No abstract

A 13-residue frog antimicrobial peptide Temporin L (TempL) possesses versatile antimicrobial activities and is considered a lead molecule for the development of new antimicrobial agents. To find out the amino acid sequences that influence the anti-microbial property of TempL, a phenylalanine zipper-like sequence was identified in it which was not reported earlier. Several alanine-substituted analogs and a scrambled peptide having the same composition of TempL were designed for evaluating the role of this motif. To investigate whether leucine residues instead of phenylalanine residues at 'a' and/or 'd' position(s) of the heptad repeat sequence could alter its antimicrobial property, several TempL analogs were synthesized after replacing these phenylalanine residues with leucine residues. Replacing phenylalanine residues with alanine residues in the phenylalanine zipper sequence significantly compromised the anti-endotoxin property of TempL. This is evident from the higher production of tumor necrosis factor-α and interleukin-6 in lipopolysaccharide (LPS)-stimulated rat bone-marrow-derived macrophage cells in the presence of its alanine-substituted analogs than TempL itself. However, replacement of these phenylalanine residues with leucine residues significantly augmented anti-endotoxin property of TempL. A single alanine-substituted TempL analog (F8A-TempL) showed significantly reduced cytotoxicity but retained the antibacterial activity of TempL, while the two single leucine-substituted analogs (F5L-TempL and F8L-TempL), although exhibiting lower cytotoxicity, were able to retain the antibacterial activity of the parent peptide. The results demonstrate how minor amino acid substitutions in the identified phenylalanine zipper sequence in TempL could yield analogs with better antibacterial and/or anti-endotoxin properties with their plausible mechanism of action.

No abstract

MD2, a 160-residue accessory glycoprotein, is responsible for the recognition and binding of Gram-negative bacterial membrane component, lipopolysaccharide (LPS). Internalization of pathogen inside the mononuclear phagocytes has also been attributed to MD2 which leads to the clearance of pathogens from the host. However, not much is known about the segments in MD2 that are responsible for LPS interaction or internalization of pathogen inside the defense cells. A 16-residue stretch (MD54) from MD2 protein has been identified that possesses a short heptad repeat sequence and four cationic residues enabling it to participate in both hydrophobic and electrostatic interactions with LPS. An MD54 analog of the same size was also designed in which a leucine residue at a heptadic position was replaced with an alanine residue. MD54 but not its analog, MMD54 induced aggregation of LPS and aided in its internalization within THP-1 monocytes. Furthermore, MD54 inhibited LPS-induced nuclear translocation of NF-κB in PMA-treated THP-1 and TLR4/MD2/CD14-transfected HEK-293T cells and the production of pro-inflammatory cytokines. In addition, in in vivo experiments, MD54 showed marked protection and survival of mice against LPS-induced inflammation and death. Overall, we have identified a short peptide with heptad repeat sequence from MD2 that can cause aggregation of LPS and abet in its internalization within THP-1 cells, resulting in attenuation of LPS-induced pro-inflammatory responses in vitro and in vivo.

To become clinically effective, antimicrobial peptides (AMPs) should be non-cytotoxic to host cells. Piscidins are a group of fish-derived AMPs with potent antimicrobial and antiendotoxin activities but limited by extreme cytotoxicity. We conjectured that introduction of cationic residue(s) at the interface of polar and non-polar faces of piscidins may control their insertion into hydrophobic mammalian cell membrane and thereby reducing cytotoxicity. We have designed several novel analogs of piscidin-1 by substituting threonine residue(s) with L and D-lysine residue(s). L/D-lysine-substituted analogs showed significantly reduced cytotoxicity but exhibited either higher or comparable antibacterial activity akin to piscidin-1. Piscidin-1-analogs demonstrated higher efficacy than piscidin-1 in inhibiting lipopolysaccharide (LPS)-induced pro-inflammatory responses in THP-1 cells. T15,21K-piscidin-1 (0.5 mg/Kg) and T15,21dK-piscidin-1 (1.0 mg/Kg) demonstrated 100% survival of LPS (12.0 mg/Kg)-administered mice. High resolution NMR studies revealed that both piscidin-1 and T15,21K-piscidin-1 adopted helical structures, with latter showing a shorter helix, higher amphipathicity and cationic residues placed at optimal distances to form ionic/hydrogen bond with lipid A of LPS. Remarkably, T15,21dK-piscidin-1 showed a helix-loop-helix structure in LPS and its interactions with LPS could be sustained by the distance of separation of side chains of R7 and D-Lys-15 which is close to the inter-phosphate distance of lipid A.