Essential Calcium-binding Cluster of Leptospira LipL32 Protein for Inflammatory Responses through the Toll-like Receptor 2 Pathway

Leptospirosis is the most widespread zoonosis caused by the pathogenic Leptospira worldwide. LipL32, a 32-kDa lipoprotein, is the most abundant protein on the outer membrane of Leptospira and has an atypical poly(Asp) motif (161DDDDDGDD168). The x-ray crystallographic structure of LipL32 revealed that the calcium-binding cluster of LipL32 includes several essential residues Asp132, Thr133, Asp164, Asp165, and Tyr178. The goals of this study were to determine possible roles of the Ca2+-binding cluster for the interaction of LipL32 and Toll-like receptor 2 (TLR2) in induced inflammatory responses of human kidney cells. Site-directed mutagenesis was employed to individually mutate Ca2+-binding residues of LipL32 to Ala, and their effects subsequently were observed. These mutations abolished primarily the structural integrity of the calcium-binding cluster in LipL32. The binding assay and atomic force microscopy analysis further demonstrated the decreased binding capability of LipL32 mutants to TLR2. Inflammatory responses induced by LipL32 variants, as determined by TLR2 pathway intermediates hCXCL8/IL-8, hCCL2/MCP-1, hMMP7, and hTNF-α, were also lessened. In conclusion, the calcium-binding cluster of LipL32 plays essential roles in presumably sustaining LipL32 conformation for its proper association with TLR2 to elicit inflammatory responses in human renal cells. Background: LipL32 induces a renal cell inflammatory response through the TLR2-signaling pathway. Results: Ca2+-binding LipL32 mutants showed attenuated TLR2-mediated inflammatory responses. Conclusion: The Ca2+-binding cluster of LipL32 is essential in regulating its interaction with TLR2 for subsequent inflammatory response induction. Significance: This investigation provides significant evidence for crucial roles of the Ca2+-binding cluster of LipL32 for pathogenesis via association with TLR2.