Study On The Function And Mechanism Of CRISPR-Cas System Regulating Virulence In Salmonella From Chicken

Salmonella is recognized as one of the most common microbial pathogens worldwide.The bacterium contains the clustered regularly interspaced short palindromic repeats（CRISPR）-CRISPR-associated（Cas）systems,providing adaptive immunity against invading foreign nucleic acids.Previous studies suggested that certain bacteria employ their CRISPR-Cas systems to target their own genes,which also alter bacterial physiology and the virulence to mammals.However,whether CRISPR-Cas systems in Salmonella have similar functions in virulence and host cells infection remains unknown.Here,we detected the expression of key virulence genes of 150 Salmonella strains to briefly evaluate the level of pathogenicity,and then screened out the highly pathogenic strain carrying CRISPR-Cas system by detecting the virulence phenotypes.Then the changes of virulence phenotypes because of cas3gene deletion were analyzed and cas3-related genes were screened in the whole genome.To understand the correlation and regulation mechanism between cas3 gene and virulence in bacterial CRISPR-Cas system,this study will open a deep understanding of the regulation of virulence phenotypes of Salmonella and other bacteria infected mammal and provide theoretical basis for the prevention and treatment of Salmonella infection.Detecting the CRISPR1 and CRISPR2 locus in 150 Salmonella strains from chicken by PCR technique to analyze the distribution of CRISPR locus.It was found that the detection percentage of CRISPR1 locus was 78%,that of CRISPR2 locus was80.67%,while the proportion of CRISPR-carried Salmonella strains was 92.67%,and only 11 strains was not detected this CRISPR locus.It was shown that the strains carrying CRISPR-Cas system is majority in Salmonella,which may be results from long-term evolution in Salmonella.Next,the expression of 23 key virulence genes in these strains was detected by q RT-PCR including stn,spv R,spv B,spv C,prg H,hil A,avr A,ttr C,ssa Q,mgt C,sop B,rpo S,bcf C,mis L,fim A,flj B,lpf C,sod C1,rck,pef A,tra T,sop E1and pag C,compared with the standard strain CVCC541.It was found that the genes expression of S.Typhimurium,S.Enteritidis and S.Anatis were at a high level among these strains.Then both macrophages RAW264.7 and intestinal epithelial cell IPEC-J2 were used to identify the adhesion and invasion of Salmonella to the host cells after 3-hours infection.Then,according to the serotype and cell infection,9Salmonella strains carrying CRISPR-Cas system were selected for chicken infection to detect their animal pathogenicity,including 6 strains with high virulence in host cells numbered respectively as 211,64,92,94 and 76,2 strains with medium adhesion and invasion to host cells marked respectively as 201 and 62,one strain with strong virulence to RAW264.7 but no adhesion and invasion to IPEC-J2 was also selected.Among them,2 strains of S.Enteritidis,4 strains of S.Typhimurium,2 strains of S.Anatis and one strain of S.Indiana.After intramuscular injection,the fatality rate of strain No.172 was 33.33%,and that of the all other strains was 100%.Whereas chicken infected by Salmonella strains orally or secondly,fatality rates of 9Salmonella strains carrying the system to chickens infected were range at 0%-16.67%and 0%-100%,respectively.Of which,No.201 strain had the strongest lethality to chicken,the mortalities in orally infection and secondary infection respectively were16.67%and 100%.Next to that,No.211 strain,the mortalities were 0%and 80%,respectively.Finally,considering that S.Enteritidis No.211 strain had strong cell infection ability,we selected it for further study in cas3 gene-related function and the regulation of other genes,and called the strain as cas3 WT.The cas3 gene deletion mutantΔcas3 was constructed by twice homologous recombination using suicide plasmid p LP12;The p BAD33-CM recombinant expression plasmid of cas3 gene was constructed by seamless cloning,which was transferred intoΔcas3 mutant to obtain cas3 gene complementary strainΔcas3/p BAD33-CM-cas3（named asΔcas3/p-cas3）.The effects of cas3 gene on bacterial virulence phenotype were investigated by measuring the growth ability of cas3 WT,Δcas3 andΔcas3/p-cas3,the biofilm,the adhesion and invasion to host cells,the mortality of cells and animals and so on.The results showed that there was no significant difference in the growth rate among the three strains,and the differences of other virulence phenotypes could be analyzed appropriately.The deletion of cas3 gene led to significantly changes of certain virulence phenotypes of Salmonella,such as the decreased biofilm formation,the decreased bacterial count in host cells and the increased host cell survivability.In addition,the medium lethal dose(LD₅₀)of the three strains was calculated through the chicken infection test,the results showed that the LD₅₀ ofΔcas3 was significantly higher than that of both cas3 WT andΔcas3/p-cas3,demonstrating that the deletion of cas3 gene significantly weakened the pathogenicity of Salmonella.Above results suggested that cas3 gene has a certain effect on the virulence of Salmonella.Using next-generation sequencing（NGS）,based on Illumination Hi Seq sequencing platform,to sequence the constructed library by paired-end（PE）sequencing,and then the differentially expressed genes（DEGs）inΔcas3 compared with cas3 WT were screened,and the relationship between cas3 gene and these DEGs was analyzed.RNA-seq results showed 141 DEGs,of which 81 genes were down-regulated significantly inΔcas3,while 60 genes were significantly up-regulated.Furthermore,type III secretion system（T3SS）-related genes sip A,sip C,sip D,spt P,sop E,sop E2 encoded by Salmonella pathogenicity island（SPI-1）were down-regulated,which were mapped to the pathway“Salmonella infection and invasion of host cells”.Also,quorum sensingrelated genes lsr B,lsr G,lsr E and lsr F were upregulated,and flagellum and biofilm related genes saf A,saf B,saf C,saf D and bdm were significantly changed.Overall,cas3 may enhance the virulence of Salmonella by regulating the gene expression of quorum sensing,SPI-1-T3SS and flagellum and so on.In summary,first,based on the bacterial serotypes,the expression of virulence genes,cell infection and animal pathogenicity,we screened out a highly pathogenic Salmonella strains carrying CRISPR-Cas system.And then,constructing the cas3gene,the core gene of CRSISPR-Cas system,deletion mutant and complementary strain.Finally,the functions of CRISPR-Cas system in bacterial virulence and how it works were studied.It was found that the CRISPR-Cas system in Salmonella might use its core protein Cas3 to regulate the expression of virulence-related genes,and then enhance the biofilm formation,cell adhesion and invasion,host cell lethality and animal pathogenicity of bacteria.It provides a border scopes and deeper level in the mechanism of Salmonella virulence and is very important for the prevention and cure of Salmonella infection in clinical practice.