Immunological Activities Of The Mutant Strain With IsdA Gene Deleted In Staphylococcus Aureus

Staphylococcus aureus （S. aureus） is a Gram-positive bacterium and one of themost common organisms responsible for hospital-acquired bacterial infections. S.aureus depends on the expression of virulence factors and can lead to a number ofinfection, including skin infections, and life-threatening infectious diseases. Duringinfection, S. aureus secretes toxic hemolysins which cause the rupture of red bloodcells, resulting in the release of the primary Hb store in the body. This represents asignificant source of iron for the organism and not surprisingly, S. aureus has beenshown to utilize heme preferentially as an iron source which is necessary forestablishment and pathogenesis of S. aureus. Some studies suggest that ironregulatory factor IsdA not only intereacts with host heme but also can adhere toextracellular matrix proteins and envelope protein of human stratum corneum.Furthermore, IsdA can reduce the hydrophobicity of the bacterial cells making themresist the bactericidal action of fatty acids and peptides of human skin. In view of theimportant role of IsdA in colonization and invasion of S. aureus, the immunologicalstudy about coincubation between S. aureus isdA+/-and human epithelial cells wereprogressed.First, we constructed the S. aureus isdA deletion mutant through homologousrecombination. The upstream and downstream sequences of isdA and spc gene weregenerated by PCR from chromosomal DNA of S. aureus Newman and plasmid pGB2respectively. The homologous recombination vector pMAD isdA was constructedbased on shuttle vector pMAD （carrying the screening markers, thetemperature-sensitive replication origin, erythromycin resistance gene （erm）, andβ-lactase gene （bgaB））. Then S. aureus RN4220, a restriction deficient strain, wasused as the initial recipient for transformation and the modified vector waselectroporated into S. aureus Newman subsequently. The isdA deletion mutants wereselected by temperature sensitivity （incubated alternately at30°C and42°C）, antibiotic resistance and β-lactase activity. Results from genome PCR, real-timequantitative PCR and direct sequencing indicates that the isdA gene of the screenedmutants was deleted from chromosomal DNA of S. aureus Newman.Secondly, the human Hacat epithelial cells as a cell model were treated with S.aureus isdA+/+and isdA^-/-. The real-time PCR showed us that all S. aureus isdA+/-improve epithelial cell to secrete proimflammatory cytokines （IL-8, IL-6and TNF-α）,the chemokine MCP-1, cell surface receptors （TLR2and TLR4）, Ddectin-1and theantimicrobial peptides LL-37. Although the cytokines amount of epithelial cellstreated with isdA^-/-was lower than isdA+/+, epithelial cytokines levels of isdA^-/-groupwas higher than control group. Bisides, the ELISA results consistent with the resultsof quantitative PCR revealed that S. aureus isdA^-/-reduced the cytokines secreted byepithelial cells compared with the wild strains isdA+/+. Furthermore, the results weresupported by western blot which show that the expression levels of TLR2, TLR4,Ddectin-1and LL-37decrease in isdA^-/-group. In conclusion, the proinflammatorycytokines of epithelial cells treated with the deficient strains isdA^-/-dereased and thetranscription and protein levels were also reduced, indicated that the deletion mutantisdA^-/-invasion force declined.In this study, the invasion of S. aureus was affected by the gene knockout of thevirulence factor isdA which lay the foundation for the further study about S. aureusinfection mechanism, and develop a new way for the research of anti-S. aureus agentsand vaccine.