Molecular Mechanism Of CRISPR-Cas System Regulating Stx2 Phage Lysogenization And Biological Characteristic In Escherichia Coli

Strains of Shiga-like toxin(Stx)-producing Escherichia coli(STEC)are increasingly implicated as causative agents of human disease,including hemorrhagic colitis,hemolytic-uremic syndrome(HUS),and thrombotic thrombocytopenic purpura(TTP).STEC is characterized by a widely pathogenic Shiga toxin(Stx)gene.Shiga toxins from E.coli are classified in two major types: Stx1 and Stx2.Each group comprises several subtypes,with the Stx2 group being more pathogenic than the Stx1 group.Shiga toxin is produced by Shiga toxin-encoding genes(stx)which derived from Stx phage,moreover,Stx phage lysogenization mediated the enhancement of E.coli virulence has been a consensus.How the Stx phages escape the host immune system so that to realize lysogenizaiton remains unanswered.Clustered regularly interspaced short palindromic repeats(CRISPRs)-CRISPR associated(Cas)systems are part of an adaptive immune system in diverse bacteria and archaea,which played a vital role in resisting phage infection and proliferation.However,the mechanism of interactions between CRISPR-Cas system and Stx phage,as well as the effect of CRISPR-Cas on phage lysogenization and superinfection are still unclear.Therefore,prevention and control of high pathogenicity STEC is impeded.However,some CRISPR-Cas systems,such as the Escherichia coli type I-E system are not active under laboratory conditions,as the cas genes are silenced by the repressor protein H-NS.In such strains,a functional immune response against invading DNA is only observed in the absence of hns or in engineered strains that overexpress the CRISPR-Cas system.In this study,we focused on the E.coli CRISPR-Cas system and STEC strains which are derived from Stx phage lysogen.In order to explore the mechanism of interactions between CRISPR-Cas system and Stx phage single or double lysoge,and to prevent the emergence of virulent STEC,this study focused on the lysogenic Stx2 phage and E.coli CRISPR-Cas system.On the one hand,the CRISPR sequence diversity in the CRISPR-Cas system of clinical isolated E.coli strains were widely analyzed,and the sequence features as well as the the potential relationship between the CRISPR elements and the virulence genes were investigated.Moreover,potential application of the CRISPR spacer sequences in bacteria typing and classification were also involved.While on the other hand,the role of H-NS mutation-activated CRISPR-Cas system in modifying the effects of the Stx2 phage single or double lysogen,and in resisting lytic phage vB_EcoS_SH2 and lysogenic Stx2 phage infection were also observed.Through analyzing the role of CRISPR-Cas system in Stx phage superinfection,possible correlation between Stx phage double lysogenization and CRISPR-Cas system was determined,and interactions of the two components were elucidated.In order to explore the sequence diversity of CRISPR array in E.coli CRISPR-Cas system,CRISPR loci in E.coli K-12 and O157:H7 strains were screened and amplified by polymerase chain reaction.Results showed that all the strains carried two or more CRISPR loci,with different repeat and spacer number as well as distinct sequence.Based on the above data,CRISPR loci of 80 E.coli isolates obtained from chickens with avian pathogenic E.coli(APEC)or avian fecal commensal E.coli(AFEC)were also amplifiated and sequcenced.The strains were further analyzed for CRISPR loci and virulence factor genes to determine a possible association between their CRISPR elements and their potential virulence.After analysis of the spacer profile of CRISPR array in 52 APEC and 28 AFEC isolates,association between the spacer number and virulence factor as well as pathogenicity.As pathogenic strains,more spacers were identified in CRISPR arrays in APEC isolates,which may indicate that they acquired more foreign genes through horzontial gene transfer,and meanwhile,have more chance to get virulence factors.The results suggested that CRISPR spacers may have a positive correlation with the potential pathogenicity of the E.coli isolates.Further classification of the isolates was achieved by sorting them into nine CRISPR clusters based on their spacer profiles,which indicates a candidate typing method for E.coli.Whether the entensively exited CRISPR array and relevant CRISPR-Cas system confers immunity against bacteriophage infection and exogenous DNA invasion in E.coli remains unknown.In order to unveil this case,the hns mutant of the E.coli K-12 strain was obtained through Red recombination system.The Δhns strain revealed a significant upregulation of the transcriptions of cas genes than the wild-type strain.The hns mutant effectively inhibits foreign plasmids transformation and Min27 phage infection,nevertheless,no new spacer incorporation in the CRISPR array was detected.The results indicated that H-NS mutantion mediated CRISPR-Cas activation,which confers immunity against phage infection.To investigate the effect of CRISPR-Cas system on Stx2 phage lysogen,lysogen model and recombination Min27 phage,as well as engineered CRISPRs plasmids were constructed and correlational study were performed.Results showed that the H-NS mutation-mediated CRISPR-Cas activation may participate in regulating bacterial group behavior,and singificantly inhibits Min27 phage lysogenic and lytic infection.It was showed that the phage titer produced by the Min27 phage lysogen with the CRISPR plasmid carrying the anti-Min27 spacers was approximately 100-times lower than the Δhns lysogen with the control spacer.Moreover,Shiga toxin expression of the lysogen was blocked by the CRISPR-Cas System,resulting in less Vero cell death.It was demonstrated that the biological characteristic of Stx2 phage lysogen was affected by CRISPR-Cas system.To explore the interrelationship between different phages and CRISPR-Cas system,as well as to enrich the phage pool,a new lytic Escherichia coli phage,named vB_EcoS_SH2(SH2),was isolated.The phage plaque was shown as big,circular,transparent and neat.Morphological analysis by electron microscopy revealed that phage SH2 had an icosahedron head with 50 nm in diameter,and a systolic tail fiber with 8 nm width and 120 nm length.Genome sequencing and comparison revealed that the nucleic acid type of SH2 is dsDNA,and the DNA genome of SH2 is composed of 49,088 bp with a G+C content of 45%(GenBank accession number: KY985004).Phage morphology and BLASTp analysis revealed that SH2 belongs to the family of Caudovirales,Siphoviridae.The engineered CRISPR plasmids carrying spacers against SH2 phage and strains harboring the engineered CRISPR plasmids were generated,and experiments were performed.Results showed that the infection of lytic phage SH2 against K-12 strain was repressed by the CRISPR-Cas system,with higher bacterial cell survial rate.Furthermore,it showed that the CRISPR-Cas system showing different immunity against phage infection under different cell density.To unveil the mechanism of the interactions between CRISPR-Cas and Min27 double lysogen,recombination phage ΦMin27(ΔStx::Cat)and the subsequent double lysogen were generated,and the phages were found to be intergrated into the wrbA site of E.coli genome.The transcriptional level of cas genes in double lysogen with hns deleted were significantly upregulated when compared with single lysogen.The phage release and Shiga toxin expression of double lysogen harboring Min27 spacers were significantly inhibited.It was demonstrated that the dynamic change of lysis-lysogeny regulation genes CI,CII,Cro transcription level are critical for phage transition from lysogeny to lytic development.The CRISPR-Cas may regulate the phage release and toxin expression of the lysogen through affecting the lysis-lysogeny regulation pathway.Overexpression of Cas3 mediated great inhibition of Min27 phage multiplication in hns mutant.However,no resistance against phage infection was detected when the key domain of cas3 gene deleted in the hns mutant or wild-type strain.These results indicated that Cas3 is crucial for CRISPR-Cas immunity,and overexpression of Cas3 can result in enhancement of immunity against phage infection.In conclusion,the sequence diversity of CRISPR-Cas system and the molecular mechanism of Stx2 phage lysogenization were systematically studied in this study.Based on the H-NS mutation mediated CRISPR-Cas activation,mechanism of CRISPR-Cas targeting Min27 phage single and double lysogen through spacer matching was elucidated.The effect of CRISPR-Cas adaptive immune system on Shiga toxin production will provide new thought for prevention and control of STEC.