Evaluation of Recombinant Kpkt Cytotoxicity on HaCaT Cells: Further Steps towards the Biotechnological Exploitation Yeast Killer Toxins.

Gavino Carboni,Marilena Budroni,Ilaria Mannazzu,Severino Zara,Ivana Marova,Giacomo Zara

doi:10.3390/foods10030556

Gavino Carboni, Marilena Budroni + Show 4 more

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https://doi.org/10.3390/foods10030556

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Abstract

The soil yeast Tetrapisispora phaffii secretes a killer toxin, named Kpkt, that shows β-glucanase activity and is lethal to wine spoilage yeasts belonging to Kloeckera/Hanseniaspora, Saccharomycodes and Zygosaccharomyces. When expressed in Komagataella phaffii, recombinant Kpkt displays a wider spectrum of action as compared to its native counterpart, being active on a vast array of wine yeasts and food-related bacteria. Here, to gather information on recombinant Kpkt cytotoxicity, lyophilized preparations of this toxin (LrKpkt) were obtained and tested on immortalized human keratinocyte HaCaT cells, a model for the stratified squamous epithelium of the oral cavity and esophagus. LrKpkt proved harmless to HaCaT cells at concentrations up to 36 AU/mL, which are largely above those required to kill food-related yeasts and bacteria in vitro (0.25–2 AU/mL). At higher concentrations, it showed a dose dependent effect that was comparable to that of the negative control and therefore could be ascribed to compounds, other than the toxin, occurring in the lyophilized preparations. Considering the dearth of studies regarding the effects of yeast killer toxins on human cell lines, these results represent a first mandatory step towards the evaluation the possible risks associated to human intake. Moreover, in accordance with that observed on Ceratitis capitata and Musca domestica, they support the lack of toxicity of this toxin on non-target eukaryotic models and corroborate the possible exploitation of killer toxins as natural antimicrobials in the food and beverages industries.

Highlights

Yeast killer toxins are proteins, often glycoproteins, which recognize specific receptors on the surface of their sensitive targets and kill them through different modes of action
While previous works proved the feasibility of recombinant Kpkt production in K. phaffii and the advantages of rKpkt lyophilization, the aim of this work was to gather information on recombinant Kpkt toxicity on human cells
After having shown that recombinant Kpkt produced by rc#17 maintains its biological functions upon lyophilization, its effect on immortalized human keratinocyte HaCaT cells was tested and compared to that of its native counterpart

Summary

Introduction

Yeast killer toxins are proteins, often glycoproteins, which recognize specific receptors on the surface of their sensitive targets and kill them through different modes of action. The spectrum of action of yeast killer toxins covers spoilage microorganisms significant for the fermentative [2,3,4,5,6,7,8,9,10,11,12] and food and feed industries [13,14], but it encompasses microbial pathogens of clinical interest [15,16,17,18], and plant pathogens [19,20,21]. On the contrary non-Saccharomyces yeast killer toxins have a broader spectrum of action and some of them are active on spoilage yeasts of relevance for the wine industry [24,25]

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