Regional Dichotomy in Enteric Mucosal Immune Responses to a Persistent Mycobacterium avium ssp. paratuberculosis Infection.

Chronic enteric Mycobacterium avium ssp. paratuberculosis (MAP) infections are endemic in ruminants globally resulting in significant production losses. The mucosal immune responses occurring at the site of infection, specifically in Peyer's patches (PP), are not well-understood. The ruminant small intestine possesses two functionally distinct PPs. Discrete PPs function as mucosal immune induction sites and a single continuous PP, in the terminal small intestine, functions as a primary lymphoid tissue for B cell repertoire diversification. We investigated whether MAP infection of discrete vs. continuous PPs resulted in the induction of significantly different pathogen-specific immune responses and persistence of MAP infection. Surgically isolated intestinal segments in neonatal calves were used to target MAP infection to individual PPs. At 12 months post-infection, MAP persisted in continuous PP (n = 4), but was significantly reduced (p = 0.046) in discrete PP (n = 5). RNA-seq analysis revealed control of MAP infection in discrete PP was associated with extensive transcriptomic changes (1,707 differentially expressed genes) but MAP persistent in continuous PP elicited few host responses (4 differentially expressed genes). Cytokine gene expression in tissue and MAP-specific recall responses by mucosal immune cells isolated from PP, lamina propria and mesenteric lymph node revealed interleukin (IL)22 and IL27 as unique correlates of protection associated with decreased MAP infection in discrete PP. This study provides the first description of mucosal immune responses occurring in bovine discrete jejunal PPs and reveals that a significant reduction in MAP infection is associated with specific cytokine responses. Conversely, MAP infection persists in the continuous ileal PP with minimal perturbation of host immune responses. These data reveal a marked dichotomy in host-MAP interactions within the two functionally distinct PPs of the small intestine and identifies mucosal immune responses associated with the control of a mycobacterial infection in the natural host.

An anti-infective synthetic peptide with dual antimicrobial and immunomodulatory activities.

Antibiotic-resistant infections are predicted to kill 10 million people per year by 2050, costing the global economy $100 trillion. Therefore, there is an urgent need to develop alternative technologies. We have engineered a synthetic peptide called clavanin-MO, derived from a marine tunicate antimicrobial peptide, which exhibits potent antimicrobial and immunomodulatory properties both in vitro and in vivo. The peptide effectively killed a panel of representative bacterial strains, including multidrug-resistant hospital isolates. Antimicrobial activity of the peptide was demonstrated in animal models, reducing bacterial counts by six orders of magnitude, and contributing to infection clearance. In addition, clavanin-MO was capable of modulating innate immunity by stimulating leukocyte recruitment to the site of infection, and production of immune mediators GM-CSF, IFN-γ and MCP-1, while suppressing an excessive and potentially harmful inflammatory response by increasing synthesis of anti-inflammatory cytokines such as IL-10 and repressing the levels of pro-inflammatory cytokines IL-12 and TNF-α. Finally, treatment with the peptide protected mice against otherwise lethal infections caused by both Gram-negative and -positive drug-resistant strains. The peptide presented here directly kills bacteria and further helps resolve infections through its immune modulatory properties. Peptide anti-infective therapeutics with combined antimicrobial and immunomodulatory properties represent a new approach to treat antibiotic-resistant infections.

Endosomal pH modulation by peptide-gold nanoparticle hybrids enables potent anti-inflammatory activity in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays a central role in the pathophysiology of many acute and chronic human inflammatory diseases, and pharmacological regulation of TLR responses is anticipated to be beneficial in many inflammatory conditions. Currently there are no specific TLR inhibitors in clinical use. To overcome this challenge, we have developed a nano-based TLR inhibitor (peptide-gold nanoparticle hybrids) that inhibits a broad spectrum of TLR responses. Through mechanistic studies, we established that specific peptide decorated-gold nanoparticles that display high cellular uptake in phagocytic immune cells modulate endosomal pH, leading to significant attenuation of signaling through multiple TLRs. Using a global transcriptomic approach, we defined the broad anti-inflammatory activity of the nanoparticle in human peripheral blood mononuclear cells. In vivo studies confirmed the beneficial immunomodulatory activity since treatment with the nanoparticle significantly reduced weight loss, improved the disease activity index, and ameliorated colonic inflammation in a murine model of intestinal inflammation. This work enhances our fundamental understanding of the role of peptide coatings on the nanoparticle surface in regulating innate immune signaling, and identifies specific peptide decorated nanoparticles that may represent a novel class of anti-inflammatory therapeutics for human inflammatory diseases.

Identification of Synthetic and Natural Host Defense Peptides with Leishmanicidal Activity.

Leishmaniaparasites are a major public health problem worldwide. Effective treatment of leishmaniasis is hampered by the high incidence of adverse effects to traditional drug therapy and the emergence of resistance to current therapeutics. A vaccine is currently not available. Host defense peptides have been investigated as novel therapeutic agents against a wide range of pathogens. Here we demonstrate that the antimicrobial peptide LL-37 and the three synthetic peptides E6, L-1018, and RI-1018 exhibit leishmanicidal activity against promastigotes and intramacrophage amastigotes ofLeishmania donovaniandLeishmania major We also report that theLeishmaniaprotease/virulence factor GP63 confers protection toLeishmaniafrom the cytolytic properties of alll-form peptides (E6, L-1018, and LL-37) but not thed-form peptide RI-1018. The results suggest that RI-1018, E6, and LL-37 are promising peptides to develop further into components for antileishmanial therapy.

Conditional-ready mouse embryonic stem cell derived macrophages enable the study of essential genes in macrophage function.

The ability to differentiate genetically modified mouse embryonic stem (ES) cells into functional macrophages provides a potentially attractive resource to study host-pathogen interactions without the need for animal experimentation. This is particularly useful in instances where the gene of interest is essential and a knockout mouse is not available. Here we differentiated mouse ES cells into macrophages in vitro and showed, through a combination of flow cytometry, microscopic imaging, and RNA-Seq, that ES cell-derived macrophages responded to S. Typhimurium, in a comparable manner to mouse bone marrow derived macrophages. We constructed a homozygous mutant mouse ES cell line in the Traf2 gene that is known to play a role in tumour necrosis factor-α signalling but has not been studied for its role in infections or response to Toll-like receptor agonists. Interestingly, traf2-deficient macrophages produced reduced levels of inflammatory cytokines in response to lipopolysaccharide (LPS) or flagellin stimulation and exhibited increased susceptibility to S. Typhimurium infection.

Transcription of the oprF gene of Pseudomonas aeruginosa is dependent mainly on the SigX sigma factor and is sucrose induced.

The OprF porin is the major outer membrane protein of Pseudomonas aeruginosa. OprF is involved in several crucial functions, including cell structure, outer membrane permeability, environmental sensing, and virulence. The oprF gene is preceded by the sigX gene, which encodes the poorly studied extracytoplasmic function (ECF) sigma factor SigX. Three oprF promoters were previously identified. Two intertwined promoters dependent on σ(70) and SigX are located in the sigX-oprF intergenic region, whereas a promoter dependent on the ECF AlgU lies within the sigX gene. An additional promoter was found in the cmpX-sigX intergenic region. In this study, we dissected the contribution of each promoter region and of each sigma factor to oprF transcription using transcriptional fusions. In Luria-Bertani (LB) medium, the oprF-proximal region (sigX-oprF intergenic region) accounted for about 80% of the oprF transcription, whereas the AlgU-dependent promoter had marginal activity. Using the sigX mutant PAOSX, we observed that the SigX-dependent promoter was largely predominant over the σ(70)-dependent promoter. oprF transcription was increased in response to low NaCl or high sucrose concentrations, and this induced transcription was strongly impaired in the absence of SigX. The lack of OprF itself increased oprF transcription. Since these conditions led to cell wall alterations, oprF transcription could be activated by signals triggered by perturbation of the cell envelope.

Enhanced immune responses and protection by vaccination with respiratory syncytial virus fusion protein formulated with CpG oligodeoxynucleotide and innate defense regulator peptide in polyphosphazene microparticles.

Although respiratory syncytial virus (RSV) is the leading cause of serious respiratory tract disease in children, to date no RSV vaccine is available. To produce an effective subunit vaccine, a truncated secreted version of the F protein (ΔF) was expressed in mammalian cells, purified and shown to form trimers. The ΔF protein was then formulated with a CpG oligodeoxynucleotide (ODN) and an innate defense regulator (IDR) peptide in polyphosphazene microparticles (ΔF-MP). Mice immunized either intramuscularly (IM) or intranasally (IN) with ΔF-MP developed significantly higher levels of virus-neutralizing antibodies in the sera and lungs, as well as higher numbers of IFN-γ secreting cells than mice immunized with the ΔF protein alone. In contrast, the IM delivered ΔF induced high production of IL-5 while the IN delivered ΔF did not elicit a measurable immune response. After RSV challenge, essentially no virus and no evidence of immunopathology were detected in mice immunized with ΔF-MP regardless of the route of delivery. While the mice immunized IM with ΔF alone also showed reduced virus replication, they developed enhanced levels of pulmonary IgE, IL-4, IL-5, IL-13 and eotaxin, as well as eosinophilia after challenge. The level of protection induced by the ΔF-MP formulation was equivalent after IM and IN delivery. The efficacy and safety of the ΔF-MP formulation was confirmed in cotton rats, which also developed enhanced immune responses and were fully protected from RSV challenge after vaccination with ΔF-MP. In conclusion, formulation of recombinant ΔF with CpG ODN and IDR peptide in polyphosphazene microparticles should be considered for further evaluation as a safe and effective vaccine against RSV.

'If you listen to me properly, I feel good': a qualitative examination of patient experiences of dietetic consultations.

BACKGROUND: There is considerable interest in healthcare research regarding communication skills and some debate surrounding the effectiveness of a patient-centred approach to care. Understanding patient experiences of consultations can help indicate how consultations can be modified to improve effectiveness. At present, there is little research exploring patient experience of dietetic consultations. The present study aimed to achieve a better understanding of patients' experiences of dietetic consultations using qualitative analysis. METHODS: Patients undergoing consultations with a dietitian were invited to discuss their experience of the consultation with a research dietitian who was not involved in their care. Individual interviews and focus groups were conducted and analysed using the Framework approach. RESULTS: Seventeen patients participated and described their experiences of consultations, which were varied and influenced by factors such as information given (resources, explanation, repetition, consistency); their dietitian's approach (prescriptive or nonprescriptive, use of behaviour change skills), behaviour (listening skills, body language) and appointment (expectations, involvement of the multidisciplinary team, length of time); and their own internal experience (confidence, guilt, frustration). Patients agreed that certain factors, such as good communication and rapport, receiving effective and reliable information and resources, and nonjudgmental, regular support, were important factors in creating a positive experience of their consultation. However, they differed in what they believed constituted these factors. CONCLUSIONS: Patients like dietitians to adopt a patient-centred approach, which might be either patient- or practitioner-led, and to take account of what they wanted from consultations, adapting these to meet their individual requirements.

Mechanism of action and limited cross-resistance of new lipopeptide MX-2401.

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 Ca²âº-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.

Deciphering the mode of action of the synthetic antimicrobial peptide Bac8c.

Bac8c (RIWVIWRR-NH(2)) is an 8-amino-acid peptide derived from Bac2A (RLARIVVIRVAR-NH(2)), a C3A/C11A variant of the naturally occurring bovine peptide, bactenecin (also known as bovine dodecapeptide), the smallest peptide with activity against a range of pathogenic Gram-positive and Gram-negative bacteria, as well as yeast. The effects of Bac8c on Escherichia coli were examined by studying its bacteriostatic and bactericidal properties, demonstrating its effects on proton motive force generation, and visually analyzing (via transmission electron microscopy) its effects on cells at different concentrations, in order to probe the complexities of the mechanism of action of Bac8c. Results were consistent with a two-stage model for the Bac8c mode of action. At sublethal concentrations (3 µg/ml), Bac8c addition resulted in transient membrane destabilization and metabolic imbalances, which appeared to be linked to inhibition of respiratory function. Although sublethal concentrations resulted in deleterious downstream events, such as methylglyoxal formation and free radical generation, native E. coli defense systems were sufficient for full recovery within 2 h. In contrast, at the minimal bactericidal concentration (6 µg/ml), Bac8c substantially but incompletely depolarized the cytoplasmic membrane within 5 min and disrupted electron transport, which in turn resulted in partial membrane permeabilization and cell death.

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.

Strategies for the discovery and advancement of novel cationic antimicrobial peptides.

Multi-drug resistant bacteria are appearing at an alarming rate and impose significant burdens on healthcare systems worldwide. Cationic peptides have shown great promise as broad spectrum antimicrobial agents with a demonstrated ability to kill resistant bacteria, however, issues such as protease susceptibility and toxicity issues have delayed their clinical development. This review summarizes recent progress in the advancement of cationic antimicrobial peptides for the treatment of bacterial infections. The major focus of the discussion relates to recent advances in the areas of screening and in silico modeling. A selection of novel strategies that diverge from classical linear α-peptide antimicrobials is also discussed. A diverse array of candidate structures will be key to the ultimate development of a stable platform for clinical development. The ability to accurately predict peptide activity in silico and in a high-throughput manner should benefit all classes of cationic antimicrobial peptides and provide a larger set of candidate structures for clinical evaluation.

The novel adjuvant combination of CpG ODN, indolicidin and polyphosphazene induces potent antibody- and cell-mediated immune responses in mice.

The need to enhance the immunogenicity of purified subunit antigens and modulate resulting immune responses has prompted the development of new adjuvants. Here, the ability of CpG oligodeoxynucleotides (ODN), a bovine host defence peptide indolicidin, and polyphosphazene to synergistically combine and enhance innate and adaptive immune responses was examined in mice. In vitro, the adjuvant combination of CpG ODN, indolicidin and polyphosphazene (CpG/indol/PP) enhanced the secretion of TNF-alpha, IL-12p40, and IL-6 by bone marrow-derived DCs (BMDCs) when compared to the individual components. When co-formulated with ovalbumin (OVA), CpG/indol/PP formed antigen-adjuvant complexes, and enhanced antibody and cell-mediated responses in mice, via both MHC I and II pathways, promoting a more balanced antibody-mediated and type 1-biased cell-mediated immune response. Furthermore, substitution of the proline residues of indolicidin with arginine increased the synergistic adjuvant effect of the peptide, and induced significantly higher IgG1 and IgG2a titers and IFN-gamma secretion, as well as increased uptake by antigen presenting cells. These results clearly demonstrate that the use of a combination of CpG ODN, indolicidin, and polyphosphazene as adjuvant can significantly enhance an antigen-specific immune response.

Bovine lactoferrin and lactoferricin interfere with intracellular trafficking of Herpes simplex virus-1.

Although both lactoferrin (Lf), a component of the innate immune system of living organisms, and its N-terminal pepsin cleavage product lactoferricin (Lfcin) have anti-herpes activity, the precise mechanisms by which Lf and Lfcin bring about inhibition of herpes infections are not fully understood. In the present study, experiments were carried out to characterize the activity of bovine Lf and Lfcin (BLf and BLfcin) against the Herpes simplex virus-1 (HSV-1). HSV-1 cellular uptake and intracellular trafficking were studied by immunofluorescence microscopy. In comparison to the untreated infected control cells, both the BLf- and BLfcin-treated cells showed a significant reduction in HSV-1 cellular uptake. The few virus particles that were internalized appeared to have a delayed intracellular trafficking. Thus, in addition to their interference with the uptake of the virus into host cells, Lf and Lfcin also exert their antiviral effect intracellularly.

Cationic host defence peptides: innate immune regulatory peptides as a novel approach for treating infections.

An increase in antibiotic resistance and the emergence of new pathogens has led to an urgent need for alternative approaches to infection management. Immunomodulatory molecules that do not target the pathogen directly, but rather selectively enhance and/or alter host defence mechanisms, are attractive candidates for therapeutic development. Natural cationic host defence peptides represent lead molecules that boost innate immune responses and selectively modulate pathogen-induced inflammatory responses. This review discusses recent evidence exploring the mechanisms of cationic host defence peptides as innate immune regulators, their role in the interface of innate and adaptive immunity, and their potential application as beneficial therapeutics in overcoming infectious diseases.

Immunomodulatory properties of defensins and cathelicidins.

Host defence peptides are a conserved component of the innate immune response in all complex life forms. In humans, the major classes of host defence peptides include the alpha- and beta-defensins and the cathelicidin, hCAP-18/LL-37. These peptides are expressed in the granules of neutrophils and by a wide variety of tissue types. They have many roles in the immune response including both indirect and direct antimicrobial activity, the ability to act as chemokines as well as induce chemokine production leading to recruitment of leukocytes to the site of infection, the promotion of wound healing and an ability to modulate adaptive immunity. It appears that many of these properties are mediated though direct interaction of peptides with the cells of the innate immune response including monocytes, dendritic cells, T cells and epithelial cells. The importance of these peptides in immune responses has been demonstrated since animals defective in the expression of certain host defence peptides show greater susceptibility to bacterial infections. In the very few instances in which human patients have been demonstrated to have defective host defence peptide expression, these individuals suffer from frequent infections. Although studies of the immunomodulatory properties of these peptides are in their infancy, there is a growing body of evidence suggesting that the immunomodulatory properties of these small, naturally occurring molecules might be harnessed for development as novel therapeutic agents.

Contribution of the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems to Mg2+-induced gene regulation in Pseudomonas aeruginosa.

When grown in divalent cation-limited medium, Pseudomonas aeruginosa becomes resistant to cationic antimicrobial peptides and polymyxin B. This resistance is regulated by the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems. To further characterize Mg(2+) regulation in P. aeruginosa, microarray transcriptional profiling was conducted to compare wild-type P. aeruginosa grown under Mg(2+)-limited and Mg(2+)-replete conditions to isogenic phoP and pmrA mutants grown under Mg(2+)-limited conditions. Under Mg(2+)-limited conditions (0.02 mM Mg(2+)), approximately 3% of the P. aeruginosa genes were differentially expressed compared to the expression in bacteria grown under Mg(2+)-replete conditions (2 mM Mg(2+)). Only a modest subset of the Mg(2+)-regulated genes were regulated through either PhoP or PmrA. To determine which genes were directly regulated, a bioinformatic search for conserved binding motifs was combined with confirmatory reverse transcriptase PCR and gel shift promoter binding assays, and the results indicated that very few genes were directly regulated by these response regulators. It was found that in addition to the previously known oprH-phoP-phoQ operon and the pmrHFIJKLM-ugd operon, the PA0921 and PA1343 genes, encoding small basic proteins, were regulated by Mg(2+) in a PhoP-dependent manner. The number of known PmrA-regulated genes was expanded to include the PA1559-PA1560, PA4782-PA4781, and feoAB operons, in addition to the previously known PA4773-PA4775-pmrAB and pmrHFIJKLM-ugd operons.

Effects of single D-amino acid substitutions on disruption of beta-sheet structure and hydrophobicity in cyclic 14-residue antimicrobial peptide analogs related to gramicidin S.

Gramicidin S (GS) is a 10-residue cyclic beta-sheet peptide with lytic activity against the membranes of both microbial and human cells, i.e. it possesses little to no biologic specificity for either cell type. Structure-activity studies of de novo-designed 14-residue cyclic peptides based on GS have previously shown that higher specificity against microbial membranes, i.e. a high therapeutic index (TI), can be achieved by the replacement of a single L-amino acid with its corresponding D-enantiomer [Kondejewski, L.H. et al. (1999) J. Biol. Chem. 274, 13181]. The diastereomer with a D-Lys substituted at position 4 caused the greatest improvement in specificity vs. other L to D substitutions within the cyclic 14-residue peptide GS14, through a combination of decreased peptide amphipathicity and disrupted beta-sheet structure in aqueous conditions [McInnes, C. et al. (2000) J. Biol. Chem. 275, 14287]. Based on this information, we have created a series of peptide diastereomers substituted only at position 4 by a D- or L-amino acid (Leu, Phe, Tyr, Asn, Lys, and achiral Gly). The amino acids chosen in this study represent a range of hydrophobicities/hydrophilicities as a subset of the 20 naturally occurring amino acids. While the D- and L-substitutions of Leu, Phe, and Tyr all resulted in strong hemolytic activity, the substitutions of hydrophilic D-amino acids D-Lys and D-Asn in GS14 at position 4 resulted in weaker hemolytic activity than in the L-diastereomers, which demonstrated strong hemolysis. All of the L-substitutions also resulted in poor antimicrobial activity and an extremely low TI, while the antimicrobial activity of the D-substituted peptides tended to improve based on the hydrophilicity of the residue. D-Lys was the most polar and most efficacious substitution, resulting in the highest TI. Interestingly, the hydrophobic D-amino acid substitutions had superior antimicrobial activity vs. the L-enantiomers although substitution of a hydrophobic D-amino acid increases the nonpolar face hydrophobicity. These results further support the role of hydrophobicity of the nonpolar face as a major influence on microbial specificity, but also highlights the importance of a disrupted beta-sheet structure on antimicrobial activity.

Clinical development of cationic antimicrobial peptides: from natural to novel antibiotics.

Over the past decade, levels of bacterial resistance to antibiotics have risen dramatically and "superbugs" resistant to most or all available agents have appeared in the clinic. Thus there is a growing need to discover and introduce new drugs. One potential source of novel antibiotics is the cationic antimicrobial peptides, which have been isolated from most living entities as components of their non-specific defenses against infectious organisms. Based on these natural templates, scores of structurally diverse antimicrobial cationic peptides have been designed, manufactured both chemically and biologically, and tested for activity against specific pathogens. A few of these peptide antibiotics have entered clinical trials to date, with mixed success. However, their diverse portfolio of structures, activity spectra, biological activities, and modes of action, provide substantial potential.

Exploring relationships between mimic configuration, peptide conformation and biological activity in indolizidin-2-one amino acid analogs of gramicidin S.

Indolizidin-2-one amino acids (I2aas, 6S- and 6R-1) possessing 6S- and 6R-ring-fusion stereochemistry were introduced into the antimicrobial peptide gramicidin S (GS) to explore the relationships between configuration, peptide conformation and biological activity. Solution-phase and solid-phase techniques were used to synthesize three analogs with I2aa residues in place of the d-Phe-Pro residues at the turn regions of GS: [(6S)-I2aa4-5,4'-5']GS (2), [Lys2,2',(6S)-I2aa4-5,4'-5']GS (3) and [(6R)-I2aa4-5,4'-5']GS (4). Although conformational analysis of [I2aa4-5,4'-5']GS analogs 2-4 indicated that both ring-fusion stereoisomers of I2aa gave peptides with CD and NMR spectral data characteristic of GS, the (6S)-I2aa analogs 2 and 3 exhibited more intense CD curve shapes, as well as greater numbers of nonsequential NOE between opposing Val and Leu residues, relative to the (6R)-I2aa analog 4, suggesting a greater propensity for the (6S)-diastereomer to adopt the beta-turn/antiparallel beta-pleated sheet conformation. In measurements of antibacterial and antifungal activity, the (6S)-I2aa analog 2 exhibited significantly better potency than the (6R)-I2aa diastereomer 4. Relative to GS, [(6S)-I2aa4-5,4'-5']GS (2) exhibited usually 1/2 to 1/4 antimicrobial activity as well as 1/4 hemolytic activity. In certain cases, antimicrobial and hemolytic activities of GS were shown to be dissociated through modification at the peptide turn regions with the (6S)-I2aa diastereomer. The synthesis and evaluation of GS analogs 2-4 has furnished new insight into the importance of ring-fusion stereochemistry for turn mimicry by indolizidin-2-one amino acids as well as novel antimicrobial peptides.