Dermatomycoses and inflammation: The adaptive balance between growth, damage, and survival.

Dermatomycosis is characterized by both superficial and subcutaneous infections of keratinous tissues and mucous membranes caused by a variety of fungal agents, the two most common classes being dermatophytes and yeasts. Overall, the stepwise process of host infection is similar among the main dermatomycotic species; however, the species-specific ability to elicit a host reaction upon infection is distinct. Yeasts such as Candida albicans elicit a relatively low level of host tissue damage and inflammation during pathogenic infection, while dermatophytes may induce a higher level of tissue damage and inflammatory reaction. Both pathogens can, however, manipulate the host's immune response, ensuring survival and prolonging chronic infection. One common element of most dermatomycotic infections is the disease burden caused by inflammation and associated signs and symptoms, such as erythema, burning and pruritus. There is a strong clinical rationale for the addition of a topical corticosteroid agent to an effective antimycotic therapy, especially in patients who present with inflammatory dermatomycoses (e.g., tinea inguinalis). In this review, we aim to compare the pathogenesis of common dermatomycotic species, including Candida yeasts (Candida albicans), dermatophytes (Trichophyton, Epidermophyton or Microsporum species), and other pathogenic yeasts (Malassezia), with a special focus on unique species-specific aspects of the respective infection processes, the interaction between essential aspects of pathogenic infection, the different roles of the host inflammatory response, and the clinical consequences of the infection-related tissue damage and inflammation. We hope that a broader understanding of the various mechanisms of dermatomycoses may contribute to more effective management of affected patients.

Game theoretical modelling of survival strategies of Candida albicans inside macrophages.

The polymorphic fungus Candida albicans can live as an aggressive pathogen that causes a wide variety of diseases in humans. Host resistance against these infections is mediated predominantly by phagocytes, namely neutrophils and macrophages. This report provides two game theoretical models of ingested C. albicans cells in macrophages. Two strategies are available for each pathogenic yeast cell: avoiding lysis transiently (called silencing) or forming hyphae and escaping (called piercing because the macrophage is pierced from inside). In dependence on parameter values, two different outcomes can be derived from the model: when the difference of the costs of the two strategies is low, all fungal cells inside a macrophage will play the piercing strategy, while in the high-cost case, a mixed population of piercing and silencing cells is the only stable solution. Further, the role of the SAP gene family encoding secreted proteinases and the Sap proteins is investigated with the help of known studies and is put in relation to the costs of the strategies, the most important parameter of this model. Our results are in agreement with wet-lab results presented by other groups and the model parameters can be estimated from experimental data.

Candida glabrata persistence in mice does not depend on host immunosuppression and is unaffected by fungal amino acid auxotrophy.

Candida glabrata has emerged as an important fungal pathogen of humans, causing life-threatening infections in immunocompromised patients. In contrast, mice do not develop disease upon systemic challenge, even with high infection doses. In this study we show that leukopenia, but not treatment with corticosteroids, leads to fungal burdens that are transiently increased over those in immunocompetent mice. However, even immunocompetent mice were not capable of clearing infections within 4 weeks. Tissue damage and immune responses to microabscesses were mild as monitored by clinical parameters, including blood enzyme levels, histology, myeloperoxidase, and cytokine levels. Furthermore, we investigated the suitability of amino acid auxotrophic C. glabrata strains for in vitro and in vivo studies of fitness and/or virulence. Histidine, leucine, or tryptophan auxotrophy, as well as a combination of these auxotrophies, did not influence in vitro growth in rich medium. The survival of all auxotrophic strains in immunocompetent mice was similar to that of the parental wild-type strain during the first week of infection and was only mildly reduced 4 weeks after infection, suggesting that C. glabrata is capable of utilizing a broad range of host-derived nutrients during infection. These data suggest that C. glabrata histidine, leucine, or tryptophan auxotrophic strains are suitable for the generation of knockout mutants for in vivo studies. Notably, our work indicates that C. glabrata has successfully developed immune evasion strategies enabling it to survive, disseminate, and persist within mammalian hosts.

Analysis of differentially expressed genes associated with tryptophan-dependent pigment synthesis in M. furfur by cDNA subtraction technology.

Malassezia species are associated with pityriasis versicolor (PV) and its depigmented variant pityriasis versicolor alba (PVa), widespread fungal skin infections in humans. The pathogenesis of PV and PVa remains unclear, including their clinical and histological symptoms such as hyper- and depigmentation, reduced responsiveness to ultraviolet radiation and lack of inflammatory reaction despite high fungal load. Pigments produced by M. furfur are possibly involved in the pathogenesis of PV. In vitro, M. furfur produces a wide range of pigments and fluorochromes when cultured with tryptophan as the sole nitrogen source. We have begun to analyse the molecular basis of pigment production by searching for genes associated with tryptophan-based pigment production. A suppression subtractive hybridization (SSH) protocol was used to identify genes expressed in M. furfur cells producing pigments, but not in non-induced cells. SSH was performed 3 and 5 h after onset of pigment induction. Up-regulation of genes in the pigment-producing cells was confirmed by reverse northern analysis. More than 1,500 cDNA sequences of both the indicated time points were analysed. We identified a wide variety of genes associated with metabolism and several genes with unknown function are specifically expressed during pigment production. Furthermore, a fraction of genes possibly involved in different steps of the newly discovered indolic pathway of M. furfur were expressed in pigment producing cells. These data provide the first molecular insight into pigment production of M. furfur.

[Characterization of the interaction between Candida albicans and host cells: In vitro model using reconstituted human skin and mucosa]. / Charakterisierung der Interaktion zwischen Candida albicans und Wirtszellen. In-vitro-Modelle auf der Basis von rekonstituierter humaner Haut/Schleimhaut.

Basic research on the biology and immunology of microbial infection requires appropriate model systems. Currently most such studies involve animal studies which are a focus of ethical controversy. Possible alternatives, especially for localized infections, are provided by models using in vitro reconstituted human epithelium or epidermis (RHE). In recent years, these model systems have been successfully established to evaluate the effectiveness of topical anti-infectives, to characterize the role of fungal virulence factors, and to study the immune responses during localized Candida albicans infections. Most recently, these models have been supplemented with immune cells such as lymphocytes, polymorphonuclear leukocytes, mast cells or dendritic cells, to study their role during the course of infection and to characterize the interaction between the skin barrier and accessory immune cells. Although the most experience is with Candida albicans RHE infections, such model systems can also be used to study infections with other fungi or bacteria.

In vitro investigations on the mode of action of the hydroxypyridone antimycotics rilopirox and piroctone on Candida albicans.

Rilopirox and piroctone belong to the class of hydroxypyridone antimycotics. This class is not related to other antimycotics. In contrast to azole antimycotics and polyene antimycotics the mode of action of hydroxypyridone antimycotics is not fully understood. Inhibition of cellular uptake of essential compounds as well as loss of other compounds seems to be only a secondary effect of a primary not known action of these drugs. The antifungal effect in vitro depends on the medium used. The hyphal induction of Candida albicans is inhibited by hydroxypyridone antimycotics, but this effect is compensated by iron ions. A damage of the cell membrane and a direct influence on adenosine triphosphate synthesis, respectively, do not seem to be part of the mode of action. But there are clear hints that reactive oxygen species (ROS) and available metabolic activity are important parts of the mode of action of the hydroxypyridone antimycotics rilopirox and piroctone.

Systemic fungal infections caused by Candida species: epidemiology, infection process and virulence attributes.

Candida species, in particular C. albicans, represent a major threat to immunocompromised patients. Able to exist as a commensal on mucosal surfaces of healthy individuals, these opportunistic fungi frequently cause superficial infections of mucosae and skin. Furthermore, in hospital settings, Candida species may cause life-threatening invasive infections in a growing population of vulnerable patients. In fact, candidaemia is associated with the highest crude mortality of all bloodstream infections. Candida cells may enter the bloodstream by direct penetration from epithelial tissues, due to damage of barriers in the body caused by surgery, polytrauma or drug treatment, or may spread from biofilms produced on medical devices. From the bloodstream, cells may infect almost all organs but appear to prefer certain organs depending upon the route of infection. The exact mechanisms by which Candida cells survive the challenge of the blood environment and escape from the bloodstream to cause deep-seated infections have not yet been elucidated, but various investigations are reviewed. It is clear, however, that Candida must have particular attributes which enable the organism to survive and grow within the environment of healthy individuals and to invade tissues in the immunocompromised host. Most studies have focussed on C. albicans and this review will therefore summarise work on the various known virulence factors and methods used to identify further virulence attributes of this fungus.

CandidaDB: a genome database for Candida albicans pathogenomics.

CandidaDB is a database dedicated to the genome of the most prevalent systemic fungal pathogen of humans, Candida albicans. CandidaDB is based on an annotation of the Stanford Genome Technology Center C.albicans genome sequence data by the European Galar Fungail Consortium. CandidaDB Release 2.0 (June 2004) contains information pertaining to Assembly 19 of the genome of C.albicans strain SC5314. The current release contains 6244 annotated entries corresponding to 130 tRNA genes and 5917 protein-coding genes. For these, it provides tentative functional assignments along with numerous pre-run analyses that can assist the researcher in the evaluation of gene function for the purpose of specific or large-scale analysis. CandidaDB is based on GenoList, a generic relational data schema and a World Wide Web interface that has been adapted to the handling of eukaryotic genomes. The interface allows users to browse easily through genome data and retrieve information. CandidaDB also provides more elaborate tools, such as pattern searching, that are tightly connected to the overall browsing system. As the C.albicans genome is diploid and still incompletely assembled, CandidaDB provides tools to browse the genome by individual supercontigs and to examine information about allelic sequences obtained from complementary contigs. CandidaDB is accessible at http://genolist.pasteur.fr/CandidaDB.

[Mode of action of ciclopiroxolamine on Candida albicans]. / Wirkungsweise von Ciclopiroxolamin auf Candida albicans.

Candida albicans is a common pathogen causing both superficial and invasive mycoses. The hydroxypyridone ciclopiroxolamine belongs to antimycotic drugs used for treatment of superficial mycoses. Whereas the mode of action of other antimycotics, for example the azoles, is well known, the specific action of ciclopiroxolamine is poorly understood. There are hints, that ciclopiroxolamine acts as a potential chelating agent and influences some cellular processes by chelating metal ions. Consequently, the antimycotic effectiveness of ciclopiroxolamine could be due to a general reduced viability of the fungus, or due to a higher sensitivity of a fungus against the human immune system as well as due to a reduced capability to produce some specific virulence factors that are indispensable for infection. Nevertheless, iron metabolism seems to play a major role in its effect.

The KEX2 gene of Candida glabrata is required for cell surface integrity.

Candida glabrata has emerged as one of the most common causes of candidosis. In order to identify factors that are necessary for viability and pathogenicity of this fungal pathogen, we analysed the role of the KEX2 gene, which codes for a regulatory endoproteinase that is known to process certain virulence factors in Candida albicans. The KEX2 gene from C. glabrata was cloned and found to have 51% and 62% identity and high structural similarities to the homologous counterparts in C. albicans and Saccharomyces cerevisiae. KEX2 was expressed at all time points investigated during growth in complex medium. In order to investigate the role of this putative regulatory proteinase, Kex2-deficient mutants were produced. In addition to known kex2 phenotypes, such as pH and calcium hypersensitivity, the mutants grew in cellular aggregates and were found to be hypersensitive to several antifungal drugs that target the cell membrane, including azoles, amorolfine and amphotericin B. Ultrastructural investigation after exposure to low doses of itraconazole showed azole-specific alterations such as enlarged vacuoles and proliferation of the cytoplasmatic membrane in the kex2 mutants, but not in the control strains. In contrast, antifungals such as 5-flucytosine and hydroxypyridones inhibited growth of the kex2 mutants and the control strains to the same extent. In an in vitro model of oral candidosis, kex2 mutants showed reduced tissue damage in the presence of itraconazole compared with the control infections. These data suggest that Kex2 is involved in the processing of proteins that are essential for cell surface integrity of C. glabrata.

Glucosylceramide synthases, a gene family responsible for the biosynthesis of glucosphingolipids in animals, plants, and fungi.

Glucosylceramides are membrane lipids in most eukaryotic organisms and in a few bacteria. The physiological functions of these glycolipids have only been documented in mammalian cells, whereas very little information is available of their roles in plants, fungi, and bacteria. In an attempt to establish appropriate experimental systems to study glucosylceramide functions in these organisms, we performed a systematic functional analysis of a glycosyltransferase gene family with members of animal, plant, fungal, and bacterial origin. Deletion of such putative glycosyltransferase genes in Candida albicans and Pichia pastoris resulted in the complete loss of glucosylceramides. When the corresponding knock-out strains were used as host cells for homologous or heterologous expression of candidate glycosyltransferase genes, five novel glucosylceramide synthase (UDP-glucose:ceramide glucosyltransferase) genes were identified from the plant Gossypium arboreum (cotton), the nematode Caenorhabditis elegans, and the fungi Magnaporthe grisea, Candida albicans, and P. pastoris. The glycosyltransferase gene expressions led to the biosynthesis of different molecular species of glucosylceramides that contained either C18 or very long chain fatty acids. The latter are usually channeled exclusively into inositol-containing sphingolipids known from Saccharomyces cerevisiae and other yeasts. Implications for the biosynthesis, transport, and function of sphingolipids will be discussed.

Invasion of Candida albicans correlates with expression of secreted aspartic proteinases during experimental infection of human epidermis.

Secreted aspartic proteinases (Saps) encoded by 10 genes of Candida albicans are important virulence factors for different types of candidiasis. Distinct SAP genes have previously been shown to contribute to tissue damage in a model of oral candidiasis. In this study a progressive SAP expression in the order SAP1 and SAP2 > SAP8 > SAP6 > SAP3 was observed in an in vitro model of cutaneous candidiasis based on reconstituted human epidermis. Transcripts of SAP1 and SAP2 were detected during initial invasion of the stratum corneum by C. albicans. Deeper, extensive penetration of the corneal layer was accompanied by additional SAP8 mRNA. SAP6 expression occurred concomitantly with germ tube formation and extensive hyphal growth in the strata granulosum, spinosum, and basale. Ultrastructural studies using specific polyclonal antibodies directed against the gene products of SAP1-3 and SAP4-6 revealed predominant expression of Sap1-3. The protective effect of the aspartic proteinase inhibitor pepstatin A during infection of the epidermis and an attenuated virulence phenotype of SAP-deficient mutants suggest that the observed SAP expression correlates with tissue damage in the skin.

Secreted lipases of Candida albicans: cloning, characterisation and expression analysis of a new gene family with at least ten members.

Extracellular lipolytic activity enabled the human pathogen Candida albicans to grow on lipids as the sole source of carbon. Nine new members of a lipase gene family (LIP2-LIP10) with high similarities to the recently cloned lipase gene LIP1 were cloned and characterised. The ORFs of all ten lipase genes are between 1281 and 1416 bp long and encode highly similar proteins with up to 80% identical amino acid sequences. Each deduced lipase sequence has conserved lipase motifs, four conserved cysteine residues, conserved putative N-glycosylation sites and similar hydrophobicity profiles. All LIP genes, except LIP7, also encode an N-terminal signal sequence. LIP3-LIP6 were expressed in all media and at all time points of growth tested as shown by Northern blot and RT-PCR analyses. LIP1, LIP3, LIP4, LIP5, LIP6 and LIP8 were expressed in medium with Tween 40 as a sole source of carbon. However, the same genes were also expressed in media without lipids. Two other genes, LIP2 and LIP9, were only expressed in media lacking lipids. Transcripts of most lipase genes were detected during the yeast-to-hyphal transition. Furthermore, LIP5, LIP6, LIP8 and LIP9 were found to be expressed during experimental infection of mice. These data indicate lipid-independent, highly flexible in vitro and in vivo expression of a large number of LIP genes, possibly reflecting broad lipolytic activity, which may contribute to the persistence and virulence of C. albicans in human tissue.

[Extracellular hydrolytic enzymes and their relevance during Candida albicans infections]. / Extrazelluläre hydrolytische Enzyme und ihre Relevanz bei Candida-albicans-Infektionen.

Candida albicans cannot only infect skin and mucosa, but can also cause life threatening systemic candidosis. While natural barriers and the immune system of healthy individuals normally prevent such infections, virulence factors exist that enable C. albicans to survive on surfaces and the permit the fungus to invade tissues and organs in immunocompromised patients. Adhesions factors, morphological flexibility and hydrolytic enzymes belong to this group of virulence factors.C.albicans appears to be able to use these specific virulence attributes at distinct stages of an infection or in different types of candidosis. For example, distinct adhension factors are important for the persistence of C. albicans on mucosal epithelial cells, while other factors are necessary for the adhesion to endothelial tissue. The differential expression of specific virulence factors at different stages of an infection could be the reason why C. albicans not only has single genes for extracellular hydrolytic enzymes, but gene families. Both secreted aspartate proteinases (Saps) and secreted lipases (Lips) from C. albicans are encoded by at least 10 different genes. This high number of similar genes might empower C. albicans with the ability to secrete a specific and appropriate enzymatic response at distinct stages of an infection. For both gene families differential expression has been shown in vitro and in vivo, which would be reasonable for such an adaptation. Expression studies revealed that distinct SAP and LIP genes were expressed under conditions when potential subtrates ( proteins or lipids) were not present in the growth medium. Such expression patterns would imply that these genes may have functions other than simply providing nutrients for the fungus. The specific transcription of single SAP genes during the course of an infection suggests that these genes may have specific functions during different stages of an infection. In fact, inhibition studies and the use of mutants with targeted gene disruptions showed that distinct SAP genes (SAP1-3) are important durning infections of skin and mucosa, while others (SAP4-6) are most relevant for systemic infections.

Characterization of UDP-glucose:ceramide glucosyltransferases from different organisms.

Cerebrosides are typical membrane lipids of many organisms. They occur in plants, fungi, animals, humans and some prokaryotes. Almost all of our knowledge on the physiological functions of cerebrosides results from experimental data obtained with mammalian cells. However, very little is known about the roles played by these lipids in plants and fungi. To initiate such investigations we have cloned and characterized a ceramide glucosyltransferase from the yeast Candida albicans. Functional expression of this gene in Saccharomyces cerevisiae led to the accumulation of new glycolipids which were not present in wild-type baker's yeast. They were identified by MS and NMR spectroscopy as beta-D-glucopyranosyl ceramides. The ceramide moieties of these cerebrosides comprised phytosphinganine and mainly long-chain (C(26)) alpha-hydroxy fatty acids in amide linkage. We also generated a ceramide glucosyltransferase-knock-out strain of C. albicans which was devoid of cerebrosides. The viability of this mutant showed that for this organism glucosyl ceramides are not essential for vegetative growth on complete or minimal media. In addition, we have cloned and functionally expressed one of the three putative glucosylceramide synthases from Caenorhabditis elegans, as well as a corresponding enzyme from Pichia pastoris.

Secreted aspartic proteinase (Sap) activity contributes to tissue damage in a model of human oral candidosis.

Secreted aspartic proteinases (Saps) are important virulence factors during Candida albicans mucosal or disseminated infections. A differential expression of individual SAP genes has been shown previously in a model of oral candidosis based on reconstituted human epithelium (RHE), and in the oral cavity of patients. In this study, the ultrastructural localization of distinct groups of Sap isoenzymes expressed during RHE infection was shown by immunoelectron microscopy using specific polyclonal antibodies directed against the gene products of SAP1-3 and SAP4-6. Large amounts of Sap1-3 antigen were found within C. albicans yeast and hyphal cell walls, often predominantly in close contact with epithelial cells, whereas lower quantities of Sap4-6 were detected in hyphal cells. To elucidate the relevance of the expressed Saps during oral infections, we examined the effect of the aspartic proteinase inhibitor, pepstatin A, during infection of the RHE. The extent of lesions caused by the strain SC5314 was found to be strongly reduced by the inhibitor, indicating that proteinase activity contributes to tissue damage in this model. To clarify which of the SAP genes are important for tissue necrosis, the histology of RHE infection with Deltasap1, Deltasap2, Deltasap3, Deltasap4-6 and three Deltasap1/3 double mutants were examined. Although tissue damage was not blocked completely with these mutants, an attenuated phenotype was observed for each of the single sap null mutants, and was more strongly attenuated in the Deltasap1/3 double null mutants. In contrast, the lesions caused by the Deltasap4-6 triple mutant were at least as severe as those caused by SC5314. During infection with the mutants, we observed that the SAP gene expression pattern of the Deltasap1 and the Deltasap1/3 mutants was altered in comparison with the wild-type strain. Expression of SAP5 was observed only during infection with the Deltasap1/3 mutant, whereas upregulation of SAP2 and SAP8 transcripts was observed in the Deltasap1 and the Deltasap1/3 mutants. These results suggest that Sap1-3, but not Sap4-6, contribute to tissue damage in this model. Furthermore, C. albicans may compensate for the deletion of certain SAP genes by upregulation of alternative SAP genes.

Germ tubes and proteinase activity contribute to virulence of Candida albicans in murine peritonitis.

Peritonitis with Candida albicans is an important complication of bowel perforation and continuous ambulatory peritoneal dialysis. To define potential virulence factors, we investigated 50 strains of C. albicans in a murine peritonitis model. There was considerable variation in their virulence in this model when virulence was measured as release of organ-specific enzymes into the plasma of infected mice. Alanine aminotransferase (ALT) and alpha-amylase (AM) were used as parameters for damage of the liver and pancreas, respectively. The activities of ALT and AM in the plasma correlated with invasion into the organs measured in histologic sections and the median germ tube length induced with serum in vitro. When the activity of proteinases was inhibited in vivo with pepstatin A, there was a significant reduction of ALT and AM activities. This indicates that proteinases contributed to virulence in this model. Using strains of C. albicans with disruption of secreted aspartyl proteinase gene SAP1, SAP2, SAP3, or SAP4 through SAP6 (collectively referred to as SAP4-6), we showed that only a Deltasap4-6 triple mutant induced a significantly reduced activity of ALT in comparison to the reference strain. In contrast to the Deltasap1, Deltasap2, and Deltasap3 mutants, the ALT induced by the Deltasap4-6 mutant could not be further reduced by pepstatin A treatment, which indicates that Sap4-6 may contribute to virulence in this model.

Effects of the human immunodeficiency virus (HIV) proteinase inhibitors saquinavir and indinavir on in vitro activities of secreted aspartyl proteinases of Candida albicans isolates from HIV-infected patients.

The effects of therapeutically relevant concentrations of the human immunodeficiency virus (HIV) proteinase inhibitors saquinavir and indinavir on the in vitro proteinase activity of Candida albicans were investigated with isolates from HIV-infected and uninfected patients with oral candidiasis. After exposure to the HIV proteinase inhibitors, proteinase activity was significantly reduced in a dose-dependent manner. These inhibitory effects, which were similar to that of pepstatin A, and the reduced virulence phenotype in experimental candidiasis after application of saquinavir indicate the usefulness of these HIV proteinase inhibitors as potential anticandidal agents.

Cloning and functional expression of UGT genes encoding sterol glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum.

Sterol glucosides, typical membrane-bound lipids of many eukaryotes, are biosynthesized by a UDP-glucose:sterol glucosyltransferase (EC 2. 4.1.173). We cloned genes from three different yeasts and from Dictyostelium discoideum, the deduced amino acid sequences of which all showed similarities with plant sterol glucosyltransferases (Ugt80A1, Ugt80A2). These genes from Saccharomyces cerevisiae (UGT51 = YLR189C), Pichia pastoris (UGT51B1), Candida albicans (UGT51C1), and Dictyostelium discoideum (ugt52) were expressed in Escherichia coli. In vitro enzyme assays with cell-free extracts of the transgenic E. coli strains showed that the genes encode UDP-glucose:sterol glucosyltransferases which can use different sterols such as cholesterol, sitosterol, and ergosterol as sugar acceptors. An S. cerevisiae null mutant of UGT51 had lost its ability to synthesize sterol glucoside but exhibited normal growth under various culture conditions. Expression of either UGT51 or UGT51B1 in this null mutant under the control of a galactose-induced promoter restored sterol glucoside synthesis in vitro. Lipid extracts of these cells contained a novel glycolipid. This lipid was purified and identified as ergosterol-beta-D-glucopyranoside by nuclear magnetic resonance spectroscopy. These data prove that the cloned genes encode sterol-beta-D-glucosyltransferases and that sterol glucoside synthesis is an inherent feature of eukaryotic microorganisms.