CRISPR系统靶向自身基因对淋病奈瑟菌活性和致病性的影响及调控机制

项目名称： CRISPR系统靶向自身基因对淋病奈瑟菌活性和致病性的影响及调控机制

项目编号： No.81471906

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 李国才

作者单位： 扬州大学

项目金额： 80万元

中文摘要： 淋病奈瑟菌耐药性严重、感染率高且无可用疫苗，亟需全面理解其生物学特性与致病机制，以便采取准确的防治措施以消除淋病的危害。CRISPR是近年发现的细菌获得性免疫系统，可抵抗噬菌体或质粒等外源核酸对细菌的再次侵染。但有趣的是，我们前期研究发现淋病奈瑟菌CRISPR却可靶向自身染色体基因。本课题选取淋病奈瑟菌标准菌株和重要临床分离株为对象，通过同源重组技术敲除野生株的CRISPR系统，借此分析CRISPR系统对其靶基因表达的影响，以及由此导致的淋病奈瑟菌活性和致病性的变化；构建含CRISPR系统表达载体的菌株为供体菌，以含靶基因的菌株为受体菌，利用接合转化实验研究CRISPR系统的转移率，以及接合株中CRISPR系统和靶基因的碱基突变及核酸片段缺失，阐明CRISPR系统和自身靶基因间的相互调控机制。研究结果将加深对CRISPR系统结构与功能的认识，为淋病防治提供新的理论和实践依据。

中文关键词： 淋病奈瑟菌；CRISPR；自身免疫；活性；致病性

英文摘要： Neisseria gonorrhoeae causes gonorrhea that is the second most common bacterial sexually transmitted disease (STD). Infection of gonococci increases the risks of HIV transmission. No vaccine is available against gonorrhea and therapy depends still on antibiotics. However, widespread emergence of gonococci resisting to currently used antimicrobials threatens to herald an era of untreatable gonorrhea, worldwide. Therefore, comprehensive understanding gonococcal biological characteristics and pathogenesis is the key to prevent and control gonococcal infections. Clustered regularly interspaced palindromic repeats (CRISPR) is a recently discovered bacterial acquired immune system existing in the genomes of about 90% of archaea and about 40% of bacteria. In these loci, repeats are separated by spacers homologous to invading genetic elements, such as viruses (phages) and plasmids. Immunity is achieved by transcription of CRISPR spacers into small antisense CRISPR RNAs (crRNAs) that are ultimately responsible for the sequence specific identification and destruction of the invader. Our preliminary analysis indicates that there are CRISPR loci not only in the sequenced genomes of the 3 N. gonorrhoeae standard strains, but also in our collected clinical isolates. It is interesting that most of gonococcal CRISPR spacers can be homologous to genes in the genomes of the same bacterial strains. We postulate that the structures and functions of gonococcal CRISPR systems are in a state of flux. CRISPR can be acquired and spread through horizontal gene transfer, ascend when the targeted and destructed genes encoded materials are not beneficial for gonococcal infections (e.g., immunogens which stimulate human hosts to produce strong immune responses). However, gonococcal CRISPR loci can lose or become nonfunctional when the targeted genes are essential for adaptive evolution. To verify the hypothesis, this project plans to construct CRISPRs deletion mutants of some N. gonorrhoeae standard strains and clinical isolates by homologous recombination technology. The mutants will be used to analyze the effects of CRISPR system on the expression of the targeted genes and the resultant altering of gonococcal characteristics and pathogenesis. The recombinant gonococcal strains containing CRISPRs expression vector will be constructed as donors to conjugate with recipient strains that contain targeted genes. The rates of the CRISPR expression vectors transferring between the donors and the recipients are to be explored. We will also explore the variations and damages of CRISPRs and the targeted genes in the transconjugants. This is the first study on mechanisms of the interaction between gonococcal CRISPR systems and the targeted own genes. The research results will deepen our understanding of the structure and function of CRISPR systems, provide us with new directions and technical supports for the prevention and therapy of gonorrhea.

英文关键词： Neisseria gonorrhoeae;CRISPR;autoimmunity;biological properties;pathogenicities