The Genetic Polymorphism Of CRISPR System And Its Functions Of Immune And Regulation In Shigella

Shigella is the causative agent of shigellosis, with nearly 167 million cases and more than one million deaths annually worldwide, mostly of children less than 5 years of age. It is prevalent in less developed countries because of poorer sanitation conditions. Four species or groups of Shigella were recognized and categorized during the 1940 s. Each can be further subdivided into one or more serotypes based on the O-antigen structures of the membrane-associated lipopolysaccharide: S. dysenteriae(17 serotypes), S. flexneri(16 serotypes), S. boydii(18 serotypes), and S. sonnei(one serotype). Shigella was thought to be closely related to Escherichia coli, and in recent years, increasing evidence has confirmed the relatedness of these two species. Phylogenetic studies have suggested that Shigella forms a single pathovar of E. coli.The clustered regularly interspaced short palindromic repeats(CRISPR) structure was first described in E. coli in 1987. It is characterized by 24–47-bp DNA direct repeats, separated by variable 21–72bp sequences called spacers. Spacers are thought mainly to derive from invading DNA, and often have a specific arrangement in different bacteria. A “leader sequence” and cas(CRISPR-associated sequence) genes are often located adjacent to the CRISPR locus, and encode the proteins required for CRISPR function. Before their physiological role was understood, CRISPR-Cas systems were found to be a useful tool for typing bacterial diversity. Subtyping methods based on analyses of the spacers of the CRISPR loci have been developed for Mycobacterium tuberculosis, Yersinia pestis and Salmonella. Two decades after its discovery, CRISPR was defined as an adaptive RNA-mediated immune system that imparts sequence-specific immunity against mobile genetic elements such as bacteriophage and plasmids. With the deepening of the study, it is found that it not only has the immune function, but also has regulatory functions in bacteria. Mounting evidence shows that different CRISPR-Cas systems can modulate various processes in bacteria, such as the genetic regulation of group behavior and virulence.The objective of this study is to analyze the genetic polymorphism of CRISPR system in Shigella, including the CRISPR locus and its adjacent CRISPR-Cas proteins, to test if it is suitable for CRISPR to be a target for the rapid detection and molecular typing of Shigella. Based on the full understanding of the genetic polymorphism of CRISPR in Shigella, we aim to study and verify its immune function and then explore whether the CRISPR system in Shigella have regulation function.To analyze the polymorphism of CRISPR in Shigella, we amplified and sequenced six CRISPR loci within 237 strains belonging to the four Shigella species and 13 E. coli strains. Through the above analysis, we have a full understanding of the genetic polymorphism of CRISPR system in the different types of Shigella. The results also showed that CRISPR in Shigella has special replacement characteristics of R-S units and a special hairpin structure at the terminal direct repeat. There are some differences in CRISPR between different subtypes of Shigella and the differences are conserved in the corresponding subtypes. We also found that CRISPR-CRf locus is specific in Shigella. So we established a new method for the rapid detection and molecular typing of Shigella based on the above results.By compare the sequences of CRISPR loci and Cas proteins in Shigella and E. coli, we found the coexistence of most CRISPR loci between these two species. The coexistence of CRISPR loci is an evidence of the evolutionary relatedness of Shigella species and E. coli. To investigate this evolutionary relatedness further, phylogenetic analysis of the CRISPR loci of Shigella subtypes and E. coli was conducted. The results show that the evolutionary distance between different subtypes of Shigella and E. coli is different. This suggests that although the different types of Shigella strains maintain a convergent evolution at the phenotypic level, the generally recognized subgroups of Shigella do not represent natural groupings. We also found the evidences of CRIPSR’s conventional vertical inheritance and horizontal transfer. All the results above suggest that CRISPR can be used as a tool for the evolution analysis of Shigella and other species.Through the amplification and comparative analysis of the Cas protein gene clusters of Shigella and E. coli, we found that the CRISPR system in Shigella belongs to I-E type according to the classification of CRISPR. Two insertion sequences, IS600 and ISSfl2, are inserted into the S. sonnei Cas protein gene cluster. In order to observe if the CRISPR system in S. sonnei has the normal immune function, we knock the two insertion sequences out with the method of traceless knockout. The HNS gene, which inhibits the function of CRISPR, was knocked out too. Then, with the target sequences in it, we transformed the plasmids into the wild type and mutant strains. The results show that the mutant strains, which restored the integrated structure of CRISPR, have the shearing force to the target plasmid while the strains with insertion sequence in CRISPR do not have the force. The outcomes showed that the presence of the insertion sequence affects the normal immune function of the CRISPR system. This result for the first time confirmed that the insertion sequence can silence the immune function of CRISPR system.On the basis of the recovery of the immune function of CRISPR, we studied the regulation function of CRISPR system. We have performed homology analysis to detect the targets of CRISPR, the result show that one spacer showed similarity to the glgP gene in the chromosomal sequence of S. sonnei. To explore the self-target of CRISPR system in S. sonnei, we analyzed the expression of glgP gene, which is the self-target of CRISPR in wild and mutant strains and the expression level of this gene was decreased in the mutation strain △ISD-HNS. And then we analyzed the mutant strains at phenotypic levels at first. The results showed the mutation strain △ISD, which restored the complete structure of the CRISPR, has a decreasing growth rate. After abolishing the inhibitory effect of the HNS gene, the mutation strain △ISD-HNS’s growth rate decreased more significantly. At the same time, we also analyzed the biochemical index of the mutation strains and found that indicators related to growth and metabolism declined in the mutation strains with complete CRISPR structure. All of these results showed that the CRISPR system had a certain inhibitory effect on the growth of Shigella after the recovery of its structure though the incomplete matching between spacer and target sequence, suggesting the regulation mode of CRISPR system in bacteria. In order to further understand the change in mutant strains at protein level, we used comparative proteomics based on iTRAQ technology. The results showed mutant strain △ISD has more kinds of proteins down regulated than others. By the assay of GO cluster analysis and KEGG pathway analysis, we know that most of the different proteins were related to the bacteria energy metabolism. It proved that the recovery of the CRISPR system structure in S. sonnei affected many kinds of protein gene expression and confirmed the correlation between CRISPR system and metabolic regulation in Shigella.In summery, through a comprehensive and systematic analysis, we have a full understanding of the CRISPR system in Shigella. Our findings confirmed that the CRISPR in Shigella can be used as a target for the rapid detection and molecular typing. The phylogenetic analysis of the CRISPR loci of Shigella subtypes and E. coli showed that the generally recognized subgroups of Shigella do not represent natural groupings. It is also proved that the CRISPR system can be used as a tool to study the evolutionary relationship between Shigella, Escherichia coli and other bacteria. By restoring the complete structure of CRISPR in Shigella, the normal immune function was verified. Analysis of phenotype and comparative proteomics of mutant strains suggest the regulatory function of CRISPR in Shigella, which provides the direction of the investigation on the regulatory function of CRISPR system in bacteria.