Research On Function Of Small RNA In Shigella

Shigella spp., commonly called Bacillus dysenteriae, are gram-negative, facultative anaerobes, non-spore forming pathogens. The pathogenesis of S. flexneri is based on its ability to invade and replicate within the colonic epithelium, which results in severe inflammation and epithelial destruction. Shigella include 4 groups and 37 serotypes.In our country the predominant group of Shigella spp is S. flexneri (86%) and the predominant serotype is S. flexneri 2a (80%). Upon infection, humans develop severe abdominal cramps, fever, and frequent passage of bloody stools. According to statistics of WHO, Shigellosis caused 1.5million deaths and over 164 million cases each year with the majority of cases occurring in the children of developing countries. While in China, there were about 20 million cases each year, and S. flexneri 2a was responsible for 50-70% of those cases. The traditional treatment to dysentery is the use of antibiotics. But more than 95% strains have the resistance to various antibiotics. The best treatment is the use of vaccine, but few are known about pathogenesis of Shigella and the immune response of host till now. Thus it is important to research on S. flexneri 2a for our sanitary and epidemic prevention.Prokaryotic small RNAs (sRNAs) that are not ribosomal transfer or messenger RNAs were initially identified in the intergenic regions of genome. SRNA is a kind of newly discovered 50-400 nt small RNAs that do not encode proteins. In recent years, many computational strategies have identified hundreds of sRNA candidates in prokaryote and these sRNA have many kinds of biological functions.They conduce bacteria to regulate physiological systems and accommodate the change of surroundings .Thus research on the function of sRNA is very important. Small RNA maybe represent a new style of Gene Expression Regulation.Now people have realized that sRNAs have participated many gene regulations of organisms in bacteria, such as translational activation, translational suppression, housekeeping sRNAs, quorum sensing, protein degradation ,ion dynamic equilibrium, glycometabolism and expression regulations of growth-dependend outer membrane proteins, the sRNAs are considered the regulators "over" the genomes. Although now more sRNAs have been found in bacteria rapidly, most of their functions are still unknown. Many researchers analyzed that bacteria maybe regulate their physiological system by sRNAs to accustomed to survive in rapidly changing environment. Therefore understanding these sRNAs may open a new research area, because they may represent a new level of the regulation of gene expression.Functional analysis indicate sRNA is a uncared-for aspect in bacteria expression regulation.Rencent research on the sRNA's functions supply the blank of ions dynamic equilibrium, glycometabolism, growth-dependend outer membrane protein expression regulation.Study on the function of sRNA can promote the study of physiology, phyletic evolution, pathogenic mechanism in S. flexneri 2a. The research on expression of sRNA also can provide a new approach for vaccine of shigella. Studies show sRNA in bacterias play an important role in bacteria metabolism, environment , quorum -sensing and bacteria virulence expression.First, according to the characters of the detected E.coli sRNAs, we use bioinformatics methods :sequence alignment between genome , secondary structure of RNA and un-dependency terminator analysis to predict sRNA in pCP301.We also use Northern blot to validate the predicted sRNA.We got 6 sRNA in pCP 301 by machine learing-based prediction method and choose 5 sRNA in S. flexneri 2a genome from internet and literatures(http://www.sanger.ac.uk).In order to clarify the function of sRNA in shigella, the deletion mutant S. flexneri 2a 301ΔsRNA::Kan were constructed with modifiedλ-Red recombination system. The growth curve of the sRNA mutants and the wild-type presented non-significant difference in LB medium and ferrum limitated medium.Compared with wild strain, the metabolism and growth presented non-significant difference.Furthermore, we evaluated the effect of sRNA to the virulence of S. flexneri. In this study ,we use cell culturing (in vitro) and animal tests (in vivo) to evaluate the function of sRNA. HeLa cell line was used for invasion test of sRNA mutants.Sereny test with guinea pig and lung invasion test with Balb/c mouse were used, respectively, to verify the virulent of wild type and mutants. It had been demonstrated that the three sRNA mutants retained the ability to invade mammalian cells in sereny test and tissue model, on the other hand, ln3 RNA mutant evoking time and inflammation intensity were less efficient than those of wild-type but invasiveness of C0719 sRNA and IS061 sRNA mutants were more efficient than that of wild-type.The comparative proteomics includes the proteomic research of different cultivation, the proteomic research of different strains and the proteomic research between mutants and wild-type. In our study, 2D-gel electrophoresis was used to identify the differential expression of the mutant and the wild-type strain at various growth phases. Moreover, different protein spots between sRNA mutants and wild- type were excised from gels, in-gel digested,and then indentified by using atrix- assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/ TOF) in combination with Mascot search program. It had been found that through the comparative proteomics of eight mutants in stationary phase of 37℃and sf301ΔIS061 in exponential phase of 37℃,we found 31 differential expressed protein spots.We analyzed the functions of these proteins and found the deletion of these sRNAs didn't make differential expression between the mutants and the wild-type strain.But we also found a differential expressed protein OspC3 that may influence the virulence of Shigella and another differential expressed protein OmpA,whose expression may be regulate by sRNAs.In conclusion,we have predicted 6 sRNAs in pCP301 and use Northern blot to validated them;we have got 8 sRNA mutants and analyzed the pathogenesis of sRNA mutants by using cell culturing (in vitro) and animal tests;we also combine with com- parative proteomic to analyze the function of sRNA's expression regulation. The sRNA mutants in this research form the basis and provide valuable information for future sRNA function analysis.