Luciano Neves de Medeiros

Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy.

Plant defensins are cysteine-rich cationic peptides, components of the innate immune system. The antifungal sensitivity of certain exemplars was correlated to the level of complex glycosphingolipids in the membrane of fungi strains. Psd1 is a 46 amino acid residue defensin isolated from pea seeds which exhibit antifungal activity. Its structure is characterized by the so-called cysteine-stabilized alpha/beta motif linked by three loops as determined by two-dimensional NMR. In the present work we explored the measurement of heteronuclear Nuclear Overhauser Effects, R1 and R2 (15)N relaxation ratios, and chemical shift to probe the backbone dynamics of Psd1 and its interaction with membrane mimetic systems with phosphatidylcholine (PC) or dodecylphosphocholine (DPC) with glucosylceramide (CMH) isolated from Fusarium solani. The calculated R2 values predicted a slow motion around the highly conserved among Gly12 residue and also in the region of the Turn3 His36-Trp38. The results showed that Psd1 interacts with vesicles of PC or PC:CMH in slightly different forms. The interaction was monitored by chemical shift perturbation and relaxation properties. Using this approach we could map the loops as the binding site of Psd1 with the membrane. The major binding epitope showed conformation exchange properties in the mus-ms timescale supporting the conformation selection as the binding mechanism. Moreover, the peptide corresponding to part of Loop1 (pepLoop1: Gly12 to Ser19) is also able to interact with DPC micelles acquiring a stable structure and in the presence of DPC:CMH the peptide changes to an extended conformation, exhibiting NOE mainly with the carbohydrate and ceramide parts of CMH.

Evaluation of the membrane lipid selectivity of the pea defensin Psd1

Psd1, a 46 amino acid residues defensin isolated from the pea Pisum sativum seeds, exhibits anti-fungal activity by a poorly understood mechanism of action. In this work, the interaction of Psd1 with biomembrane model systems of different lipid compositions was assessed by fluorescence spectroscopy. Partition studies showed a marked lipid selectivity of this antimicrobial peptide (AMP) toward lipid membranes containing ergosterol (the main sterol in fungal membranes) or specific glycosphingolipid components, with partition coefficients (K(p)) reaching uncommonly high values of 10(6). By the opposite, Psd1 does not partition to cholesterol-enriched lipid bilayers, such as mammalian cell membranes. The Psd1 mutants His36Lys and Gly12Glu present a membrane affinity loss relative to the wild type. Fluorescence quenching data obtained using acrylamide and membrane probes further clarify the mechanism of action of this peptide at the molecular level, pointing out the potential therapeutic use of Psd1 as a natural antimycotic agent.

Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins

ABSTRACT Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma in DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. We first identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1 binding partner by using a yeast two-hybrid system. This interaction was further assessed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay. The NS1-VN complex was also detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the DENV2 NS1 protein, either by itself or by interacting with VN, hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 proteins produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system. IMPORTANCE Dengue is the most important arthropod-borne viral disease nowadays and is caused by dengue virus (DENV). The flavivirus NS1 glycoprotein has been characterized functionally as a complement evasion protein that can attenuate the activation of the classical, lectin, and alternative pathways. The present study describes a novel mechanism by which DENV NS1 inhibits the terminal complement pathway. We identified the terminal complement regulator vitronectin (VN) as a novel DENV NS1 binding partner, and the NS1-VN complex was detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the NS1-VN complex inhibited membrane attack complex (MAC) formation, thus interfering with the complement terminal pathway. Interestingly, NS1 itself also inhibited MAC activity, suggesting a direct role of this protein in the inhibition process. Our findings imply a role for NS1 as a terminal pathway inhibitor of the complement system.

Psd1 binding affinity toward fungal membrane components as assessed by SPR: The role of glucosylceramide in fungal recognition and entry

Psd1 is a plant defensin that has antifungal activity against several pathogenic and nonpathogenic fungi. Previous analysis of Psd1 chemical shift perturbations by nuclear magnetic resonance (NMR) spectroscopy demonstrated that this defensin interacts with phospholipids and the sphingolipid glucosylceramide isolated from Fusarium solani (GlcCerFusarium solani). In this study, these interactions were evaluated by real‐time surface plasmon resonance (SPR) analysis. The data obtained demonstrated that Psd1 could bind more strongly to small unilamellar vesicles (SUV)‐containing GlcCerFusarium solani than to SUV that was composed of phosphatidylcholine (PC) alone or was enriched with GlcCer that had been isolated from soybeans. An increase in the SPR response after cholesterol or ergosterol incorporation in PC‐SUV was detected; however, SUV composed of PC:Erg (7:3; molar:molar) became unstable in the presence of Psd1, suggesting membrane destabilization. We also observed a lack of Psd1 internalization in Candida albicans strains that were deficient in the glucosyl ceramide synthase gene. Together, these data indicate that GlcCer is essential for Psd1 anchoring in the fungal plasma membrane as well as internalization.

Arrest of oogenesis in the bug Rhodnius prolixus challenged with the fungus Aspergillus niger is mediated by immune response-derived PGE2.

In this work we characterized the immune response of the insect Rhodnius prolixus to a direct injection into the hemocoel of the non-entomopathogenic fungus Aspergillus niger, and evaluated its consequences on host oogenesis. These animals were able to respond by mounting effective cellular and humoral responses to this fungus; these responses were shown, however, to have reproductive fitness costs, as the number of eggs laid per female was significantly reduced. The disturbance of egg formation during infectious process correlated with an elevation in the titer of hemolymph prostaglandin E2 48 h post-challenge. Administration of Zymosan A as an immunogenic non-infectious challenge produced similar effects on phenoloxidase and prophenoloxidase activities, oocyte development and prostaglandin E2 titer, precluding the hypothesis of an effect mediated by fungal metabolites in animals challenged with fungus. Ovaries at 48 h post-challenge showed absence of vitellogenic ovarian follicles, and the in vivo administration of prostaglandin E2 or its receptor agonist misoprostol, partially reproduced this phenotype. Together these data led us to hypothesize that immune-derived prostaglandin E2 raised from the insect response to the fungal challenge is involved in disturbing follicle development, contributing to a reduction in host reproductive output and acting as a host-derived adaptive effector to infection.

Microscopic and molecular characterization of ovarian follicle atresia in Rhodnius prolixus Stahl under immune challenge

In this work we characterized the degenerative process of ovarian follicles of the bug Rhodnius prolixus challenged with the non-entomopathogenic fungus Aspergillus niger. An injection of A. niger conidia directly into the hemocoel of adult R. prolixus females at the onset of vitellogenesis caused no effect on host lifespan but elicited a net reduction in egg batch size. Direct inspection of ovaries from the mycosed insects revealed that fungal challenge led to atresia of the vitellogenic follicles. Light microscopy and DAPI staining showed follicle shrinkage, ooplasm alteration and disorganization of the monolayer of follicle cells in the atretic follicles. Transmission electron microscopy of thin sections of follicle epithelium also showed nuclei with condensed chromatin, electron dense mitochondria and large autophagic vacuoles. Occurrence of apoptosis of follicle cells in these follicles was visualized by TUNEL labeling. Resorption of the yolk involved an increase in protease activities (aspartyl and cysteinyl proteases) which were associated with precocious acidification of yolk granules and degradation of yolk protein content. The role of follicle atresia in nonspecific host-pathogen associations and the origin of protease activity that led to yolk resorption are discussed.

Psd1 Effects on Candida albicans Planktonic Cells and Biofilms

Candida albicans is an important human pathogen, causing opportunistic infections. The adhesion of planktonic cells to a substrate is the first step for biofilm development. The antimicrobial peptide (AMP) Psd1 is a defensin isolated from Pisum sativum seeds. We tested the effects of this AMP on C. albicans biofilms and planktonic cells, comparing its activity with amphotericin B and fluconazole. Three C. albicans variants were studied, one of them a mutant deficient in glucosylceramide synthase, conferring resistance to Psd1 antifungal action. Atomic force microscopy (AFM) was used to assess morphological and biomechanical changes on fungal cells. Surface alterations, with membrane disruption and leakage of cellular contents, were observed. Cytometry assays and confocal microscopy imaging showed that Psd1 causes cell death, in a time and concentration-dependent manner. These results demonstrate Psd1 pleiotropic action against a relevant fungal human pathogen, suggesting its use as natural antimycotic agent.

Expression of Pisum sativum defensin 1 (Psd1) in shaking flasks and bioreactor cultivations of recombinant Pichia pastoris at different pHs

Abstract - The methylotrophic yeast Pichia pastoris was used to produce the recombinant Pisum sativum defensin (r Ps d1), a small peptide from pea seeds that has a high level of antifungal activity. The plasmidr Ps d1/pPIC9 was integrated into the yeast genome and methanol was used to induce expression and secretionof the recombinant Ps d1, at 30qC in a fed-batch mode. The effects of different pH conditions and processscale-up were evaluated using a Monod-type model where dissolved oxygen was considered the limitingsubstrate. Parameter estimation showed that the process could be improved by expressing r Ps d1 in a 1000 mLbioreactor at pH 4. Structural and functional analyses revealed that the recombinant Ps d1 is very similar to thenative one. Keywords : Pichia pastoris , Pisum sativum Defensin 1, recombinant DNA, fed-batch, modeling, pH. INTRODUCTION Pichia pastoris is a methylotrophic yeastemployed as the heterologous expression system formany different proteins (Romanos, 1995; Chen et al.,1997; Cereghino and Cregg, 2000). One of the mostimportant system characteristics is the yeast’s abilityto grow to high biomass concentrations and tosecrete high concentrations of proteins (Romanos,1995; Chiruvolu et al., 1997). Moreover, recombinant