The Genetic Diversity Of Spacer Sequences In CRISPR Loci And Function Analysis Of CRISPR/Cas System In Salmonella

Salmonella,a Gram-negative facultative anaerobic intracellular pathogen in Enterobacteriaceae,is a leading cause of foodborne disease,which leads to serious economic loss and major public health burden worldwide.In the epidemiological investigation of Salmonella,serotype identification and bacteria typing are both very important steps,so how to identify the serotypes of the isolates and subtype them accurately within a short period of time become a challenge in the epidemic surveillance and outbreak investigation systemCRISPR/Cas(Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated proteins)system is an adaptive immune system in prokaryotes.CRISPR loci comprise direct repeats separated by unique spacers,which are generally considered to be acquired from exogenous genetic material.Based on its adaptive feature,spacer sequences are developed to be used as a molecular marker to reflect the interaction between bacteria and foreign genetic material,which can be applied in bacterial typing and evolutionary studies.In this study,we analyzed the spacer diversity of 1,367 Salmonella isolates belonging to 13 serotypes,to explore the relationship between spacers and the collection background of the isolates,and establish the CRISPR typing method to study the evolutionary relationship of the isolates within the same serotype.Furthermore,we explored the potential function of CRISPR/Cas system in Salmonella1.Genetic diversity analysis of S.Pullorum and S.Enteritidis based on characterization and evolution of spacer sequences in CRISPR lociWe analyzed the genetic diversity of spacer sequences in 655 S.Pullorum isolates and 329 S.Enteritidis isolates.For S.Pullorum isolates,11 spacers were detected including 3 spacers in CRISPR1 locus and 8 spacers in CRISPR2 locus respectively.The 655 S.Pullorum isolates were divided into 20 Pullorum Sequence Types(PSTs)with PST7(74%,486/655)to be the predominant type.While in S.Enteritidis,22 spacers(10 spacers in CRISPR1 locus,12 spacers in CRISPR2 locus)were identified and divided the 319 isolates into 18 Enteritidis Sequence types(ESTs)with EST2(41%,136/329)to be the predominant type.To some extent,the composition of the spacer sequences in S.Pullorum can reflect the geographical collection information of the strains,while the composition of the spacer sequences in S.Enteritidis can distinguish the strains from different host sources.Furthermore,96 S.Pullorum isolates and 49 S.Enteritidis isolates were sequenced and subjected to Whole genome single nucleotide polymorphism typing(WGST).By comparing the results of two typing methods,they showed high consistency,which proved the feasibility of using CRISPR typing as an alternative tool to reveal the bacterial evolutionary characteristics.2.Genetic diversity analysis of S.Typhimurium and the monophasic variant S.4,[5],12:i:-based on characterization and evolution of spacer sequences in CRISPR lociWe analyzed the spacer diversity in 173 strains of S.Typhimurium(62 isolates)and its monophasic variants(111 isolates),where 67 spacers were detected including 31 in CRISPR1 locus and 36 in CRISPR2 locus,respectively.The CRISPR typing divided these isolates into 34 Typhimurium Sequence Types(TSTs)with TST4 to be the most frequent type,containing 79.3%of monophasic variants,and 12.9%of S.Typhimurium strains.Except for one TST4 isolate with a new ST type,the remaining 95 TST4 isolates belonged to the ST34,which is shared by isolates mainly from pig(76 isolates)and human(19 isolates).The TST4-ST34 S.Typhimurium and its monophasic variants shared by pig and human isolates provided that pig is the main reservoir for evolution of S.Typhimurium and its monophasic variants,which could be transmitted to human through the contaminated pork meat.The CRISPR typing also provided the information of specific TST types to S.Typhimurium(TST 9,13-34)and its monophasic variant(TST1,2,3,7,8,10,11,12)mainly belonged to Lineage II and Lineage IB-2,respectively.While TST4,TST5,and TST6 were shared by both serotypes.All of these findings indicated that S.Typhimurium and its monophasic variant are closely related in pig and human,but they are developing to two different directions with the times going on.In addition,only 7%(5/67)spacers were found to share high homology with Salmonella phages and plasmids.No novel spacer was detected in these isolates,indicating that the relative conservation characteristics of CRISPR/Cas system in S.Typhimurium and its monophasic variants.It is speculated that the CRISPR/Cas system in Salmonella doesn't have the ability to acquire new spacers any more.3.Genetic diversity of spacer sequences in different serotypes of SalmonellaBy analyzing the spacer diversity of 210 Salmonella isolates belonging to 9 serotypes(S.Meleagridis,S.Anatum,S.London,S.Agona,S.Indiana,S.Infantis,S.Hadar,S.Fillmore,S.Simi),we found perfect correspondence between the spacer contents and the serotypes.Except for the same CRISPR type shared by one S.Fillmore isolate and one S.Hadar,other CRISPR types showed perfect correspondence to the serotype.Therefore,spacers can not only be used for subtyping different isolates of the same Salmonella serotype,but also distinguish isolates belonging to different serotypes.Thus CRISPR typing has a good application prospect to identify the serotypes of the isolates and also differentiate these isolates in molecular typing level.4.Function exploration of the CRISPR/Cas system in SalmonellaThe CRISPR/Cas system is widely distributed in Salmonella with the highly conserved structure among different isolates,while few research was reported about its role in Salmonella.Depending on the analysis of spacer sequences in Salmonella,we found the diversity of spacer content was caused by the replication,deletion,and single-nucleotide mutations of spacers without acquisition of new spacers,so we speculated that the CRISPR/Cas system was silenced in Salmonella as that in E.coli.The expression of cas genes was detected to be very low in laboratory condition,but the increased expression can be detected when leuO was overexpressed.Comparison of the ability to resist against exogenous plasmids between cas gene deletion strains and wild type strain,the CRISPR/Cas system of Salmonella was not able to inhibit the invasion of exogenous genetic materials.In addition,the strain with all of the cas genes deleted showed no significant differences in fitness cost,infection to cells,and virulence in animals,when compared with the wild type strain.All of these findings demonstrated that the absence of the CRISPR/Cas system had no significant effect on the growth and infection ability of Salmonella.