Regulatory Function Of CRISPR-Cas System On Streptococcus Agalactiae Virulence

Streptococcus agalacitae also known as group B streptococcus is a gram-positive human-animal-fish zoonotic bacterium,which cause neonatal meningitis in human,mastitis in dairy cattle and meningitis and septicemia in fish.In this decade,the threaten for the aquiculture industry is becoming more and more serious,the infection caused by S.agalacitae usually has the high mortality with greatly impacted the development of aquaculture industry in China.CRISPR-Cas system is broadly encoding in lots of bacteria and archaea,it's consisted of CRISPR(clustered regularly interspaced short palindromic repeats)locus and Cas(CRISPR-associated)protein.CRISPR-Cas system is an adaptive bacterial immune system which helps defending exogenous nucleic acids elements.However,more and more researches indicated that beyond the immune function CRISPR-Cas system also regulates endogenous genes expression and impact the virulence in several pathogens.In order to investigate the function of CRISPR-Cas system in S.agalactiae,several CRISPR-Cas system associated gene deletion mutants were constructed by using homologus recombination method,furthermore,RNA-seq and dRNA-seq were used to reveal the transcriptional character and the relationship between Type ?-A CRISPR-Cas system and bacterial virulence,this study provides essential theoretical knowledge for the further research of Type ?-A CRISPR-Cas system in S.agalactiae or other pathogens.1.The impact of CRISPR-Cas system on bacterial virulence in S.agalactiaeThe CRISPRfinder analysis shows that S.agalactiae GD201008-001 strain harbors a Type ?-A CRISPR-Cas system in its genome,this system is consists of a CRISPR locus contains 8 spacers and 9 repeats,and 4 different cas gene as cas9,cas1,cas2 and csn2.To investigate the function of those elements of system,cas1,cas2,csn2,CRISPR,all cas genes and CRISPR-Cas system deletion mutants were constructed.Cas9 deletion mutant and complementary strain were constructed in previous study.All deletion had no impact on the growth of bacteria in a rich nutrition environment,however,when growth under the stress environments,those strains performed very differently.When under an oxidation environment,all gene deletion mutants exhibited an enhanced growth speed compared to wild type,but cas9,csn2,cas and CRISPR-Cas deletion mutants were more sensitive to acid environment.And all deletion mutants were more fragile to the DNA damage environment except the cas1 deletion strain.The deletion of cas9,csn2,cas and CRISPR-Cas system also repressed the adhesion and invasion ability to mice brain microvascular endothelium cell,but the deletion of cas2 only repress the adhesin capability but had no impact the bacteria invading the endothelium cells.Besides that,deletion of cas9,cas,CRISPR-Cas also depressed the anti-phagocytosis ability and repressed the intraocular survival rate in macrophages.The mice infection model showed that cas9,CRISPR,cas and CRISPR-Cas deletion mutants showed significantly decrease bacterial virulence,especially the CRISPR mutants only killed 10%of infected mice compared to 100%mortality of wild type infection.And the zebrafish model also showed the LD50 in zebrafish of cas9 mutant and CRISPR mutant were 79-fold and 154-fold higher than wild type.The results of mice meningitis analysis model revealed that the deletion of CRISPR weakened the bacteria breach the blood brain barrier,therefor showed a lower level of brain infection ability.2.The RNA-seq analysis of cas9 and CRISPR deletion mutantsTo better understand how cas9 and CRISPR impact the bacteria virulence,RNA-seq analysis was used to compare the difference between cas9,CRISPR mutants and wild type strain.The analysis showed in cas9 deletion there're 100 gene showed more than 1-fold changes on transcription level when compared to wild type,47 genes of these were down regulated and 53 were up regulated.The GO analysis showed the function of most genes riched in catalytically activity molecules,and in CRISPR mutant the different expressed gene with a-fold changes were 849,477 genes were up regulated after the CRISPR deletion and 372 genes were down regulated.Those genes' function mostly riched in transferase and carbohydrate ramification metabolism.when narrowed down the gene expression changes from 1-fold changes to 2-fold changes.There're 29 genes were found in cas9 deletion mutants,20 up regulated genes and 9 genes were down regulated.And in CRISPR mutant,236 genes showed at least 2-fold changes compare to wild type strain,159 of them were up regulated and 77 genes were down regulated.Interestingly,26 genes were overlapped between cas9 and CRISPR mutants which means those 26 genes probably were regulated by both cas9 and CRISPR.When looked at the 29 differently expressed genes in cas9 mutant,among all 20 down regulated genes,16 of them were located in prophage lambdaSa04.LambdaSa04 is the only prophage encoded in GD201008-001 strain,it is 29.3kb long and contains 28 conding genes.For the 9 up regulated genes in cas9 mutant,RNA-RNA interaction analysis was performed to study the potential connection between those RNAs and crRNA.Among all those genes,only transcriptional regulator regR mRNA could partly complemented with the spacers of CRISPR,and the RNA stability assay showed that Cas9 had negative impact on the stability of regR mRNA.In the list of CRISPR regulated genes,27 of 159 up-regulated genes were regulator genes,the MEME analysis revealed that there's a highly conserved motif in all those regulator genes.This 21nt-long motif can partly complement with the CRISPR repeats.3.The impact of cas9 regulated genes on bacterial virulenceThe RNA-seq data showed that in Acas9 16 of 20 down-regulated genes located in prophage lambdaSa04 and regR genes was the only one up-regulated genes that showed potential interaction possibility with crRNA.To investigate whether the repressed bacterial virulence in Acas9 were caused by lambdaSa04 and regR,lambdaSa04 deletion mutant and regR associated gene modified strains were construct.The mice infection model showed deletion of prophage lambdaSa04 couldn't impact the bacterial virulence,neither in lethality nor bacteai burden in the tissues.The analysis on regR showed it could repress the activity of hyaluronidase which is an important virulence factor of S.agalactiae,and the deletion of regR in cas9 mutant also increased 10%lethality in mice and recovered the bacteria burden in blood and brain,indicating that the Cas9 could repress the regR to increase the activity of hyaluronidase which could contribute to bacterial virulence.4.The impact of CRISPR regulated genes on bacterial virulenceThe previously RNA-seq data showed that,in S.agalactiae GD201008-001 the deletion of CRISPR locus caused 236 genes significantly up or down regulated,interestingly,among all those genes there're 27 genes were transcriptional regulator genes including the up regulated two components system CovR/S(control of virulence).CovR/S system negatively regulated bacterial virulence in some S.agalactiae strains were already well studied.In order to verify whether the up regulated covR/S could also repress the virulence in GD201008-001,covR/S genes were deleted in the wild type and CRISPR mutant background.The deletion of covR/S caused the clone changed the color from white to orange,meanwhile,the hemolytic activity was also significantly increased in strains lacking of covR/S.The RT-PCR results showed deletion of covR/S also up regulated the expression of ?-hemolysin coding gene cylE.However,the virulence analysis resulted indicated that the deletion of covR/S didn't enhance the virulence but lightly decreased.Compared to wild type strain covR/S deletion mutants also caused all the infected mice dead but it cost 20 more hours,and the bacteria burden in spleen,blood and brain also lower than wild type.Therefore,the decreased virulence in CRISPR mutant was not because of up regulated covR/S.The proinflammatory cytokine transcriptional level results showed that the CRISPR deletion mutant trigerd increased level of IL-6 in spleen and brain of infected mice,however the covR/S mutant repressed the cytokine.In the CRISPR regulated candidate genes there's a lipoprotein gene sag0671 and the RT-PCR revealed that this gene was not regulated by covR/S.The mice macrophage cytokine transcriptional level assay indicated that the enhanced IL-6 by CRISPR mutant was primarily depended on this lipoprotein and the TLR2 played a very important role in this process.5.The transcriptional character of Type ?-A CRISPR-Cas system in S.agalactiaeGD201008-001 strain has a very close relationship with human isolate A909,most genes in those two strains are highly conserved,and the identity of their genomes were over 99%.S.agalactiae A909 shared the completely same cas gene and CRISPR repeats with GD201008-001.To investigate the transcription and processing of Type ?-A CRISPR-Cas system in S.agalactiae,dRNA-seq was performed on A909.After analysis,1597 TSS were predicted for 2134 coding sequence,and 51.3%TSS were belong to primary TSS(pTSS).And the MEME analysis for the promotor region showed in 63.7%promotor region had the conserved ?70 binding site TATAATA,howere,the classical TTGACA-35 box was only found in 10.1%promotor regions.The data of CRISPR-Cas system indicated that all cas gene formed an operon that shared two TSS located at the upstream of cas9 gene.All mature crRNAs were 42nt long and were processed from the same transcript,and the promotor for CRISPR locus was between csn2 and the first repeats.On the reverse strand of the cas9 stream,there was a sRNA which can partly complemented with the crRNA repeats,this sRNA is the tracrRNA.The precursor tracrRNA were 171nt or 90nt and the precursor tracrRNA can bind with pro-crRNA and then processed into mature tracrRNA and crRNA.The results of Northern blot revealed that,in both A909 and GD201008-001 the deletion of cas9 had a very strong impact on the stability of crRNA,and the deletion of CRISPR could impact the process of precursor tracrRNA casue the richment of precursor tracrRNA in cell.The CoIP showed the Cas9 can strongly bind crRNA and tracrRNA form a complex,but in CRISPR mutant,because lacking of mature tracrRNA and crRNAs,cas9 can substituent binding precursor tracrRNA which might have a regulatory function.The deletion of tracrRNA in CRISPR mutant background significantly enhanced the virulence and the RT-PCR also showed tracrRNA regulated some genes,like cpsB,cpsJ,cpsC and covR/S which were considered as CRISPR regulated genes.In conclusion,the CRISPR-Cas systems share highly conserved transcriptional characters in S.agalactaie A909 stranin and GD201008-001 strain.The transcription of crRNA is independent of cas operon.The mature crRNAs contain spacers and partly repeats which are processed from precursor crRNA.The tracrRNA locates at the upstream of cas9 gene and encoded on the reverse strand,which contains an anti-repeats sequence that can hybrid with crRNA and form a double strand RNA stracture.In S.agalactiae,Cas9 plays very important role in maintaining the stability of crRNA and tracrRNA,which can also bind with them to form a complex molecular.crRNA involves in the processing and maturation of tracrRNA.The deletion of crRNA will cause the enrichment of precursor tracrRNA and increase the chance of binding Cas9,which represses various endogeneous genes in bacteria and causes the decreased virulence.