Effects of Charge Location on the Absorptions and Lifetimes of Protonated Tyrosine Peptides in Vacuo

Nearby charges affect the electronic energy levels of chromophores, with the extent of the effect being determined by the magnitude of the charge and degree of charge-chromophore separation. The molecular configuration dictates the charge chromophore distance. Hence, in this study, we aim to assess how the location of the charge influences the absorption of a set of model protonated and diprotonated peptide ions, and whether spectral differences are large enough to be identified. The studied ions were the dipeptide YK, the tripeptide KYK (Y = tyrosine; K = lysine) and their complexes with 18-crown-6-ether (CE). The CE targets the ammonium group by forming internal ionic hydrogen bonds and limits the folding of the peptide. In the tripeptide, the distance between the chromophore and the backbone ammonium is enlarged relative to that in the dipeptide. Experiments were performed in an electrostatic ion storage ring using a tunable laser system, and action spectra based on lifetime measurements were obtained in the range from 210 to 310 nm. The spectra are all quite similar though there seems to be some changes in the absorption band between 210 and 250 nm, while in the lower energy band all ions had a maximum absorption at similar to 275 nm. Lifetimes after photoexcitation were found to shorten upon protonation and lengthen upon CE complexation, in accordance with the increased number of degrees of freedom and an increase in activation energies for dissociation as the mobile proton model is no longer operative.

A complete lipopolysaccharide inner core oligosaccharide is required for resistance of Burkholderia cenocepacia to antimicrobial peptides and bacterial survival in vivo

Burkholderia cenocepacia is an important opportunistic pathogen of patients with cystic fibrosis. This bacterium is inherently resistant to a wide range of antimicrobial agents, including high concentrations of antimicrobial peptides. We hypothesized that the lipopolysaccharide (LPS) of B. cenocepacia is important for both virulence and resistance to antimicrobial peptides. We identified hldA and hldD genes in B. cenocepacia strain K56-2. These two genes encode enzymes involved in the modification of heptose sugars prior to their incorporation into the LPS core oligosaccharide. We constructed a mutant, SAL1, which was defective in expression of both hldA and hldD, and by performing complementation studies we confirmed that the functions encoded by both of these B. cenocepacia genes were needed for synthesis of a complete LPS core oligosaccharide. The LPS produced by SAL1 consisted of a short lipid A-core oligosaccharide and was devoid of O antigen. SAL1 was sensitive to the antimicrobial peptides polymyxin B, melittin, and human neutrophil peptide 1. In contrast, another B. cenocepacia mutant strain that produced complete lipid A-core oligosaccharide but lacked polymeric O antigen was not sensitive to polymyxin B or melittin. As determined by the rat agar bead model of lung infection, the SAL1 mutant had a survival defect in vivo since it could not be recovered from the lungs of infected rats 14 days postinfection. Together, these data show that the B. cenocepacia LPS inner core oligosaccharide is needed for in vitro resistance to three structurally unrelated antimicrobial peptides and for in vivo survival in a rat model of chronic lung infection.

Novel macrocyclic and acyclic cationic lipids for gene transfer: Synthesis and in vitro evaluation

The synthesis and in vitro evaluation of four cationic lipid gene delivery vectors, characterized by acyclic or macrocyclic, and saturated or unsaturated hydrophobic regions, is described. The synthesis employed standard protocols, including ring-closing metathesis for macrocyclic lipid construction. All lipoplexes studied, formulated from plasmid DNA and a liposome composed of a synthesized lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol as co-lipid, exhibited plasmid DNA binding and protection from DNase I degradation, and concentration dependent cytotoxicity using Chinese hamster ovary-K1 cells. The transfection efficiency of formulations with cholesterol outperformed those with DOPE, and in many cases the EPC/cholesterol control, and formulations with a macrocyclic lipid (+/- 10:1) outperformed their acyclic counterparts (+/- 3:1).

PRIMARY STRUCTURES AND BIOLOGICAL-ACTIVITIES OF SUBSTANCE-P-RELATED PEPTIDES FROM THE BRAIN OF THE DOGFISH, SCYLIORHINUS-CANICULA

Two peptides with substance-P-like immunoreactivity were isolated in pure form from an extract of the brain of the elasmobranch fish, Scyliorhinus canicula (european common dogfish). One peptide was identical to scyliorhinin I, previously identified in dogfish intestine, and the second was the undecapeptide Lys-Pro-Arg-Pro-Gly-Gln-Phe-Phe-Gly-Leu-Met-CONH2 which is structurally similar to mammalian substance P Scyliorhinin II or a peptide analogous to mammalian neurokinin A were not detected in the extract. Synthetic dogfish substance P ([Lys1, Arg3, Gly5]substance P) was approximately threefold more potent than mammalian substance P (K(d) = 0.21 +/- 0.11 nM versus K(d)= 0.74 +/- 0.17 nM; mean +/- SD; n = 6) in inhibiting the binding of I-125-labelled substance P to neurokinin (NK1) receptors in rat submandibular gland membranes. The vasodilator action of tachykinins in mammals is mediated primarily through interaction with NK1 receptors. Bolus intravenous injections of [Lys1, Arg3, Gly5]substance P (100 pmol) and scyliorhinin I (100 pmol) produced appreciable (>4 kPa) decreases in arterial blood pressure in the rat whereas intravenous injections of up to 5 nmol of the peptides into conscious, unrestrained dogfish produced no change in arterial blood pressure, pulse amplitude or heart rate. Injections of greater amounts of the peptides (10-50 nmol) produced a slight increase (400-667 Pa) in blood pressure. The data indicate that mammalian-type NK1 tachykinin receptors are not involved in cardiovascular regulation in elasmobranch fish.

Plasma somatostatin and gastrointestinal peptides in Alzheimer's disease and vascular dementia

Few markers distinguish between different dementia types. As dementia affects many body systems outside the central nervous system, we investigated gastrointestinal regulatory peptides as possible disease markers in Alzheimer's Disease (AD) and vascular dementia (VaD). Subjects with mild-to-moderate dementia were diagnosed as probable AD and VaD according to defined criteria. Gastrointestinal peptides were stimulated using a standardized meal test, administered after an overnight fast to 58 dementia patients (40 AD, 18 VaD) and 47 controls matched for age and sex. Blood samples were taken at designated time intervals, and basal and stimulated plasma concentrations of eleven peptides were determined by radio-immunoassay. Results were analysed using the Kruskal-Wallis one-way analysis of variance; the Mann-Whitney U test was used in post hoc analysis where appropriate. There were significant differences in somatostatin levels but in none of the other peptides. Basal somatostatin was significantly increased in VaD compared to controls (p

Defense peptides secreted by helminth pathogens: antimicrobial and/or immunomodulator molecules?

Host defense peptides (HDPs) are an evolutionarily conserved component of the innate immune response found in all living species. They possess antimicrobial activities against a broad range of organisms including bacteria, fungi, eukaryotic parasites, and viruses. HDPs also have the ability to enhance immune responses by acting as immunomodulators. We discovered a new family of HDPs derived from pathogenic helminth (worms) that cause enormous disease in animals and humans worldwide. The discovery of these peptides was based on their similar biochemical and functional characteristics to the human defense peptide LL-37. We propose that these new peptides modulate the immune response via molecular mimicry of mammalian HDPs thus providing a mechanism behind the anti-inflammatory properties of helminth infections.

Can we trust mass spectrometry for determination of arsenic peptides in plants:comparison of LC-ICP-MS and LC-ES-MS/ICP-MS with XANES/EXAFS in analysis of Thunbergia alata

The weakest step in the analytical procedure for speciation analysis is extraction from a biological material into an aqueous solution which undergoes HPLC separation and then simultaneous online detection by elemental and molecular mass spectrometry (ICP-MS/ES-MS). This paper describes a study to determine the speciation of arsenic and, in particular, the arsenite phytochelatin complexes in the root from an ornamental garden plant Thunbergia alata exposed to 1 mg As L(-1) as arsenate. The approach of formic acid extraction followed by HPLC-ES-MS/ICP-MS identified different As(III)-PC complexes in the extract of this plant and made their quantification via sulfur (m/z 32) and arsenic (m/z 75) possible. Although sulfur sensitivity could be significantly increased when xenon was used as collision gas in ICP-qMS, or when HR-ICP-MS was used in medium resolution, the As:S ratio gave misleading results in the identification of As(III)-PC complexes due to the relatively low resolution of the chromatography system in relation to the variety of As-peptides in plants. Hence only the parallel use of ES-MS/ICP-MS was able to prove the occurrence of such arsenite phytochelatin complexes. Between 55 and 64% of the arsenic was bound to the sulfur of peptides mainly as As(III)(PC(2))(2), As(III)(PC(3)) and As(III)(PC(4)). XANES (X-ray absorption near-edge spectroscopy) measurement, using the freshly exposed plant root directly, confirmed that most of the arsenic is trivalent and binds to S of peptides (53% As-S) while 38% occurred as arsenite and only 9% unchanged as arsenate. EXAFS data confirmed that As-S and As-O bonds occur in the plants. This study confirms, for the first time, that As-peptides can be extracted by formic acid and chromatographically separated on a reversed-phase column without significant decomposition or de-novo synthesis during the extraction step.

Medusins: a new class of antimicrobial peptides from the skin secretions of phyllomedusine frogs

Natural drug discovery represents an area of research with vast potential. The investigation into the use of naturally-occurring peptides as potential therapeutic agents provides a new “chemical space” for the procurement of drug leads. Intensive and systematic studies on the broad-spectrum antimicrobial peptides found in amphibian skin secretions are of particular interest in the quest for new antibiotics to treat multiple drug-resistant bacterial infections. Here we report the molecular cloning of the biosynthetic precursor-encoding cDNAs and respective mature peptides representing a novel group of antimicrobial peptides from the skin secretions of representative species of phyllomedusine leaf frogs: the Central American red-eyed leaf frog (Agalychnis callidryas), the South American orange-legged leaf frog (Phyllomedusa hypochondrialis) and the Giant Mexican leaf frog, (Pachymedusa dacnicolor). Each novel peptide possessed the highly-conserved sequence, LGMIPL/VAISAISA/SLSKLamide, and each exhibited activity against the Gram-positive bacterium, Staphylococcus aureus and the yeast, Candida albicans, but all were devoid of haemolytic effects at concentrations up to and including the MICs for both organisms. The novel peptide group were named medusins, derived from the name of the hylid frog sub-family, Phyllomedusinae, to which all species investigated belong. These data clearly demonstrate that comparative studies of the skin secretions of phyllomedusine frogs can continue to produce novel peptides that have the potential to be leads in the development of new and effective antimicrobials.

Molecular basis of Yersinia enterocolitica temperature-dependent resistance to antimicrobial peptides

Antimicrobial peptides (APs) belong to the arsenal of weapons of the innate immune system against infections. In the case of gram-negative bacteria, APs interact with the anionic lipid A moiety of the lipopolysaccharide (LPS). In yersiniae most virulence factors are temperature regulated. Studies from our laboratory demonstrated that Yersinia enterocolitica is more susceptible to polymyxin B, a model AP, when grown at 37°C than at 22°C (J. A. Bengoechea, R. Díaz, and I. Moriyón, Infect. Immun. 64:4891-4899, 1996), and here we have extended this observation to other APs, not structurally related to polymyxin B. Mechanistically, we demonstrate that the lipid A modifications with aminoarabinose and palmitate are downregulated at 37°C and that they contribute to AP resistance together with the LPS O-polysaccharide. Bacterial loads of lipid A mutants in Peyer's patches, liver, and spleen of orogastrically infected mice were lower than those of the wild-type strain at 3 and 7 days postinfection. PhoPQ and PmrAB two-component systems govern the expression of the loci required to modify lipid A with aminoarabinose and palmitate, and their expressions are also temperature regulated. Our findings support the notion that the temperature-dependent regulation of loci controlling lipid A modifications could be explained by H-NS-dependent negative regulation alleviated by RovA. In turn, our data also demonstrate that PhoPQ and PmrAB regulate positively the expression of rovA, the effect of PhoPQ being more important. However, rovA expression reached wild-type levels in the phoPQ pmrAB mutant background, hence indicating the existence of an unknown regulatory network controlling rovA expression in this background.

Efficacy of cecropin A-melittin peptides on a sepsis model of infection by pan-resistant Acinetobacter baumannii

Pan-resistant Acinetobacter baumannii have prompted the search for therapeutic alternatives. We evaluate the efficacy of four cecropin A-melittin hybrid peptides (CA-M) in vivo. Toxicity was determined in mouse erythrocytes and in mice (lethal dose parameters were LD(0), LD(50), LD(100)). Protective dose 50 (PD(50)) was determined by inoculating groups of ten mice with the minimal lethal dose of A. baumannii (BMLD) and treating with doses of each CA-M from 0.5 mg/kg to LD(0). The activity of CA-Ms against A. baumannii was assessed in a peritoneal sepsis model. Mice were sacrificed at 0 and 1, 3, 5, and 7-h post-treatment. Spleen and peritoneal fluid bacterial concentrations were measured. CA(1-8)M(1-18) was the less haemolytic on mouse erythrocytes. LD(0) (mg/kg) was 32 for CA(1-8)M(1-18), CA(1-7)M(2-9), and Oct-CA(1-7)M(2-9), and 16 for CA(1-7)M(5-9). PD(50) was not achieved with non-toxic doses (= LD(0)). In the sepsis model, all CA-Ms were bacteriostatic in spleen, and decreased bacterial concentration (p <0.05) in peritoneal fluid, at 1-h post-treatment; at later times, bacterial regrowth was observed in peritoneal fluid. CA-Ms showed local short-term efficacy in the peritoneal sepsis model caused by pan-resistant Acinetobacter baumannii.

Klebsiella pneumoniae OmpA confers resistance to antimicrobial peptides

A Klebsiella pneumoniae ompA mutant was more susceptible to antimicrobial peptides (APs) than the wild type. Susceptibility did not result from surface changes other than the absence of OmpA. Our data suggest that OmpA is implicated in the activation of yet-unknown systems dedicated to ameliorating AP cytotoxicity.

Capsule polysaccharide is a bacterial decoy for antimicrobial peptides

Antimicrobial peptides (APs) are important host weapons against infections. Nearly all APs are cationic and their microbicidal action is initiated through interactions with the anionic bacterial surface. It is known that pathogens have developed countermeasures to resist these agents by reducing the negative charge of membranes, by active efflux and by proteolytic degradation. Here we uncover a new strategy of resistance based on the neutralization of the bactericidal activity of APs by anionic bacterial capsule polysaccharide (CPS). Purified CPSs from Klebsiella pneumoniae K2, Streptococcus pneumoniae serotype 3 and Pseudomonas aeruginosa increased the resistance to polymyxin B of an unencapsulated K. pneumoniae mutant. Furthermore, these CPSs increased the MICs of polymyxin B and human neutrophil alpha-defensin 1 (HNP-1) for unencapsulated K. pneumoniae, Escherichia coli and P. aeruginosa PAO1. Polymyxin B or HNP-1 released CPS from capsulated K. pneumoniae, S. pneumoniae serotype 3 and P. aeruginosa overexpressing CPS. Moreover, this material also reduced the bactericidal activity of APs. We postulate that APs may trigger in vivo the release of CPS, which in turn will protect bacteria against APs. We found that anionic CPSs, but not cationic or uncharged ones, blocked the bactericidal activity of APs by binding them, thereby reducing the amount of peptides reaching the bacterial surface. Supporting this, polycations inhibited such interaction and the bactericidal activity was restored. We postulate that trapping of APs by anionic CPSs is an additional selective virulence trait of these molecules, which could be considered as bacterial decoys for APs.

Quinolones sensitize gram-negative bacteria to antimicrobial peptides

The treatment of infections caused by bacteria resistant to the vast majority of antibiotics is a challenge worldwide. Antimicrobial peptides (APs) make up the front line of defense in those areas exposed to microorganisms, and there is intensive research to explore their use as new antibacterial agents. On the other hand, it is known that subinhibitory concentrations of antibiotics affect the expression of numerous bacterial traits. In this work we evaluated whether treatment of bacteria with subinhibitory concentrations of quinolones may alter the sensitivity to APs. A 1-h treatment of Klebsiella pneumoniae with 0.25 x the MIC of ciprofloxacin rendered bacteria more sensitive to polymyxins B and E, human neutrophil defensin 1, and beta-defensin 1. Levofloxacin and nalidixic acid at 0.25 x the MICs also increased the sensitivity of K. pneumoniae to polymyxin B, whereas gentamicin and ceftazidime at 0.25 x the MICs did not have such an effect. Ciprofloxacin also increased the sensitivities of K. pneumoniae ciprofloxacin-resistant strains to polymyxin B. Two other pathogens, Pseudomonas aeruginosa and Haemophilus influenzae, also became more sensitive to polymyxins B and E after treatment with 0.25 x the MIC of ciprofloxacin. Incubation with ciprofloxacin did not alter the expression of the K. pneumoniae loci involved in resistance to APs. A 1-N-phenyl-naphthylamine assay showed that ciprofloxacin and levofloxacin increased the permeabilities of the K. pneumoniae and P. aeruginosa outer membranes, while divalent cations antagonized this action. Finally, we demonstrated that ciprofloxacin and levofloxacin increased the binding of APs to the outer membrane by using dansylated polymyxin B.