The Roles Of Lipase GehB In Staphylococcus Aureus Infected Host Acute Wounds

Objective:Lipase Geh B,as an important pathogenic enzyme secreted by Staphylococcus aureus,plays an important role in the pathogenesis of many diseases.Acute wound is susceptible to bacterial infection due to skin defect.Staphylococcus aureus is one of the common isolated bacteria.Clinically,some acute wound infections caused by Staphylococcus aureus are featured by delayed healing.However,it is still unclear whether Geh B is involved in wound healing of S.aureus-infected host.In this study,Geh B polyclonal antibodies were prepared by using the expressed and purified Geh B recombinant proteins.Furthermore,a Geh B-knockout strain,complement strain and plasmid control strain was constructed,the Lpp recombinant proteins were prepared with S.aureus,and a acute wound murine model was createdby infecting with diverse S.aureus strains.In addition,the roles of Geh B in the acute wounds of S.aureus-infected mice were explored in vivo and in vitro.Methods:（a）Preparation of recombinant lipase Geh B and its polyclonal antibodies.Escherichia coli（E.coli）expression vector p ET28a-geh B was constructed,and Geh B-His recombinant proteins were expressed with the p ET28a-geh B-transformed E.coli BL21.The target proteins were purified using Ni-affinity chromatography.The polyclonal antibodies were prepared by immunizing mice with Geh B-His recombinant proteins.（b）Construction of Geh B-knockout S.aureus strain and the complementary strain.The p BT2-Δgeh B knockout plasmid was constructed and the target gene in S.aureus USA300 was knocked out by homologous recombination.The complementary strain was constructed using a p-geh B plasmid.The constructed strains were identified using Western blot.The Lpp-GST recombinant proteins were expressed and purified through the S.aureus expression system.（c）The role of S.aureus in reducing bacterial stimulation of the natural TLR2-dependent immune response by Geh B hydrolyzing lipoproteins.To measure the effects of bacteria and supernatant on the stimulation of macrophage responses,overnight S.aureus and culture cell-free supernatant was applied to the macrophages and the cytokine levels were determined by an ELISA kit.To determine whether Geh B-His treated Lpp-GST（Lpp-GST*）changes macrophage responses relative to wild-type Lpps,the macrophages were stimulated with lipidated Lpp-GST and Lpp-GST*,and the cell supernatants were collected and the cytokine levels were determined by ELISA kit.（d）The effect of Geh B on acute wound healing of S.aureus-infected mice.The wounds in center of the back of BALB/c and C57BL/6TLR2^-/-mice were createdand infected with S.aureus strains of interest.One group of infected mice was observed for wound healing,bacterial burden and weight,and the other group of infected mice skin wound tissues were collected and examined by histopathological assays.Results:In this study,the polyclonal antibodies against lipase Geh B were successfully prepared.Western blot confirmed the successful construction of Geh B-knockout strain USA300Δgeh B,complementary strain USA300Δgeh B/p-geh B and an empty p LI50plasmid-transformed USA300 served as negative control.The Lpp-GST recombinant proteins were prepared using S.aureus expression system.Additionally,it was confirmed at the cellular level that bacterial supernatant of Geh B-knockout strain stimulated higher level of inflammatory factor TNF-αin mouse mononuclear macrophages than that of the wild-type USA300 strain.Moreover,Geh B-treated Lpp-GST*significantly reduced the TNF-αexpression in RAW264.7 cells compared with lipidated Lpp-GST,suggesting that lipase Geh B can reduce natural TLR2-dependent immune response by inactivating Lpp.Finally,the animal experiments confirmed that Geh B is TLR2-dependent in delaying the healing of acute wounds in mice infected with Staphylococcus aureus.Significantly increased inflammatory cell recruitment and subcutaneous adipocytes and decreased bacterial load were observed in wound tissues of mice infected with S.aureus USA300Δgeh B strain compared with those challenged by wild-type USA300 and USA300Δgeh B/p-geh B strains.Conclusion:（a）S.aureus lipase Geh B can inactivate Lpps,reduce TLR2 receptor-mediated innate immune response,and decrease immune cell recruitment.（b）S.aureus Geh B may inhibit adipocyte differentiation by hydrolyzing triglycerides in lipid droplets in adipocytes.（c）Reduced leukocyte infiltration and limited adipocyte differentiation may promote bacterial survival and hinder S.aureus-caused cutaneous wound healing.The results of this study demonstrate that Geh B plays an important role as an immunomodulator in acute wound infection caused by S.aureus,and intervention of Geh B may be an effective strategy for the treatment of acute wound infection caused by S.aureus.