Protective Efficacy Of Humoral And CD4~+ T Cellular Immunity Induced By Staphylococcus Aureus MntC

Staphylococcus aureus?S.aureus?is an opportunistic pathogen,which can cause different types of diseases from mild skin infections to life-threatening sepsis worldwide.In the past,S.aureus-caused infection was commonly treated with antibiotics in clinical practice.Owing to the emergence and transmission of multidrug-resistant strains,antibiotic treatment shows a non-ideal effect.Developing an impactful immunotherapy especially vaccine against S.aureus infections,is increasingly encouraged and supported.S.aureus manganese transport protein C?MntC?has been found to be highly expressed on the surface of the cell membrane and proven to be conserved across the staphylococcal species,and thus could confer immune protection against S.aureus and S.epidermidis.Up to now,there are a number of examples shown both humoral immunity and CD4⁺T cell immunity activated obviously after S.aureus infection.However,specific antibodies and CD4⁺T cell-mediated immunoprotection induced by MntC of S.aureus remains unclear.In this study,mntc gene of S.aureus was amplified using PCR,and PCR products were inserted into the pET-28a vector.The recombinant plasmid was induced to expression in E.coli BL21?DE3?by IPTG,and the recombinant MntC protein were purified.The BALB/c mice were intramuscularly inoculated with 50?g MntC emulsified with equal-volume of complete Freund's adjuvant.The booster inoculation was given three weeks later with the same protein in incomplete Freund's adjuvant.The mice inoculated with PBS emulsified with equal-volume of adjuvant was as the negative control.Then,the immunized mice were infected by intraperitoneal injection with three strains of different capsular serotypes S.aureus Newman,Wood 46 or HLJ23-1.The survival rate of MntC-immunized mice was substantially increased compared to adjuvant control.In addition,we analyzed the IgG titer in the sera and cytokines in the CD4⁺T cells using ELISA and flow cytometry,and found that MntC-induced high-level of IgG in sera,and the IgG subtypes tended to be IgG1 and that CD4⁺T cell significantly proliferated and more polarized to Th1 and Th17 cells,producing high-level of IFN-?,IL-17A.Furthermore,we investigated the mechanism of anti-infection by opsonophagocytic killing assay,cytokine neutralization and passive immunization.MntC-specific sera effectively promoted phagocytosis of S.aureus by macrophages.Passive immunization with antisera obviously decreased bacterial CFUs in organs and the death rate by challenge,and antisera together with CD4⁺T cells conferred better protection.In the cytokine neutralization,neutralization of IL-17 alone eliminated the protective efficacy mediated by Mnt C,and neutralization of IFN-?had no effect on reduction of bacterial CFUs.Meanwhile the bacterial CFUs in organs of Th17 recipient mice was considerably lower than in IFN-?recipient mice.Finally we identified a CD4⁺T cell epitope that induces IL-17 high expression by bioinformatics prediction,protein structure analysis and cytokine detection,which is ₂₂₇KHKLKHLLVETSVD KKAMES₂₄₆46 in MntC.To sum up,the results demonstrate that both MntC specific antibodies and IL-17 are indispensable in protective immunity against S.aureus infection.