OmpA-like protein Loa22 from Leptospira interrogans serovar Lai is cytotoxic to cultured rat renal cells and promotes inflammatory responses

Leptospirosis renal disease is one of the common clinical manifestations of leptospirosis, including acute renal failure and tubulointerstitial nephritis. Outer membrane protein A-like protein Loa22 is a lipoprotein from Leptospira interrogans and has been suggested to be a corresponding virulence factor. However, the role of Loa22 in leptospiral nephropathy is not yet understood. In the present study, we constructed a vector and artificially expressed Loa22 in Escherichia coli BL21(DE)pLysS cells. After extensive purification, along with a GST tag protein control, Loa22 protein was used to test the cytotoxicity in cultured rat proximal tubule cells (NRK52E) and examine its effects on the induction of inflammatory responses. Using morphological examination, 2,3-bis(2-methoxy-4nitro-5-sulfophenyl)-5-[( phenylamino)carbonyl]-2H-tetrazoium hydrixide absorbance, lactate dehydrogenase assays and an analysis of apoptosis via flow cytometry, it was found that Loa22 protein mediates a direct cytotoxic effect on NRK52E cells in a dose-dependent manner. Using real-time PCR, western blotting and immunofluorescence, it was found that Loa22 protein upregulates the expression of toll-like receptor 2 (TLR2), induces nitric oxide synthase and promotes the production of nitric oxide (NO) and monocyte chemoattractant protein-1 (MCP-1) by NRK52E cells. Additionally, using a TLR2 blocking antibody, it was found that enhanced NO and MCP-1 production by NRK52E cells after Loa22 stimulation requires the activation of TLR2. Collectively, our data suggested that Loa22 is a critical virulence factor of L. interrogans and is involved in the leptospiral nephropathy through mediating direct cytotoxicity and enhancing inflammatory responses.