Molecular mechanism underlying the TLR4 antagonistic and antiseptic activities of papiliocin,an insect innate immune response molecule

Antimicrobial peptides are innate immune molecules playing essential roles in insects, which lack the adaptive immune system. Insects possess Toll9, the innate pattern-recognition receptor highly similar to the mammalian Toll-like receptor 4 (TLR4), which is involved in recognizing lipopolysaccharide (LPS). TLR4 is an important therapeutic target, as it causes uncontrolled immune response in sepsis; therefore, identification of TLR4-targeting molecules is imperative. Papiliocin, an insect cecropin derived from the larvae of the swallowtail butterfly, possesses potent antibacterial activities against gram-negative bacteria. We investigated the molecular mechanism underlying the TLR4-antagonistic and antiseptic activities of papiliocin. Binding analysis, docking simulation, and flow cytometry showed that papiliocin inhibited LPS-induced TLR4 signaling by directly binding to TLR4/MD-2 and causing rapid dissociation of LPS from the TLR4/MD-2 complex. R13 and R16 in the N-terminal helix, conserved in insect cecropins, were the key binding sites at the TLR4/MD-2 interface, along with the flexible hinge region, which promoted the interaction of the hydrophobic carboxyl-terminal helix with the MD-2 pocket to competitively inhibit the LPS–TLR4/MD-2 interaction. Papiliocin, an antiendotoxin molecule and TLR4 inhibitor, rescued the pathology of Escherichia coli–induced sepsis in mice more effectively and with lower nephrotoxicity compared to polymyxin B. Our results provide insight into the key structural components and mechanism underlying the TLR4-antagonistic activities of papiliocin, which is essential for the innate immune response of the insect against microbial infection. Papiliocin may be useful for developing a multifunctional alternative to polymyxin B for treating gram-negative sepsis.

Toll-like receptors (TLRs) are innate immune receptors that recognize pathogen-associated molecular patterns to protect the host from invading pathogens (1). TLR4 is one of the most critical pattern-recognition receptors in the TLR family that recognizes lipopolysaccharide (LPS) released from the outer membrane of gram-negative bacteria to elicit innate immune response (2). Subsequently, an LPS-binding protein attracts LPS and facilitates CD14-dependent transfer of LPS to TLR4 via the adaptor protein MD-2, resulting in dimerization of the TLR4/MD-2 complex. The dimer mediates translocation of nuclear factor-kappa B (NF-κB), ultimately resulting in the production of proinflammatory cytokines. Therefore, uncontrolled LPS-induced inflammatory TLR4 signaling can cause acute sepsis (3). Sepsis induced by multidrug-resistant gram-negative bacteria, such as carbapenem-resistant bacteria, is difficult to eradicate and causes serious health issues (4, 5), as carbapenems such as imipenem, doripenem, and meropenem are generally the final choices for treating infections caused by gram-negative bacteria. As management of carbapenem-resistant Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae including Klebsiella pneumoniae and Escherichia coli is extremely difficult, the World Health Organization has prioritized the development of methods for effectively treating infections caused by these pathogens (6, 7). Research in this field has mainly focused on developing antiseptic molecules that can either clear bacterial LPS or competitively target the binding of LPS to TLR4/MD-2, resulting in inhibition of the TLR4 signaling pathway (8–10). Therefore, molecules with dual effects can be advantageous for inhibiting systemic TLR4-mediated inflammatory sepsis.Antimicrobial peptides (AMPs) are important natural components of the innate immune system in various organisms (11) and typically kill pathogens by permeabilizing their membranes or targeting intracellular components (12). In addition, AMPs can modulate the host immune system via multiple pathways (13). Therefore, AMPs have emerged as effective molecules against multidrug-resistant bacteria and potential alternatives to conventional antibiotics for treating gram-negative infections (14). Polymyxin B (PMB) and colistin are cyclic cationic AMPs, which are used as a last resort for treating gram-negative infections (15). PMB prevents gram-negative sepsis by removing bacterial LPS. However, increased resistance, nephrotoxicity, and neurotoxicity associated with PMB have limited its use in clinical practice (16).Insects are extremely resistant to microbial infections owing to their strong innate immune system, which includes the production of AMPs (17). In insects, Toll was initially identified in Drosophila melanogaster as an integral membrane receptor (18). Insect Toll is highly similar to mammalian TLR, and Toll/TLRs are considered key regulators of the innate immune system in both insects and mammals (19). A recent study showed that in the silkworm, Bombyx mori, Toll9 recognizes LPS by interacting with two MD-2–related lipid recognition domains, named Toll9/MD-2A or Toll9/MD-2B, indicating their functional and evolutionary similarity with mammalian TLR4/MD-2 proteins (20, 21). Insect Toll9 may be a pattern-recognition immune receptor similar to mammalian TLR4 in complex with MD-2 during LPS recognition and signaling (21). Cecropins are a group of widely studied AMPs that play important roles in the innate immune response of insects (22–24). In 1981, Steiner et al. reported that cecropins are produced from the hemolymph of bacterially challenged diapausing pupae of the giant silk moth, Hyalophora cecropia (25). Since then, several cecropin-like peptides have been identified in insects such as B. mori (26) and D. melanogaster (24). In B. mori, LPS can activate the expression of AMPs such as cecropin B, moricin, lebocin3, and attacin1 (21, 27). Some insect cecropins have shown potent antibacterial activities and in vivo antiseptic activities, confirming their therapeutic potential (23, 28–31).Papiliocin, an AMP belonging to the insect cecropin family, was isolated from the larvae of the swallowtail butterfly, Papilio xuthus (32). We previously showed that papiliocin has broad-spectrum antibacterial activity—particularly against gram-negative bacteria—in which it disrupts the bacterial membrane, similar to other insect cecropins (33–37). We determined the solution structure of papiliocin, which contains two α-helices: an amphipathic N-terminal helix from R1 to K21 and a hydrophobic carboxyl-terminal helix from A25 to V37, separated by a hinge region (33). Most insect cecropins share this helix–hinge–helix structure with high sequence homology. Furthermore, papiliocin inhibits nitric oxide (NO) production and may suppress tumor necrosis factor (TNF)-α via innate defense response mechanisms, which involve the TLR4 pathway in LPS-stimulated RAW 264.7 cells (33). We also found that aromatic residues (W2 and F5), as well as the amphipathic N-terminal helix, play important roles in the membrane permeabilization and anti-inflammatory activities of papiliocin (34, 35). Therefore, using the sequence of the N-terminal helix of papiliocin, short peptide antibiotics, such as the papiliocin–magainin hybrid peptide and a 12-mer peptide, which exhibited a diverse range of antimicrobial activities against gram-negative infections, were designed (36, 37). However, the detailed mechanism underlying TLR4 signaling inhibition and the role of the conserved hydrophobic carboxyl-terminal helix remain unclear.Considering the emerging role of TLR4 in the progression of gram-negative bacterial infections to sepsis and the urgent need to find safe antiseptic alternatives to PMB for clinical use, we investigated the molecular mechanism of action of papiliocin as a TLR4 inhibitor using binding analysis, docking simulation, saturation transfer difference (STD) NMR, and flow cytometry. This study demonstrates the role of the conserved structural components of insect cecropins involved in direct binding to TLR4/MD-2, preventing its dimerization and thereby inhibiting the LPS-stimulated TLR4 inflammatory signaling pathway. This study also provides insight into the mechanism underlying the human TLR4-antagonistic activities of papiliocin, which may be essential for understanding the functionally similar Toll9/MD-2–mediated insect innate immune response against microbial infection. The antiseptic effect and low nephrotoxicity of papiliocin were confirmed using in vivo sepsis models and compared to that of PMB, highlighting its potential as a safe alternative to PMB for treating gram-negative sepsis.