Study On SigB Gene Knockout And Its Function Of Staphylococcus Aureus Based On CRISPR/Cas9 Technology

Staphylococcus aureus is an important human pathogen that can cause a variety of serious diseases such as necrotizing pneumonia,endocarditis and toxic shock syndrome,thus seriously endangering human health.The?~B protein encoded via the sigB was considered to be a regulatory factor that could combine with RNA polymerase to form RNA polymerase holoenzyme(RNAP)to activate the expression of multiple stress-related genes when S.aureus encounters stress.The sigB gene is essential for the survival of S.aureus,so it has the potential to become a new antibacterial drug target.The current research mainly reveals that sigB plays an important role in stress resistance of bacterial such as acid resistance,heat resistance,and antioxidant.There were few reports on whether sigB was involved in other physiological regulation such as synthesis of pathogenic factors,induction of quorum induction,and virulence expression.Therefore,it urgently requires further research on sigB in S.aureus to analyze the other functions of sigB.Gene editing technology has been used as a key method to research gene function,drug resistance pathogenesis,and screen antibacterial targets.However,traditional gene editing in S.aureus has problems such as time-consuming,labor-intensive and inefficient.Currently,there is lack of an efficient gene editing technology in S.aureus.CRISPR-Cas9 was a new generation of gene editing technology after Zinc finger nucleases(ZFNs)and transcription activator-like effector nucleases(TALENs).With its advantages of flexibility,operability as well as efficiency,it has been widely used in various research fields.This study was constructed sigB gene knockout system in S.aureus via CRISPR-Cas9 gene editing technology,and constructed the corresponding sigB complement strains through the p LI50 plasmid,then revealed the functions of sigB via studying the differences in the biological characteristics of each genotype strain,it provides basic theoretical support for further research on sigB as an antibacterial target.In this study,CRISPR-Cas9 expression vector pCasSA1 targeting the sigB was constructed based on the pCasSA plasmid using the isoschizomer-heterotail restriction endonuclease one-step cloning method,then,homologous arm donors for homologous recombination repair were synthesized via overlap extension PCR,next,the donors were subcloned into the pCasSA1 plasmid via enzyme digestion and T4 ligation to obtain the pCasSA2 plasmid.Subsequently,pCasSA2 was transferred into S.aureus RN4220 strain for plasmid modification to obtain pCasSA3 plasmid.Finally,the pCasSA3 was transferred into the S.aureus Newman for sigB knockout to obtain mutant strain S.aureus?sigB-Newman.In the rescue experiment,the single-copy integration plasmid p LI50 was used to construct sigB complemented strain.First,the sigB fragment was subcloned into the p LI50 plasmid,and then modified via S.aureus?sigB-RN4220.Subsequently,the modified plasmid was transferred into S.aureus?sigB-Newman to obtain complemented strain?sigB/sigB-Newman.Next,we investigated the phenotypic differences in growth,biofilm formation,hemolytic ability,induce autolysis ability,acid and alkali resistance of wild strains Newman,sigB-deficient strain,and sigB complemented strain as well as combined with the relative expression data on related genes such as biofilm,hemolysis,virulence factors,pathogenic factors,and quorum sensing at different growth stages.Finally,the sigB function was analyzed as follows:1.sigB could improve the biofilm formation and hemolytic ability of S.aureus,and it also involved in regulating bacterial virulence expression.2.sigB could inhibit the autolysis of S.aureus induced via Triton X-100.3.sigB was involved in the regulation of S.aureus acid resistance mechanism,but no obvious evidence that it was involved in the S.aureus alkali resistance mechanism.4.sigB could fully up-regulated the expression of virulence factors,adhesion factors,quorum sensing,and biofilm-related genes during the logarithmic growth plateau stage in S.aureus.This study demonstrated that targeted inactivation of sigB could reduce negative factors such as virulence expression,biofilm formation,and pathogenicity in the later stages of bacterial growth.It provides correct ideas and theoretical support for studying sigB as an antibacterial target for new drugs.In addition,the sigB knockout system via CRISPR/Cas9 of S.aureus and its complement system constructed in this study lay the foundation for the study of sigB functions in different S.aureus,it also provides a technical platform for further research on other gene functions,drug resistance mechanisms,pathogenic infection mechanisms and development of attenuated vaccines in S.aureus.