Antifungal properties of Canavalia ensiformis urease and derived peptides

Abstract

Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea into ammonia and CO(2). These proteins have insecticidal and fungicidal effects not related to their enzymatic activity. The insecticidal activity of urease is mostly dependent on the release of internal peptides after hydrolysis by insect digestive cathepsins. Jaburetox is a recombinant version of one of these peptides, expressed in Escherichia coli. The antifungal activity of ureases in filamentous fungi occurs at submicromolar doses, with damage to the cell membranes. Here we evaluated the toxic effect of Canavalia ensiformis urease (JBU) on different yeast species and carried out studies aiming to identify antifungal domain(s) of JBU. Data showed that toxicity of JBU varied according to the genus and species of yeasts, causing inhibition of proliferation, induction of morphological alterations with formation of pseudohyphae, changes in the transport of H(+) and carbohydrate metabolism, and permeabilization of membranes, which eventually lead to cell death. Hydrolysis of JBU with papain resulted in fungitoxic peptides (~10 kDa), which analyzed by mass spectrometry, revealed the presence of a fragment containing the N-terminal sequence of the entomotoxic peptide Jaburetox. Tests with Jaburetox on yeasts and filamentous fungi indicated a fungitoxic activity similar to ureases. Plant ureases, such as JBU, and its derived peptides, may represent a new alternative to control medically important mycoses as well as phytopathogenic fungi, especially considering their potent activity in the range of 10(-6)-10(-7)M.

Pliable natural biocide: Jaburetox is an intrinsically disordered insecticidal and fungicidal polypeptide derived from jack bean urease

“…Indeed, the presence of this tag does not interfere with the biological insecticidal [17] and fungicidal [20] activities of this urease-derived peptide. The expression protocol of Jaburetox was optimized in this work to typically yield 30 mg of Jaburetox per liter of culture.…”

“…parapsilosis, Ca. tropicalis, Kluyveromyces marxianus, Pichia membranisfaciens and Saccharomyces cerevisiae), in addition to its entomotoxic activity [20]. In order to understand the mode of action of Jaburetox-2Ec, molecular modeling studies were carried out that suggested the existence of a b-hairpin motif at the C-terminal portion [17,19].…”

Uso potencial y limitantes de la leguminosa Canavalia ensiformis en la salud y productividad de los ovinos

“…Es probable que la integridad y funcionalidad de los hongos ruminales se afectada por los FAN de canavalia, debido a que C. ensiformis produce isoformas de ureasa con acción insecticida y fungicida (Becker-Ritt et al 2007). La ureasa más abundante es JBURE-I, seguida por la canatoxina y JBURE-IIB, la toxicidad de la ureasa de canavalia ocurre en dosis micromolares y varía con el género y especie de hongo, causando inhibición de la proliferación, alteraciones morfológicas en la pared y membrana, cambios en el transporte de H + y en la permeabilidad de la membrana, lo que induce a la muerte celular (Postal et al 2012).…”

Jaburetox: update on a urease-derived peptide

“…The data obtained by Martinelli et al [14] brought important contributions to the understanding of the JBTX’s mechanism of action, suggesting that it represents a new type of active peptide in membranes, with insecticidal and fungitoxic properties. The antifungal activity of JBTX against yeasts occurs at higher doses (9 to18 μM) than those observed for JBU (0.27 μM), suggesting that other regions of the protein could probably be involved in this activity [11]. …”