Characterization of a Myeloid Differentiation Factor 2-Derived Peptide that Facilitates THP-1 Macrophage-Mediated Phagocytosis of Gram-Negative Bacteria.

Myeloid differentiation factor 2 (MD2), the TLR4 coreceptor, has been shown to possess opsonic activity and has been implicated in phagocytosis and intracellular killing of Gram-negative bacteria. However, any MD2 protein segment involved in phagocytosis of Gram-negative bacteria is not yet known. A short synthetic MD2 segment, MD54 (amino acid regions 54 to 69), was shown to interact with a Gram-negative bacterial outer membrane component, LPS, earlier. Furthermore, the MD54 peptide induced aggregation of LPS and facilitated its internalization in THP-1 cells. Currently, it has been investigated if MD2-derived MD54 possesses any opsonic property and role in phagocytosis of Gram-negative bacteria. Remarkably, we observed that MD54 facilitated agglutination of Gram-negative bacteria, Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC BAA-427), but not of Gram-positive bacteria, Bacillus subtilis (ATCC 6633) and Staphylococcus aureus (ATCC 25923). The MD54-opsonized Gram-negative bacteria internalized within PMA-treated THP-1 cells and were killed over a longer incubation period. However, both internalization and intracellular killing of the MD54-opsonized Gram-negative bacteria within THP-1 phagocytes were appreciably inhibited in the presence of a phagocytosis inhibitor, cytochalasin D. Furthermore, MD54 facilitated the clearance of Gram-negative bacteria E. coli (ATCC 25922) and P. aeruginosa (ATCC BAA-427) from the infected BALB/c mice whereas an MD54 analog, MMD54, was inactive. Overall, for the first time, the results revealed that a short MD2-derived peptide can specifically agglutinate Gram-negative bacteria, act as an opsonin for these bacteria, and facilitate their phagocytosis by THP-1 phagocytes. The results suggest that the MD54 segment could have a crucial role in MD2-mediated host-pathogen interaction involving the Gram-negative bacteria.

A TLR4-derived non-cytotoxic, self-assembling peptide functions as a vaccine adjuvant in mice.

Vaccination is devised/formulated to stimulate specific and prolonged immune responses for long-term protection against infection or disease. A vaccine component, namely adjuvant, enhances antigen recognition by the host immune system and thereby stimulates its cellular and adaptive responses. Especially synthetic Toll-like receptor (TLR) agonists having self-assembling properties are considered as good candidates for adjuvant development. Here, a human TLR4-derived 20-residue peptide (TR-433), present in the dimerization interface of the TLR4-myeloid differentiation protein-2 (MD2) complex, displayed self-assembly and adopted a nanostructure. Both in vitro studies and in vivo experiments in mice indicated that TR-433 is nontoxic. TR-433 induced pro-inflammatory responses in THP-1 monocytes and HEK293T cells that were transiently transfected with TLR4/CD14/MD2 and also in BALB/c mice. In light of the self-assembly and pro-inflammatory properties of TR-433, we immunized with a mixture of TR-433 and either ovalbumin or filarial antigen trehalose-6-phosphate phosphatase (TPP). A significant amount of IgG titers was produced, suggesting adjuvanting capability of TR-433 that was comparable with that of Freund's complete adjuvant (FCA) and appreciably higher than that of alum. We found that TR-433 preferentially activates type 1 helper T cell (Th1) response rather than type 2 helper T cell (Th2) response. To our knowledge, this is the first report on the identification of a short TLR4-derived peptide that possesses both self-assembling and pro-inflammatory properties and has significant efficacy as an adjuvant, capable of activating cellular responses in mice. These results indicate that TR-433 possesses significant potential for development as a new adjuvant in therapeutic application.

An MD2-derived peptide promotes LPS aggregation, facilitates its internalization in THP-1 cells, and inhibits LPS-induced pro-inflammatory responses.

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.

Selective phenylalanine to proline substitution for improved antimicrobial and anticancer activities of peptides designed on phenylalanine heptad repeat.

Introducing cell-selectivity in antimicrobial peptides (AMPs) without compromising the antimicrobial and anti-endotoxin properties is a crucial step towards the development of new antimicrobial agents. A peptide designed on phenylalanine heptad repeat possesses significant cytotoxicity along with desired antimicrobial and anti-endotoxin properties. Amino acid substitutions at 'a' and/or 'd' positions of heptad repeats of AMPs could alter their helical structure in mammalian membrane-mimetic environments and cytotoxicity towards mammalian cells. Since proline is a helix breaker, effects of selective proline substitution(s) at 'a' and/or 'd' positions of a 15-residue peptide designed on phenylalanine heptad repeat (FR-15) were investigated. Proline-substituted FR-15 variants were highly selective toward bacteria and fungi over hRBCs and murine 3T3 cells and also retained their antibacterial activities at high salt, serum and elevated temperatures. These non-cytotoxic variants also inhibited LPS-induced production of pro-inflammatory cytokines/chemokines in human monocytes, THP-1, RAW 264.7 and in BALB/c mice. The two non-cytotoxic variants (FR8P and FR11P) showed potent anti-cancer activity against highly metastatic human breast cancer cell line MDA-MB-231 with IC50 values less than 10µM. At sub-IC50 concentrations, FR8P and FR11P also showed anti-migratory and anti-invasive effects against MDA-MB-231 cells. FR8P and FR11P induced cellular apoptosis by triggering intrinsic apoptotic pathway through depolarization of mitochondrial membrane potential and activation of caspases. Overall the results demonstrated the utilization of selective phenylalanine to proline substitution in a heptad repeat of phenylalanine residues for the design of cell-selective, broad-spectrum AMPs with significant anti-cancer properties. STATEMENT OF SIGNIFICANCE: We have demonstrated a methodology to design cell-selective potent antimicrobial and anti-endotoxin peptides by utilizing phenylalanine zipper as a template and replacement of phenylalanine residue(s) from "a" and/or "d" position(s) with proline residue(s) produced non-cytotoxic AMPs with improved antibacterial properties against the drug-resistant strains of bacteria. The work showed that the 'a' and 'd' positions of the phenylalanine heptad repeat could be replaced by an appropriate amino acid to control cytotoxicity of the peptide without compromising its potency in antimicrobial and anti-endotoxin properties. The direct bacterial membrane targeting mechanism of proline substituted analogs of parent peptide makes difficult for bacteria to grow resistance against them. The peptides designed could be lead molecules in the area of sepsis as they possess significant anti-LPS activities for in vitro and in vivo. Interestingly since cancer cells and bacterial cell membranes possess the structural resemblances, the cancer cells are also targets for these peptides making them lead molecules in this field. However, unlike in bacteria where the peptides showed membrane permeabilization property to lyse them, the peptides induced apoptosis in MDA-MB-231 breast cancer cells to inhibit their proliferation and growth. The results are significant because it reveals that "a" and "d" positions of a phenylalanine zipper can be utilized as switches to design cell-selective, antimicrobial, anti-endotoxin and anticancer peptides.

Piscidin-1-analogs with double L- and D-lysine residues exhibited different conformations in lipopolysaccharide but comparable anti-endotoxin activities.

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.

Modulation of anti-endotoxin property of Temporin L by minor amino acid substitution in identified phenylalanine zipper sequence.

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.

Variants of self-assembling peptide, KLD-12 that show both rapid fracture healing and antimicrobial properties.

KLD-12 (KLD) is a 12-residue self-assembling peptide that can adopt nano-structures and is known for its tissue-engineering properties. Our objective was to introduce antimicrobial attribute to KLD which would help in preventing secondary infection associated with external application of such tissue engineering materials. Considering the net charge of KLD-12, varying number of cationic arginine residues were added to its N-terminus. KLD variants showed appreciable bactericidal properties without any significant increase in cytotoxicity against tested mammalian cells. Further, these variants adopted ß-sheet structures and self-assembled into nano-structures comparable to that of KLD. Interestingly, the KLD variants with two (KLD-2R) and three (KLD-3R) arginine residues added to its N-terminus showed significant osteogenic effect which was comparable or better than the original peptide as evident from the alkaline phosphatase activity assay, mineralized nodule formation and expression of different osteogenic genes. Particularly, application of KLD-2R in rats to the site of a drill-hole (0.8 mm diameter) that was created in the femur metaphysis displayed significantly higher bone regeneration compared to that of KLD. The results demonstrate a simple way to improve biological property of a self-assembling peptide with tissue engineering property.