Stx噬菌体突破宿主菌CRISPR免疫系统导致多重溶原的分子互作机制

The CRISPR (clustered regularly interspaced short palindromic repeats, CRISPR), is identified as an adaptive immune system against lytic phages, plasmids, and other extrachromosomal elements in Bacteria. However, the mechanism of interaction between Stx phages superinfection and the host CRISPR is still not clear, which prevent the effective control of pathogenic Shiga toxin-producing Escherichia coli (STEC). This proposal will focus on how the Stx phage breaking through the host CRISPR and realizing polylysogenization. Deletion mutation of CRISPR region including CRISPR loci, cas genes, or leader sequences, and some genes related to phage lysogenization will be constructed respectively as well as some insertion mutant with phage protospacer, PAM (Proto-spacer Adjacent Motif) sequences and integration site. Then the superinfection model of the mutant strains with Stx phage will be established, and the level of lysogenization will be evaluated. The transcription of immune related genes located in prophage of the polylysogens will also be analyzed. Thus the influence of CRISPR system on Stx phages superinfection will be determined. Furthermore, protein differential expression profiling of lysogens before and after CRISPR system mutation will be investigated by the comparative proteomics technique. Proteins interacted with the differential expressed proteins will be explored using co-immunoprecipitation, GST drop-down and confirmed using bacterial two hybrid methods. Once the assumptive interaction protein is found, the mutant of the gene encoded the protein will be constructed, and all the mutants will be investigated for the general biological characteristics and some pathogenic characteristics such as biofilm formation and cell adhesion, etc. Combined all the results, interaction pathways of the differential expressed proteins will be revealed and the molecular mechanism of the Stx phage superinfection against host CRISPR immune system will be elucidated. All these will be advantage to tracking STEC molecular evolution characteristic, revealing virulent variation mechanisms, preventing and controlling the emergence of new pathogens.

CRISPR是细菌的适应性免疫系统，可抵御裂解性噬菌体和质粒等外源基因入侵，但Stx噬菌体突破该系统导致多重溶原的机制尚不清楚,制约了有效防控STEC。本项目分别构建CRISPR位点、cas基因、前导序列、噬菌体溶原相关基因的缺失突变株，以及噬菌体前间隔序列、邻近基序、整合位点的插入突变株，建立Stx噬菌体超感染模型，评价各菌株的溶原水平及多重溶原菌的生物学特性，确定Stx噬菌体超感染与CRISPR相关性。基于比较蛋白质组学技术，获得CRISPR突变前后溶原株的蛋白差异表达谱。采用免疫共沉淀、GST下拉、细菌双杂交等方法，挖掘与差异表达蛋白互作的蛋白，并构建互作蛋白的基因突变株，验证蛋白通路所发挥的生物学活性。从而诠释差异表达蛋白的分子作用路径，阐明Stx噬菌体突破宿主CRISPR导致多重溶原的分子互作机制，为探寻STEC进化标记、解析毒力变异机制、防控新病原菌出现提供新的理论依据。

CRISPR-Cas系统是细菌的适应性免疫系统，可抵御噬菌体等外源基因入侵，但Stx噬菌体突破该系统导致超感染的机制、以及该系统调控溶原菌株的噬菌体释放和Stx毒素表达的机制尚不清楚，制约了有效防控STEC。本项目聚焦大肠杆菌及其CRISPR-Cas系统，分析Stx2噬菌体溶原前后大肠杆菌的蛋白表达差异，获得四种蛋白均与FeS合成亚单位相关，即NfuA、FdoH、SdhB和FtnA蛋白，其中NfuA可维持噬菌体溶原株的生长，且保障Stx2噬菌体溶原株的稳定。进一步解析CRISPR-Cas在Stx2噬菌体溶原中的作用，发现临床分离的APEC的CRISPR序列中含有更多的间隔序列，表明这些菌株在水平基因转移过程中获得了外源基因，因而可能获得了更多的毒力因子，CRISPR间隔序列的多少与菌株毒力因子正相关。那么CRISPR-Cas系统在Stx噬菌体溶原中发挥怎样的作用呢？首先构建H-NS基因缺失株以激活CRISPR-Cas功能，在hns缺失的大肠杆菌中， Cas基因转录水平明显上调，且有效抵御噬菌体感染及外源质粒入侵，表明hns缺失解除了CRISPR-Cas系统的抑制作用，活化了菌株的适应性免疫功能。激活的CRISPR-Cas系统能有效抑制Min27噬菌体的溶原和裂解性感染，同时外源CRISPR质粒转化hns缺失株的转化效率受到明显抑制，表明H-NS可介导调控CRISPR-Cas在抵抗噬菌体感染和携带相同间隔序列的外源质粒入侵中发挥重要作用。通过构建重组CRISPR质粒，获得携带重组CRISPR质粒的单一或双重溶原株。Stx噬菌体Min27单一溶原株在丝裂霉素诱导状态下，其上清中噬菌体的释放及Stx毒素表达均受到明显抑制，表明H-NS介导激活CRISPR系统，从而抑制含有相同间隔序列的溶原株噬菌体释放及毒素表达；而双重溶原株中，激活的CRISPR-Cas系统对噬菌体的诱导释放、Stx毒素转录水平及毒素释放的抑制更为明显。进一步研究发现，Cas3蛋白的过表达有助于增强溶原株的存活率，同时减少溶原株转换为裂解状态释放子代噬菌体；同时噬菌体溶原裂解相关基因可能参与CRISPR-Cas系统介导的对Stx噬菌体释放及毒素表达的抑制过程。研究结果阐明了STEC中CRISPR-Cas系统的部分分子调控机制，为探寻STEC进化标记、解析毒力变异机制、防控新病原菌出现提供新的理论依据。