Low pH enhances the action of maximin H5 against Staphylococcus aureus and helps mediate lysylated phosphatidylglycerol induced resistance

Maximin H5 (MH5) is an amphibian antimicrobial peptide specifically targeting Staphylococcus aureus. At pH 6, the peptide showed an increased ability to penetrate (∆П = 6.2 mN m-1) and lyse (lysis = 48 %) S. aureus membrane mimics, which incorporated physiological levels of lysylated phosphatidylglycerol (Lys-PG, 60 %) as compared to pH 7 (∆П = 5.6 mN m-1 and lysis = 40 % at pH 7) where levels of Lys-PG are lower (40 %). The peptide therefore appears to have optimal function at pH levels known to be optimal for the organism’s growth. MH5 killed S. aureus (minimum inhibitory concentration = 90 µM) via membranolytic mechanisms that involved the stabilization of α-helical structure (circa 45-50 %) and which showed similarities to the ‘Carpet’ mechanism based on its ability to increase the rigidity (Cs-1 = 109.94 mN m-1) and thermodynamic stability (∆Gmix = -3.0) of physiologically relevant S. aureus membrane mimics at pH 6. Based on theoretical analysis this mechanism may involve the use of a tilted peptide structure and efficacy was noted to vary inversely with the Lys-PG content of S. aureus membrane mimics for each pH studied (R2 circa 0.97), which led to the suggestion that under biologically relevant conditions, low pH helps mediate Lys-PG induced resistance in S. aureus to MH5 antibacterial action. The peptide showed a lack of haemolytic activity (< 2 % haemolysis) and merits further investigation as a potential template for development as an anti-staphylococcal agent in medically and biotechnically relevant areas

Characterisation of three alpha-helical antimicrobial peptides from the venom of Scorpio maurus palmatus.

Scorpion venoms provide a rich source of anti-microbial peptides. Here we characterise three from the venom of Scorpion maurus palmatus. Smp13 is biologically inactive, despite sharing homology with other antimicrobial peptides, probably because it lacks a typically charged structure. Both Smp-24 and Smp-43 have broad spectrum antimicrobial activity, disrupting bacterial membranes. In addition, there is evidence that Smp24 may inhibit DNA synthesis in Bacillus subtilis. Smp24 haemolysed red blood cells but in contrast, Smp43 was non-haemolytic. The introduction of a flexible Gly-Val-Gly hinge into the middle of Smp24 did not alter the haemolytic activity of Smp24 (as might have been predicted from earlier studies with Pandinin2 (Pin2), although C-terminal truncation of Smp-24 reduced its haemolytic activity, in agreement with earlier Pin 2 studies. Smp24 and its derivatives, as well as Smp-43, were all cytotoxic (ATP release assay) toward mammalian HepG2 liver cells. Our results highlight the beneficial effect of helical-hinge-helical conformation on promoting prokaryotic selectivity of long chain scorpion AMPs, as well as the importance of examining a wide range of mammalian cell types in cytotoxicity testing

Antimicrobial Peptides and Skin: A Paradigm of Translational Medicine

Antimicrobial peptides (AMPs) are small, cationic, amphiphilic peptides with broad-spectrum microbicidal activity against both bacteria and fungi. In mammals, AMPs form the first line of host defense against infections and generally play an important role as effector agents of the innate immune system. The AMP era was born more than 6 decades ago when the first cationic cyclic peptide antibiotics, namely polymyxins and tyrothricin, found their way into clinical use. Due to the good clinical experience in the treatment of, for example, infections of mucus membranes as well as the subsequent understanding of mode of action, AMPs are now considered for treatment of inflammatory skin diseases and for improving healing of infected wounds. Based on the preclinical findings, including pathobiochemistry and molecular medicine, targeted therapy strategies are developed and first results indicate that AMPs influence processes of diseased skin. Importantly, in contrast to other antibiotics, AMPs do not seem to propagate the development of antibiotic-resistant micro-organisms. Therefore, AMPs should be tested in clinical trials for their efficacy and tolerability in inflammatory skin diseases and chronic wounds. Apart from possible fields of application, these peptides appear suited as an example of the paradigm of translational medicine for skin diseases which is today seen as a `two-way road' - from bench to bedside and backwards from bedside to bench. Copyright (c) 2012 S. Karger AG, Base

Side Chain Hydrophobicity Modulates Therapeutic Activity and Membrane Selectivity of Antimicrobial Peptide Mastoparan-X

The discovery of new anti-infective compounds is stagnating and multi-resistant bacteria continue to emerge, threatening to end the "antibiotic era". Antimicrobial peptides (AMPs) and lipo-peptides such as daptomycin offer themselves as a new potential class of antibiotics; however, further optimization is needed if AMPs are to find broad use as antibiotics. In the present work, eight analogues of mastoparan-X (MPX) were investigated, having side chain modifications in position 1, 8 and 14 to modulate peptide hydrophobicity. The self-association properties of the peptides were characterized, and the peptide-membrane interactions in model membranes were compared with the bactericidal and haemolytic properties. Alanine substitution at position 1 and 14 resulted in higher target selectivity (red blood cells versus bacteria), but also decreased bactericidal potency. For these analogues, the gain in target selectivity correlated to biophysical parameters showing an increased effective charge and reduction in the partitioning coefficient for membrane insertion. Introduction of an unnatural amino acid, with an octyl side chain by amino acid substitution, at positions 1, 8 and 14 resulted in increased bactericidal potency at the expense of radically reduced membrane target selectivity. Overall, optimized membrane selectivity or bactericidal potency was achieved by changes in side chain hydrophobicity of MPX. However, enhanced potency was achieved at the expense of selectivity and vice versa in all cases

Adaptive Evolution of Escherichia coli to an α-Peptide/β-Peptoid Peptidomimetic Induces Stable Resistance.

Antimicrobial peptides (AMPs) and synthetic analogues thereof target conserved structures of bacterial cell envelopes and hence, development of resistance has been considered an unlikely event. However, recently bacterial resistance to AMPs has been observed, and the aim of the present study was to determine whether bacterial resistance may also evolve against synthetic AMP analogues, e.g. α-peptide/β-peptoid peptidomimetics. E. coli ATCC 25922 was exposed to increasing concentrations of a peptidomimetic (10 lineages), polymyxin B (10 lineages), or MilliQ water (4 lineages) in a re-inoculation culturing setup covering approx. 500 generations. All 10 lineages exposed to the peptidomimetic adapted to 32 × MIC while this occurred for 8 out of 10 of the polymyxin B-exposed lineages. All lineages exposed to 32 × MIC of either the peptidomimetic or polymyxin B had a significantly increased MIC (16-32 ×) to the selection agent. Five transfers (≈ 35 generations) in unsupplemented media did not abolish resistance indicating that resistance was heritable. Single isolates from peptidomimetic-exposed lineage populations displayed MICs against the peptidomimetic from wild-type MIC to 32 × MIC revealing heterogeneous populations. Resistant isolates showed no cross-resistance against a panel of membrane-active AMPs. These isolates were highly susceptible to blood plasma antibacterial activity and were killed when plasma concentrations exceeded ≈ 30%. Notably, MIC of the peptidomimetic against resistant isolates returned to wild-type level upon addition of 25% plasma. Whole-genome sequencing of twenty isolates from four resistant lineages revealed mutations, in murein transglycosylase D (mltD) and outer-membrane proteins, which were conserved within and between lineages. However, no common resistance-conferring mutation was identified. We hypothesise that alterations in cell envelope structure result in peptidomimetic resistance, and that this may occur via several distinct mechanisms. Interestingly, this type of resistance result in a concomitant high susceptibility towards plasma, and therefore the present study does not infer additional concern for peptidomimetics as future therapeutics

Raman Spectroscopy of Synthetic Antimicrobial Frog Peptides Magainin 2a and PGLa

Magainin and PGLa are 23- and 21-residue peptides isolated from the skin of the African clawed frog Xenopus lueuis. They protect the frog from infection and exhibit a broad-spectrum antimicrobial activity in vitro. The mechanism of this activity involves the interaction of magainin with microbial membranes. We have measured the secondary structure and membrane-perturbing ability of these peptides to obtain information about this mechanism. Our results show that mgn2a forms a helix with an average length of less than 20 Å upon binding to liposomes. At high concentrations (50 mg/mL) mgn2a spontaneously solubilizes phosphatidylcholine liposomes at temperatures above the gel-liquid-crystalline phase transition. Mgn2a appears to bind to the surface of liposomes made of negatively charged lipids without spontaneously penetrating the bilayer. Finally, mgn2a and PGLa interact together with liposomes in a synergistic way that enhances the helix content of one or both of the peptides and allows the peptides to more easily penetrate the bilayer. PGLa mixed with a small nonperturbing amount of magainin 2 amide is 25-43 times as potent as PGLa alone at inducing the release of carboxyfluorescein from liposomes. The results suggest that the mechanism of antimicrobial activity does not involve a channel formed by transmembrane helical peptides

Genome-wide mapping of cystitis due to Streptococcus agalactiae and Escherichia coli in mice identifies a unique bladder transcriptome that signifies pathogen-specific antimicrobial defense against urinary tract infection

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli; however, Gram-positive organisms, including Streptococcus agalactiae, or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize the bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35-year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; quantitative reverse transcriptase PCR (qRT-PCR) was used to analyze selected gene responses identified in array data sets. A surprisingly small significant-gene list of 172 genes was identified at 24 h; this compared to 2,507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2 h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2 h. Bioinformatics analyses, including integrative system-level network mapping, revealed multiple activated biological pathways in the GBS bladder transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens

L-isoleucine-supplemented Oral Rehydration Solution in the Treatment of Acute Diarrhoea in Children: A Randomized Controlled Trial

Antimicrobial peptides represent an important component of the innate immune defenses of living organisms, including humans. They are broad-spectrum surface-acting agents secreted by the epithelial cells of the body in response to infection. Recently, L-isoleucine and its analogues have been found to induce antimicrobial peptides. The objectives of the study were to examine if addition of L-isoleucine to oral rehydration salts (ORS) solution would reduce stool output and/or duration of acute diarrhoea in children and induce antimicrobial peptides in intestine. This double-blind randomized controlled trial was conducted at the Dhaka Hospital of ICDDR,B. Fifty male children, aged 6-36 months, with acute diarrhoea and some dehydration, attending the hospital, were included in the study. Twenty-five children received L-isoleucine (2 g/L)-added ORS (study), and 25 received ORS without L-isoleucine (control). Stool weight, ORS intake, and duration of diarrhoea were the primary outcomes. There was a trend in reduction in mean±standard deviation (SD) daily stool output (g) of children in the L-isoleucine group from day 2 but it was significant on day 3 (388±261 vs 653±446; the difference between mean [95% confidence interval (CI) (-)265 (−509, −20); p=0.035]. Although the cumulative stool output from day 1 to day 3 reduced by 26% in the isoleucine group, it was not significant. Also, there was a trend in reduction in the mean±SD intake of ORS solution (mL) in the L-isoleucine group but it was significant only on day 1 (410±169 vs 564±301), the difference between mean (95% CI) (-)154 (-288, −18); p=0.04. The duration (hours) of diarrhoea was similar in both the groups. A gradual increase in stool concentrations of ß-defensin 2 and 3 was noted but they were not significantly different between the groups. L-isoleucine-supplemented ORS might be beneficial in reducing stool output and ORS intake in children with acute watery diarrhoea. A further study is warranted to substantiate the therapeutic effect of L-isoleucine

Classification and function of small open reading frames

Small open reading frames (smORFs) of 100 codons or fewer are usually - if arbitrarily - excluded from proteome annotations. Despite this, the genomes of many metazoans, including humans, contain millions of smORFs, some of which fulfil key physiological functions. Recently, the transcriptome of Drosophila melanogaster was shown to contain thousands of smORFs of different classes that actively undergo translation, which produces peptides of mostly unknown function. Here, we present a comprehensive analysis of smORFs in flies, mice and humans. We propose the existence of several functional classes of smORFs, ranging from inert DNA sequences to transcribed and translated cis-regulators of translation and peptides with a propensity to function as regulators of membrane-associated proteins, or as components of ancient protein complexes in the cytoplasm. We suggest that the different smORF classes could represent steps in gene, peptide and protein evolution. Our analysis introduces a distinction between different peptide-coding classes of smORFs in animal genomes, and highlights the role of model organisms for the study of small peptide biology in the context of development, physiology and human disease