Identification And Functional Study Of Type Ⅲ-A CRISPR-Cas System In Clinical Isolates Of Staphylococcus Aureus

Staphylococcus aureus is an important human pathogen,which can cause a variety of infectious diseases.Under pressure from host and the environment,S.aureus evolves constantly to increases its fitness,so that it can withstand the bactericidal factors from eukaryotic immune system and the environment.One of the main adaptations is to acquire virulence and antibiotic resistance genes from mobile genetic elements such as plasmids,transposons,and phages via lateral gene transfer.The CRISPR-Cas(clustered regularly interspaced short palindromic repeats[CRISPR]-CRISPR associated proteins[Cas])system can provide prokaryotes with immunity against invading mobile genetic elements(MGEs)such as phages and plasmids.To date,only a few S.aureus strains containing CRISPR-Cas system have been identified,but no functional study in these strains was reported.In Staphylococcus epidermidis RP62a,which is a close relative of S.aureus,the immunity function of typeⅢ-A CRISPR-Cas system was verified.Furthermore,some type Ⅰ and type Ⅱ systems were found to be involved in the regulation of bacterial physiology and virulence.The distribution of CRISPR-Cas system in Chinese clinical isolates,the immunity and other potential functions of S.aureus CRISPR-Cas system had not been reported before this study.This dissertation mainly consists of three parts:Firstly,we designed degenerate primers of four cas gene for PCR screening based on three reported CRISPR-Cas system.As a result,six strains containing type Ⅲ-A CRISPR-Cas system from about six hundred clinical strains of S.aureus were obtained,and subsequently next generation sequencing was performed.Five of the six newly identified strains have similar CRISPR sequences with known strains,but the MLST and spa typing results showed their far phylogenetic distance,which suggests that CRISPR-Cas system may spread via lateral transfer apart from vertical transmission.Among the reported and five newly identified MRSA strains,the CRISPR-Cas loci are near their SCCmec elements,which implies the site for CRISPR-Cas system integration may be near the SCCmec insertion site.Analysis of CRISPR sequences revealed that repeat of staphylococcal type Ⅲ-A CRISPR-Cas system is highly conserved,and the homologous sequences of spacers are mostly found in the template strand of lytic region in staphylococcal phages.Furthermore,comparison of the six strains revealed that the number of resistance and virulence related genes in AH1 with the least spacers is much greater than those in other five strains,this may be associated with the CRISPR-Cas system for its function in preventing MGEs transference.At the same time,we investigate the immunity function and mechanism of typeⅢ-A CRISPR-Cas system in strain AH1.Plasmid challenge assays indicated that S.aureus CRISPR-Cas system can prevent transfer of plasmid with homologous sequences to spacers in a transcription-dependent manner.The recognition of target needs sequence complementarity between crRNA and target RNA,in which the base paring at 5’ end of is much more important than that at 3’ end.The immunity function of CRISPR-Cas system also depends on Cas proteins,among which the endonuclease Cas6 and CSM components are involved.Furthermore,to investigated the unclassical function of CRISPR-Cas system in clinical isolates of S.aureus,we performed RNA-seq analyses on the wild type and CRISPR knockout strains of AH1,AH3 and SH3.The high throughput data revealed that their CRISPRs have affected different genes.As to strain AH3,knockout of CRISPR caused changes in transcription of gene clusters related to nitrogen metabolism and virulence.Results from Real-time PCR had confirmed the changes of virulence genes spa and sspABC,as well as regulartor sarS at transcription level.Through above studies,we have verified the immunity function of CRISPR-Cas system in clinical isolates of S.aureus.Target recognition was found to be dependent on the complementarity between crRNA and target RNA,while Cas6 and CSM complex were proved to be necessarry for immunity function.As to unconventional function,we found that CRISPR-Cas system can modulate the expression of virulence genes,but the mechanism needs to be further investigated.