Subcutaneous GAPDH vaccine in mice induces a proficient innate immune response.

Background: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on the surface of , coded with gapC, is a glycolytic enzyme that was reported to be a moonlighting protein and virulence factor.

Objective: This study assessed GAPDH as a potential immunization candidate protein to prevent streptococcus infections.

Methods: Mice were vaccinated subcutaneously with recombinant GAPDH and challenged with . They were then evaluated using histological methods. rGAPDH of mouse bone marrow-derived dendritic cells (BMDCs) was evaluated using immunoblotting, reverse transcription quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay methods.

Results: Vaccination with rGAPDH improved the survival rates and decreased the bacterial burdens in the mammary glands compared to the control group. The mechanism by which rGAPDH vaccination protects against was investigated. experiments showed that rGAPDH boosted the generation of interleukin-10 and tumor necrosis factor-α. Treatment of BMDCs with TAK-242, a toll-like receptor 4 inhibitor, or C29, a toll-like receptor 2 inhibitor, reduced cytokines substantially, suggesting that rGAPDH may be a potential ligand for both TLR2 and TLR4. Subsequent investigations showed that rGAPDH may activate the phosphorylation of MAPKs and nuclear factor-κB.

Conclusions: GAPDH is a promising immunization candidate protein for targeting virulence and enhancing immune-mediated protection. Further investigations are warranted to understand the mechanisms underlying the activation of BMDCs by rGAPDH in a TLR2- and TLR4-dependent manner and the regulation of inflammatory cytokines contributing to mastitis pathogenesis.