Outer membrane differences between pathogenic and environmental Yersinia enterocolitica biogroups probed with hydrophobic permeants and polycationic peptides

Sensitivities to polycationic peptides and EDTA were compared in Yersinia enterocolitica pathogenic and environmental biogroups. As shown by changes in permeability to the fluorescent hydrophobic probe N-phenylnaphthylamine (NPN), the outer membranes (OMs) of pathogenic and environmental strains grown at 26 degrees C in standard broth were more resistant to poly-L-lysine, poly-L-ornithine, melittin, cecropin P1, polymyxin B, and EDTA than Escherichia coli OMs. At 37 degrees C, OMs of pathogenic biogroups were resistant to EDTA and polycations and OMs of environmental strains were resistant to EDTA whereas E. coli OMs were sensitive to both EDTA and polycations. Similar results were found when testing deoxycholate sensitivity after polycation exposure or when isogenic pairs with or without virulence plasmid pYV were compared. With bacteria grown without Ca++ available, OM permeability to NPN was drastically increased in pathogenic but not in environmental strains or E. coli. Under these conditions, OMs of pYV+ and pYV- cells showed small differences in NPN permeability but differences in polycation sensitivity could not be detected by fluorimetry. O:1,6 (environmental type) lipopolysaccharide (LPS), but not O:3 or O:8 LPS, was markedly rough at 37 degrees C, and this could explain the differences in polycation sensitivity. LPSs from serotypes O:3 and O:8 grown at 37 degrees C were more permeable to NPN than O:1,6 LPS, and O:8 LPS was resistant to polycation-induced permeabilization. These data suggest that LPSs relate to some but not all the OM differences described. It is hypothesized that the different OM properties of environmental and pathogenic biogroups reflect the adaptation of the latter biogroups to pathogenicity.

Yersinia pseudotuberculosis and Yersinia pestis are more resistant to bactericidal cationic peptides than Yersinia enterocolitica

The action of bactericidal polycationic peptides was compared in Yersinia spp. by testing peptide binding to live cells and changes in outer membrane (OM) morphology and permeability. Moreover, polycation interaction with LPS was studied by measuring the dependence of dansylcadaverine displacement and zeta potential on polycation concentration. When growth at 37 degrees C, Yersinia pestis and Yersinia pseudotuberculosis bound less polymyxin B (PMB) than pathogenic or non-pathogenic Yersinia enterocolitica, regardless of virulence plasmid expression. Y. pseudotuberculosis OMs were unharmed by PMB concentrations causing extensive OM blebbing in Y. enterocolitica. The permeability to lysozyme caused by PMB was greater in Y. enterocolitica than in Y. pseudotuberculosis or Y. pestis and differences increased at 37 degrees C. Similar observations were made with other polycations using a polymyxin/novobiocin permeability assay. With LPS of cells grown at 26 degrees C, polycation binding was highest for Y. pseudotuberculosis and lowest for Y. pestis, with Y. enterocolitica yielding intermediate results which were lower for pathogenic than for non-pathogenic strains. With LPS of cells grown at 37 degrees C, polycation binding remained unchanged for Y. pestis and pathogenic Y. enterocolitica, increased for non-pathogenic Y. enterocolitica and decreased for Y. pseudotuberculosis to Y. pestis levels. Polycation binding related in part to differences in charge density (zeta potential) of LPS aggregates, suggesting similar effects at bacterial surfaces. It is suggested that species and temperature differences in polycation resistance relate to infection route, invasiveness and intracellular multiplication of Yersinia spp.

Purification, characterization and molecular cloning of chymotrypsin inhibitor peptides from the venom of Burmese Daboia russelii siamensis

One novel Kunitz BPTI-like peptide designated as BBPTI-1, with chymotrypsin inhibitory activity was identified from the venom of Burmese Daboia russelii siamensis. It was purified by three steps of chromatography including gel filtration, cation exchange and reversed phase. A partial N-terminal sequence of BBPTI-1, HDRPKFCYLPADPGECLAHMRSF was obtained by automated Edman degradation and a Ki value of 4.77. nM determined. Cloning of BBPTI-1 including the open reading frame and 3' untranslated region was achieved from cDNA libraries derived from lyophilized venom using a 3' RACE strategy. In addition a cDNA sequence, designated as BBPTI-5, was also obtained. Alignment of cDNA sequences showed that BBPTI-5 exhibited an identical sequence to BBPTI-1 cDNA except for an eight nucleotide deletion in the open reading frame. Gene variations that represented deletions in the BBPTI-5 cDNA resulted in a novel protease inhibitor analog. Amino acid sequence alignment revealed that deduced peptides derived from cloning of their respective precursor cDNAs from libraries showed high similarity and homology with other Kunitz BPTI proteinase inhibitors. BBPTI-1 and BBPTI-5 consist of 60 and 66 amino acid residues respectively, including six conserved cysteine residues. As these peptides have been reported to have influence on the processes of coagulation, fibrinolysis and inflammation, their potential application in biomedical contexts warrants further investigation. © 2013 Elsevier Inc.

Asprich Peptides Are Occluded in Calcite and Permanently Disorder Biomineral Crystals

Macromolecules are a minority but important component of the minerals formed by living organisms, or biominerals. The role these macromolecules play at the early stages of biomineral formation, as well as their long-term and long-range effects on the mature biomineral, is poorly understood. A 42-amino acid peptide, asp2, was derived from the Asprich family of proteins. In this study we present X-ray absorption near-edge structure spectroscopy and X-ray photoelectron emission microscopy data from the asp2 peptide, the calcite (CaCO3) crystals, and the peptide + crystal composites. The results clearly show that asp2 is occluded in fully formed biomineral crystals and slightly but permanently disorders the crystal structure at short- and long-range distances.

Local and systemic immune responses in mice to intranasal delivery of peptides representing bovine respiratory syncytial virus epitopes encapsulated in poly (dl-lactide-co-glycolide) microparticles

The potential of a microparticulate vaccine delivery system in eliciting a specific mucosal antibody response in the respiratory tract of mice was evaluated. Two vaccine candidate peptides representing epitopes from the G attachment and F fusion antigens from bovine respiratory syncytial virus (BRSV) were encapsulated into poly(dl- lactide co-glycolide) biodegradable microparticles. The encapsulation process did not denature the entrapped peptides as verified by detection of peptide-specific antibodies in mucosal secretions by ELISA using peptide as antigen. Following intranasal immunisation, the encapsulated peptides induced stronger upper and lower respiratory tract specific-IgA responses, respectively, than the soluble peptide forms. Moreover, a strong peptide-specific cell-mediated immune response was measured in splenocytes in vitro from the mice inoculated with the encapsulated peptides compared to their soluble form alone indicating that migration of primed T cells had taken place from the site of mucosal stimulation in the upper respiratory tract to the spleen. These results act as a foundation for vaccine efficacy studies in large animal BRSV challenge models.

Fragmentation of Neutral Amino Acids and Small Peptides by Intense, Femtosecond Laser Pulses

High power femtosecond laser pulses have unique properties that could lead to their application as ionization or activation sources in mass spectrometry. By concentrating many photons into pulse lengths approaching the timescales associated with atomic motion, very strong electric field strengths are generated, which can efficiently ionize and fragment molecules without the need for resonant absorption. However, the complex interaction between these pulses and biomolecular species is not well understood. To address this issue, we have studied the interaction of intense, femtosecond pulses with a number of amino acids and small peptides. Unlike previous studies, we have used neutral forms of these molecular targets, which allowed us to investigate dissociation of radical cations without the spectra being complicated by the action of mobile protons. We found fragmentation was dominated by fast, radical-initiated dissociation close to the charge site generated by the initial ionization or from subsequent ultrafast migration of this charge. Fragments with lower yields, which are useful for structural determinations, were also observed and attributed to radical migration caused by hydrogen atom transfer within the molecule.

Two peptides, TsAP-1 and TsAP-2, from the venom of the Brazilian yellow scorpion, Tityus serrulatus: evaluation of their antimicrobial and anticancer activities

Here we report two novel 17-mer amidated linear peptides (TsAP-1 and TsAP-2) whose structures were deduced from cDNAs cloned from a venom-derived cDNA library of the Brazilian yellow scorpion, Tityus serrulatus. Both mature peptides were structurally-characterised following their location in chromatographic fractions of venom and synthetic replicates of each were subjected to a range of biological assays. The peptides were each active against model test micro-organisms but with different potencies. TsAP-1 was of low potency against all three test organisms (MICs 120-160µM), whereas TsAP-2 was of high potency against the Gram-positive bacterium, Staphylococcus aureus (MIC 5µM) and the yeast, Candida albicans (10µM). Haemolytic activity of TsAP-1 was low (4% at 160µM) and in contrast, that of TsAP-2 was considerably higher (18% at 20µM). Substitution of four neutral amino acid residues with Lys residues in each peptide had dramatic effects on their antimicrobial potencies and haemolytic activities, particularly those of TsAP-1. The MICs of the enhanced cationic analogue (TsAP-S1) were 2.5µM for S.aureus/C.albicans and 5µM for E.coli but with an associated large increase in haemolytic activity (30% at 5µM). The same Lys residue substitutions in TsAP-2 produced a dramatic effect on its MIC for E.coli lowering this from >320µM to 5µM. TsAP-1 was ineffective against three of the five human cancer cell lines tested while TsAP-2 inhibited the growth of all five. Lys residue substitution of both peptides enhanced their potency against all five cell lines with TsAp-S2 being the most potent with IC50 values ranging between 0.83 and 2.0 µM. TsAP-1 and TsAP-2 are novel scorpion venom peptides with broad spectrum antimicrobial and anticancer cell activities the potencies of which can be significantly enhanced by increasing their cationicity.

Solid phase synthesis of Smac/DIABLO-derived peptides using a 'Safety-Catch' resin:Identification of potent XIAP BIR3 antagonists

The N-terminal sequence of the Smac/DIABLO protein is known to be involved in binding to the BIR3 domain of the anti-apoptotic proteins IAPs, antagonizing their action. Short peptides and peptide mimetics based on the first 4-residues of Smac/DIABLO have been demonstrated to re-sensitize resistant cancer cells, over-expressing IAPs, to apoptosis. Based on the well-defined structural basis for this interaction, a small focused library of C-terminal capped Smac/DIABLO-derived peptides was designed in silico using docking to the XIAP BIR3 domain. The top-ranked computational hits were conveniently synthesized employing Solid Phase Synthesis (SPS) on an alkane sulfonamide 'Safety-Catch' resin. This novel approach afforded the rapid synthesis of the target peptide library with high flexibility for the introduction of various C-terminal amide-capping groups. The library members were obtained in high yield (>65%) and purity (>85%), upon nucleophilic release from the activated resin by treatment with various amine nucleophiles. In vitro caspase-9 activity reconstitution assays of the peptides in the presence of the recombinant BIR3-domain of human XIAP (500nM) revealed N-methylalanyl-tertiarybutylglycinyl-4-(R)-phenoxyprolyl-N-biphenylmethyl carboxamide (11a) to be the most potent XIAP BIR3 antagonist of the series synthesized inducing 93% recovery of caspase-9 activity, when used at 1µM concentration. Compound (11a) also demonstrated moderate cytotoxicity against the breast cancer cell lines MDA-MB-231 and MCF-7, compared to the Smac/DIABLO-derived wild-type peptide sequences that were totally inactive in the same cell lines.

Bradykinin-related peptides (BRPs) from skin secretions of three genera of phyllomedusine leaf frogs and their comparative pharmacological effects on mammalian smooth muscles

While bradykinin has been identified in the skin secretions from several species of amphibian, bradykinin-related peptides (BRPs) are more common constituents. These peptides display a plethora of primary structural variations from the type peptide which include single or multiple amino acid substitutions, N- and/or C-terminal extensions and post-translational modifications such as proline hydroxylation and tyrosine sulfation. Such modified peptides have been reported in species from many families, including Bombinatoridae, Hylidae and Ranidae. The spectrum of these peptides in a given species is thought to be reflective of its predator profile from different vertebrate taxa. Here we report the isolation of BRPs and parallel molecular cloning of their respective biosynthetic precursor-encoding cDNAs from the skin secretions of the Mexican leaf frog (Pachymedusa dacnicolor), the Central American red-eyed leaf frog (Agalychnis callidryas) and the South American orange-legged leaf frog (Phyllomedusa hypochondrialis). Additionally, the eight different BRPs identified were chemically synthesized and screened for bioactivity using four different mammalian smooth muscle preparations and their effects and rank potencies were found to be radically different in these with some acting preferentially through bradykinin B1-type receptors and others through B2-type receptors.

Ultrashort Cationic Naphthalene-Derived Self-Assembled Peptides as Antimicrobial Nanomaterials

Self-assembling dipeptides conjugated to naphthalene show considerable promise as nanomaterial structures, biomaterials, and drug delivery devices. Biomaterial infections are responsible for high rates of patient mortality and morbidity. The presence of biofilm bacteria, which thrive on implant surfaces, are a huge burden on healthcare budgets, as they are highly resistant to current therapeutic strategies. Ultrashort cationic self-assembled peptides represent a highly innovative and cost-effective strategy to form antibacterial nanomaterials. Lysine conjugated variants display the greatest potency with 2% w/v NapFFKK hydrogels significantly reducing the viable Staphylococcus epidermidis biofilm by 94%. Reducing the size of the R-group methylene chain on cationic moieties resulted in reduction of antibiofilm activity. The primary amine of the protruding R-group tail may not be as readily available to interact with negatively charged bacterial membranes. Cryo-SEM, FTIR, CD spectroscopy, and oscillatory rheology provided evidence of supramolecular hydrogel formation at physiological pH (pH 7.4). Cytotoxicity assays against murine fibroblast (NCTC 929) cell lines confirmed the gels possessed reduced cytotoxicity relative to bacterial cells, with limited hemolysis upon exposure to equine erythrocytes. The results presented in this paper highlight the significant potential of ultrashort cationic naphthalene peptides as future biomaterials.

Attempting to rewrite History:Challenges with the analysis of histidine-phosphorylated peptides

A significant number of proteins in both eukaryotes and prokaryotes are known to be post-translationally modified by the addition of phosphate, serving as a means of rapidly regulating protein function. Phosphorylation of the amino acids serine, threonine and tyrosine are the focus of the vast majority of studies aimed at elucidating the extent and roles of such modification, yet other amino acids, including histidine and aspartate, are also phosphorylated. Although histidine phosphorylation is known to play extensive roles in signalling in eukaryotes, plants and fungi, roles for phosphohistidine are poorly defined in higher eukaryotes. Characterization of histidine phosphorylation aimed at elucidating such information is problematic due to the acid-labile nature of the phosphoramidate bond, essential for many of its biological functions. Although MSbased strategies have proven extremely useful in the analysis of other types of phosphorylated peptides, the chromatographic procedures essential for such approaches promote rapid hydrolysis of phosphohistidinecontaining peptides. Phosphate transfer to non-biologically relevant aspartate residues during MS analysis further complicates the scenario. © 2013 Biochemical Society.

Cationicity-Enhanced Analogues of the Antimicrobial Peptides, AcrAP1 and AcrAP2, from the Venom of the Scorpion, Androctonus crassicauda, Display Potent Growth Modulation Effects on Human Cancer Cell Lines

The non disulphide-bridged peptides (NDBPs) of scorpion venoms are attracting increased interest due to their structural heterogeneity and broad spectrum of biological activities. Here, two novel peptides, named AcrAP1 and AcrAP2, have been identified in the lyophilised venom of the Arabian scorpion, Androctonus crassicauda, through “shotgun” molecular cloning of their biosynthetic precursor-encoding cDNAs. The respective mature peptides, predicted from these cloned cDNAs, were subsequently isolated from the same venom sample using reverse phase HPLC and their identities were confirmed by use of mass spectrometric techniques. Both were found to belong to a family of highly-conserved scorpion venom antimicrobial peptides - a finding confirmed through the biological investigation of synthetic replicates. Analogues of both peptides designed for enhanced cationicity, displayed enhanced potency and spectra of antimicrobial activity but, unlike the native peptides, these also displayed potent growth modulation effects on a range of human cancer cell lines. Thus natural peptide templates from venom peptidomes can provide the basis for rational analogue design to improve both biological potency and spectrum of action. The diversity of such templates from such natural sources undoubtedly provides the pharmaceutical industry with unique lead compounds for drug discovery.

Isotretinoin therapy changes the expression of antimicrobial peptides in acne vulgaris

In acne vulgaris, antimicrobial peptides (AMPs) could play a dual role; i.e., protective by acting against Propionibacterium acnes, pro-inflammatory by acting as signalling molecules. The cutaneous expression of 15 different AMPs was investigated in acne patients; furthermore, the impact of isotretinoin therapy on AMP expression was analysed in skin biopsies from 13 patients with acne vulgaris taken before, during and after a 6-month treatment cycle with isotretinoin using quantitative real-time polymerase chain reaction. Cutaneous expression of the AMPs cathelicidin, human β-defensin-2 (HBD-2), lactoferrin, lysozyme, psoriasin (S100A7), koebnerisin (S100A15), and RNase 7 was upregulated in untreated acne vulgaris, whereas α-defensin-1 (HNP-1) was downregulated compared to controls. While relative expression levels of cathelicidin, HBD-2, lactoferrin, psoriasin (S100A7), and koebnerisin (S100A15) decreased during isotretinoin treatment, only those of cathelicidin and koebnerisin returned to normal after 6 months of isotretinoin therapy. The increased expression of lysozyme and RNase 7 remained unaffected by isotretinoin treatment. The levels of granulysin, RANTES (CCL5), perforin, CXCL9, substance P, chromogranin B, and dermcidin were not regulated in untreated acne patients and isotretinoin had no effect on these AMPs. In conclusion, the expression of various AMPs is altered in acne vulgaris. Isotretinoin therapy normalizes the cutaneous production of distinct AMPs while the expression of others is still increased in healing acne. Considering the antimicrobial and pro-inflammatory role of AMPs, these molecules could serve as specific targets for acne therapy and maintenance of clinical remission.