Leptospiral outer membrane lipoprotein LipL32 binding on toll-like receptor 2 of renal cells as determined with an atomic force microscope.
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.
Hsu
Shen-Hsing
SH
Department 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.
Lo
Yueh-Yu
YY
Tung
Jung-Yu
JY
Ko
Yi-Ching
YC
Sun
Yuh-Ju
YJ
Hung
Cheng-Chieh
CC
Yang
Chih-Wei
CW
Tseng
Fan-Gang
FG
Fu
Chien-Chung
CC
Pan
Rong-Long
RL
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007249:Inflammation; D007668:Kidney / Q000382:microbiology* / Q000473:pathology; D007919:Leptospira / Q000737:chemistry / Q000378:metabolism / Q000472:pathogenicity*; D007922:Leptospirosis / Q000209:etiology / Q000382:microbiology; D008074:Lipoproteins / Q000737:chemistry / Q000378:metabolism*; D018625:Microscopy, Atomic Force / Q000379:methods; D011485:Protein Binding; D021382:Protein Sorting Signals; D051195:Toll-Like Receptor 2 / Q000378:metabolism*; D051197:Toll-Like Receptor 4; D014774:Virulence
D001425:Bacterial Outer Membrane Proteins; C406589:LipL32 protein, Leptospira; D008074:Lipoproteins; D021382:Protein Sorting Signals; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4