The Role Of Staphylococcus Aureus Virulence Factor PVL, SpA And Coa In Osteomyelits

Background and Objective: Osteomyelitis is a common and frequently-occurringorthopedics, which is acute or chronic inflammation of bone, periosteum and surroundingtissues that caused by bacteria. Staphylococcus aureus is the major pathogen of osteomyelitis,accounting for 80% of all cases of osteomyelitis. The pathogenicity caused by Staphylococcusaureus is a complex process, and the strength of its pathogenicity depends on toxins andenzymes. Panton-Valentine leucocidin(PVL) is a unique exotoxin that secreted byStaphylococcus aureus, belonging to two-component toxin family and composed of theLukS-PV and LukF-PV. PVL kills the whilte cells through forming the pores on the cellmembrane. Many studies showed that PVL plays an important role in necrotic skin lesionsand necrotic pneumonia caused by Staphylococcus aureus. Staphylococcal protein A(SpA) isa surface protein of cell wall of Staphylococcus aureus, which could bind to a variety of Fc ofmammalian IgG, having variety of biological activity including anti-phagocytosis, promotingcell division, hypersensitivity, and the damage to platelets. Coagulase(Coa) is an importantvirulence factor of Staphylococcus aureus, which could protect against pathogens not beswallowed and antibody binding effect. Although PVL, SpA and Coa has been proved to beimportant pathogenic factors of Staphylococcus aureus, but their role in osteomyelitisgenerated from bone infections caused by Staphylococcus aureus remains unclear. Therefore,in this study we investigated the effects of PVL, SpA and Coa on the proliferation, apoptosis,metabolism and differentiation of osteoblast, and receptor activator for nuclear factor-κBligand(RANK-L) by constructing overexpression plasmids and gene deletion plasmids ofStaphylococcus aureus virulence factors PVL, SpA and Coa combined with a variety ofdetection methods to explore the role of Staphylococcus aureus virulence factors PVL, SpAand Coa in osteomyelitis and possible regulation mechanisms.Methods: 1) The recombinant plasmids of PVL, SpA and Coa were constructed usingRT-PCR and digestion-ligation cloning method, and their gene deletion plasmids wereconstructed by homologous recombination. The gene deletion plasmids were transfected intoStaphylococcus aureus with the electroporation method, and the PVL, SpA and Coa deletionmutants of Staphylococcus aureus were screened using the temperature and pressure oferythromycin-resistant. 2) The influence of PVL, SpA and Coa on the proliferation andapoptosis of osteoblast were observed using MTT assay and Annexin V-FITC / PI doublestaining flow cytometry, and their effect on the activities of caspase-3 and caspase-9 weredetermined using western blotting. 3) MC3T3-E1 cells were infected with Staphylococcusaureus with overexpression plasmids or gene deletion plasmids of PVL, SpA and Coa, and theexpression levels of collagen, OPN and OC affected by PVL, SpA and Coa were observedusing RT-PCR. 4) The situation of mineralized of nodules was observed using alizarin red andvon kossa, and the activity of alkaline phosphatase(ALP) was determined using ALP assaykit. 5) MC3T3-E1 cells were infected with Staphylococcus aureus with overexpressionplasmids or gene deletion plasmids of PVL, SpA and Coa, and the expression of RANK-Lwere determined using ELISA.Results: 1) The recombinant plasmids of Staphylococcus aureus virulence factors PVL,SpA and Coa pMD19-T-PVL, pMD19-T-SpA and pMD19-T-Coa and their gene deletionplasmids pMAD △ PVL, pMAD △ SpA and pMAD △ Coa were successfully constructedusing recombinant DNA technology and homologous recombination, and the PVL, SpA andCoa gene deletion mutants PVL(-), SpA(-) and Coa(-) were successfully obtained. 2) 24 hafter infection, the proliferation inhibition rates of MC3T3-E1 cells infected withStaphylococcus aureus containing the recombinant plasmids of PVL, SpA and Coa were29.2%, 42.8% and 32.7%, which increased compared with the group infected withStaphylococcus aureus(28.2%), while the proliferation inhibition rates of MC3T3-E1 cellsinfected with Staphylococcus aureus containing the gene deletion plasmids of PVL, SpA andCoa decreased compared with the group infected with Staphylococcus aureus(12.3%,12.1%and 17.2%, respectively). With longer duration of infection, the inhibitory effects of thegroups infected with Staphylococcus aureus with recombinant plasmids significantlyincreased, while the inhibitory effects of the groups infected with Staphylococcus aureuscontaining gene deletion plasmids significantly decreased. Compared with the group infectedwith control Staphylococcus aureus, the apoptosis rate of MC3T3-E1 cells infected withStaphylococcus aureus with the recombinant plasmids of PVL, SpA and Coa was significantlyimproved, and the apoptosis rate of MC3T3-E1 cells infected with Staphylococcus aureuscontaining the gene deletion plasmids of PVL, SpA and Coa slightly decreased. Theexpression of pro-caspase-3 significantly decreased in cells infected with Staphylococcusaureus containing the recombinant plasmids of PVL, SpA and Coa(P<0.05), and theexpression of cleaved-caspase-3 markedly increased. The expression of pro-caspase-3 slightlyincreased in cells infected with Staphylococcus aureus containing the gene deletion plasmidsof PVL, SpA and Coa(P<0.05), and the expression of cleaved-caspase-3 slightly declined,but there was no statistically significance between two groups. The expression levels ofpro-caspase-9 and cleaved-caspase-9 are consistent with the expression levels ofpro-caspase-3 and cleaved-caspase-9. 3) The mRNA levels of collagen I, OPN and OC wereinhibited by Staphylococcus aureus virulence factor PVL, SpA and Coa. 4) Compared withthe uninfected group, the precipitated calcium in the cells infected with Staphylococcusaureus containing the recombinant plasmids of PVL, SpA and Coa was significantly reduced(P<0.05), and the activity of ALP was also significantly reduced(P<0.05). Compared withthe group with Staphylococcus aureus infection, the precipitated calcium in the cells infectedwith Staphylococcus aureus containing the overexpression plasmids of PVL, SpA and Coawas also significantly reduced(P<0.05), and the activity of ALP was also significantlyreduced(P<0.05); 5) The expression of RANK-L in the uninfected group is very low.Compared with the uninfected group, the expression of RANK-L significantly increased inthe cells infected with Staphylococcus aureus containing the recombinant plasmids or genedeletion plasmids of PVL, SpA and Coa, with statistical significance(P<0.05). Comparedwith the medium-infected group, the expression of RANK-L in the Staphylococcus aureusinfected group significantly increased(P <0.05).Conclusion: 1) Staphylococcus aureus virulence factor PVL, SpA and Coa could inhibitthe proliferation of MC3T3-E1 cells in time-dependent manner, and could promote theapoptosis of the cells. PVL, SpA and Coa may induced the apoptosis of MC3T3-E1 cellsthrough the mitochondrial pathway. 2) PVL, SpA and Coa could inhibit the mRNA levels ofcollagen I, OPN and OC, which affect the normal metabolism of osteoblasts. 3) PVL, SpAand Coa could inhibit the mineralization of osteoblasts and the activity of ALP, which affectthe differentiation of osteoblasts. 4) PVL, SpA and Coa could promote the expression ofRANK-L. Taken together, the worse degree of osteomyelitis may be increased by PVL, SpAand Coa through affecting the process of bone formation and bone resorption, breaking thebalance of bone metabolism, which lead to osteoporosis.