Peptidoglycan Mediates Loa22 and Toll-like Receptor 2 Interactions in Pathogenic Leptospira

Abstract

Leptospirosis is an overlooked zoonotic disease caused by pathogenic Leptospira. The kidney is the major organ infected by Leptospira which causes tubulointerstitial nephritis. Leptospira outer membrane components contain several virulence factors that play important roles in the pathogenesis of leptospirosis. Among them, OmpA-like protein Loa22 is essential for leptospiral virulence. However, the pathogenic mechanisms of tubulointerstitial nephritis involving this virulence factor are still unclear and need further investigation. In this study, pull-down assays suggested that Toll-like receptor 2 (TLR2) proteins interacted with Loa22 from Leptospira outer membrane extractions. Combination of Atomic force microscopy (AFM) and side-directed mutagenesis suggested that Loa22 exhibited high affinity for Leptospira peptidoglycan (LPGN) and the residues of Loa22 were involved in LPGN interaction. Mutation of two key residues within the OmpA-like domain of Loa22, Asp122 and Arg143, significantly attenuated their relative affinities for LPGN indicating that these two residues were responsible for LPGN binding. Thus Loa22 OmpA domain was responsible for interacting with LPGN and the two indicated residues may participate in binding to LPGN. Recombinant Loa22 (rLoa22) protein was further complexed with LPGN and incubated with HEK293-TLR2 cells to monitor inflammatory responses. Inflammatory responses were provoked by rLoa22-LPGN complexes, but not rLoa22 alone, involved CXCL8/IL8, hCCL2/MCP-1, and hTNF- activation. Confocal microscopy further identified the co-localization of Loa22-LPGN complexes and TLR2 receptors on HEK293-TLR2 cell surface. The affinity between Loa22-LPGN complexes and TLR2 were further confirmed and measured by AFM and ELISA. Downstream signals from TLR2 including p38, ERK, and JNK were observed by western blotting induced by Loa22-LPGN complexes. In summary, this study identified LPGN in leptospira mediates interactions between Loa22 and TLR2 and induces downstream signals to trigger inflammatory responses. Interactions between Loa22-LPGN-TLR2 reveal a novel binding mechanism for the innate immune system and infection induced by leptospira.\n\nAuthor summaryLeptospirosis is one of the most overlooked zoonotic diseases caused by pathogenic Leptospira in warm and humid regions worldwide. With the infection by Leptospira, many organs are invaded and can result in multiple-organ failure (Weils syndrome). Kidney is the major organ infected by pathogenic Leptospira, which would manifest as tubulointerstitial nephritis. In this study, we focused on the outer membrane lipoprotein Loa22 (Leptospiral OmpA-like domain 22) from pathogenic Leptospira which triggers inflammatory responses on renal tubular cell. Protein domain prediction indicated that Loa22 contains an important domain termed OmpA-like domain and the function of this domain is peptidoglycan (PGN) binding. From sequence alignments of Loa22 with other OmpA proteins, two important amino acids, Asp122 and Arg143, were found to be highly conserved. The role of the two conserved residues in AbOmpA (OmpA protein in A. baumannii) and Pal (peptidoglycan-associated lipoprotein in E. coli) proteins are important for PGN binding. These two residues in Loa22 were altered by site-directed mutagenesis to obtain D122A and R143A variants. In pull-down and AFM analysis, the binding capacities of Loa22 variants to Leptospira PGN (LPGN) were significantly decreased as compared to rLoa22WT, indicating that the two residues are involved in LPGN binding. Furthermore, recombinant Loa22 and its variants in the absence or presence of LPGN, were incubated with HEK293-TLR2 cells, to confirm the role of LPGN in triggering inflammatory responses involving CXCL8/IL8, hCCL2/MCP-1, and hTNF-. These factors are involved in downstream signaling of inflammatory responses through Toll-like receptor 2 (TLR2). In addition, confocal microscopy was employed to observe the co-localization of Loa22-LPGN complexes and TLR2 receptors on HEK293-TLR2 cell surfaces. Finally, the interaction forces between rTLR2 and rLoa22-LPGN complexes were measured by AFM and ELISA to conclude the necessary role of LPGN in rLoa22-TLR2 complex formation. In summary, these results demonstrate that the interaction of Loa22 protein with the important cell wall component, PGN, concomitantly triggered inflammatory responses of host cells through interaction with TLR2.