Identification of a Killer Toxin from Wickerhamomyces anomalus with β-Glucanase Activity

Abstract

The yeast Wickerhamomyces anomalus has several applications in the food industry due to its antimicrobial potential and wide range of biotechnological properties. In particular, a specific strain of Wickerhamomyces anomalus isolated from the malaria mosquito Anopheles stephensi, namely WaF17.12, was reported to secrete a killer toxin with strong anti-plasmodial effect on different developmental stages of Plasmodium berghei; therefore, we propose its use in the symbiotic control of malaria. In this study, we focused on the identification/characterization of the protein toxin responsible for the observed antimicrobial activity of the yeast. For this purpose, the culture medium of the killer yeast strain WaF17.12 was processed by means of lateral flow filtration, anion exchange and gel filtration chromatography, immunometric methods, and eventually analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on this concerted approach, we identified a protein with a molecular weight of approximately 140 kDa and limited electrophoretic mobility, corresponding to a high molecular weight β-glucosidase, as confirmed by activity tests in the presence of specific inhibitors.

Keywords: Wickerhamomyces anomalus; killer toxin; malaria; symbiotic control.

Figure 1

(A): elution profile obtained from the anion-exchange chromatography (DEAE) performed on the concentrated broth of the yeast WaF17.12. (B): immunodetection of the W. anomalus KT on peak 1 and peak 2 with the mAbKT4 antibody.

Figure 2

(A): superimposition of the profile obtained from the gel filtration chromatography (post-DEAE) with fractions obtained from independent analyses (peaks 1–5). (B): immunodetection of the W. anomalus KT in peaks 1–5 obtained after gel filtration using the mAbKT4 antibody.

Figure 3

Killing activity of the isolated β-glucanase against the susceptible WaUM3 strain. The addition of castanospermine and Ni²⁺, two β-glucanase inhibitors, strongly affects the activity of the WaF17.12 killer protein. (A) shows data on WaUM3 cells viability upon 12 h treatments. Values represent the mean ± S.D. of results obtained from three separate experiments. # indicates p < 0.01 compared to control and * indicates p < 0.01 compared to KT. (B) shows data obtained from flow cytometry analysis of treated WaUM3 cells stained with PI. Percentages indicate PI-positive cells. Data shown are representative of three separate experiments.

Figure 4

Three-dimensional representation of the structure of the β-glucanase isolated from W. anomalus F17.12 obtained by folding-assisted modeling and of the predicted complex thereof with castanospermine. Secondary structures of the enzyme are visualized in (A) (α-helices, light blue; β-sheets, violet), and Asn glycosylation sites are shown as solid blue sticks and summarized in the table inset of (B). Global and catalytic site local electrostatic potential surfaces (calculated with the Adaptive Poisson–Boltzmann Solver Tool—PyMol) are presented in (C) and (D), respectively. (E) close-up of the residues of the catalytic pocket involved in the interaction with castanospermine: catalytic Asp-299 is shown as a solid red stick, and all other residues predicted to form H-bonds with castanospermine (Arg-111, Lys-216, Tyr-267, Trp300, and Glu-523) are shown as solid magenta sticks. All images were rendered with PyMOL.