Detection Of CRISPR/Cas System Of Shigella And Analysis Its Relationship With Virulence Genes

Acute dysentery caused by Shigella spp. Its pathogenicity depends on the effects of the invasion and toxicity of products of virulence genes on the epithelial cells of intestinal mucosa. Some virulence genes existed on the plasmid or Pathogencity Island, so these genes could be transferred through HGT and results in higher toxitic strains. CRISPR/Cas system is a novel prokaryotic immunity-like system. As for CRISPR/Cas system plays an important role in the integration and acquisition of the genetic material, so this system achieves extensive attention. To data, research on CRISPR/Cas system of Shigella and its relationship with virulences genes is limited. ObjectiveThis study explores the distribution and characteration of CRISPR/Cas system of Shigella and analyzes the relationship between the CRISPR and the distribution of virulence genes. Method1. The primers were designed by Primer designing tool on line according to the sequence of S.sonnei 53 G, Ss046 and S.flexneri 2457 T.2. PCR and DNA sequencing were used for the detection of CRISPR and cas genes. The bioinformatics software was used to analyze the sequence of CRISPR and cas genes.3. The virulence genes of Shigella were detected by PCR. The relativity between the presence of CRISPR and virulence genes were analyzed byχ2 –test. Result1. The primers designed for CRISPR/Cas system of Shigella were specifically amplified and Insertion sequence of cas genes was also amplified.2. Three CRISPRs were identified among 59 Shigella strains. CRISPR1/Cas system is detected in 47 strains of Shigella and about 2/3 of them could be detected IS elements. The positive rate of CRISPR2 is 84.7%. CRISPR3 were present in almost all strains(49/59).3. The repeat sequences were degernated. A total of 33 distinct spacer sequences were identified. Analysis of CRISPR sequences show that CRISPR have little change which makes CRISPR poor geotyping markers. The spacers in CRISPR3 match the I-F cas genes and no cas gene was present around this region.4. The sequence of cas gene may influences the number of spacer sequence. IS elements were presented in some strains; IS600 was detected in cse2 gene; ISSfl2 was presented in cas6 e gene; IS629 was identified in cse1 gene.5. The positive rates of ipa H, ial, ipa BCD, ics A, ics P and vir A were 100%, 100%, 86.4%, 100%, 98.3% and 98.3%. The strong combination of virulence genes is ipa H+ial+ipa BCD+ics A+ics P which account for 84.7% of all types.6. No statistical significant difference was found in the ipa BCD gene between the CRISPR positive strains and CRISPR negative strains. There was no statistical significant difference between the number of spacer sequence of CRISPR and the ipa BCD genes. No statistical significant difference was found in the ipa BCD gene between the IS-present strains and IS-absent strains in the CRISPR1/Cas system. Conclusion1. Three CRISPR were identified among 59 Shigella strains and CRISPR were widely distributed in Shigella. CRISPR3 may be an anti-CRISPR I-F system.2. The sequence of cas gene influence the number of spacer sequence. IS elements were identified in cas genes in some strains.3. Some of repeat sequences found in the CRISPR were degenerated. Analysis of CRISPR sequences show that CRISPR have little change which makes CRISPR poor genotyping markers.4. The positive rates of virulence genes are high. Analysis of CRISPR did not reveal a potential link between their presence and the virulence genes of Shigella strains.