Leptospiral Outer Membrane Lipoprotein LipL32 Binding on Toll-like Receptor 2 of Renal Cells As Determined with an Atomic Force Microscope

Abstract

Leptopirosis is a renal disease caused by pathogenic Leptospira that primarily infects the renal proximal tubules, consequently resulting in severe tubular injuries and malfunctions. The protein extracted from the outer membrane of this pathogenic strain contains a major component of a 32 kDa lipoprotein (LipL32), which is absent in the counter membrane of nonpathogenic strains and has been identified as a crucial factor for host cell infection. Previous studies showed that LipL32 induced inflammatory responses and interacted with the extracellular matrix (ECM) of the host cell. However, the exact relationship between LipL32-mediated inflammatory responses and ECM binding is still unknown. In this study, an atomic force microscope with its tip modified by purified LipL32 was used to assess the interaction between LipL32 and cell surface receptors. Furthermore, an antibody neutralization technique was employed to identify Toll-like receptor 2 (TLR2) but not TLR4 as the major target of LipL32 attack. The interaction force between LipL32 and TLR2 was measured as approximately 59.5 +/- 8.7 pN, concurring with the theoretical value for a single-pair molecular interaction. Moreover, transformation of a TLR deficient cell line with human TLR2 brought the interaction force from the basal level to approximately 60.4 +/- 11.5 pN, confirming unambiguously TLR2 as counter receptor for LipL32. The stimulation of CXCL8/IL-8 expression by full-length LipL32 as compared to that without the N-terminal signal peptide domain suggests a significant role of the signal peptide of the protein in the inflammatory responses. This study provides direct evidence that LipL32 binds to TLR2, but not TLR4, on the cell surface, and a possible mechanism for the virulence of leptospirosis is accordingly proposed.

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

“…DNA Construction and Mutagenesis-The lipl32 gene (782 bp) was cloned from pathogenic Leptospira shermani genomic DNA with Pfu-Turbo DNA polymerase (Stratagene, La Jolla, CA) according to previous investigations (10,23). The gene was amplified by PCR and inserted into a pRSET-c vector (Invitrogen).…”

“…TLR2 heterodimerizes with TLR1 or -6 to interact with ligands. LipL32 was found to initiate the signaling cascade by interacting with TLR2 but not with TLR4 (23,24).…”

Differential In Vivo Gene Expression of Major Leptospira Proteins in Resistant or Susceptible Animal Models

“…Because LipL32 also increases the permeability and apoptosis of human umbilical vein endothelial cells (19), it can be hypothesized that LipL32 is required for invasion only in the early stages of the infection through endothelial cells but is not required and therefore is repressed once Leptospira bacteria disseminate into the systemic circulation. Moreover, LipL32 was shown to interact with Toll-like receptor 2 (TLR-2), leading to the induced expression of inflammatory chemokine interleukin-8 (IL-8) (9). Thus, a decrease in lipL32 expression might reflect a dissimulation strategy to avoid recognition by the host immune system.…”

Substrate-induced Changes in Domain Interaction of Vacuolar H+-Pyrophosphatase

“…AFM and SMFS Experiments-A commercial atomic force microscope (Nanoscope III; Digital Instruments, Santa Barbara, CA) with E type scanner and PicoForce module were used in this study (18,19). The gold-coated silicon nitride (Si 3 N 4 ) cantilevers (60 m long) were utilized, and the nominal spring constant and resonance frequency were 30 pN/nm and 8 kHz in water, respectively.…”

“…The worm-like chain (WLC) fit of each force peak provided the contour length that reflected the amount of amino acids unfolded and stretched (24). The contour lengths and the corresponding rupture forces obtained were statistically analyzed using IgorPro 5 built-in and custom procedures (18,19). The SPIP (Image Metrology ApS, Denmark) software was used for data analysis, and values of extraction force were thus determined with SigmaPlot software (SPSS, Chicago).…”