The Function And Regulatory Mechanism Of Type Ⅲ-A CRISPR-Cas System In Staphylococcus Aureus

Staphylococcus aureus(S.aureus)is a Gram-positive bacterium that is widely dispersed in nature and is considered as an important clinical and food-borne pathogen.S.aureus commonly causes servere infectious diseases in humans and animals,including skin and soft tissue infections,pneumonia,sepsis,and arthritis.Livestock products can serve as a source of S.aureus zoonotic infections,and handling or consuming contaminated food can potentially result in its transmission to humans,thus causing food poisoning.Therefore,regular surveillance of S.aureus in food can effectively reduce the risk and incidence of foodborne diseases.CRISPR-Cas system is an adaptive immune system in prokaryotes that defends against the invasion of foreign nucleic acids,such as plasmids and phages(viruses).It is estimated that approximately 42%of bacteria and 85%of archaea harbor CRISPR-Cas systems.Based on the composition of cas genes,CRISPR-Cas systems are classified into two classes,six types(Ⅰ-Ⅵ)with 50 subtypes.Most CRISPR-Cas systems identified in S.aurers belong to the Type Ⅲ-A system.Until now,the Type Ⅲ-A CRISPR-Cas system is not prevalent in S.aureus,with only 0.94%of clinical strains reported to harbor this system.However,the characterization,functional activity and transcriptional regulation mechanism of this system have not been elucidated in S.aureus.In this study,we examined the prevalence and molecular characterization of S.aureus isolated from retail chicken meat in China,and screened for CRISPR-Cas system-positive strains from chicken and human-derived isolates.We used the clinical Methicillin-resistant Staphylococcus aureus(MRSA)strain TZ0912 as a model to investigate the functional activity and regulatory mechanism of the Type Ⅲ-A CRISPR-Cas system.1.Identification of Type Ⅲ-A CRISPR-Cas system in S.aureusA total of 507 samples were randomly collected from retail chicken meat in Chongqing,Huaian and Yangzhou between July 2016 and November 2016.We identified S.aureus isolates from the samples and performed antimicrobial susceptibility testing and molecular typing of S.aureus isolates.A total of 104 S.aureus isolates were obtained,including eight MRS A isolates,at a MRS A detection rate of 1.6%.Antimicrobial susceptibility testing revealed that 91.4%of the isolates were resistant to penicillin,followed by 64.4%to tetracycline and 53.9%to erythromycin.spa typing showed that t127 and t091 were the predominant spa types among the 104 S.aureus isolates,while ST88 was the most common clone among the eight MRSA isolates.In addition,SCCmec typing indicated that most MRS A isolates carried SCCmec Ⅳa mobile genetic element.We also screened for CRISPR-Cas system-positive strains from the chicken and human-derived isolates.In the present study,we identified seven strains harbor a Type Ⅲ-A CRISPR-Cas system with a frequency of 3.3%.Among these strains,only one strain(named TZ0912)was isolated from human and was identified as MRSA,while the remaining six strains were isolated from chicken meat.The 16s rDNA analysis further suggested that these six strains were Staphylococcus argenteus of ST2250,which is closely related to S.aureus,as previously reported.Finally,we analyzed all available whole genome sequences of S.aureus from the NCBI database.We identified 35 S.aureus genomes carrying the complete Type Ⅲ-A CRISPR-Cas system.Most of the strains were isolated from human,and the CRISPR-Cas system was prevalent in S.aureus CC8 clones.2.Molecular characterization and functional activity of Type Ⅲ-A CRISPR-Cas system in S.aureusWe used the clinical MRSA TZ0912 strain as a model and performed whole genome sequencing analysis.The results showed that the TZ0912 strain harbors a complete TypeⅢ-A CRISPR-Cas system,which is located within a mobile genetic element:the SCCmec Ⅴresistance cassette,indicating that the CRISPR-Cas system could be horizontally transferred among Staphylococcus isolates.We compared the efficiency of transformation of the targeted plasmid pCR1SP1,pCR1SP6 and pCR1SP14 in the wild type and ACRISPR Acas strains.The results showed that the Type Ⅲ-A CRISPR-Cas system in the TZ0912 strain could effectively eliminate the targeted plasmid,with the highest efficiency for pCR1SP1 and the lowest efficiency for pCR1SP14,indicating that the efficiency depended on the location of the targeting spacer within the CRISPR array.The RNA-seq results further suggested that the crRNA expression showed a gradient decline,with the highest abundance for leader-proximal crRNAs and the lowest abundance for leader-distal crRNAs.During phage philPLA-RODI infection,the Type Ⅲ-A CRISPR-Cas system in the TZ0912 strain provided immunity against phage attack only when the spacer targeted the early genes expressed by phage,whereas it could not protect TZ0912 from phage attack when the spacer targeted the middle/late genes expressed by phage.Taken together,the Type Ⅲ-A CRISPR-Cas system in TZ0912 is active and can defend against the invasion of foreign nucleic acids.3.Screening and identification of transcriptional regulators of Type Ⅲ-A CRISPR-Cas system in S.aureusUsing P-galactosidase assays,RNA-seq and qRT-PCR,we identified that the leader sequence(termed Pleader)located before the CRISPR 1 array contained a promoter for the CRISPR 1 array,and the 298th to 597th nucleotide sequence(termed Pcas)located within the cas1 gene served as the promoter for the cas gene cluster.To identify the direct transcriptional regulators of the Type Ⅲ-A CRISPR-Cas system,we performed DNA pull-down assays with the Pleader and Pcas sequence as a bait,respectively.Mass spectrometry analysis showed that SarA and ArcR were captured by both Pleader and Pcas sequences in the DNA pull-down assays.By measuring cas genes expression,we found that SarA inhibited the transcription of CRISPR 1,but promoted cas genes expression.In comparison,ArcR acts as a positive regulator of CRISPR 1 and cas genes.EMSA results further confirmed that SarA and ArcR can specifically bind to Pleader and Pcas sequences,suggesting that SarA and ArcR directly regulate Type Ⅲ-A CRISPR-Cas expression.The results of plasmid invasion and phage infection assays showed that SarA and AcrR could activate the interference activity of the Type Ⅲ-A CRISPR-Cas system.Subsequently,we identified the specific binding sites for SarA and ArcR in the Pleader and Pcas sequences by EMSA.Our results showed that SarA preferentially binds to AT-rich sequences in the Pleader and Pcas sequences,and that ArcR can specifically bind to Pleader-26+24 and Pcas-156-127 sequences.The above results demonstrate that the two regulators SarA and ArcR can directly initiate the transcription of cas genes and crRNAs,and thus promoting the interference activity of the Type Ⅲ-A CRISPR-Cas system against invasive plasmids or phages.4.Regulatory mechanism of quorum sensing on the Type Ⅲ-A CRISPR-Cas system in S.aureusIn Gram-negative bacteria,quorum sensing(QS)can positively regulate the CRISPR-Cas expression and activity.However,it is currently unknown whether QS regulates CRISPR-Cas systems in Gram-positive bacteria.Therefore,we knocked out the QS agr system encoding genes(agrACDB)and measured the cas genes expression by qRT-PCR.The results suggest that the agr system inhibits Type Ⅲ-A CRISPR-Cas expression.We further determined that the loss of the agr system global regulator AgrA had the same effect on cas genes expression as the deletion of the entire agr system,suggesting that AgrA mediates QS regulation of the Type Ⅲ-A CRISPR-Cas system.EMSA results showed that AgrA could not bind to the Pleader and Pcas sequences,indicating that AgrA indirectly inhibits Type Ⅲ-A CRISPR-Cas expression.The results of plasmid invasion and phage infection assays indicated that AgrA could inhibit the interference activity of Type Ⅲ-A CRISPR-Cas system.Furthermore,we confirmed that AgrA directly represses the expression of Type Ⅲ-A CRISPR-Cas system positive regulators SarA and ArcR using qRT-PCR and EMSA.Finally,we found that AgrA-mediated cas genes repression depended on SarA and ArcR.Together with the results of plasmid invasion and phage infection assays,we confirmed that AgrA represses Type Ⅲ-A CRISPR-Cas expression and interference activity by downregulating SarA and ArcR regulators in S.aureus.