Anselmo J. Otero-González

Antimicrobial peptides from marine invertebrates as a new frontier for microbial infection control

Antimicrobial peptides are widely expressed in organisms and have been linked to innate and acquired immunities in vertebrates. These compounds are constitutively expressed and rapidly induced at different cellular levels to interact directly with infectious agents and/or modulate immunoreactions involved in defense against pathogenic microorganisms. In invertebrates, antimicrobial peptides represent the major humoral defense system against infection, showing a diverse spectrum of action mechanisms, most of them related to plasma membrane disturbance and lethal alteration of microbial integrity. Marine invertebrates are widespread, extremely diverse, and constantly under an enormous microbial challenge from the ocean environment, itself altered by anthropic influences derived from industrialization and transportation. Consequently, this study reexamines the peptides isolated over the past 2 decades from different origins, bringing phyla not previously reviewed up to date. Moreover, a promising novel use of antimicrobial peptides as effective drugs in human and veterinary medicine could be based on their unusual properties and synergic counterparts as immune response humoral effectors, in addition to their direct microbicidal activity. This has been seen in many other marine proteins that are sufficiently immunogenic to humans, not necessarily in terms of antibody generation but as inflammation promoters and recruitment agents or immune enhancers. —Otero‐González, A. J., Magalhães, B. S., Garcia‐Villarino, M., López‐Abarrategui, C., Sousa, D. A., Dias, S. C., Franco, O. L. Antimicrobial peptides from marine invertebrates as a new frontier for microbial infection control. FASEB J. 24, 1320–1334 (2010). www.fasebj.org

Deciphering the magainin resistance process of Escherichia coli strains in light of the cytosolic proteome.

ABSTRACT Antimicrobial peptides (AMPs) are effective antibiotic agents commonly found in plants, animals, and microorganisms, and they have been suggested as the future of antimicrobial chemotherapies. It is vital to understand the molecular details that define the mechanism of action of resistance to AMPs for a rational planning of the next antibiotic generation and also to shed some light on the complex AMP mechanism of action. Here, the antibiotic resistance of Escherichia coli ATCC 8739 to magainin I was evaluated in the cytosolic subproteome. Magainin-resistant strains were selected after 10 subsequent spreads at subinhibitory concentrations of magainin I (37.5 mg · liter−1), and their cytosolic proteomes were further compared to those of magainin-susceptible strains through two-dimensional electrophoresis analysis. As a result, 41 differentially expressed proteins were detected by in silico analysis and further identified by tandem mass spectrometry de novo sequencing. Functional categorization indicated an intense metabolic response mainly in energy and nitrogen uptake, stress response, amino acid conversion, and cell wall thickness. Indeed, data reported here show that resistance to cationic antimicrobial peptides possesses a greater molecular complexity than previously supposed, resulting in cell commitment to several metabolic pathways.

Exploring the pharmacological potential of promiscuous host-defense peptides: from natural screenings to biotechnological applications

In the last few years, the number of bacteria with enhanced resistance to conventional antibiotics has dramatically increased. Most of such bacteria belong to regular microbial flora, becoming a real challenge, especially for immune-depressed patients. Since the treatment is sometimes extremely expensive, and in some circumstances completely inefficient for the most severe cases, researchers are still determined to discover novel compounds. Among them, host-defense peptides (HDPs) have been found as the first natural barrier against microorganisms in nearly all living groups. This molecular class has been gaining attention every day for multiple reasons. For decades, it was believed that these defense peptides had been involved only with the permeation of the lipid bilayer in pathogen membranes, their main target. Currently, it is known that these peptides can bind to numerous targets, as well as lipids including proteins and carbohydrates, from the surface to deep within the cell. Moreover, by using in vivo models, it was shown that HDPs could act both in pathogens and cognate hosts, improving immunological functions as well as acting through multiple pathways to control infections. This review focuses on structural and functional properties of HDP peptides and the additional strategies used to select them. Furthermore, strategies to avoid problems in large-scale manufacture by using molecular and biochemical techniques will also be explored. In summary, this review intends to construct a bridge between academic research and pharmaceutical industry, providing novel insights into the utilization of HDPs against resistant bacterial strains that cause infections in humans.

Functional characterization of a synthetic hydrophilic antifungal peptide derived from the marine snail Cenchritis muricatus

Antimicrobial peptides have been found in mollusks and other sea animals. In this report, a crude extract of the marine snail Cenchritis muricatus was evaluated against human pathogens responsible for multiple deleterious effects and diseases. A peptide of 1485.26 Da was purified by reversed-phase HPLC and functionally characterized. This trypsinized peptide was sequenced by MS/MS technology, and a sequence (SRSELIVHQR), named Cm-p1 was recovered, chemically synthesized and functionally characterized. This peptide demonstrated the capacity to prevent the development of yeasts and filamentous fungi. Otherwise, Cm-p1 displayed no toxic effects against mammalian cells. Molecular modeling analyses showed that this peptide possible forms a single hydrophilic α-helix and the probable cationic residue involved in antifungal activity action is proposed. The data reported here demonstrate the importance of sea animals peptide discovery for biotechnological tools development that could be useful in solving human health and agribusiness problems.

LPS inmobilization on porous and non-porous supports as an approach for the isolation of anti-LPS host-defense peptides

Lipopolysaccharides (LPSs) are the major molecular component of the outer membrane of Gram-negative bacteria. This molecule is recognized as a sign of bacterial infection, responsible for the development of local inflammatory response and, in extreme cases, septic shock. Unfortunately, despite substantial advances in the pathophysiology of sepsis, there is no efficacious therapy against this syndrome yet. As a consequence, septic shock syndrome continues to increase, reaching mortality rates over 50% in some cases. Even though many preclinical studies and clinical trials have been conducted, there is no Food and Drug Administration-approved drug yet that interacts directly against LPS. Cationic host-defense peptides (HDPs) could be an alternative solution since they possess both antimicrobial and antiseptic properties. HDPs are small, positively charged peptides which are evolutionarily conserved components of the innate immune response. In fact, binding to diverse chemotypes of LPS and inhibition of LPS-induced pro-inflammatory cytokines from macrophages have been demonstrated for different HDPs. Curiously, none of them have been isolated by their affinity to LPS. A diversity of supports could be useful for such biological interaction and suitable for isolating HDPs that recognize LPS. This approach could expand the rational search for anti-LPS HDPs.

Magnetic nanoparticles: new players in antimicrobial peptide therapeutics.

Antimicrobial peptides are distributed in all forms of life presenting activity against bacteria, fungi, viruses, parasites and cancer. In spite of the tremendous potential of these molecules, very few of them have been successfully developed into therapeutics. The major problems associated with this new class of antimicrobials are molecule stability, toxicity in host cells and production cost. A novel strategy to overcome these obstacles is conjugation to nanomaterials. Magnetic nanoparticles have been widely studied in biomedicine due to their physicochemical properties. The conjugation of antimicrobial peptides to magnetic nanoparticles could combine the best properties of both, generating an improved antimicrobial nanoparticle. Here we provide an overview and discuss the potential application of magnetic nanoparticles conjugated to antimicrobial peptides in overcoming diseases.

Structural and functional evaluation of the palindromic alanine-rich antimicrobial peptide Pa-MAP2.

Recently, several peptides have been studied regarding the defence process against pathogenic microorganisms, which are able to act against different targets, with the purpose of developing novel bioactive compounds. The present work focuses on the structural and functional evaluation of the palindromic antimicrobial peptide Pa-MAP2, designed based on the peptide Pa-MAP from Pleuronectes americanus. For a better structural understanding, molecular modelling analyses were carried out, together with molecular dynamics and circular dichroism, in different media. Antibacterial activity against Gram-negative and positive bacteria was evaluated, as well as cytotoxicity against human erythrocytes, RAW 264.7, Vero and L6 cells. In silico docking experiments, lipid vesicle studies, and atomic force microscopy (AFM) imaging were carried out to explore the activity of the peptide. In vivo studies on infected mice were also done. The palindromic primary sequence favoured an α-helix structure that was pH dependent, only present on alkaline environment, with dynamic N- and C-terminals that are stabilized in anionic media. Pa-MAP2 only showed activity against Gram-negative bacteria, with a MIC of 3.2 μM, and without any cytotoxic effect. In silico, lipid vesicles and AFM studies confirm the preference for anionic lipids (POPG, POPS, DPPE, DPPG and LPS), with the positively charged lysine residues being essential for the initial electrostatic interaction. In vivo studies showed that Pa-MAP2 increases to 100% the survival rate of mice infected with Escherichia coli. Data here reported indicated that palindromic Pa-MAP2 could be an alternative candidate for use in therapeutics against Gram-negative bacterial infections.

Cm-p5: an antifungal hydrophilic peptide derived from the coastal mollusk Cenchritis muricatus (Gastropoda: Littorinidae)

Antimicrobial peptides form part of the first line of defense against pathogens formany organisms. Current treatments for fungal infections are limited by drug toxicity and pathogen resistance. Cm‐p5 (SRSE‐LIVHQRLF), a peptide derived from the marine mollusk Cenchritis muricatus peptide Cm‐p1, has a significantly increased fungistatic activity against pathogenic Candida albicans (minimal inhibitory concentration, 10 μg/ml; EC50, 1.146 μg/ml) while exhibiting low toxic effects against a cultured mammalian cell line. Cm‐p5 as characterized by circular dichroism and nuclear magnetic resonance revealed an α‐helical structure in membrane‐mimetic conditions and a tendency to random coil folding in aqueous solutions. Additional studies modeling Cm‐p5 binding to a phosphatidylserine bilayer in silico and isothermal titration calorimetry using lipid monophases demonstrated that Cm‐p5 has a high affinity for the phospholipids of fungal membranes (phosphatidylserine and phosphatidylethanolamine), only moderate interactions with a mammalian membrane phospholipid, low interaction with ergosterol, and no interaction with chitin. Adhesion of Cm‐p5 to living C. albicans cells was confirmed by fluorescence microscopy with FITC‐labeled peptide. In a systemic candidiasis model in mice, intraperitoneal administration of Cm‐p5 was unable to control the fungal kidney burden, although its low amphiphaticity could be modified to generate new derivatives with improved fungicidal activity and stability.—López‐Abarrategui, C., McBeth, C., Mandai, S. M., Sun, Z. J., Heffron, G., Alba‐Menéndez, A., Migliolo, L., Reyes‐Acosta, O., Garcia‐Villarino, M., Nolasco, D. O., Falcão, R., Cherobim, M. D., Dias, S. C., Brandt, W., Wessjohann, L., Starnbach, M., Franco, O. L., Otero‐González, A. J. Cm‐p5: an antifungal hydrophilic peptide derived from the coastal mollusk Cenchritis muricatus (Gastropoda: Littorinidae). FASEB J. 29, 3315‐3325 (2015). www.fasebj.org

Identification of a Novel Antimicrobial Peptide from Brazilian Coast Coral Phyllogorgia dilatata

The marine ecosystem is able to provide enormous biomolecule diversity that could be used for treatment of various diseases. In this highly competitive environment, organisms need chemical barriers to reduce or avoid microorganism contamination. Among the molecules that protect these animals the antimicrobial peptides (AMPs) are included. In the present study, crude extracts of coral coral specimens Carijoa riisei, Muriceopsis sulphurea, Neospongodes atlantica, Palythoa caribeorum, Phyllogorgia dilatata and Plexaurella grandiflora were challenged against multiple Grampositive and -negative bacteria showing different activities. P. dilatata crude extract showed the antibacterial activity, and was ammonium-sulfate (0-40%) fractionated, being able to control the growth of K. pneumoniae, S. flexineri and S. aureus. Rich-fraction was further purified by using Amicon® Ultra Centrifugal 10 kDa associated with reversed-phase HPLC chromatography (C18), producing the peptide named Pd-AMP1. Pd-AMP1 was able to inhibit S. aureus development. Mass spectrometry analyses showed a monoisotopic mass of 5372.66 Da and N-terminal sequence showed no significant match with databank. In this view, the prospecting of protein biomolecules and biotechnological potential from marine animals is still little explored and may serve as an alternative to common antibiotics.

A heterogeneous enzymatic assay for quantification of Plasmepsin II activity and the evaluation of its inhibitors.

The emergence and worldwide spreading of Plasmodium falciparum strains that shown to be resistant to traditional drugs is considered a very serious health problem, given the high mortality and morbidity rate of Malaria. In the search for new drugs against this parasite, Hb hydrolyzing enzymes, such as Plasmepsin II (Plm II), have been classified as very promising targets for therapeutic attacks. In this work, it is developed a cheap and high-throughput heterogeneous enzymatic assay for measuring Plasmepsin II activity in order to use it as a tool in the discovery of new inhibitors of this enzyme. In this assay, Plasmepsin II acts upon a solid-phase bound synthetic peptide (DU2) whose sequence comprises the cleavage site F(33)-L(34) present in Hb alpha-chain. The peptide surface density is quantified by means of a classical ELISA-based procedure. In order to estimate the kinetic constants of the system and to quantify both, enzymatic and inhibitory activity, it was used a model for the kinetics of enzyme quasi-saturable systems previously developed by our group, that fitted very well to the experimental data. It was used Pepstatin as a model inhibitor of Plasmepsin II and the resulting dose-response relation agreed with the expected behavior for the Pepstatin-Plasmepsin II pair under the employed experimental conditions.