Innate Immunity and Leptospira Associated Renal Fibrosis：Genomic, Structural and Functional Mechanistic Study

Leptospirosis is a widespread zoonosis which occurs frequently in Taiwan. Kidneys are the preferential site where leptospira accumulates, in the interstitium and in renal tubule epithelial cells. Renal invasion may lead to tubulointerstitial nephritis responsible for acute or chronic renal failure. Our previous findings indicated that leptospiral outer membrane proteins (OMP) from pathogenic Leptospira shermani were specifically recognized by Toll-like receptors 2 (TLR2), induced activation of the NF- kappa B and MAP kinase p38 pathway, and thereby the up-regulation of proinflammatory cytokines/chemokines. We have demonstrated the stimulatory effect of Leptospira OMP on extracellular matrix (ECM) production by a TGF-beta1/Smad -dependent pathway in human proximal renal tubule cells and further implied the role of leptospira infection on the process of tubulointerstitial fibrosis. In this proposal, we intend to study further whether Leptospira OMP stimulate ECM production in the renal tubule cell is related to innate immunity pathway. Both TLR dependent and independent pathways (NLRs) will be examined. Furthermore, the structural and functional relationship between Leptospira OMP and host cell response in terms of ECM production will be examined by site-mutagenesis of a major MOP LipL32. Finally, the role of other OMPs that may be important to host response through prediction of newly disclosed genomic sequence of Leptospira shermani, a major serovar encountered in Taiwan, will be studied. Our study would be important in understanding the pathogen-host interaction that would induce renal fibrosis in the kidney epithelial cells and will further understand the pathogen derived protein in the pathogenesis of renal fibrosis in relation to innate immunity from structural and functional studies. Year 1. 1. to clarify whether Leptospira santorosai serovar Shermani OMP may induce ECM (Fibronectin, Collagen typeI and type IV, Proteoglycans, ECM degradation enzymes ex. MMP2, MMP7 and MMP9) accumulation in renal tubule cells. 2. to determine if the Leptospira santorosai serovar Shermani OMP-induced ECM production requires the interaction between TLRs and NLRs. Year 2. 1. Mutational expression of major OMP LipL32: N-terminal, intermediate, and C-terminal portions of the LipL32 2. Structure and functional study of LipL32: To define which subfragments of the major OMP LipL32 correspond to the interaction to innate immunity receptor and to the increase of ECM. Year 3. 1. To characterize putative surface-exposed Leptospira proteins in silico 2. To construct the recombinant plasmid for protein expression in E.coli and purification. 3. To examine cellular localization of putative surface-exposed proteins in Leptospira santarosai serovar Shermani 4. To assess the stimulating ability of putative surface-exposed proteins to ECM components 5. To evaluate whether putative surface-exposed proteins are present during infection Finally, this study would disclose important pathogen related OMP that would be pertinent to understanding the pathogenesis of renal fibrosis in Taiwan.

Project ID:PC10202-0249
External Project ID:NSC100-2314-B182-031-MY3