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Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins
Published on: July 1, 2011
Leptospiral outer membrane lipoprotein LipL32 binding on toll-like receptor 2 of renal cells as determined with an
Shen-Hsing Hsu1, Yueh-Yu Lo, Jung-Yu Tung
1Department of Life Science and Institute of Bioinformatics and Structural Biology, College of Life Science, National Tsing Hua University, Hsin Chu 30013, Taiwan, Republic of China.
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.
Insights
Leptospira outer membrane protein LipL32 directly binds to Toll-like receptor 2 (TLR2), not TLR4, on host cells. This interaction triggers inflammatory responses, contributing to the virulence of leptospirosis renal disease.
Area of Science:
- Infectious Diseases
- Immunology
- Microbiology
Background:
- Leptospirosis is a renal disease caused by pathogenic Leptospira, targeting renal proximal tubules and causing severe injury.
- The 32 kDa outer membrane lipoprotein (LipL32) is crucial for pathogenic Leptospira infection and induces inflammatory responses.
- The precise mechanism linking LipL32's inflammatory effects and its interaction with host cells, specifically extracellular matrix (ECM) and cell surface receptors, remains unclear.
Purpose of the Study:
- To investigate the direct interaction between LipL32 and host cell surface receptors.
- To identify the specific host receptor(s) targeted by LipL32.
- To elucidate the role of LipL32 in initiating inflammatory responses during leptospirosis.
Main Methods:
- Utilized atomic force microscopy (AFM) with LipL32-modified tips to measure interaction forces with cell surface receptors.
- Employed antibody neutralization techniques to identify target receptors, specifically differentiating between Toll-like receptor 2 (TLR2) and TLR4.
- Confirmed receptor binding by measuring interaction forces and assessing inflammatory marker (CXCL8/IL-8) expression in TLR-deficient cell lines.
Main Results:
- Atomic force microscopy revealed a specific binding interaction between LipL32 and cell surface receptors.
- Antibody neutralization identified Toll-like receptor 2 (TLR2) as the primary target of LipL32, with minimal interaction with TLR4.
- Measured interaction force between LipL32 and TLR2 was approximately 59.5 ± 8.7 pN, consistent with single-molecule interactions.
- Restoring TLR2 in deficient cells re-established the interaction force, confirming TLR2 as the specific counter-receptor.
- The N-terminal signal peptide domain of LipL32 was found to be significant for stimulating CXCL8/IL-8 expression, indicating its role in inflammation.
Conclusions:
- Leptospira's outer membrane protein LipL32 directly binds to Toll-like receptor 2 (TLR2) on host cells, but not TLR4.
- This LipL32-TLR2 interaction is a key mechanism driving inflammatory responses in leptospirosis.
- The findings propose a novel mechanism for leptospirosis pathogenesis, highlighting LipL32-TLR2 signaling as a virulence factor.
