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The glucocorticoid betamethasone (BM) is frequently employed in clinical practice because of its anti-inflammatory and immunosuppressive properties. In this study, we investigated the effect of BM (1 and 2 mM) on the ability of Candida albicans to adhere to, invade and damage oral, intestinal or vaginal epithelial cells, as well as to elicit cytokine and chemokine release. BM at 2 mM concentration stimulated adherence of C. albicans to vaginal cells and facilitated the invasion of intestinal and vaginal epithelia without influencing the growth rate of invading C. albicans hyphae at any type of epithelia and BM concentrations tested. In addition, BM at 2 mM concentration also augmented C. albicans-initiated cell damage of oral and intestinal cells. Furthermore, BM exposure decreased IL-6 cytokine and IL-8 chemokine release from oral and vaginal epithelial cells and also IL-6 release from intestinal epithelium after infection with C. albicans. These observations suggest that high-dose applications of BM may predispose patients to various epithelial C. albicans infections.

Vulvovaginal candidiasis, a superficial infection caused predominantly by the pathogenic fungus Candida albicans, is frequently treated with clotrimazole. Some drug formulations contain lactate for improved solubility. Lactate may modify C. albicans physiology and drug sensitivity by serving as a carbon source for the fungus and/or affecting local pH. Here, we explored the effects of lactate, in combination with pH changes, on C. albicans proliferation, morphology and clotrimazole sensitivity. Moreover, we determined the influence of growth phase and morphology per se on drug sensitivity. We showed that utilization of lactate as a carbon source did not promote fast fungal proliferation or filamentation. Lactate had no influence on clotrimazole-mediated killing of C. albicans in standard fungal cultivation medium but had an additive effect on the fungicidal clotrimazole action under in vitro vagina-simulative conditions. Moreover, clotrimazole-mediated killing was growth-phase and morphology dependent. Post-exponential cells were resistant to the fungicidal action of clotrimazole, whilst logarithmic cells were sensitive, and hyphae showed the highest susceptibility. Finally, we showed that treatment of pre-formed C. albicans hyphae with sublethal concentrations of clotrimazole induced a reversion to yeast-phase growth. As C. albicans hyphae are considered the pathogenic morphology during mucosal infections, these data suggest that elevated fungicidal activity of clotrimazole against hyphae plus clotrimazole-induced hyphae-to-yeast reversion may help to dampen acute vaginal infections by reducing the relative proportion of hyphae and thus shifting to a non-invasive commensal-like population. In addition, lactate as an ingredient of clotrimazole formulations may potentiate clotrimazole killing of C. albicans in the vaginal microenvironment.

Cytosolic alpha-mannosidases are glycosyl hydrolases that participate in the catabolism of cytosolic free N-oligosaccharides. Two soluble alpha-mannosidases (E-I and E-II) belonging to glycosyl hydrolases family 47 have been described in Candida albicans. We demonstrate that addition of pepstatin A during the preparation of cell homogenates enriched alpha-mannosidase E-I at the expense of E-II, indicating that the latter is generated by proteolysis during cell disruption. E-I corresponded to a polypeptide of 52 kDa that was associated with mannosidase activity and was recognized by an anti-alpha1,2-mannosidase antibody. The N-mannan core trimming properties of the purified enzyme E-I were consistent with its classification as a family 47 alpha1,2-mannosidase. Differential density-gradient centrifugation of homogenates revealed that alpha1,2-mannosidase E-I was localized to the cytosolic fraction and Golgi-derived vesicles, and that a 65 kDa membrane-bound alpha1,2-mannosidase was present in endoplasmic reticulum and Golgi-derived vesicles. Distribution of alpha-mannosidase activity in a kex2Delta null mutant or in wild-type protoplasts treated with monensin demonstrated that the membrane-bound alpha1,2-mannosidase is processed by Kex2 protease into E-I, recognizing an atypical cleavage site of the precursor. Analysis of cytosolic free N-oligosaccharides revealed that cytosolic alpha1,2-mannosidase E-I trims free Man8GlcNAc2 isomer B into Man7GlcNAc2 isomer B. This is believed to be the first report demonstrating the presence of soluble alpha1,2-mannosidase from the glycosyl hydrolases family 47 in a cytosolic compartment of the cell.

A quantitative real-time RT-PCR system was established to identify which secreted aspartyl proteinase (SAP) genes are most highly expressed and potentially contribute to Candida albicans infection of human epithelium in vitro and in vivo. C. albicans SC5314 SAP1-10 gene expression was monitored in organotypic reconstituted human epithelium (RHE) models, monolayers of oral epithelial cells, and patients with oral (n=17) or vaginal (n=17) candidiasis. SAP gene expression was also analysed in Deltasap1-3, Deltasap4-6, Deltaefg1 and Deltaefg1/cph1 mutants to determine whether compensatory SAP gene regulation occurs in the absence of distinct proteinase gene subfamilies. In monolayers, RHE models and patient samples SAP9 was consistently the most highly expressed gene in wild-type cells. SAP5 was the only gene significantly upregulated as infection progressed in both RHE models and was also highly expressed in patient samples. Interestingly, the SAP4-6 subfamily was generally more highly expressed in oral monolayers than in RHE models. SAP1 and SAP2 expression was largely unchanged in all model systems, and SAP3, SAP7 and SAP8 were expressed at low levels throughout. In Deltasap1-3, expression was compensated for by increased expression of SAP5, and in Deltasap4-6, expression was compensated for by SAP2: both were observed only in the oral RHE. Both Deltasap1-3 and Deltasap4-6 mutants caused RHE tissue damage comparable to the wild-type. However, addition of pepstatin A reduced tissue damage, indicating a role for the Sap family as a whole in inducing epithelial damage. With the hypha-deficient mutants, RHE tissue damage was significantly reduced in both Deltaefg1/cph1 and Deltaefg1, but SAP5 expression was only dramatically reduced in Deltaefg1/cph1 despite the absence of hyphal growth in both mutants. This indicates that hypha formation is the predominant cause of tissue damage, and that SAP5 expression can be hypha-independent and is not solely controlled by the Efg1 pathway but also by the Cph1 pathway. This is believed to be the first study to fully quantify SAP gene expression levels during human mucosal infections; the results suggest that SAP5 and SAP9 are the most highly expressed proteinase genes in vivo. However, the overall contribution of the Sap1-3 and Sap4-6 subfamilies individually in inducing epithelial damage in the RHE models appears to be low.

While searching for regulators of virulence attributes of the human-pathogenic fungus Candida albicans, a gene was identified similar to the genes encoding the mammalian phospholipase A2-activating protein (PLAP) and the Saccharomyces cerevisiae protein Doa1, which is known to play a key role during ubiquitin (Ub)-dependent protein degradation. All three proteins contain WD-repeats. Both PLAP and CaDoa1 contain a mellitin-like sequence with a central 'KVL'. This mellitin-like sequence was shown to be necessary for full function of CaDoa1. CaDOA1 was expressed under all conditions investigated. Gene disruption of CaDOA1 caused phenotypes including modified colony morphologies, temperature sensitivity, reduced secretion of hydrolytic enzymes and hypersensitivity to various compounds such as propranolol, butanol, caffeine, chelators, azoles, nocodazole and cadmium. Strikingly, mutants lacking DOA1 were filamentous and grew as pseudohyphae and true hyphae under conditions that normally support yeast growth. Transcriptional profiling of Deltadoa1 indicated that several genes associated with Ub-mediated proteolysis, including CDC48 and UBI4, are upregulated. These data suggest that DOA1 of C. albicans, like its orthologue in S. cerevisiae, is associated with Ub-mediated proteolysis and has multiple functions. However, some functions of CaDoa1 seem to be unique for C. albicans. These results support the hypothesis that Ub-mediated proteolysis plays an important role in the regulation of morphology in C. albicans.

To study invasion of the human fungal pathogen Candida albicans, several infection models have been established. This study describes the successful establishment of an ex vivo haemoperfused liver as a model to study invasion of C. albicans. Perfused organs from pigs could be kept functional for up to 12 h. By comparing a non-invasive and invasive strain of C. albicans and by following a time course of invasion, it was shown that the invasion process in the perfused liver infection model is very similar to the in vivo situation after intraperitoneal infection of mice. The advantage of this set-up compared with other models of invasion is discussed.

Malassezia furfur is a dimorphic fungus and a member of the normal cutaneous microflora of humans. However, it is also a facultative pathogen, associated with a wide range of skin diseases. One unusual feature of M. furfur is an absolute dependency on externally provided lipids which the fungus hydrolyses by lipolytic activity to release fatty acids necessary for both growth and pathogenicity. In this study, the cloning and characterization of the first gene encoding a secreted lipase of M. furfur possibly associated with this activity are reported. The gene, MfLIP1, shows high sequence similarity to other known extracellular lipases, but is not a member of a lipase gene family in M. furfur. MfLIP1 consists of 1464 bp, encoding a protein with a molecular mass of 54.3 kDa, a conserved lipase motif and an N-terminal signal peptide of 26 aa. By using a genomic library, two other genes were identified flanking MfLIP1, one of them encoding a putative secreted catalase, the other a putative amine oxidase. The cDNA of MfLIP1 was expressed in Pichia pastoris and the biochemical properties of the recombinant lipase were analysed. MfLip1 is most active at 40 degrees C and the pH optimum was found to be 5.8. The lipase hydrolysed lipids, such as Tweens, frequently used as the source of fatty acids in M. furfur media, and had minor esterase activity. Furthermore, the lipase is inhibited by different bivalent metal ions. This is the first molecular description of a secreted lipase from M. furfur.

Phospholipases C are known to be important regulators of cellular processes but may also act as virulence factors of pathogenic microbes. At least three genes in the genome of the human-pathogenic fungus Candida albicans encode phospholipases with conserved phospholipase C (Plc) motifs. None of the deduced protein sequences contain N-terminal signal peptides, suggesting that these phospholipases are not secreted. In contrast to its orthologue in Sacharomyces cerevisiae, CaPLC1 seems to be an essential gene. However, a conditional mutant with reduced transcript levels of CaPLC1 had phenotypes similar to Plc1p-deficient mutants in S. cerevisiae, including reduced growth on media causing increased osmotic stress, on media with a non-glucose carbon source, or at elevated or lower temperatures, suggesting that CaPlc1p, like the Plc1p counterpart in S. cerevisiae, may be involved in multiple cellular processes. Furthermore, phenotypic screening of the heterozygous DeltaCaplc1/CaPLC1 mutant showed additional defects in hyphal formation. The loss of CaPLC1 cannot be compensated by two additional PLC genes of C. albicans (CaPLC2 and CaPLC3) encoding two almost identical phospholipases C with no counterpart in S. cerevisiae but containing structural elements found in bacterial phospholipases C. Although the promoter sequences of CaPLC2 and CaPLC3 differed dramatically, the transcriptional pattern of both genes was similar. In contrast to CaPLC1, CaPLC2 and CaPLC3 are not essential. Although Caplc2/3 mutants had reduced abilities to produce hyphae on solid media, these mutants were as virulent as the wild-type in a model of systemic infection. These data suggest that C. albicans contains two different classes of phospholipases C which are involved in cellular processes but which have no specific functions in pathogenicity.

Candida dubliniensis is a pathogenic yeast species closely related to Candida albicans. However, it is less frequently associated with human disease and displays reduced virulence in animal models of infection. Here comparative genomic hybridization was used in order to assess why C. dubliniensis is apparently less virulent than C. albicans. In these experiments the genomes of the two species were compared by co-hybridizing C. albicans microarrays with fluorescently labelled C. albicans and C. dubliniensis genomic DNA. C. dubliniensis genomic DNA was found to hybridize reproducibly to 95.6 % of C. albicans gene-specific sequences, indicating a significant degree of nucleotide sequence homology (> 60 %) in these sequences. The remaining 4.4 % of sequences (representing 247 genes) gave C. albicans/C. dubliniensis normalized fluorescent signal ratios that indicated significant sequence divergence (< 60 % homology) or absence in C. dubliniensis. Sequence divergence was identified in several genes (confirmed by Southern blot analysis and sequence analysis of PCR products) with putative virulence functions, including the gene encoding the hypha-specific human transglutaminase substrate Hwp1p. Poor hybridization of C. dubliniensis genomic DNA to the array sequences for the secreted aspartyl proteinase-encoding gene SAP5 also led to the finding that SAP5 was absent in C. dubliniensis and that this species possesses only one gene homologous to SAP4 and SAP6 of C. albicans. In addition, divergence and absence of sequences in several gene families was identified, including a family of HYR1-like GPI-anchored proteins, a family of genes homologous to a putative transcriptional activator (CTA2) and several ALS genes. This study has confirmed the close relatedness of C. albicans and C. dubliniensis and has identified a subset of unique C. albicans genes that may contribute to the increased prevalence and virulence of this species.

The immune response and the anticandidal activity of keratinocytes and polymorphonuclear leukocytes (PMNs) play a key role in host defence against localized Candida albicans infection. An established model of oral candidosis based on reconstituted human oral epithelium (RHE) was supplemented with PMNs to study the effect of these immune cells during experimental oral candidosis. Infection of RHE with C. albicans induced a strong expression of the chemokine interleukin-8 (IL-8) and the cytokine granulocyte-macrophages colony-stimulating factor (GM-CSF), and a moderate stimulation of interleukin-1alpha (IL-1alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) by keratinocytes. This immune response was associated with chemoattraction of PMNs to the site of infection, whereas uninfected RHE failed to induce cytokine expression or to attract PMNs. Growth of the pathogen and tissue damage of C. albicans-infected RHE were significantly reduced when PMNs were applied to the apical epithelial surface or when PMNs migrated through a perforated basal polycarbonate filter of the model. Notably, protection against epithelial tissue damage was also observed when PMNs were placed on the basal side of non-perforated filters, which prevented PMN migration into the RHE. Addition of PMNs enhanced a Th1-type immune response (IFN-gamma, TNF-alpha), down-regulated the expression of the Th2-type cytokine interleukin-10 (IL-10), and was associated with protection against Candida-induced tissue damage. This PMN-supplemented model of oral candidosis mimics the in vivo situation, and provides a promising tool for studying the immunological interactions between keratinocytes and C. albicans, as well as the influence of PMNs on C. albicans pathogenesis.

The transition of Candida albicans from a yeast to a hyphal form is controlled by several transcriptional factors, including the key regulators Cph1 and Efg1, and is considered an important virulence attribute. These factors, especially Efg1, regulate the expression of hyphal-associated genes e.g. SAP4-SAP6. In order to investigate the relevance of these transcriptional regulators for hyphal-independent SAP genes, recently constructed cph1 and efg1 single mutants and a cph1/efg1 double mutant lacking these factors were tested during interaction with oral epithelium and polymorphonuclear neutrophils. In contrast to the parental wild-type strain and the cph1 mutant, the efg1 and the cph1/efg1 mutants did not produce hyphal forms in all experiments and were less capable of damaging epithelial cells and neutrophil granulocytes. The attenuated epithelial lesions of these mutants were correlated not only with reduced expression of the hyphal-associated gene SAP4, but also with the lack of SAP1 and SAP3 expression previously shown to be important for oral infections. An efg1 mutant strain carrying a plasmid-borne copy of the EFG1 gene regained hyphal growth, damage of keratinocytes, granulocytes and the expression of SAP1 and SAP3. Although efg1 and cph1/efg1 mutants did not produce germ tubes during infection, expression of the hyphal-associated genes SAP5 and SAP6 was not completely abolished. A reduced capacity to stimulate an epithelial immune response manifested by a delayed onset of IL-1beta, IL-8 and TNF expression was only observed in the cph1/efg1-infected tissue. These results provide further evidence for a combined regulation of different virulence factors, such as dimorphism and expression of SAP genes. Furthermore, it could be demonstrated that the lack of Efg1 also caused reduced expression of hyphal-independent SAP genes. Both the EFG1 and the CPH1 gene products are necessary for adequate induction of an immune response.

The inhibitory effect of human immunodeficiency virus (HIV) proteinase inhibitors amprenavir and saquinavir and antifungal agents terbinafine, ketoconazole, amphotericin B and ciclopiroxolamine on aspartyl proteinases (Saps) secreted by Candida albicans was tested in an in vitro spectophotometric assay. As expected, both HIV proteinase inhibitors showed a significant inhibitory effect on Sap activity, which was comparable to that of the classical aspartyl proteinase inhibitor pepstatin A (P < 0.001). Antifungal drugs such as ketoconazole, terbinafine and amphotericin B had no, or only minor, inhibitory effects on proteolytic activity. In contrast, a significant reduction in Sap activity could be demonstrated during treatment with the antifungal agent ciclopiroxolamine (P < 0.001). These results point to a multiple effect of this antimycotic agent and might explain the reduced adherence of C. albicans to human epithelial cells at subinhibitory doses.

The phosphatidylcholine-specific phospholipase D1 (PLD1) in Saccharomyces cerevisiae is involved in vesicle transport and is essential for sporulation. The gene encoding the homologous phospholipase D1 from Candida albicans (PLD1) was used to study the role of PLD1 in this pathogenic fungus. In vitro and in vivo expression studies using Northern blots and reverse transcriptase-PCR showed low PLD1 mRNA levels in defined media supporting yeast growth and during experimental infection, while enhanced levels of PLD1 transcripts were detected during the yeast to hyphal transition. To study the relevance of PLD1 during yeast and hyphal growth, an essential part of the gene was deleted in both alleles of two isogenic strains. In vitro PLD1 activity assays showed that pld1 mutants produced no detectable levels of phosphatidic acid, the hydrolytic product of PLD1 activity, and strongly reduced levels of diacylglycerol, the product of lipid phosphate phosphohydrolase, suggesting no or a negligible background PLD1 activity in the pld1 mutants. The pld1 mutants showed no growth differences compared to the parental wild-type in liquid complex and minimal media, independent of the growth temperature. In addition, growth rates of pld1 mutants in media with protein as the sole source of nitrogen were similar to growth rates of the wild-type, indicating that secretion of proteinases was not reduced. Chlamydospore formation was normal in pld1 mutants. When germ tube formation was induced in liquid media, pld1 mutants showed similar rates of yeast to hyphal transition compared to the wild-type. However, no hyphae formation was observed on solid Spider medium, and cell growth on cornmeal/Tween 80 medium indicated aberrant morphogenesis. In addition, pld1 mutants growing on solid media had an attenuated ability to invade the agar. In a model of oral candidosis, pld1 mutants showed no attenuation of virulence. In contrast, the mutant was less virulent in two different mouse models. These data suggest that PLD1 is not essential for growth and oral infections. However, they also suggest that a prominent part of the phosphatidic acid and diacylglycerol pools is produced by PLD1 and that the level of these components is important for morphological transitions under certain conditions in C. albicans.

Secreted aspartic proteinases (Saps) contribute to the virulence of Candida albicans in systemic animal models of infection. Seven genes encoding Saps (SAP1-SAP7) have been identified to date but evidence suggested the existence of additional SAP genes. The screening of a C. albicans lambda EMBL3 genomic library for the presence of other SAP genes was undertaken. Two new genes, SAP8 and SAP9, were isolated. The N-terminal amino acid sequence deduced from SAP8 downstream of a Kex2p-like cleavage site corresponds to the N-terminal amino acid sequence of the 41 kDa Sap isolated and characterized previously. SAP8 mRNA was expressed preferentially in yeasts at 25 degrees C after 6 and 9 h growth in BSA-containing medium. SAP9 encodes an aspartic proteinase with a Kex2p-like cleavage site and contains a putative glycophosphatidylinositol-anchor signal at the C-terminus. Although the SAP9 gene product has not yet been isolated from cultures of C. albicans, transcripts of SAP9 were observed preferentially in later growth phases when SAP8 expression had decreased.