The intraspecies diversity of C. albicans triggers qualitatively and temporally distinct host responses that determine the balance between commensalism and pathogenicity.

The host immune status is critical for preventing opportunistic infections with Candida albicans. Whether the natural fungal diversity that exists between C. albicans isolates also influences disease development remains unclear. Here, we used an experimental model of oral infection to probe the host response to diverse C. albicans isolates in vivo and found dramatic differences in their ability to persist in the oral mucosa, which inversely correlated with the degree and kinetics of immune activation in the host. Strikingly, the requirement of interleukin (IL)-17 signaling for fungal control was conserved between isolates, including isolates with delayed induction of IL-17. This underscores the relevance of IL-17 immunity in mucosal defense against C. albicans. In contrast, the accumulation of neutrophils and induction of inflammation in the infected tissue was strictly strain dependent. The dichotomy of the inflammatory neutrophil response was linked to the capacity of fungal strains to cause cellular damage and release of alarmins from the epithelium. The epithelium thus translates differences in the fungus into qualitatively distinct host responses. Altogether, this study provides a comprehensive understanding of the antifungal response in the oral mucosa and demonstrates the relevance of evaluating intraspecies differences for the outcome of fungal-host interactions in vivo.

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.

Multilocus sequence typing reveals intrafamilial transmission and microevolutions of Candida albicans isolates from the human digestive tract.

Candida albicans is a human commensal that is also responsible for superficial and systemic infections. Little is known about the carriage of C. albicans in the digestive tract and the genome dynamics that occur during commensalisms of this diploid species. The aim of this study was to evaluate the prevalence, diversity, and genetic relationships among C. albicans isolates recovered during natural colonization of the digestive tract of humans, with emphasis on Crohn's disease patients who produce anti-yeast antibodies and may have altered Candida sp. carriage. Candida sp. isolates were recovered from 234 subjects within 25 families with multiple cases of Crohn's disease and 10 control families, sampled at the oral and fecal sites. Prevalences of Candida sp. and C. albicans carriage were 53.4% and 46.5%, respectively, indicating frequent commensal carriage. No differences in prevalence of carriage could be observed between Crohn's disease patients and healthy subjects. Multilocus sequence typing (MLST) of C. albicans isolates revealed frequent colonization of a subject or several members of the same family by genetically indistinguishable or genetically close isolates. These latter isolates differed by loss-of-heterozygosity events at one or several of the MLST loci. These loss-of-heterozygosity events could be due to either chromosome loss followed by duplication or large mitotic recombination events between complementary chromosomes. This study was the first to jointly assess commensal carriage of C. albicans, intrafamilial transmission, and microevolution. The high frequency of each of these events suggests that the digestive tract provides an important and natural niche for microevolutions of diploid C. albicans through the loss of heterozygosity.

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.

Collaborative consensus for optimized multilocus sequence typing of Candida albicans.

A panel of 86 different Candida albicans isolates was subjected to multilocus sequence typing (MLST) in two laboratories to obtain sequence data for 10 published housekeeping gene fragments. Analysis of data for all possible combinations of five, six, seven, eight, and nine of the fragments showed that a set comprising the fragments AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13, and ZWF1b was the smallest that yielded 86 unique diploid sequence types for the 86 isolates. This set is recommended for future MLST with C. albicans.

Usefulness of multilocus sequence typing for characterization of clinical isolates of Candida albicans.

Molecular characterization of Candida albicans isolates is essential for understanding the epidemiology of nosocomial infections caused by this yeast. Here, we investigated the potential value of multilocus sequence typing (MLST) for characterizing epidemiologically related or unrelated C. albicans strains of various clinical origins. Accordingly, we sequenced the internal regions (loci) of six selected housekeeping genes of 40 C. albicans clinical isolates and 2 reference strains. In all, 68 polymorphic nucleotide sites were identified, of which 65 were found to be heterozygous in at least one isolate. Ten to 24 different genotypes were observed at the different loci, resulting, when combined, in 39 unique genotype combinations or diploid sequence types (DSTs). When MLST was applied to 26 epidemiologically unrelated isolates and the 2 reference strains, it allowed the identification of 27 independent DSTs, thus demonstrating a discriminatory power of 99.7. Using multidimensional scaling together with the minimum spanning tree method to analyze interstrain relationships, we identified six groups of genetically related isolates on the basis of bootstrap values of greater than 900. Application of MLST to 14 epidemiologically related isolates showed that those recovered from patients in the same hospital ward during the same 3 months had specific DSTs, although 73% of these isolates were genetically very close. This suggests that MLST can trace minute variations in the sequences of related isolates. Overall, MLST proved to be a highly discriminatory and stable method for unambiguous characterization of C. albicans.

Transcript profiling in Candida albicans reveals new cellular functions for the transcriptional repressors CaTup1, CaMig1 and CaNrg1.

The pathogenic fungus, Candida albicans contains homologues of the transcriptional repressors ScTup1, ScMig1 and ScNrg1 found in budding yeast. In Saccharomyces cerevisiae, ScMig1 targets the ScTup1/ScSsn6 complex to the promoters of glucose repressed genes to repress their transcription. ScNrg1 is thought to act in a similar manner at other promoters. We have examined the roles of their homologues in C. albicans by transcript profiling with an array containing 2002 genes, representing about one quarter of the predicted number of open reading frames (ORFs) in C. albicans. The data revealed that CaNrg1 and CaTup1 regulate a different set of C. albicans genes from CaMig1 and CaTup1. This is consistent with the idea that CaMig1 and CaNrg1 target the CaTup1 repressor to specific subsets of C. albicans genes. However, CaMig1 and CaNrg1 repress other C. albicans genes in a CaTup1-independent fashion. The targets of CaMig1 and CaNrg1 repression, and phenotypic analyses of nrg1/nrg1 and mig1/mig1 mutants, indicate that these factors play differential roles in the regulation of metabolism, cellular morphogenesis and stress responses. Hence, the data provide important information both about the modes of action of these transcriptional regulators and their cellular roles. The transcript profiling data are available at http://www.pasteur.fr/recherche/unites/RIF/transcriptdata/.

NRG1 represses yeast-hypha morphogenesis and hypha-specific gene expression in Candida albicans.

We have characterized CaNrg1 from Candida albicans, the major fungal pathogen in humans. CaNrg1 contains a zinc finger domain that is conserved in transcriptional regulators from fungi to humans. It is most closely related to ScNrg1, which represses transcription in a Tup1-dependent fashion in Saccharomyces cerevisiae. Inactivation of CaNrg1 in C.albicans causes filamentous and invasive growth, derepresses hypha-specific genes, increases sensitivity to some stresses and attenuates virulence. A tup1 mutant displays similar phenotypes. However, unlike tup1 cells, nrg1 cells can form normal hyphae, generate chlamydospores at normal rates and grow at 42 degrees C. Transcript profiling of 2002 C.albicans genes reveals that CaNrg1 represses a subset of CaTup1-regulated genes, which includes known hypha-specific genes and other virulence factors. Most of these genes contain an Nrg1 response element (NRE) in their promoter. CaNrg1 interacts specifically with an NRE in vitro. Also, deletion of two NREs from the ALS8 promoter releases it from Nrg1-mediated repression. Hence, CaNrg1 is a transcriptional repressor that appears to target CaTup1 to a distinct set of virulence-related functions, including yeast-hypha morphogenesis.

Molecular and physiological characterization of the NAD-dependent glycerol 3-phosphate dehydrogenase in the filamentous fungus Aspergillus nidulans.

In filamentous fungi, glycerol biosynthesis has been proposed to play an important role during conidiospore germination and in response to a hyperosmotic shock, but little is known about the genes involved. Here, we report on the characterization of the major Aspergillus nidulans glycerol 3-phosphate dehydrogenase (G3PDH)-encoding gene, gfdA. G3PDH is responsible for the conversion of dihydroxyacetone phosphate (DHAP) into glycerol 3-phosphate (G3P), which is subsequently converted into glycerol by an as yet uncharacterized phosphatase. Inactivation of gfdA does not abolish glycerol biosynthesis, showing that the other pathway from DHAP, via dihydroxyacetone (DHA), to glycerol is also functional in A. nidulans. The gfdA null mutant displays reduced G3P levels and an osmoremediable growth defect on various carbon sources except glycerol. This growth defect is associated with an abnormal hyphal morphology that is reminiscent of a cell wall defect. Furthermore, the growth defect at low osmolarity is enhanced in the presence of the chitin-interacting agent calcofluor and the membrane-destabilizing agent sodium dodecyl sulphate (SDS). As inactivation of gfdA has no impact on phospholipid biosynthesis or glycolytic intermediates levels, as might be expected from reduced G3P levels, a previously unsuspected link between G3P and cell wall integrity is proposed to occur in filamentous fungi.

A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans.

The construction of mutant fungal strains is often limited by the poor efficiency of homologous recombination in these organisms. Higher recombination efficiencies can be obtained by increasing the length of homologous DNA flanking the transformation marker, although this is a tedious process when standard molecular biology techniques are used for the construction of gene replacement cassettes. Here, we present a two-step technology which takes advantage of an Escherichia coli strain expressing the phage lambda Red(gam, bet, exo) functions and involves (i) the construction in this strain of a recombinant cosmid by in vivo recombination between a cosmid carrying a genomic region of interest and a PCR-generated transformation marker flanked by 50 bp regions of homology with the target DNA and (ii) genetic exchange in the fungus itself between the chromosomal locus and the circular or linearized recombinant cosmid. This strategy enables the rapid establishment of mutant strains carrying gene knock-outs with efficiencies >50%. It should also be appropriate for the construction of fungal strains with gene fusions or promoter replacements.

Neutral trehalases catalyse intracellular trehalose breakdown in the filamentous fungi Aspergillus nidulans and Neurospora crassa.

A cAMP-activatable Ca2+-dependent neutral trehalase was identified in germinating conidia of Aspergillus nidulans and Neurospora crassa. Using a PCR approach, A. nidulans and N. crassa genes encoding homologues of the neutral trehalases found in several yeasts were cloned and sequenced. Disruption of the AntreB gene encoding A. nidulans neutral trehalase revealed that it is responsible for intracellular trehalose mobilization at the onset of conidial germination, and that this phenomenon is partially involved in the transient accumulation of glycerol in the germinating conidia. Although trehalose mobilization is not essential for the completion of spore germination and filamentous growth in A. nidulans, it is required to achieve wild-type germination rates under carbon limitation, suggesting that intracellular trehalose can partially contribute the energy requirements of spore germination. Furthermore, it was shown that trehalose accumulation in A. nidulans can protect germinating conidia against an otherwise lethal heat shock. Because transcription of the treB genes is not increased after a heat shock but induced upon heat shock recovery, it is proposed that, in filamentous fungi, mobilization of trehalose during the return to appropriate growth is promoted by transcriptional and post-translational regulatory mechanisms, in particular cAMP-dependent protein kinase-mediated phosphorylation.

Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5'-monophosphate decarboxylase.

A homologous transformation system for the opportunistic fungal pathogen Aspergillus fumigatus was developed. It is based on the A. fumigatus pyrG gene, encoding orotidine 5'-monophosphate decarboxylase, which was cloned and sequenced. Transformation of both Aspergillus (Emericella) nidulans and A. fumigatus pyrG mutant strains by the use of protoplasts or electroporation established the functionality of the cloned gene. DNA sequencing of the A. fumigatus pyrG1 mutant allele revealed that it encodes a truncated, non-functional, PyrG protein. Transformation of an A. fumigatus pyrG1 mutant with a plasmid carrying the novel pyrG2 allele constructed by in vitro mutagenesis yielded prototrophic transformants following recombination between both mutation sites. Analysis of transformants carrying the entire plasmid showed that up to 45% of integration had occurred at the pyrG locus. This provides a tool to target defined genetic constructs at a specific locus in the A. fumigatus genome in order to study gene regulation and function.

Molecular characterization of the Aspergillus nidulans treA gene encoding an acid trehalase required for growth on trehalose.

Aspergillus nidulans conidiospores contain high levels of the non-reducing disaccharide trehalose. We show that upon induction of conidiospore germination, the trehalose pool is rapidly degraded and a glycerol pool is transiently accumulated. A trehalase with an acidic pH optimum was purified from conidiospores. Characterization of the treA gene encoding this trehalase shows that it is homologous to Saccharomyces cerevisiae vacuolar acid trehalase, the product of the ATH1 gene, and to two related proteins of unknown function identified in Mycobacterium tuberculosis and Mycobacterium leprae. A. nidulans mutants that lack acid trehalase activity were constructed by gene replacement at the treA locus. Analysis of these mutants suggests that the treA gene product is localized in the conidiospore wall, is required for growth on trehalose as a carbon source, and is not involved in the mobilization of the intracellular pool of trehalose. Therefore, it is proposed that a cytoplasmic regulatory trehalase is controlling this latter process.

Attenuated virulence of uridine-uracil auxotrophs of Aspergillus fumigatus.

Aspergillus fumigatus mutants that are deficient in the de novo UMP biosynthesis pathway because of a mutation in the pyrG gene encoding orotidine-5'-phosphate decarboxylase (and therefore auxotrophic for uridine or uracil) were evaluated in a murine model of invasive aspergillosis. These mutants were entirely nonpathogenic, and mutant conidia remained ungerminated in alveolar macrophages. Both the germination and virulence defects could be restored by supplementing the drinking water of the animals with uridine. DNA-mediated transformation of one of the pyrG mutants with the Aspergillus niger pyrG gene also restored virulence. These results suggest that uridine and uracil are limiting in the lung environment, thus preventing conidium germination and hence virulence of the pyrG mutants.

Selection of multiple disruption events in Aspergillus fumigatus using the orotidine-5'-decarboxylase gene, pyrG, as a unique transformation marker.

A 8.6-kb disruption cassette, referred to here as a pyrG-blaster and consisting of the Aspergillus niger pyrG gene flanked by a direct repeat that encodes the neomycin phosphotransferase of transposon Tn5 was constructed. Following transformation of a uridine/uracil auxotrophic pyrG strain of A. fumigatus, genomic insertions of the pyrG-blaster were obtained either by targeted gene replacement at the rodA locus, resulting in the formation of hydrophilic spores, or by ectopic integration. In both cases, recombination between the two elements of the direct repeat could be selected in the presence of 5-fluoro-orotic acid and resulted in the excision of the A. niger pyrG gene, producing A. fumigatus uridine/uracil auxotrophs that retained their additional mutant phenotype because of the persistence of one of the two elements of the direct repeat at the site of insertion of the pyrG-blaster. Selection for uracil/uridine prototrophy can therefore be used again to disrupt another gene.

Purification and characterization of SAR1p, a small GTP-binding protein required for transport vesicle formation from the endoplasmic reticulum.

SEC12 encodes an integral membrane glycoprotein essential for vesicle formation from the endoplasmic reticulum (ER) in yeast. The SAR1 gene was discovered as a multicopy suppressor of a sec12ts strain and encodes a 21-kDa GTP-binding protein also required for protein transport from the ER to the Golgi apparatus (Nakano, A., and Muramatsu, M. (1989) J. Cell Biol. 109, 2677-2691). We have purified Sar1p to apparent homogeneity from cells harboring a galactose-regulated recombinant SAR1. Purified Sar1p binds guanine nucleotides specifically and exhibits GTPase activity (0.001 min-1). Nucleotide exchange and hydrolysis rates are greatly increased in the presence of Mg2+ and nonionic detergents or phospholipids. An assay that measures the formation of a vesicle intermediate in ER to Golgi transport was devised that is dependent on the addition of purified Sar1p. This assay employs membranes prepared from wild-type cells and cytosol fractions depleted of Sar1p due to overproduction of Sec12p or by gel filtration chromatography. The gel-filtered cytosol requires the addition of Sar1p and GTP to support vesicle budding. Sar1p prebound with GTP gamma S inhibits Sar1p function in the vesicle formation assay. The results indicate a role for Sar1p in vesicle budding from the ER and suggest that GTP hydrolysis by Sar1p is required for this event.

Fission yeast and a plant have functional homologues of the Sar1 and Sec12 proteins involved in ER to Golgi traffic in budding yeast.

Sec12p and Sar1p are required for the formation of transport vesicles generated from the endoplasmic reticulum (ER) in the yeast Saccharomyces cerevisiae. Sec12p is an ER type II membrane protein that mediates the membrane attachment of the GTP-binding Sar1 protein. The SAR1 gene is a multi-copy suppressor of a thermosensitive sec12 mutation. In an attempt to identify functional homologues of Sec12p and Sar1p from other eukaryotic organisms, we screened cDNA expression libraries derived from the fission yeast Schizosaccharomyces pombe and from the plant Arabidopsis thaliana for complementation of the sec12ts mutation. Four individual cDNAs were isolated, two of which encode the S. pombe and A. thaliana homologues of Sar1p. The three Sar1 proteins are 67% identical on average. The two other cDNAs encode type II membrane proteins which were designated Stl1p for the S. pombe protein and Stl2p for the A. thaliana protein (Stl stands for Sec12p-like). Both proteins have NH2-terminal cytoplasmic domains which resemble that of Sec12p: they are similar in size and present a significant degree of amino acid identity with the cytoplasmic domain of Sec12p. In contrast, the lumenal domains of Sec12p, Stl1p and Stl2p are very different in size and do not show any appreciable homology. That Stl1p and Stl2p are functional homologues of Sec12p was confirmed by showing that expression of either cloned gene complements a sec12 null mutation. Our results indicate that some of the mechanisms regulating vesicle formation at the ER are conserved not only in yeasts, but also in plants.