Leptospirosis Renal Disease Virulent Factors---From Bacterial Genome, Protein Structural Biology to Animal Model Verification

Leptospirosis is a most common zoonotic disease worldwide, particularly in tropical and sub-tropical regions where flooding frequently occurs. In Taiwan, the Leptospirosis occurred in 2003 after the typhoon Morakot, with 203 confirmed cases. Leptospira infection induces tubulointerstitial nephritis and acute kidney injury. When Leptospira infects animals, kidneys are the preferential site where Leptospira accumulates, in the interstitium and also in renal tubule epithelial cells. The mechanisms of Leptospira pathogenesis are unclear and the virulent factors in Leptospira need further identification. 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 also 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 found renal injury in residents in southern Taiwan with previous infection of leptospira. In this study, we will focus on the other virulent factors such as Loa22, Lsa24, Lsa63, and LRR proteins and investigate the structural and functional relationship of these virulent factors, especially the disease mechanisms relative to innate immunity and the effect of kidneys in Leptospirosis animal model. We will also search for TLR2 interaction outer membrane proteins that interact with kidney epithelial cells. This study would disclose Leptospira pathogen related Leptospiral virulent factors that would be pertinent to the pathogenesis in kidney. We will also study crystal structures of these virulent factors to understand the disease mechanism at host-pathogen interaction. In structure-function analyses should allow many of these questions to be resolved in Leptospiral infection in kidney. Year 1: Developing construction, heterologous overexpression and purification systems for mass production of important Leptospira outer membrane lipoprotein, such as Loa22, Lsa24, Lsa63, LRR proteins, etc. 1. Construction and heterologous overexpression of virulent factors from different serovar of Leptospira spp. 2. The lipoprotein enriched membrane fraction isolation. 3. Determination of the molecular size and interaction force. 4. To establish an acute leptspirosis model in hamsters Year 2: Determining the molecular shape, architecture, and size of Leptospira virulent factors and their dynamic changes 1. The secondary structure of the Leptospira virulent factors. 2. The structural morphology of Leptospira virulent factors. 3. Identification of essential residues, motifs, domains of Leptospira virulent factors. 4.Determination of conformational change and protein dynamics by ensemble measurement. 5. To measure the effect of virulent factos in inflammation and extracelluar matrix gene expression in renal tubular cells 6. To establish a chronic leptspirosis model in mice Year 3: Exploring TLR2 interacting proteins of the Leptospira spp. 1. Searching TLR2 interaction proteins of Leptopsiral outer membrane. 2. Identification of the TLR2 interaction proteins from Leptopsiral outer membrane. 3. To evaluate the kidney expression of virulent factors in Leptospira infected animal model. 4. To evaluate the effect of Leptospira recombinant proteins virulent factos in animal model.

Project ID:PC10401-0619
External Project ID:MOST103-2314-B182-020-MY3