Melissa Elliott

Synthetic Cationic Peptide IDR-1002 Provides Protection against Bacterial Infections through Chemokine Induction and Enhanced Leukocyte Recruitment

With the rapid rise in the incidence of multidrug resistant infections, there is substantial interest in host defense peptides as templates for production of new antimicrobial therapeutics. Natural peptides are multifunctional mediators of the innate immune response, with some direct antimicrobial activity and diverse immunomodulatory properties. We have previously developed an innate defense regulator (IDR) 1, with protective activity against bacterial infection mediated entirely through its effects on the immunity of the host, as a novel approach to anti-infective therapy. In this study, an immunomodulatory peptide IDR-1002 was selected from a library of bactenecin derivatives based on its substantially more potent ability to induce chemokines in human PBMCs. The enhanced chemokine induction activity of the peptide in vitro correlated with stronger protective activity in vivo in the Staphylococcus aureus-invasive infection model, with a >5-fold reduction in the protective dose in direct comparison with IDR-1. IDR-1002 also afforded protection against the Gram-negative bacterial pathogen Escherichia coli. Chemokine induction by IDR-1002 was found to be mediated through a Gi-coupled receptor and the PI3K, NF-κB, and MAPK signaling pathways. The protective activity of the peptide was associated with in vivo augmentation of chemokine production and recruitment of neutrophils and monocytes to the site of infection. These results highlight the importance of the chemokine induction activity of host defense peptides and demonstrate that the optimization of the ex vivo chemokine-induction properties of peptides is a promising method for the rational development of immunomodulatory IDR peptides with enhanced anti-infective activity.

Screening and Characterization of Surface-Tethered Cationic Peptides for Antimicrobial Activity

There is an urgent need to coat the surfaces of medical devices, including implants, with antimicrobial agents to reduce the risk of infection. A peptide array technology was modified to permit the screening of short peptides for antimicrobial activity while tethered to a surface. Cellulose-amino-hydroxypropyl ether (CAPE) linker chemistry was used to synthesize, on a cellulose support, peptides that remained covalently bound during biological assays. Among 122 tested sequences, the best surface-tethered 9-, 12-, and 13-mer peptides were found to be highly antimicrobial against bacteria and fungi, as confirmed using alternative surface materials and coupling strategies as well as coupling through the C and N termini of the peptides. Structure-activity modeling of the structural features determining the activity of tethered peptides indicated that the extent and positioning of positive charges and hydrophobic residues were influential in determining activity.

Structural Studies of a Peptide with Immune Modulating and Direct Antimicrobial Activity

The structure and function of the synthetic innate defense regulator peptide 1018 was investigated. This 12 residue synthetic peptide derived by substantial modification of the bovine cathelicidin bactenecin has enhanced innate immune regulatory and moderate direct antibacterial activities. The solution state NMR structure of 1018 in zwitterionic dodecyl phosphocholine (DPC) micelles indicated an α-helical conformation, while secondary structures, based on circular dichroism measurements, in anionic sodium dodecyl sulfate (SDS) and phospholipid vesicles (POPC/PG in a 1:1 molar ratio) and simulations revealed that 1018 can adopt a variety of folds, tailored to its different functions. The structural data are discussed in light of the ability of 1018 to potently induce chemokine responses, suppress the LPS-induced TNF-α response, and directly kill both Gram-positive and Gram-negative bacteria.

Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli.

Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of microbes, and/or modulating host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites, but there are substantial roadblocks to their therapeutic application. High manufacturing costs associated with amino acid precursors have limited the delivery of inexpensive therapeutics through industrial-scale chemical synthesis. Conversely, the production of peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial activity of these peptides and their susceptibility to proteolytic degradation, while subsequent purification of recombinant peptides often requires multiple steps and has not been cost-effective. Here we have developed methodologies appropriate for large-scale industrial production of HDPs; in particular, we describe (i) a method, using fusions to SUMO, for producing high yields of intact recombinant HDPs in bacteria without significant toxicity and (ii) a simplified 2-step purification method appropriate for industrial use. We have used this method to produce seven HDPs to date (IDR1, MX226, LL37, CRAMP, HHC-10, E5 and E6). Using this technology, pilot-scale fermentation (10L) was performed to produce large quantities of biologically active cationic peptides. Together, these data indicate that this new method represents a cost-effective means to enable commercial enterprises to produce HDPs in large-scale under Good Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans.

A novel vaccine adjuvant comprised of a synthetic innate defence regulator peptide and CpG oligonucleotide links innate and adaptive immunity.

There has been an increased demand for the development of novel vaccine adjuvants that lead to enhanced induction of protection from infectious challenges and development of immunological memory. A novel vaccine adjuvant was developed comprising a complex containing CpG oligonucleotide and the synthetic cationic innate defence regulator peptide HH2 that has enhanced immune modulating activities. The complex of HH2 and the CpG oligonucleotide 10101 was a potent inducer of cytokine/chemokine expression ex vivo, retained activity following extended storage, had low associated cytotoxicity, and upregulated surface marker expression in dendritic cells, a critical activity for a vaccine adjuvant. Immunization of mice with a coformulation of the HH2-CpG complex and pertussis toxoid significantly enhanced the induction of toxoid-specific antibody titres when compared to toxoid alone, inducing high titres of IgG1 and IgG2a, typical of a balanced Th1/Th2 response, and also led to high IgA titres. This study demonstrates the potential application of the HH2-CpG complex as a vaccine adjuvant.

ROS-deficient monocytes have aberrant gene expression that correlates with inflammatory disorders of chronic granulomatous disease

Chronic granulomatous disease is an immunodeficiency caused by an inability to produce reactive oxygen species. While the mechanism of hyper-sensitivity to infection is well understood in CGD, the basis for debilitating inflammatory disorders that arise in the absence of evident infection has not been fully explained. Herein it is demonstrated that resting and TLR-activated monocytes from individuals with CGD expressed significantly higher levels of inflammatory mediators than control cells; the expression in CGD cells resembled normal cells stimulated with lipopolysaccharide. The lack of acute illness, infection or circulating endotoxin in the blood of the CGD patients at the time of sampling was consistent with infection-free inflammation. The enhanced expression of inflammatory mediators correlated with elevated expression of NF-kappaB and was dependent on ERK1/2 signalling. The results are consistent with the hypothesis that ROS are anti-inflammatory mediators that control gene expression and potentially limit the development of sterile inflammatory disorders.

Mechanism of Action and Limited Cross-Resistance of New Lipopeptide MX-2401

ABSTRACT MX-2401 is a semisynthetic calcium-dependent lipopeptide antibiotic (analogue of amphomycin) in preclinical development for the treatment of serious Gram-positive infections. In vitro and in vivo, MX-2401 demonstrates broad-spectrum bactericidal activity against Gram-positive organisms, including antibiotic-resistant strains. The objective of this study was to investigate the mechanism of action of MX-2401 and compare it with that of the lipopeptide daptomycin. The results indicated that although both daptomycin and MX-2401 are in the structural class of Ca2+-dependent lipopeptide antibiotics, the latter has a different mechanism of action. Specifically, MX-2401 inhibits peptidoglycan synthesis by binding to the substrate undecaprenylphosphate (C55-P), the universal carbohydrate carrier involved in several biosynthetic pathways. This interaction resulted in inhibition, in a dose-dependent manner, of the biosynthesis of the cell wall precursors lipids I and II and the wall teichoic acid precursor lipid III, while daptomycin had no significant effect on these processes. MX-2401 induced very slow membrane depolarization that was observed only at high concentrations. Unlike daptomycin, membrane depolarization by MX-2401 did not correlate with its bactericidal activity and did not affect general membrane permeability. In contrast to daptomycin, MX-2401 had no effect on lipid flip-flop, calcein release, or membrane fusion with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (POPG) liposomes. MX-2401 adopts a more defined structure than daptomycin, presumably to facilitate interaction with C55-P. Mutants resistant to MX-2401 demonstrated low cross-resistance to other antibiotics. Overall, these results provided strong evidence that the mode of action of MX-2401 is unique and different from that of any of the approved antibiotics, including daptomycin.

Effect of BMAP-28 Antimicrobial Peptides on Leishmania major Promastigote and Amastigote Growth: Role of Leishmanolysin in Parasite Survival

Background Protozoan parasites, such as Leishmania, still pose an enormous public health problem in many countries throughout the world. Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies. Several innovative approaches are under investigation, including the utilization of host defence peptides (HDPs) as emerging anti-parasitic therapies. HDPs are characterised by their small size, amphipathic nature and cationicity, which induce permeabilization of cell membranes, whilst modulating the immune response of the host. Recently, members of the cathelicidin family of HDPs have demonstrated significant antimicrobial activities against various parasites including Leishmania. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28) has broad antimicrobial activities and confers protection in animal models of bacterial infection or sepsis. We tested the effectiveness of the use of BMAP-28 and two of its isomers the D-amino acid form (D-BMAP-28) and the retro-inverso form (RI-BMAP-28), as anti-leishmanial agents against the promastigote and amastigote intracellular Leishmania major lifecycle stages. Methodology/Principal Findings An MTS viability assay was utilized to show the potent antiparasitic activity of BMAP-28 and its protease resistant isomers against L. major promastigotes in vitro. Cell membrane permeability assays, caspase 3/7, Tunel assays and morphologic studies suggested that this was a late stage apoptotic cell death with early osmotic cell lysis caused by the antimicrobial peptides. Furthermore, BMAP-28 and its isomers demonstrated anti-leishmanial activities against intracellular amastigotes within a macrophage infection model. Conclusions/Significance Interestingly, D-BMAP-28 appears to be the most potent antiparasitic of the three isomers against wild type L. major promastigotes and amastigotes. These exciting results suggest that BMAP-28 and its protease resistant isomers have significant therapeutic potential as novel anti-leishmanials.

Short cationic antimicrobial peptides interact with ATP.

ABSTRACT The mode of action of short, nonhelical antimicrobial peptides is still not well understood. Here we show that these peptides interact with ATP and directly inhibit the actions of certain ATP-dependent enzymes, such as firefly luciferase, DnaK, and DNA polymerase. α-Helical and planar or circular antimicrobial peptides did not show such interaction with ATP.

Manipulation of innate immunity by a bacterial secreted peptide: Lantibiotic nisin Z is selectively immunomodulatory

Innate immunity is triggered by a variety of bacterial molecules, resulting in both protective and potentially harmful pro-inflammatory responses. Further, innate immunity also provides a mechanism for the maintenance of homeostasis between the host immune system and symbiotic or non-pathogenic microorganisms. However, the bacterial factors that mediate these protective effects have been incompletely defined. Here, it was demonstrated that the lantiobiotic nisin Z is able to modulate host immune responses and mediate protective host immunity. Nisin Z induced the secretion of the chemokines MCP-1, IL-8 and Gro-α, and significantly reduced TNF-α induction in response to bacterial LPS in human PBMC. The results correlated with the ability of nisin Z to confer protection against both the Gram-positive organism Staphylococcus aureus, and the Gram-negatives Salmonella enterica sv. Typhimurium and Escherichia coli in murine challenge models. Mechanistic studies revealed that nisin Z modulates host immunity through similar mechanisms as natural host defense peptides, engaging multiple signal transduction pathways and growth factor receptors. The results presented herein demonstrate that, in addition to nisin Z, other bacterial cationic peptides and, in particular, the lantibiotics, could represent a new class of secreted bacterial molecule with immunomodulatory activities.