Leptospirosis is a worldwide zoonotic disease caused by the bacteria Leptospira interrogans that escapes host immune defenses by multiple immune evasion strategies. One immune evasion strategy involves the glycolytic enzyme LiGAPDH, which acts as a moonlighting protein by binding to C5a of the complement system. The aim of this study was to explore the structural basis for the LiGAPDH-C5a interaction using site-directed mutagenesis (L34A, L98A, K193A) and then expressing and purifying the proteins. Six LiGAPDH mutants were made (three single site and three double site mutants), expressed, and subsequently purified using IMAC and SEC. The single site mutants maintained solubility and structural stability, while the double site mutants had a decrease in solubility which suggests that there was a cumulative destabilization due to both hydrophobic and electrostatic interactions. Docking simulations demonstrated limited variation in the selected docking scores among the wild-type and single mutants, supporting a qualitative model in which the interaction is broad and largely electrostatic in nature. Comparative analysis with other pathogenic species suggested that similar GAPDH–C5a interaction features are conserved, supporting electrostatic complementarity as a potential immune evasion mechanism. These data improve our understanding of immune evasion by bacteria and build a strong basis for targeted therapeutic strategies for leptospirosis.
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