David Pulido

AMPA: an automated web server for prediction of protein antimicrobial regions

SUMMARY AMPA is a web application for assessing the antimicrobial domains of proteins, with a focus on the design on new antimicrobial drugs. The application provides fast discovery of antimicrobial patterns in proteins that can be used to develop new peptide-based drugs against pathogens. Results are shown in a user-friendly graphical interface and can be downloaded as raw data for later examination. AVAILABILITY AMPA is freely available on the web at http://tcoffee.crg.cat/apps/ampa. The source code is also available in the web. CONTACT marc.torrent@upf.edu; david.andreu@upf.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Exploring new biological functions of amyloids: bacteria cell agglutination mediated by host protein aggregation.

Antimicrobial proteins and peptides (AMPs) are important effectors of the innate immune system that play a vital role in the prevention of infections. Recent advances have highlighted the similarity between AMPs and amyloid proteins. Using the Eosinophil Cationic Protein as a model, we have rationalized the structure-activity relationships between amyloid aggregation and antimicrobial activity. Our results show how protein aggregation can induce bacteria agglutination and cell death. Using confocal and total internal reflection fluorescence microscopy we have tracked the formation in situ of protein amyloid-like aggregates at the bacteria surface and on membrane models. In both cases, fibrillar aggregates able to bind to amyloid diagnostic dyes were detected. Additionally, a single point mutation (Ile13 to Ala) can suppress the protein amyloid behavior, abolishing the agglutinating activity and impairing the antimicrobial action. The mutant is also defective in triggering both leakage and lipid vesicle aggregation. We conclude that ECP aggregation at the bacterial surface is essential for its cytotoxicity. Hence, we propose here a new prospective biological function for amyloid-like aggregates with potential biological relevance.

Antimicrobial Peptide Action on Parasites

Diseases caused by protozoan parasites can pose a severe thread to human health and are behind some serious neglected tropical diseases like malaria and leishmaniasis. Though several different drugs have been developed in order to eradicate these diseases, a successful candidate has not yet been discovered. Among the most active compounds tested, antimicrobial peptides (AMPs) are particularly appealing because of their wide spectrum of action. AMPs have been described to perturb protozoan homeostasis by disrupting the cellular membranes but also by interfering with key processes in the parasite metabolism. In this review we describe the diverse mechanisms of action of AMPs on protozoan targets and how they can be exploited to treat diseases. Moreover, we describe with detail the antimicrobial action of AMPs on two major parasitical infections: leishmaniasis and malaria. All the features reviewed here show that AMPs are promising drugs to target protozoan parasites and that further understanding of the mechanism of action of these compounds will lead to improved drugs that could be worth to test in a clinical phase.

Lipopolysaccharide neutralization by antimicrobial peptides: a gambit in the innate host defense strategy.

Antimicrobial peptides (AMPs) are nowadays understood as broad multifunctional tools of the innate immune system to fight microbial infections. In addition to its direct antimicrobial action, AMPs can modulate the host immune response by promoting or restraining the recruitment of cells and chemicals to the infection focus. Binding of AMPs to lipopolysaccharide is a critical step for both their antimicrobial action and their immunomodulatory properties. On the one hand, removal of Gram-negative bacteria by AMPs can be an effective strategy to prevent a worsened inflammatory response that may lead to septic shock. On the other hand, by neutralizing circulating endotoxins, AMPs can successfully reduce nitric oxide and tumor necrosis factor-α production, hence preventing severe tissue damage. Furthermore, AMPs can also interfere with the Toll-like receptor 4 recognition system, suppressing cytokine production and contributing to modulate the inflammatory response. Here, we review the immune system strategies devised by AMPs to avoid an exacerbated inflammatory response and thus prevent a fatal end to the host.

Two Human Host Defense Ribonucleases against Mycobacteria, the Eosinophil Cationic Protein (RNase 3) and RNase 7

ABSTRACT There is an urgent need to develop new agents against mycobacterial infections, such as tuberculosis and other respiratory tract or skin affections. In this study, we have tested two human antimicrobial RNases against mycobacteria. RNase 3, also called the eosinophil cationic protein, and RNase 7 are two small cationic proteins secreted by innate cells during host defense. Both proteins are induced upon infection displaying a wide range of antipathogen activities. In particular, they are released by leukocytes and epithelial cells, contributing to tissue protection. Here, the two RNases have been proven effective against Mycobacterium vaccae at a low micromolar level. High bactericidal activity correlated with their bacterial membrane depolarization and permeabilization activities. Further analysis on both protein-derived peptides identified for RNase 3 an N-terminus fragment that is even more active than the parental protein. Also, a potent bacterial agglutinating activity was unique to RNase 3 and its derived peptide. The particular biophysical properties of the RNase 3 active peptide are envisaged as a suitable reference for the development of novel antimycobacterial drugs. The results support the contribution of secreted RNases to the host immune response against mycobacteria.

Structural determinants of the eosinophil cationic protein antimicrobial activity

Abstract Antimicrobial RNases are small cationic proteins belonging to the vertebrate RNase A superfamily and endowed with a wide range of antipathogen activities. Vertebrate RNases, while sharing the active site architecture, are found to display a variety of noncatalytical biological properties, providing an excellent example of multitask proteins. The antibacterial activity of distant related RNases suggested that the family evolved from an ancestral host-defence function. The review provides a structural insight into antimicrobial RNases, taking as a reference the human RNase 3, also named eosinophil cationic protein (ECP). A particular high binding affinity against bacterial wall structures mediates the protein action. In particular, the interaction with the lipopolysaccharides at the Gram-negative outer membrane correlates with the protein antimicrobial and specific cell agglutinating activity. Although a direct mechanical action at the bacteria wall seems to be sufficient to trigger bacterial death, a potential intracellular target cannot be discarded. Indeed, the cationic clusters at the protein surface may serve both to interact with nucleic acids and cell surface heterosaccharides. Sequence determinants for ECP activity were screened by prediction tools, proteolysis and peptide synthesis. Docking results are complementing the structural analysis to delineate the protein anchoring sites for anionic targets of biological significance.

Antimicrobial Action and Cell Agglutination by the Eosinophil Cationic Protein Are Modulated by the Cell Wall Lipopolysaccharide Structure

ABSTRACT Antimicrobial proteins and peptides (AMPs) are essential effectors of innate immunity, acting as a first line of defense against bacterial infections. Many AMPs exhibit high affinity for cell wall structures such as lipopolysaccharide (LPS), a potent endotoxin able to induce sepsis. Hence, understanding how AMPs can interact with and neutralize LPS endotoxin is of special relevance for human health. Eosinophil cationic protein (ECP) is an eosinophil secreted protein with high activity against both Gram-negative and Gram-positive bacteria. ECP has a remarkable affinity for LPS and a distinctive agglutinating activity. By using a battery of LPS-truncated E. coli mutant strains, we demonstrate that the polysaccharide moiety of LPS is essential for ECP-mediated bacterial agglutination, thereby modulating its antimicrobial action. The mechanism of action of ECP at the bacterial surface is drastically affected by the LPS structure and in particular by its polysaccharide moiety. We have also analyzed an N-terminal fragment that retains the whole protein activity and displays similar cell agglutination behavior. Conversely, a fragment with further minimization of the antimicrobial domain, though retaining the antimicrobial capacity, significantly loses its agglutinating activity, exhibiting a different mechanism of action which is not dependent on the LPS composition. The results highlight the correlation between the proteins antimicrobial activity and its ability to interact with the LPS outer layer and promote bacterial agglutination.

Towards the rational design of antimicrobial proteins

The ribonuclease (RNase) A superfamily lineage includes distant members with antimicrobial properties, suggesting a common ancestral host‐defense role. In an effort to identify the minimal requirements for the eosinophil cationic protein (ECP or RNase 3) antimicrobial properties we applied site‐directed mutagenesis on its closest family homolog, the eosinophil‐derived neurotoxin (EDN or RNase 2). Both eosinophil secretion proteins are involved in human immune defense, and are reported as being among the most rapidly evolving coding sequences in primates. Previous studies in our laboratory defined two regions at the N–terminus involved in the protein antimicrobial action, encompassing residues 8–16 and 34–36. Here, we demonstrate that switching two single residues is enough to provide EDN with ECP antipathogen properties. That is, the EDN double‐mutant Q34R/R35W displays enhanced bactericidal activity, particularly towards Gram‐negative bacteria, and a significant increase in its affinity towards the bacterial outer membrane lipopolysaccharides. Moreover, we confirmed the direct contribution of residue W35 in lipopolysaccharide binding, membrane interaction and permeabilization processes. Furthermore, additional T13 to I substitution provides EDN with an exposed hydrophobic patch required for protein self‐aggregation and triggers bacterial agglutination, thereby increasing the final antimicrobial activity by up to 20–fold. Our results highlight how single selected mutations can reshape the entire protein function. This study provides an example of how structure‐guided protein engineering can successfully reproduce an evolution selection process towards the emergence of new physiological roles.

The sulfate-binding site structure of the human eosinophil cationic protein as revealed by a new crystal form

The human eosinophil cationic protein (ECP), also known as RNase 3, is an eosinophil secretion protein that is involved in innate immunity and displays antipathogen and proinflammatory activities. ECP has a high binding affinity for heterosaccharides, such as bacterial lipopolysaccharides and heparan sulfate found in the glycocalix of eukaryotic cells. We have crystallized ECP in complex with sulfate anions in a new monoclinic crystal form. In this form, the active site groove is exposed, providing an alternative model for ligand binding studies. By exploring the protein-sulfate complex, we have defined the sulfate binding site architecture. Three main sites (S1-S3) are located in the protein active site; S1 and S2 overlap with the phosphate binding sites involved in RNase nucleotide recognition. A new site (S3) that is unique to ECP is one of the key anchoring points for sulfated ligands. Arg 1 and Arg 7 in S3, together with Arg 34 and Arg 36 in S1, form the main basic clusters that assist in the recognition of ligand anionic groups. The location of additional sulfate bound molecules, some of which contribute to the crystal packing, may mimic the binding to extended anionic polymers. In conclusion, the structural data define a binding pattern for the recognition of sulfated molecules that can modulate the role of ECP in innate immunity. The results reveal the structural basis for the high affinity of ECP for glycosaminoglycans and can assist in structure-based drug design of inhibitors of the protein cytotoxicity to host tissues during inflammation.

EL ÉXITO DE LAS PYMES EN COLOMBIA: UN ESTUDIO DE CASOS EN EL SECTOR SALUD

El objetivo principal del estudio es identificar y analizar los factores que determinan el exito de las pymes en Colombia, tanto en lo referido a los factores internos como a lo externos, tomando como marco teorico de referencia la Teoria de Recursos y Capacidades y la Teoria Economica Institucional. Se realizo un estudio cualitativo profundizando en el analisis de cuatro empresas colombianas. Los resultados mas relevantes indican que los factores que han determinado el exito de estas pymes son el conocimiento y la experiencia del empresario, la reputacion y el prestigio de la empresa, las medidas de apoyo y la especializacion de la industria, y la influencia de la comunidad en la cual desarrollan su actividad.