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Research on Function of Small RNA in Shigella

Author: LiNa
Tutor: WangHeng
School: PLA Military Academy of Medical Sciences
Course: Genetics
Keywords: Shigella flexneri 2a 301 Small RNA Expression regulation
CLC: R378
Type: Master's thesis
Year: 2009
Downloads: 200
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Abstract

Shigella bacteria known as Shigella (shigella), is a highly contagious and serious harm gram-negative enteric pathogens, can be caused by the invasion of intestinal epithelial cells in patients with the typical bacillary dysentery symptoms (fever, abdominal pain , diarrhea). Shigella four groups of 37 serotypes, the popular flora in our main flexneri (86%) the major Shigella flexneri serotype 2a (80%). The clinical Shigella infection can cause dysentery symptoms characterized by fever, dehydration, and blood in the stool. Dysentery the world, especially developing countries, infectious diseases. According to the World Health Organization announcement reported global annual bacillary dysentery patients 164,700,000 163,200,000 distributed in developing countries and 1.5 million deaths. In China, bacillary dysentery incidence is also high flexneri type 2a accounted for 50-70%. The main way to treat dysentery antibiotics, but up to 95% of the clinical isolates resistant to multiple antibiotics; optimal treatment vaccine is not very clear, but its pathogenesis and host immune protection mechanisms . The bacillary dysentery serious harm to the health of our people, the study has important significance Shigella. Flexneri 2a Shigella basic research is particularly important for our health and epidemic prevention work. In addition to the three we are very familiar with the RNA of tRNA, rRNA and mRNA in prokaryotic genome already know also contains many coding RNA regulation unconventional. The length of these RNAs for 50-400nt, usually by the area between the prokaryotic gene encoding but not translated into protein, it is called non-coding RNA, small RNA (sRNA) or non-coding RNA (ncRNA) for short. In recent years, with the continuous development of bioinformatics and molecular biology techniques in prokaryotes in vivo have been discovered hundreds of sRNA molecule the the sRNA molecule has a variety of biological functions. Small RNAs as a class of newly discovered sub-regulation of gene expression has been caused by a high degree of attention. They play a variety of regulatory functions in bacteria, helps bacteria fine regulation of physiological systems in order to adapt to the rapidly changing environment. Thus depth understanding of these sRNAs may open up a new field of study, they may represent a new level of gene expression and regulation. It is now recognized that sRNAs involved in many organisms of gene regulation, translation exercised in bacteria, such as activation, inhibition of translation, housekeeping sRNAs, quorum sensing system, degradation of the protein, ion homeostasis, sugar metabolism and growth-dependent outer membrane protein expression regulation and many other features, these sRNAs called \Bacteria found sRNAs rapidly increasing, but most of the functionality of them are still unknown, the knowledge accumulated from the current analysis, the bacteria may help of these sRNAs fine regulation of their physiological systems in order to adapt to the rapidly changing environment. Thus depth understanding these sRNAs may open up a new field of study, and because they may represent a new level of gene expression and regulation. Functional analysis showed that sRNAs bacteria-control study has been ignored. Latest recognize sRNAs physiological function has been to fill the the bacterial ion homeostasis, glucose metabolism and growth-dependent outer membrane protein expression regulation knowledge gaps. A new study of the Shigella genome sRNAs function, and is bound to promote research on bacteria physiology, phylogenetic and pathogenic mechanism, providing a new role for dysentery drug development target, Shigella vaccine development ideas. First of all, the study found E. coli sRNAs, bioinformatics, genome sequence comparison between RNA secondary structure analysis and analysis of non-dependent terminator flexneri 2a Shigella 301 virulence plasmid (pCP301) sRNAs predicted sRNAs predicted by Northern blot validation. In this study successfully predicted the 301 virulence large plasmids on the six sRNAs; Comprehensive literature and network information preliminary forecast sf301 gene group of five unknown function sRNAs: sRNAs IS061, IS102 sRNAs C0719 sRNAs tke1 sRNAs and ryfA sRNAs (the http: / / www.sanger.ac.uk). Studies prove sRNA played an important role in the regulation of bacterial metabolism, adaptation to the environment, quorum sensing, and bacterial virulence. Λ-Red recombination system In this study, using an improved built sRNAs lack sf301 8 mutant (referred sf301ΔsRNAs to) are 5 unknown function of sRNAs predicted three plasmid-encoded sRNAs and sf301 genome, and mutant the study of the physiological and biochemical characteristics. The results show that the the: 8 sRNA missing on cell metabolism and growth has little effect. Then the author evaluation the sf301ΔsRNAs sf301 virulence. The study by sf301ΔsRNAs with the wild-type strain HeLa cells competitive invasion force, invasive infection of mouse lungs competitive ability and guinea pig corneal infection experiments virulence evaluation research, validation the predicted sRNA genome have been reported but unknown function of the sRNA whether played an important role in the regulation of bacterial virulence play. The results show that: C0719 sRNA on the genome and IS061 sRNA deletion caused the a sf301 HeLa cell invasion force and enhanced mouse lung invasion force; the virulence plasmid forecast ln3 sRNA deletion makes sf301 HeLa cell invasion force and small The weakening of the rat lung invasion force. Comparing the group of proteins in the different states of bacteria is a very important aspect of the protein study, which includes with a bacteria group of proteins in the different culture conditions change, the proteomic comparison of the different strains of the same bacteria as well as the same strain of the mutant strain and the wild-type strain of the proteome of comparison. This study used proteomics techniques to compare the proteome sf301ΔsRNAs with sf301 Two-dimensional gel electrophoresis to establish a mutant of the wild strains of different growth stages and sRNAs dimensional electrophoresis profiles, and points of difference gel digestion and mass spectrometry identification. The results showed that: to compare eight mutant strains the 37 ℃ the late and sf301ΔIS061 of interim group of proteins, found that 31 kinds of proteins in mutant and wild-type strains, there is a difference, and functional analysis of these proteins. Lack of sRNAs and did not cause a significant impact on the protein expression of the Shigella, but we found that may affect the virulence of Shigella the protein OspC3 and may sRNA regulation mechanism protein OmpA, for the next study sRNA the regulation provides the conditions. In summary, this study predicted the six pCP301 sRNAs and validated; to successfully build eight sf301ΔsRNAs mutant and changes in biochemical properties, cellular and animal invasion assay revealed between sRNAs Shigella pathogenicity the relationship; proteomic techniques combined analysis of sRNAs regulation function. 8 sf301ΔsRNAs constructed in this study has laid the foundation for further study of the function of the sRNA.

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