The Functional Study Of Staphylococcus Aureus Virulence Factor PSM-a Acted On Neutrophils

Staphylococcus aureu（sS. aureus）is one of the most important zoonosis pathogens, and it isalso the most common pathogen in clinical. It can cause a wide variety of infections, includingsepsis. The highly pathogenic of S. aureus is mainly associated with the expression of thevirulence factors. In order to develop a new method for the treatment of S. aureus, manyresearchers focus the study on the virulence factors of S. aureus. Recently, an extracellularsecreted protein, called Phenol-soluble Modulin (PSM), has been discovered. It is another majorvirulence factor of S. aureus. PSM is mainly constituted by PSM-α and PSM-β. PSM-α is madeup by the four genes PSM-α1, PSM-α2, PSM-α3and PSM-α4, while PSM-β is constituted by thegenes PSM-β1and PSM-β2. Researchers have found that PSM-α can strongly lysis the immunecells and can cause bacteremia in mice.In this study, we connected the four genes of PSM-α through a linker, and cloned the targetfragment which enzyme digestion result was accurate into the expression vector pGEX-4T-1toconstruct the recombinant expression plasmid pGEX-PSM-α. The expression plasmidpGEX-PSM-α was then transformed into E.coli DE3for expressing soluble protein by changingthe induced conditions. The expressed protein was confirmed by SDS-PAGE and Western blotanalysis with mouse-specific monoclonal antibody to GST. The soluble protein was purifiedthrough Glutathione Sepharose4B affinity chromatography and digested by thrombin to obtainhigh purified target protein PSM-α.BCA method was used to assay the concentrated target protein. Different concentrations ofthe target protein were co-cultured with human neutrophils in order to study the fuctionalchanges of neutrophils when treated with PSM-α in vitro. Using the reduction assay of NBT, wedetected the expression of NADPH oxidase in neutrophil at1hour after treated with therecombinant protein at100ng/ml,1μg/ml and10μg/ml. Results showed that the recombinantprotein at100ng/ml didn’t affect the expression of NADPH oxidase signigicantly, while boththe recombinant protein at1μg/ml and10μg/ml could activate the NADPH oxidase inneutrophil significantly. The results of flow cytometry to detect the respiratory burst showed that the concentrations of PSM-α at1μg/ml and10μg/ml could increase the respiratory burst inneutrophil significantly, while the protein PSM-α at100ng/ml had no significant effect onrespiratory burst. In addition, we detected the expression of surface adhesion molecule CD11band CD18in neutrophil by flow cytometry and found that the recombinant protein PSM-α cansignificantly increase the expression of CD11b and CD18with the concentration at1μg/ml and10μg/ml, but the protein at100ng/ml couldn’t improve the expression of CD11b and CD18distinctly. We also used real-time quantitative PCR and ELISA methods to detect the secretionof cytokines IL-1β, IL-8and TNF-α at the level of transcription and post-transcription,respectively. Both of the results showed that only the expression of IL-8but not IL-1β andTNF-α was increased significantly after treated with the recombinant protein at100ng/ml,1μg/ml and10μg/ml. In conclusion, PSM-α could cause inflammation and tissue damage throughactivating the NADPH oxidase, stimulating the respiratory burst, increasing the expression ofCD11b and CD18and inducing the release of inflammatory cytokines IL-8in neutrophils.In this study, we obtained the PSM-α protein using tandem fusion expression technology.Study the immune mechanism of PSM-α acted on neutrophils can provide the basis for clinicaldiagnose and treatment of the infection caused by S. aureus and lay the foundation formanufacturing the vaccine against S. aureus which targeted the virulence factor PSM-α.