The Effect Of Staphylococcus Aureus PV-Leukocidin (PVL) On Polymorphonuclear Neutrophils

Objective: Staphylococcus aureus have been described as super bacteria which is an urgent problem in medical cases and public health. The strains producing Panton-Valentine leukocidin(PVL) which contribute to the virulence of MRSA , is widespread and cause a series of severe community associated infections. According to the clinical research, the strains producing Panton-Valentine leukocidin(PVL) has been associated with severe infections and high mortality rates, particularly in patients with necrotizing pneumonia. The significant characteristic of lung injury caused by PVL is that polymorphonuclear neutrophils (PMNs) collected in the surface of the airway, inducing the acute inflammatory response. Compelling epidemiological data point to PVL as an important virulence factor in S. aureus necroti- -zing infections, but the mechanism for inducing lung injury is not completely understood.Therefore, in the present study, we obtain recombinant PVL (rPVL) protein through highly efficient expression of recombinant expression vector. And in vitro, PVL has been shown to induce lysis of human PMNs. To explore the effect of rPVL on cytokine(IL-6, IL-8 and TNF-α) expression and the mechanism of signaling pathway in the development inflammatory mechanisms of severe pneumonia due to PVL expression. In addition, we used PMNs isolated from different species to evaluate the cytotoxic effect of PVL. All these studies may establish the basis for the further studies of its biological function and its pathogenicity in pneumonia and shed light on the treatment of PVL-associated Staphylococcus pneumonia.Methods: (1) rPVL-F and rPVL-S were induced and expressed from the recombinant plasmid , were respectively purified with affinity chroma- -tography (His-Bind Purification Kit) and detected the content of both of them.(2) PMNs isolated from normal peripheral blood were incubated by rPVL( 6nmol/L, 100nmol/L) protein for 6h, respectively.Trypan blue exclusion method were performed to observe the cell viability. The cell morpho- -logy were observed by light microscope and the microstructure of PMNs were tested by transmission electron microscopy.The rate of cell apoptosis / necrosis were detected by flow cytometry.(3) The cells were harvested for total RNA extraction and expression of IL-6, IL-8, TNF-α, TLR2, TLR4, CD14, CXCR4 and NF-κB p65 mRNA was assayed by RT-PCR. ELISA methods were performed to test the expression of IL-6, IL-8 and TNF-αin PMNs infected by rPVL. (4) PMNs isolated from human, New Zealand white rabbits, BALB/c mice and C57/BL6 mice were incubated by 20 nmol/L rPVL protein, respectively. Trypan blue exclusion method were performed to observe the cell viability. The cell morphology were observed by light microscope and the microstructure of PMNs were tested by transmission electron microscopy.The rate of cell apoptosis / necrosis were detected by flow cytometry.Results: (1) The genes of lukS-PV and lukF-PV are successfully cloned into plasmid pET28a. E. coli BL21 (DE3) containing recombinant vectors induced by IPTG causes soluble active protein express.The expression can be confirmed by SDS-PAGE and Western blotting. (2) rPVL can induce the typical apoptotic morphology in some PMNs and reduce the viability of the cells. Meanwhile, rPVL obviously promoted the rate of the apoptosis of the PMNs in vitro. Human, New Zealand white rabbits and BALB/c mice PMNs showed typical features of necrosis and apoptotic. The rates of the apoptosis and necrosis of PMNs in the rPVL treated group were significant higher than those in PBS group, respectively. However, Neither the typical apoptotic or necrosis morphology nor the change of rate of the cell apoptosis / necrosis were observed in the PMNs of C57/BL6 mice after treated with rPVL.(3) After treatment with rPVL, the level of IL-6, IL-8 and TNF from PMNs were significantly higher than the control group, and had marked concentration-dependent relations. rPVL was able to induce TLR2, TLR4, CD14 and CXCR4 mRNA expression.At the same time, rPVL could also significantly promote the NF-κB activity.Conclusion: (1) These results indicate that the recombinant plasmid (pET28a-LukF-PV and pET28a-LukS-PV) successfully expressed in E.coli respectively,which would provide a basis for analyzing the mole- -cular pathogenesis of PVL.(2) In vitro, PVL can induce PMNs death by necrosis or by apoptosis, depending on the PVL concentration. Our data clearly demonstrate that PVL acts differentially on PMNs of various species and suggests that PVL has an important cytotoxic role in human and rabbit PMNs.(3) rPVL was able to induce IL-6, IL-8 and TNF secretion and TLR2, TLR4 and NF-κB mRNA expression from PMNs. The directly induction of rPVL could promote the activation of NF-κB by TLR signal transduction pathway, then cause the expression of cytokine.