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The antimicrobial function of citral, one of the main compounds of the essential oils (EO) of the Citrus genus, and widely used by the food industry toward spoilage yeast, was previously proven. In this study, the possible mode of action of citral against yeast cells was evaluated by using a global deletome approach. Firstly, the suitability of Saccharomyces cerevisiae Σ1278b to serve as model yeast was assessed by determining its sensitivity to citral (MIC = 0.5 μL/mL). Subsequently, the complete library of Σ1278b haploid mutants deleted in 4019 non-essential genes was screened to identify potential molecular targets of citral. Finally, the deleted genes in the 590 mutants showing increased citral resistance was analyzed with an in-silico approach (Gene Ontology). The significantly enriched GO Terms were "cytoplasm", "vacuole", and "mitochondrion" (cellular components); "catalytic activity" (molecular function); "pseudohyphal growth" (biological process). For molecular function, resistant mutants were grouped into thiosulfate sulfur transferase activity, transferase activity, and oxidoreductase activity; for cellular components, resistant mutants were grouped as: cytoplasm, intracellular organelle, membrane-bounded organelle, mitochondrion, organelle membrane, and vacuole; and finally, with regard to biological process, deleted genes were grouped as: pseudohyphal growth, mitochondrion organization, lipid metabolic process, DNA recombination and repair, and proteolysis. Interestingly, many identified genes were associated with the cellular response to oxidative stress and ROS scavenging. These findings have important implications for the development of citral-based antimicrobials and the elucidation of its mechanism of action. [ABSTRACT FROM AUTHOR]

The antimicrobial function of citral, one of the main compounds of the essential oils (EO) of the Citrus genus, and widely used by the food industry toward spoilage yeast, was previously proven. In this study, the possible mode of action of citral against yeast cells was evaluated by using a global deletome approach. Firstly, the suitability of Saccharomyces cerevisiae Σ1278b to serve as model yeast was assessed by determining its sensitivity to citral (MIC = 0.5 μL/mL). Subsequently, the complete library of Σ1278b haploid mutants deleted in 4019 non-essential genes was screened to identify potential molecular targets of citral. Finally, the deleted genes in the 590 mutants showing increased citral resistance was analyzed with an in-silico approach (Gene Ontology). The significantly enriched GO Terms were “cytoplasm”, “vacuole”, and “mitochondrion” (cellular components); “catalytic activity” (molecular function); “pseudohyphal growth” (biological process). For molecular function, resistant mutants were grouped into thiosulfate sulfur transferase activity, transferase activity, and oxidoreductase activity; for cellular components, resistant mutants were grouped as: cytoplasm, intracellular organelle, membrane-bounded organelle, mitochondrion, organelle membrane, and vacuole; and finally, with regard to biological process, deleted genes were grouped as: pseudohyphal growth, mitochondrion organization, lipid metabolic process, DNA recombination and repair, and proteolysis. Interestingly, many identified genes were associated with the cellular response to oxidative stress and ROS scavenging. These findings have important implications for the development of citral-based antimicrobials and the elucidation of its mechanism of action.

The rapidly expanding utilization of lanthanides (Ln) for the development of new technologies, green energies, and agriculture has raised concerns regarding their impacts on the environment and human health. The absence of characterization of the underlying cellular and molecular mechanisms regarding their toxicity is a caveat in the apprehension of their environmental impacts. We performed genomic phenotyping and molecular physiology analyses of Saccharomyces cerevisiae mutants exposed to La and Yb to uncover genes and pathways affecting Ln resistance and toxicity. Ln responses strongly differed from well-known transition metal and from common responses mediated by oxidative compounds. Shared response pathways to La and Yb exposure were associated to lipid metabolism, ion homeostasis, vesicular trafficking, and endocytosis, which represents a putative way of entry for Ln. Cell wall organization and related signaling pathways allowed for the discrimination of light and heavy Ln. Mutants in cell wall integrity-related proteins (e.g., Kre1p, Kre6p) or in the activation of secretory pathway and cell wall proteins (e.g., Kex2p, Kex1p) were resistant to Yb but sensitive to La. Exposure of WT yeast to the serine protease inhibitor tosyl phenylalanyl chloromethyl ketone mimicked the phenotype of kex2 ∆ under Ln, strengthening these results. Our data also suggest that the relative proportions of chitin and phosphomannan could modulate the proportion of functional groups (phosphates and carboxylates) to which La and Yb could differentially bind. Moreover, we showed that kex2 ∆, kex1 ∆, kre1 ∆, and kre6 ∆ strains were all sensitive to light Ln (La to Eu), while being increasingly resistant to heavier Ln. Finally, shotgun proteomic analyses identified modulated proteins in kex2 ∆ exposed to Ln, among which several plasmalemma ion transporters that were less abundant and that could play a role in Yb uptake. By combining these different approaches, we unraveled that cell wall components not only act in Ln adsorption but are also active signal effectors allowing cells to differentiate light and heavy Ln. This work paves the way for future investigations to the better understanding of Ln toxicity in higher eukaryotes. [ABSTRACT FROM AUTHOR]

The rapidly expanding utilization of lanthanides (Ln) for the development of new technologies, green energies, and agriculture has raised concerns regarding their impacts on the environment and human health. The absence of characterization of the underlying cellular and molecular mechanisms regarding their toxicity is a caveat in the apprehension of their environmental impacts. We performed genomic phenotyping and molecular physiology analyses of Saccharomyces cerevisiae mutants exposed to La and Yb to uncover genes and pathways affecting Ln resistance and toxicity. Ln responses strongly differed from well-known transition metal and from common responses mediated by oxidative compounds. Shared response pathways to La and Yb exposure were associated to lipid metabolism, ion homeostasis, vesicular trafficking, and endocytosis, which represents a putative way of entry for Ln. Cell wall organization and related signaling pathways allowed for the discrimination of light and heavy Ln. Mutants in cell wall integrity-related proteins (e.g., Kre1p, Kre6p) or in the activation of secretory pathway and cell wall proteins (e.g., Kex2p, Kex1p) were resistant to Yb but sensitive to La. Exposure of WT yeast to the serine protease inhibitor tosyl phenylalanyl chloromethyl ketone mimicked the phenotype of kex2∆ under Ln, strengthening these results. Our data also suggest that the relative proportions of chitin and phosphomannan could modulate the proportion of functional groups (phosphates and carboxylates) to which La and Yb could differentially bind. Moreover, we showed that kex2∆, kex1∆, kre1∆, and kre6∆ strains were all sensitive to light Ln (La to Eu), while being increasingly resistant to heavier Ln. Finally, shotgun proteomic analyses identified modulated proteins in kex2∆ exposed to Ln, among which several plasmalemma ion transporters that were less abundant and that could play a role in Yb uptake. By combining these different approaches, we unraveled that cell wall components not only act in Ln adsorption but are also active signal effectors allowing cells to differentiate light and heavy Ln. This work paves the way for future investigations to the better understanding of Ln toxicity in higher eukaryotes.