addition, We reported the first identification of Shigella flexneri serotype 1c in China and also the emergence of resistance to ciprofloxacin and third-generation cephalosporins in serotype 1c the first time.In summary, we performed the bioinformatics and experiments methods to identify 4 novel s RNAs in Shigella flexneri. Two of these s RNAs play essencial functions on the states of growth, the survival ability under extreme environments and pathogenicity. The potencial targets of these two s RNAs were identified by using bioinformatic and proteomics methods. These results revealed that the mechanism of adaptation to environmental stress and into the pathogenesis of Shigella. The research on hfq function reveal that hfq play essential roles in stress and virulence. These results will extend our knowledge of the function of s RNA was useful for the research of pathogenesis of Shigella flexneri. These findings will be better for understanding the pathogenesis, and provide the theoretical basiss and novel drug target for anti-shigella infection, but also provide reference for other bacteria infection.