Molecular mechanisms underlying LPS transport by LPS recognition proteins

Abstract

The innate immune system plays a critical role in defending the host against microbial infection. Toll-like receptors (TLRs), key molecules in innate immunity, elicit immune responses upon recognition of pathogen-associated molecular patterns (PAMPs). Among 13 TLRs, TLR4 recognizes lipopolysaccharide (LPS) on the outer membrane of Gram-negative bacteria in the presence of MD-2 and induces inflammatory cytokine expression. Two accessory proteins, LBP and CD14, facilitate the LPS recognition by TLR4. However, severe bacterial infection causes exaggerated immune response, leading to multiple organ failure and death. Thus, clearance of bacteria by sensitive recognition of LPS in the early stage of infection is crucial for host survival. Although crystal structures of LBP, CD14, and TLR4-MD-2 complex have been determined, molecular mechanisms of LPS transfer from LPS aggregates to TLR4-MD-2 complex through LBP and CD14 have not been fully understood. In order to elucidate mechanisms underlying the LPS delivery, we characterized intermediate complexes using negative-stain transmission electron microscopy (TEM) and total internal reflection fluorescence microscopy (TIRFM). We visualized the binding mode of LBP to LPS micelle and identified critical residues for the binding. The electrostatic interaction between LBP and CD14 was also revealed. Single-molecule fluorescence analysis demonstrated the mechanism of LBP-mediated LPS trasnfer to CD14 and property of the interaction between LBP and CD14. Moreover, LPS delivery from CD14 to TLR4-MD2 was characterized.