Candida albicans, the single most frequently isolated human fungal pathogen, was thought to be asexual until the recent discovery of the mating-type-like locus (MTL). Homozygous MTL strains were constructed and shown to mate. Furthermore, it has been demonstrated that opaque-phase cells are more efficient in mating than white-phase cells. The similarity of the genes involved in the mating pathway in Saccharomyces cerevisiae and C. albicans includes at least one gene (KEX2) that is involved in the processing of the ␣ mating pheromone in the two yeasts. Taking into account this similarity, we searched the C. albicans genome for sequences that would encode the ␣ pheromone gene. Here we report the isolation and characterization of the gene MF␣1, which codes for the precursor of the ␣ mating pheromone in C. albicans. Two active ␣-peptides, 13 and 14 amino acids long, would be generated after the precursor molecule is processed in C. albicans. To examine the role of this gene in mating, we constructed an mf␣1 null mutant of C. albicans. The mf␣1 null mutant fails to mate as MTL␣, while MTLa mf␣1 cells are still mating competent. Experiments performed with the synthetic ␣-peptides show that they are capable of inducing growth arrest, as demonstrated by halo tests, and also induce shmooing in MTLa cells of C. albicans. These peptides are also able to complement the mating defect of an MTL␣ kex2 mutant strain when added exogenously, thereby confirming their roles as ␣ mating pheromones.Candida albicans is the most common opportunistic fungal pathogen of humans and mostly infects immunosuppressed patients (3). It inhabits diverse niches, which include the gastrointestinal tract and the vagina, and causes infection of skin, mucous membranes, and the bloodstream. Since each of these sites of infection presumably requires differences in gene expression, a great deal of effort has been spent in looking at how such adaptation occurs in Candida. Much of this effort was predicated on the characterization of C. albicans as an asexual obligate diploid.That this fungus is asexual was disproved after the C. albicans genome sequencing project revealed the presence of sequences homologous to the Saccharomyces cerevisiae MAT (mating-type) loci. The C. albicans homologues of the MAT loci, the MTL (mating-type-like) loci, were found to be heterozygous in common laboratory strains tested (17). Strains homozygous for the MTL locus were generated either by a deletion strategy (18) or by loss, induced by growth on sorbose as the sole carbon source (19), of one homologue of chromosome 5, the site of the MTL loci (29). Strains thus constructed were able to mate both under laboratory conditions (29) and in the animal host (18). These studies set the stage for dissecting the mating pathway in C. albicans.Mating in fungi has been examined in both yeasts and molds of various sorts. Certain general parts of the process seem to hold across genera and indeed across the kingdom. These include dissimilar regulatory genes (mating-type loci), soluble phe...

In this study, we compared the effects of altered membrane lipid composition on the localization of two membrane drug transporters from different superfamilies of the pathogenic yeast Candida albicans. We demonstrated that in comparison to the major facilitator superfamily multidrug transporter CaMdr1p, ATP-binding cassette transporter CaCdr1p of C. albicans is preferentially localized within detergent-resistant membrane (DRM) microdomains called 'rafts.' Both CaCdr1p and CaMdr1p were overexpressed as green fluorescent protein (GFP)-tagged proteins in a heterologous host Saccharomyces cerevisiae, wherein either sphingolipid (⌬sur4 or ⌬fen1 or ⌬ipt1) or ergosterol (⌬erg24 or ⌬erg6 or ⌬erg4) biosynthesis was compromised. CaCdr1p-GFP, when expressed in the above mutant backgrounds, was not correctly targeted to plasma membranes (PM), which also resulted in severely impaired drug resistance. In contrast, CaMdr1p-GFP displayed no sorting defect in the mutant background and remained properly surface localized and displayed no change in drug resistance. Our data clearly show that CaCdr1p is selectively recruited, over CaMdr1p, to the DRM microdomains of the yeast PM and that any imbalance in the raft lipid constituents results in missorting of CaCdr1p.

dMitochondrial dysfunction in Candida albicans is known to be associated with drug susceptibility, cell wall integrity, phospholipid homeostasis, and virulence. In this study, we deleted CaFZO1, a key component required during biogenesis of functional mitochondria. Cells with FZO1 deleted displayed fragmented mitochondria, mitochondrial genome loss, and reduced mitochondrial membrane potential and were rendered sensitive to azoles and peroxide. In order to understand the cellular response to dysfunctional mitochondria, genome-wide expression profiling of fzo1⌬/⌬ cells was performed. Our results show that the increased susceptibility to azoles was likely due to reduced efflux activity of CDR efflux pumps, caused by the missorting of Cdr1p into the vacuole. In addition, fzo1⌬/⌬ cells showed upregulation of genes involved in iron assimilation, in iron-sufficient conditions, characteristic of iron-starved cells. One of the consequent effects was downregulation of genes of the ergosterol biosynthesis pathway with a commensurate decrease in cellular ergosterol levels. We therefore connect deregulated iron metabolism to ergosterol biosynthesis pathway in response to dysfunctional mitochondria. Impaired activation of the Hog1 pathway in the mutant was the basis for increased susceptibility to peroxide and increase in reactive oxygen species, indicating the importance of functional mitochondria in controlling Hog1-mediated oxidative stress response. Mitochondrial phospholipid levels were also altered as indicated by an increase in phosphatidylserine and phosphatidylethanolamine and decrease in phosphatidylcholine in fzo1⌬/⌬ cells. Collectively, these findings reinforce the connection between functional mitochondria and azole tolerance, oxidant-mediated stress, and iron homeostasis in C. albicans.

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Saccharomyces cerevisiae cells lacking their mitochondrial DNA ( 0 cells) respond to this loss of genetic information by induction of a program of nuclear gene expression called the retrograde response. Expression of genes involved in multidrug resistance and sphingolipid biosynthesis is coordinately induced in 0 cells by the zinc cluster transcription factor Pdr3p. In this report, we identify a membrane protein involved in control of intracellular levels of a sphingolipid precursor as a transcriptional target of the Pdr3p-mediated retrograde response. These sphingolipid precursors are called long chain bases (LCBs) and increased LCB levels are growth inhibitory. This membrane protein has been designated Rsb1p and has previously been shown to act as a LCB transporter protein and to be a component of the endoplasmic reticulum. These earlier studies used an amino-terminal truncated form of Rsb1p. Here we employ a full-length form of Rsb1p and find that this protein is localized to the plasma membrane and is modified by N-linked glycosylation. Two glycosylation sites are present in the Rsb1p and both are required for normal LCB resistance. Mutational analysis of the RSB1 promoter revealed that two Pdr3p binding sites are present and both of these are required for normal retrograde induction of transcription. LCB tolerance is strongly increased in 0 cells but this increase is ablated in 0 rsb1⌬ cells. Together, these data indicate Pdr3p activation of RSB1 transcription is an important feature of the retrograde response allowing normal detoxification of an endogenous sphingolipid precursor.Sphingolipids are important lipid constituents of eukaryotic membranes. Key intermediates in the biosynthesis of sphingolipids are the long chain bases (LCBs).2 In Saccharomyces cerevisiae, these LCBs are dihydrosphingosine and phytosphingosine (PHS) (reviewed in Ref. 1). These LCBs are utilized by ceramide synthase to form ceramide that is a well known bioactive lipid (reviewed in Refs. 2 and 3). However, LCBs and their phosphorylated forms (LCBPs) have also recently been found to serve as potent signaling molecules. LCBs appear to act as inhibitors of proliferation, whereas increased LCBP levels stimulate growth (recently reviewed in Ref. 4). Control of the biological levels of these signaling lipids is an essential feature for ensuring both normal lipid composition of membranes and proper metabolic regulation.The central importance of these signaling and structural lipids is likely to explain the multitude of regulatory mechanisms modulating their levels. These include enzymes like ceramidases that break down ceramide (5, 6) and the LCBP lyase that cleaves these phosphorylated lipids into ethanolamine and a long chain aldehyde (7). More recent experiments in the yeast S. cerevisiae have identified a new membrane protein that is thought to efflux LCBs out of the cell. This protein has been designated Rsb1p (resistant to sphingoid bases) as it was identified as a high-copy suppressor of the PHS hypersensitivity of a dpl1⌬ strain (...

triggers the induction of a number of nuclear genes, including CIT2 and DLD3, via activation of the transcriptional regulatory proteins Rtg1p and Rtg3p (4 -6). A major function of the Rtg1p/Rtg3p-regulated retrograde signaling pathway is to permit adequate synthesis of amino acids that rely on the tricarboxylic acid cycle for their production.

Many azole-resistant (AR) clinical isolates of Candida albicans display increased expression of the drug transporters CDR1 and CDR2. In this study, we evaluate the molecular mechanisms that contribute to the maintenance of constitutively high CDR1 transcript levels in two matched pairs of azole-susceptible (AS) and AR clinical isolates of C. albicans. To address this, we use reporter constructs of GFP and lacZ fused either to the CDR1 promoter (P CDR1 -GFP/lacZ; transcriptional fusion) or to the CDR1 open reading frame (P CDR1 -CDR1-GFP/lacZ; translational fusion) integrated at the native CDR1 locus. It is observed that expression of the two reporter genes as a transcriptional fusion in the AR isolates is higher than that in matched AS isolates. However, the difference in the reporter activity between the AS and AR isolates is even greater for the translational fusions, indicating that the sequences within the CDR1 coding region also contribute to its increased expression in AR isolates. Further analysis of these observations by transcription run-on assays demonstrated a ϳ5-to 7-fold difference in the transcription initiation rates for the AR isolates from those for their respective matched AS isolates. Measurement of mRNA stability showed that the half-life of CDR1 mRNA in the AR isolates was threefold higher than that in the corresponding AS isolates. Our results demonstrate that both increased CDR1 transcription and enhanced CDR1 mRNA stability contribute to the overexpression of CDR1 in AR C. albicans isolates.Resistance of the human fungal pathogen Candida albicans to azole antifungals is often caused by increased expression of genes encoding multidrug efflux pumps, the ATP-binding cassette transporter genes CDR1 and CDR2 and/or the major facilitator gene CaMDR1 (1,7,23,29,34,36,37,38,44,45,46). However, the molecular mechanisms leading to the constitutive overexpression of efflux pump-encoding genes in drugresistant, clinical C. albicans isolates are only beginning to be understood. In particular, the regulation of CDR1 expression has been studied by many groups (2,3,4,5,10,11,17,20,32). Various unrelated stresses, including elevated temperature or the presence of drugs and steroids, induce a transient transcriptional activation of CDR1 in drug-susceptible C. albicans strains (20). Several cis elements in the CDR1 upstream region that affect basal as well as induced CDR1 expression have been identified. Puri et al. (32) identified four upstream activating sequences and four upstream repressing sequences in the 5Ј noncoding region of CDR1. A basal regulatory element and a negative regulatory element, in the proximal region of the promoter, have also been characterized and implicated in the basal expression of CDR1 (10, 11). A specific steroid-responsive region in the distal part of the CDR1 promoter, consisting of two progesterone-responsive sequences and one ␤-estradiolresponsive sequence, has been shown to contribute exclusively to steroid responsiveness of CDR1 (17). Another basal expression element in t...

Candida albicans, the dimorphic opportunistic human fungal pathogen, is capable of forming highly drug-resistant biofilms in the human host. Formation of biofilm is a multistep and multiregulatory process involving various adaptive mechanisms. The ability of cells in a biofilm to alter membrane lipid composition is one such adaptation crucial for biofilm development in C. albicans. Lipids modulate mixed species biofilm formation in vivo and inherent antifungal resistance associated with these organized communities. Cells in C. albicans biofilms display phase-dependent changes in phospholipid classes and in levels of lipid raft formation. Systematic studies with genetically modified strains in which the membrane phospholipid composition can be manipulated are limited in C. albicans. In this review, we summarize the knowledge accumulated on the impact that alterations in phospholipids may have on the biofilm forming ability of C. albicans in the human host. This review may provide the requisite impetus to analyze lipids from a therapeutic standpoint in managing C. albicans biofilms.