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

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.

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。

为研究淋病奈瑟菌CRISPR系统中间区序列靶向自身染色体基因的生物学意义，阐明CRISPR系统和自身靶基因间的相互调控机制，将WHO-A株进行基因组测序，其序列提交CRISPRFinder软件在线分析，发现2个Cas基因CT和TM、P1~P6和CL等7个CRISPR簇，另手工分析及实验验证发现CRISPR簇FA1。按间区序列-靶基因间存在≧18 bp匹配序列及匹配序列唯一靶基因上游的原则作进一步筛查，获得8个CRISPR簇的3个自身靶基因，即前噬菌体蛋白基因NGFG_01062、假定蛋白基因NGK_0072和菌毛相关蛋白基因NGK_2578，均可在pET-GFP质粒进行靶蛋白-GFP的融合表达。RT-PCR检测发现CRISPR簇CL可转录形成前体crRNA；转录组测序发现存在与CRISPR簇CL部分序列完全匹配的转录产物。用CRISPR/Cas9系统in vitro实验以重组大肠埃希菌分析淋病奈瑟菌CRISPR簇对自身靶基因的作用，合成CRISPR簇间区序列并插入到pCas9构建重组质粒pCas9-S，导入含靶基因表达载体pET-Targets-GFP的大肠埃希菌。随着重组菌在LB培养液中的传代，间区序列对靶基因表现出抑制效果，靶基因的转录和蛋白表达水平也明显下降。间区序列FAS1可抑制靶基因的转录从而降低目的蛋白的表达量。从总RNA中用RT-PCR可扩增出与CT或TM基因大小一致的DNA条带；WHO-A株细菌超声裂解液包被后以自制多抗可以检测到相应Cas蛋白CT或TM的表达；WHO-A株细菌细胞固定后用CT或TM抗体可检测到特异性荧光。将pCas9中的Cas9基因替换为CT或TM基因，插入Kan基因特异性sgRNA，未发现重组载体pCT/TM-KRn对pET-30a中Kan基因的抑制作用，但pET-30a向其转化的效率下降，提示WHO-Cas蛋白发挥一定作用，但作用机制可能与Cas9蛋白的核酸酶活性具有较大差别。pET30-Target对含重组载体pCas9-S大肠埃希菌的转化率均下降；克隆WHO-A株CRISPR簇CL及其潜在启动子Leader，与pCas9共转化大肠埃希菌，靶基因载体对该重组菌的转化效率下降。以上结果提示WHO-A株CRISPR系统中含Leader、CRISPR簇及Cas蛋白，可发挥对自身基因的编辑作用以及对水平基因转移的抑制作用。

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