Intragenic antimicrobial peptides (IAPs) from human proteins with potent antimicrobial and anti-inflammatory activity

Following the treads of our previous works on the unveiling of bioactive peptides encrypted in plant proteins from diverse species, the present manuscript reports the occurrence of four proof-of-concept intragenic antimicrobial peptides in human proteins, named Hs IAPs. These IAPs were prospected using the software Kamal, synthesized by solid phase chemistry, and had their interactions with model phospholipid vesicles investigated by differential scanning calorimetry and circular dichroism. Their antimicrobial activity against bacteria, yeasts and filamentous fungi was determined, along with their cytotoxicity towards erythrocytes. Our data demonstrates that Hs IAPs are capable to bind model membranes while attaining α-helical structure, and to inhibit the growth of microorganisms at concentrations as low as 1μM. Hs02, a novel sixteen residue long internal peptide (KWAVRIIRKFIKGFISNH2) derived from the unconventional myosin 1h protein, was further investigated in its capacity to inhibit lipopolysaccharide-induced release of TNF-α in murine macrophages. Hs02 presented potent anti-inflammatory activity, inhibiting the release of TNF-α in LPSprimed cells at the lowest assayed concentration, 0.1 μM. A three-dimensional solution structure of Hs02 bound to DPC micelles was determined by Nuclear Magnetic Resonance. Our work exemplifies how the human genome can be mined for molecules with biotechnological potential in human health and demonstrates that IAPs are actual alternatives to antimicrobial peptides as pharmaceutical agents or in their many other putative applications

A Linear 19-Mer Plant Defensin-Derived Peptide Acts Synergistically with Caspofungin against Candida albicans Biofilms

Public health problems are associated with device-associated biofilm infections, with Candida albicans being the major fungal pathogen. We previously identified potent antibiofilm combination treatment in which the antifungal plant defensin HsAFP1 is co-administered with caspofungin, the preferred antimycotic to treat such infections. In this study, we identified the smallest linear HsAFP1-derived peptide that acts synergistically with caspofungin or anidulafungin against C. albicans as HsLin06_18, a 19-mer peptide derived from the C-terminal part of HsAFP1. The [caspofungin + HsLin06_18] combination significantly reduced in vitro biofilm formation of Candida glabrata and C. albicans on catheters, as well as biofilm formation of a caspofungin-resistant C. albicans strain. The [caspofungin + HsLin06_18] combination was not cytotoxic and reduced biofilm formation of C. albicans in vivo using a subcutaneous rat catheter model, as compared to control treatment. Mode of action research on the [caspofungin + HsLin06_18] combination pointed to caspofungin-facilitated HsLin06_18 internalization and immediate membrane permeabilization. All these findings point to broad-spectrum antibiofilm activity of a combination of HsLin06_18 and caspofungin

Recombinant Inga Laurina Trypsin Inhibitor (ILTI) Production in Komagataella Phaffii Confirms Its Potential Anti-Biofilm Effect and Reveals an Anti-Tumoral Activity

Protease inhibitors have a broad biotechnological application ranging from medical drugs to anti-microbial agents. The Inga laurina trypsin inhibitor (ILTI) previously showed a great in vitro inhibitory effect under the adherence of Staphylococcus species, being a strong candidate for use as an anti-biofilm agent. Nevertheless, this is found in small quantities in its sources, which impairs its utilization at an industrial scale. Within this context, heterologous production using recombinant microorganisms is one of the best options to scale up the recombinant protein production. Thus, this work aimed at utilizing Komagataella phaffii to produce recombinant ILTI. For this, the vector pPIC9K+ILTI was constructed and inserted into the genome of the yeast K. phaffii, strain GS115. The protein expression was highest after 48 h using methanol 1%. A matrix-assisted laser desorption ionization–time-of-flight (MALDI–TOF) analysis was performed to confirm the production of the recombinant ILTI and its activity was investigated trough inhibitory assays using the synthetic substrate Nα-Benzoyl-D,L-arginine p-nitroanilide hydrochloride (BAPNA). Finally, recombinant ILTI (rILTI) was used in assays, showing that there was no significant difference between native and recombinant ILTI in its inhibitory activity in biofilm formation. Anti-tumor assay against Ehrlich ascites tumor (EAT) cells showed that rILTI has a potential anti-tumoral effect, showing the same effect as Melittin when incubated for 48 h in concentrations above 25 µg/mL. All together the results suggests broad applications for rILTI

The Antifungal Plant Defensin HsAFP1 Is a Phosphatidic Acid-Interacting Peptide Inducing Membrane Permeabilization

HsAFP1, a plant defensin isolated from coral bells (Heuchera sanguinea), is characterized by broad-spectrum antifungal activity. Previous studies indicated that HsAFP1 binds to specific fungal membrane components, which had hitherto not been identified, and induces mitochondrial dysfunction and cell membrane permeabilization. In this study, we show that HsAFP1 reversibly interacts with the membrane phospholipid phosphatidic acid (PA), which is a precursor for the biosynthesis of other phospholipids, and to a lesser extent with various phosphatidyl inositol phosphates (PtdInsP’s). Moreover, via reverse ELISA assays we identified two basic amino acids in HsAFP1, namely histidine at position 32 and arginine at position 52, as well as the phosphate group in PA as important features enabling this interaction. Using a HsAFP1 variant, lacking both amino acids (HsAFP1[H32A][R52A]), we showed that, as compared to the native peptide, the ability of this variant to bind to PA and PtdInsP’s is reduced (≥74%) and the antifungal activity of the variant is reduced (≥2-fold), highlighting the link between PA/PtdInsP binding and antifungal activity. Using fluorescently labelled HsAFP1 in confocal microscopy and flow cytometry assays, we showed that HsAFP1 accumulates at the cell surface of yeast cells with intact membranes, most notably at the buds and septa. The resulting HsAFP1-induced membrane permeabilization is likely to occur after HsAFP1’s internalization. These data provide novel mechanistic insights in the mode of action of the HsAFP1 plant defensin

ATIVIDADE ANTIFÚNGICA DO ÓLEO ESSENCIAL DE Syzygium aromaticum (L.) Merr. & L.M. Perry (Myrtaceae) EM Cryptococcus neoformans

Introdução/objetivos: Criptococose é uma infecçãoimportante em imunocomprometidos. O tratamento apresenta grande toxicidade,sendo a busca de novos antifúngicos importante. O objetivo deste trabalho foiavaliar a atividade antifúngica e o mecanismo de ação do óleo essencial (OE) deS. aromaticum em fungos do complexo C. neoformans. Metodologia: A atividade antifúngica foi determinada pela técnicade microdiluição em caldo1. 16 isolados de Cryptococcus foram avaliados com concentrações entre 2-1024μg/mL doOE. A leitura foi feita a partir da concentração inibitória mínima (CIM). Omecanismo de ação foi determinado após 1h de incubação do OE com Cryptococcus. Foi utilizado iodeto depropídio (1μg/mL) para visualizar a lesão da membrana fúngica e 0,5μM de FUN-1([2-cloro-4-(2,3-diidro-3-metil-(benzo-1,3-tiazol-2-il)-metilideno)-1-fenilquinolinoiodeto]), para avaliar o metabolismo, com leitura em citometro de fluxo. Aquantificação do ergosterol celular foi determinada por espectrofotometria.Após exposição com 128μg/mL do OE, as células foram incubadas com KOH, sendo oconteúdo lipídico extraído com heptano2. Resultados/discussões: S.aromaticum obteve valores de CIMs: 128-256μg/mL, apresentando boa atividadeantifúngica. No estudo do mecanismo de ação, foi possível determinar inibiçãoda síntese de ergosterol, alteração no metabolismo e lesão de membrana, sendoque o último devido alipofilicidade do OE3. Conclusões: O OE de S.aromaticum apresentou atividade antifúngica lesionando a membrana celular,alterando o metabolismo e inibindo a síntese de ergosterol

The Antifungal Plant Defensin HsAFP1 Is a Phosphatidic Acid-Interacting Peptide Inducing Membrane Permeabilization

Transplantation and autoimmunit