Lactate and Amino Acid Catabolism in the Cheese-Ripening Yeast Yarrowia lipolytica

The consumption of lactate and amino acids is very important for microbial development and/or aroma production during cheese ripening. A strain of Yarrowia lipolytica isolated from cheese was grown in a liquid medium containing lactate in the presence of a low (0.1×) or high (2×) concentration of amino acids. Our results show that there was a dramatic increase in the growth of Y. lipolytica in the medium containing a high amino acid concentration, but there was limited lactate consumption. Conversely, lactate was efficiently consumed in the medium containing a low concentration of amino acids after amino acid depletion was complete. These data suggest that the amino acids are used by Y. lipolytica as a main energy source, whereas lactate is consumed following amino acid depletion. Amino acid degradation was accompanied by ammonia production corresponding to a dramatic increase in the pH. The effect of adding amino acids to a Y. lipolytica culture grown on lactate was also investigated. Real-time quantitative PCR analyses were performed with specific primers for five genes involved in amino acid transport and catabolism, including an amino acid transporter gene (GAP1) and four aminotransferase genes (ARO8, ARO9, BAT1, and BAT2). The expression of three genes involved in lactate transport and catabolism was also studied. These genes included a lactate transporter gene (JEN1) and two lactate dehydrogenase genes (CYB2-1 and CYB2-2). Our data showed that GAP1, BAT2, BAT1, and ARO8 were maximally expressed after 15 to 30 min following addition of amino acids (BAT2 was the most highly expressed gene), while the maximum expression of JEN1, CYB2-1, and CYB2-2 was delayed (≥60 min).     

The plant defensin RsAFP2 induces cell wall stress, septin mislocalization and accumulation of ceramides in Candida albicans

The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2‐hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast‐to‐hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analysed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24‐ceramides in membranes of RsAFP2‐treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation.     

Farnesol promotes epithelial cell defense against Candida albicans through Toll-like receptor 2 expression,interleukin-6 and human β-defensin 2 production

Farnesol, a quorum-sensing molecule, regulates virulence and morphogenesis in Candida albicans and is involved in various human pathologies including oral candidiasis. Oral epithelial cells are involved in innate immunity against Candida infections via Toll-like receptors (TLRs) and inflammatory mediators. We investigated the effects of farnesol on host cells and its possible synergistic interaction with gingival epithelial cells against C. albicans infection by studying the expression of TLR2, 4 and 6. The production of IL-6, IL-8, and human β-defensins 1 and 2 was also examined using engineered human oral mucosa tissue put in contact with various concentrations of farnesol with and without C. albicans. Our findings indicate that 24 h after contact with C. albicans, epithelial cells expressed more TLR2 than did non-infected cells. The addition of exogenous farnesol upregulated the TLR2 expression by the gingival epithelial cells in the presence or absence of C. albicans. In contrast, TLR4 was down regulated when farnesol was added to the tissue with or without C. albicans. Finally, farnesol alone was shown to have no effect on TLR6, yet in the presence of both C. albicans and farnesol, TLR6 expression was down regulated. Farnesol modulated TLR2 expression by the epithelial cells following tissue contact with C. albicans. This effect was paralleled by IL-6 but not IL-8 secretion. Farnesol’s effect on innate immunity was strengthened by its capacity to increase human β-defensin 2 production, and by the efficacy of β-defensin against C. albicans growth. Overall results showed that exogenous farnesol promoted epithelial cell defense against C. albicans infection through the involvement of TLR2, IL-6, and human β-defensin 2.     

Inhibition of Candida albicans growth by brominated furanones

Candida albicans is the most virulent Candida species of medical importance, which presents a great threat to immunocompromised individuals such as HIV patients. Currently, there are only four classes of antifungal agents available for treating fungal infections: azoles, polyenes, pyrimidines, and echinocandins. The fast spread of multidrug resistant C. albicans strains has increased the demand for new antifungal drugs. In this study, we demonstrate the antifungal activity of brominated furanones on C. albicans. Studying the structure and activity of this class of furanones reveals that the exocyclic vinyl bromide conjugated with the carbonyl group is the most important structural element for fungal inhibition. Furthermore, gene expression analysis using DNA microarrays showed that 3 μg/mL of 4-bromo-5Z-(bromomethylene)-3-butylfuran-2-one (BF1) upregulated 32 C. albicans genes with functions of stress response, NADPH dehydrogenation, and small-molecule transport, and repressed 21 genes involved mainly in cell-wall maintenance. Interestingly, only a small overlap is observed between the gene expression changes caused by the representative brominated furanone (BF1) in this study and other antifungal drugs reported in literature. This result suggests that brominated furanones and other antifungal drugs may target different fungal proteins or genes. The existence of such new targets provides an opportunity for developing new agents to control fungal pathogens which are resistant to currently available drugs.     

Functional characterization of the ferroxidase, permease high-affinity iron transport complex from Candida albicans

Saccharomyces cerevisiae expresses two proteins that together support high‐affinity Fe‐uptake. These are a multicopper oxidase, Fet3p, with specificity towards Fe²⁺ and a ferric iron permease, Ftr1p, which supports Fe‐accumulation. Homologues of the genes encoding these two proteins are found in all fungal genomes including those for the pathogens, Candida albicans and Cryptococcus neoformans. At least one of these loci represents a virulence factor for each pathogen suggesting that this complex would be an appropriate pharmacologic target. However, the mechanism by which this protein pair supports Fe‐uptake in any fungal pathogen has not been elucidated. Taking advantage of the robust molecular genetics available in S. cerevisiae, we identify the two of five candidate ferroxidases likely involved in high‐affinity Fe‐uptake in C. albicans, Fet31 and Fet34. Both localize to the yeast plasma membrane and both support Fe‐uptake along with an Ftr1 protein, either from C. albicans or from S. cerevisiae. We express and characterize Fet34, demonstrating that it is functionally homologous to ScFet3p. Using S. cerevisiae as host for the functional expression of the C. albicans Fe‐uptake proteins, we demonstrate that they support a mechanism of Fe‐trafficking that involves channelling of the CaFet34‐generated Fe³⁺ directly to CaFtr1 for transport into the cytoplasm.     

Carbon source‐induced reprogramming of the cell wall proteome and secretome modulates the adherence and drug resistance of the fungal pathogen Candida albicans

The major fungal pathogen Candida albicans can occupy diverse microenvironments in its human host. During colonization of the gastrointestinal or urogenital tracts, mucosal surfaces, bloodstream, and internal organs, C. albicans thrives in niches that differ with respect to available nutrients and local environmental stresses. Although most studies are performed on glucose‐grown cells, changes in carbon source dramatically affect cell wall architecture, stress responses, and drug resistance. We show that growth on the physiologically relevant carboxylic acid, lactate, has a significant impact on the C. albicans cell wall proteome and secretome. The regulation of cell wall structural proteins (e.g. Cht1, Phr1, Phr2, Pir1) correlated with extensive cell wall remodeling in lactate‐grown cells and with their increased resistance to stresses and antifungal drugs, compared with glucose‐grown cells. Moreover, changes in other proteins (e.g. Als2, Gca1, Phr1, Sap9) correlated with the increased adherence and biofilm formation of lactate‐grown cells. We identified mating and pheromone‐regulated proteins that were exclusive to lactate‐grown cells (e.g. Op4, Pga31, Pry1, Scw4, Yps7) as well as mucosa‐specific and other niche‐specific factors such as Lip4, Pga4, Plb5, and Sap7. The analysis of the corresponding null mutants confirmed that many of these proteins contribute to C. albicans adherence, stress, and antifungal drug resistance. Therefore, the cell wall proteome and secretome display considerable plasticity in response to carbon source. This plasticity influences important fitness and virulence attributes known to modulate the behavior of C. albicans in different host microenvironments during infection.     

Functional specialization and differential regulation of short‐chain carboxylic acid transporters in the pathogen Candida albicans

The major fungal pathogen Candida albicans has the metabolic flexibility to assimilate a wide range of nutrients in its human host. Previous studies have suggested that C. albicans can encounter glucose‐poor microenvironments during infection and that the ability to use alternative non‐fermentable carbon sources contributes to its virulence. JEN1 encodes a monocarboxylate transporter in C. albicans and we show that its paralogue, JEN2, encodes a novel dicarboxylate plasma membrane transporter, subjected to glucose repression. A strain deleted in both genes lost the ability to transport lactic, malic and succinic acids by a mediated mechanism and it displayed a growth defect on these substrates. Although no significant morphogenetic or virulence defects were found in the double mutant strain, both JEN1 and JEN2 were strongly induced during infection. Jen1‐GFP (green fluorescent protein) and Jen2‐GFP were upregulated following the phagocytosis of C. albicans cells by neutrophils and macrophages, displaying similar behaviour to an Icl1‐GFP fusion. In the murine model of systemic candidiasis approximately 20–25% of C. albicans cells infecting the kidney expressed Jen1‐GFP and Jen2‐GFP. Our data suggest that Jen1 and Jen2 are expressed in glucose‐poor niches within the host, and that these short‐chain carboxylic acid transporters may be important in the early stages of infection.     

Metabolic Response of Candida albicans to Phenylethyl Alcohol under Hyphae-Inducing Conditions

Phenylethyl alcohol was one of the first quorum sensing molecules (QSMs) identified in C. albicans. This extracellular signalling molecule inhibits the hyphal formation of C. albicans at high cell density. Little is known, however, about the underlying mechanisms by which this QSM regulates the morphological switches of C. albicans. Therefore, we have applied metabolomics and isotope labelling experiments to investigate the metabolic changes that occur in C. albicans in response to phenylethyl alcohol under defined hyphae-inducing conditions. Our results showed a global upregulation of central carbon metabolism when hyphal development was suppressed by phenylethyl alcohol. By comparing the metabolic changes in response to phenylethyl alcohol to our previous metabolomic studies, we were able to short-list 7 metabolic pathways from central carbon metabolism that appear to be associated with C. albicans morphogenesis. Furthermore, isotope-labelling data showed that phenylethyl alcohol is indeed taken up and catabolised by yeast cells. Isotope-labelled carbon atoms were found in the majority of amino acids as well as in lactate and glyoxylate. However, isotope-labelled carbon atoms from phenylethyl alcohol accumulated mainly in the pyridine ring of NAD⁺/NADH and NADP^−/NADPH molecules, showing that these nucleotides were the main products of phenylethyl alcohol catabolism. Interestingly, two metabolic pathways where these nucleotides play an important role, nitrogen metabolism and nicotinate/nicotinamide metabolism, were also short-listed through our previous metabolomics works as metabolic pathways likely to be closely associated with C. albicans morphogenesis.     

Virulence and pathogenicity of a Candida albicans mutant with reduced filamentation

Deletion of DNA polymerase eta (Rad30/Polη) in pathogenic yeast Candida albicans is known to reduce filamentation induced by serum, ultraviolet, and cisplatin. Because nonfilamentous C. albicans is widely accepted as avirulent form, here we explored the virulence and pathogenicity of a rad30Δ strain of C. albicans in cell‐based and animal systems. Flow cytometry of cocultured fungal and differentiated macrophage cells revealed that comparatively higher percentage of macrophages was associated with the wild‐type than rad30Δ cells. In contrast, higher number of Polη‐deficient C. albicans adhered per macrophage membrane. Imaging flow cytometry showed that the wild‐type C. albicans developed hyphae after phagocytosis that caused necrotic death of macrophages to evade their clearance. Conversely, phagosomes kill the fungal cells as estimated by increased metacaspase activity in wild‐type C. albicans. Despite the morphological differences, both wild‐type and rad30? C. albicans were virulent with a varying degree of pathogenicity in mice models. Notably, mice with Th1 immunity were comparatively less susceptible to systemic fungal infection than Th2 type. Thus, our study clearly suggests that the modes of interaction of morphologically different C. albicans strains with the host immune cells are diverged, and host genetic background and several other attributing factors of the fungus could additionally determine their virulence.