Role for endosomal and vacuolar GTPases in Candida albicans pathogenesis.

The vacuole has crucial roles in stress resistance and adaptation of the fungal cell. Furthermore, in Candida albicans it has been observed to undergo dramatic expansion during the initiation of hyphal growth, to produce highly "vacuolated" subapical compartments. We hypothesized that these functions may be crucial for survival within the host and tissue-invasive hyphal growth. We also considered the role of the late endosome or prevacuole compartment (PVC), a distinct organelle involved in vacuolar and endocytic trafficking. We identified two Rab GTPases, encoded by VPS21 and YPT72, required for trafficking through the PVC and vacuole biogenesis, respectively. Deletion of VPS21 or YPT72 led to mild sensitivities to some cellular stresses. However, deletion of both genes resulted in a synthetic phenotype with severe sensitivity to cellular stress and impaired growth. Both the vps21Delta and ypt72Delta mutants had defects in filamentous growth, while the double mutant was completely deficient in polarized growth. The defects in hyphal growth were not suppressed by an "active" RIM101 allele or loss of the hyphal repressor encoded by TUP1. In addition, both single mutants had significant attenuation in a mouse model of hematogenously disseminated candidiasis, while the double mutant was rapidly cleared. Histological examination confirmed that the vps21Delta and ypt72Delta mutants are deficient in hyphal growth in vivo. We suggest that the PVC and vacuole are required on two levels during C. albicans infection: (i) stress resistance functions required for survival within tissue and (ii) a role in filamentous growth which may aid host tissue invasion.

Bmh1p (14-3-3) mediates pathways associated with virulence in Candida albicans.

The ability of the pathogenic fungus Candida albicans to cause disease requires rapid adaptation to changes in the host environment and to an evolving host immune response. The identification of 'virulence factors' using in vitro characterization of mutant strains has traditionally relied on a common set of phenotypic and biochemical assays (most often performed at 30 degrees C) and the subsequent correlation with their corresponding virulence in mouse models of disease. Utilizing a panel of isogenic mutants for the multifunctional signal-modulating 14-3-3 protein (Bmh1p), we have found that specific mutations affect a variety of different pathways currently associated with virulence, including those involved with the formation of filaments, as well as interaction with host immune cells. Surprisingly, our studies revealed that deficiencies in many of these pathways do not always correlate with virulence in a mouse model of disseminated infection. Mutations within the binding pocket of Bmh1p that affect the ability of the protein to efficiently bind ligand had varying effects on the results of a number of in vitro and in vivo assays. The capability, in vitro, to filament in embedment conditions, and to filament and form chlamydospores under microaerophilic conditions on cornmeal agar, does not correlate with virulence. It is likely that only a subset of hyphal signalling pathways is actually required for the establishment of infection in the disseminated mouse model. Most importantly, our results suggest that the delayed onset of log-phase [corrected] growth in vitro at 37 degrees C, and not at 30 degrees C, results in an inability of these mutants to rapidly adjust to environmental changes in vivo and may be responsible for their increased clearance and reduced virulence. It is critical, therefore, that future in vitro studies of putative virulence factors in C. albicans include careful characterization at physiological temperatures.

Reporter gene assays in Candida albicans.

Reporter systems are used in Candida albicans in three major experimental areas. These include gene expression, promoter analysis, and protein expression/localization. Heterologous expression in C. albicans is either not effective or inefficient due to the alternative codon usage in Candida, particularly CTG. Consequently, several reporter genes have been constructed by optimizing codons for expression in Candida. The reporter systems include lacZ, luciferase, and GFP. Generally, PCR site directed mutagenesis has been used to construct the modified reporter. Reporter gene vectors are not commercially available for Candida, but they can normally be requested from the laboratories that developed the constructs.

Autophagy in the pathogen Candida albicans.

Autophagy is a major cellular process that facilitates the bulk degradation of eukaryotic macromolecules and organelles, through degradation within the lysosomal/vacuole compartment. This has been demonstrated to influence a diverse array of eukaryotic cell functions including adaptation, differentiation and developmental programmes. For example, in Saccharomyces cerevisiae autophagy is required for sporulation and survival of nitrogen starvation. The opportunistic pathogen Candida albicans has the ability to colonize and cause disease within a diverse range of mammalian host sites. The ability to adapt and differentiate within the host is liable to be critical for host colonization and infection. Previous results indicated that the vacuole plays an important role in C. albicans adaptation to stress, differentiation, and survival within and injury of host cells. In this study the importance of vacuole-mediated degradation through the process of autophagy was investigated. This involved identification and deletion of ATG9, a C. albicans gene required for autophagy. The deletion strain was blocked in autophagy and the closely related cytoplasm to vacuole (cvt) trafficking pathway. This resulted in sensitivity to nitrogen starvation, but no defects in growth rate, vacuole morphology or resistance to other stresses. This indicates that the mutant has specific defects in autophagy/cvt trafficking. Given the importance of autophagy in the development and differentiation of other eukaryotes, it was surprising to find that the atg9Delta mutant was unaffected in either yeast-hypha or chlamydospore differentiation. Furthermore, the atg9Delta mutant survived within and killed a mouse macrophage-like cell line as efficiently as control strains. The data suggest that autophagy plays little or no role in C. albicans differentiation or during interaction with host cells.

Random mutagenesis of an essential Candida albicans gene.

A method for the analysis of Candida albicans gene function, which involves random mutagenesis of the open reading frame, is described. This method is especially suited for the study of essential and multi-functional genes, with several advantages over regulatable promoters more commonly used to study essential gene function. These advantages include expression from the endogenous promoter, which should yield a more appropriate transcript expression and abrogate the need for shifts in carbon or amino acid sources necessary with the use of regulatable promoters. Furthermore, there is potential for isolating individual functions of multi-functional genes. To verify this experimental approach, we randomly mutated the essential C. albicans gene, BMH1. The resulting "pool" of putative mutant alleles was then introduced into a BMH1/bmh1Delta strain of C. albicans, such that only the mutagenized BMH1 sequences could be expressed. Transformants were screened for rapamycin sensitivity, defects in filamentation on M199 agar, and growth at 42 degrees C. In this way, we identified six non-lethal mutant alleles of BMH1 with altered amino acid sequences. Further phenotypic analysis of these mutant strains enabled us to segregate individual functions of C. albicans BMH1. The relative merits of Escherichia coli versus PCR-mediated mutagenesis are discussed.

Mutant alleles of the essential 14-3-3 gene in Candida albicans distinguish between growth and filamentation.

The opportunistic fungal pathogen Candida albicans has the ability to exploit diverse host environments and can either reside commensally or cause disease. In order to adapt to its new environment it must respond to new physical conditions, nutrient sources, and the host immune response. This requires the co-regulation of multiple signalling networks. The 14-3-3 family of proteins is highly conserved in all eukaryotic species. These proteins regulate signalling pathways involved in cell survival, the cell cycle, and differentiation, and effect their functions via interactions with phosphorylated serines/threonines. In C. albicans there is only one 14-3-3 protein, Bmh1p, and it is required for vegetative growth and optimal filamentation. In order to dissect separate functions of Bmh1p in C. albicans, site-directed nucleotide substitutions were made in the C. albicans BMH1 gene based on studies in other species. Putative temperature-sensitive, ligand-binding and dimerization mutants were constructed. In addition two mutant strains identified through random mutagenesis were analysed. All five mutant strains demonstrated varying defects in growth and filamentation. This paper begins to segregate functions of Bmh1p that are required for optimal growth and the different filamentation pathways. These mutant strains will allow the identification of 14-3-3 target interactions and correlate the individual functions of Bmh1p to cellular processes involved in pathogenesis.

An intravaginal live Candida challenge in humans leads to new hypotheses for the immunopathogenesis of vulvovaginal candidiasis.

Acute and recurrent vulvovaginal candidiasis (VVC) remains a significant problem in women of childbearing age. While clinical studies of women with recurrent VVC (RVVC) and animal models have provided important data about a limited protective role of adaptive immunity, there remains a paucity of information on the protective mechanisms or factors associated with susceptibility to infection. In the present study, an intravaginal live Candida challenge in healthy adult women showed a differential susceptibility to symptomatic VVC, where 3 (15%) of 19 women with no history of VVC acquired a symptomatic infection compared to 6 (55%) of 11 women with an infrequent history of VVC. Furthermore, these studies revealed that protection against infection is noninflammatory while symptomatic infection correlates with a vaginal infiltration of polymorphonuclear neutrophils (PMNs) and a high vaginal fungal burden. Thus, the presence of symptomatic infection appears more dependent on host factors than on properties of the organism. Finally, vaginal lavage fluid from women with a symptomatic infection, but not those asymptomatically colonized, promoted the chemotaxis of PMNs. These results suggest that rather than RVVC/VVC being caused by an aberrant adaptive immune response, symptoms that define infection appear to be due to an aggressive innate response by PMNs.

Candida albicans VPS11 is required for vacuole biogenesis and germ tube formation.

The Candida albicans vacuole has previously been observed to undergo rapid expansion during the emergence of a germ tube from a yeast cell, to occupy the majority of the parent yeast cell. Furthermore, the yeast-to-hypha switch has been implicated in the virulence of this organism. The class C vps (vacuolar protein sorting) mutants of Saccharomyces cerevisiae are defective in multiple protein delivery pathways to the vacuole and prevacuole compartment. In this study C. albicans homologues of the S. cerevisiae class C VPS genes have been identified. Deletion of a C. albicans VPS11 homologue resulted in a number of phenotypes that closely resemble those of the class C vps mutants of S. cerevisiae, including the absence of a vacuolar compartment. The C. albicans vps11Delta mutant also had much-reduced secreted lipase and aspartyl protease activities. Furthermore, vps11Delta strains were defective in yeast-hypha morphogenesis. Upon serum induction of filamentous growth, mutants showed delayed emergence of germ tubes, had a reduced apical extension rate compared to those of control strains, and were unable to form mature hyphae. These results suggest that Vps11p-mediated trafficking steps are necessary to support the rapid emergence and extension of the germ tube from the parent yeast cell.

Translation elongation-3-like factors: are they rational antifungal targets?

The occurrence of fungal infection has escalated significantly in recent years and is expected to continue to increase for the foreseeable future. Unfortunately, only a limited number of antifungal drugs are currently available partially due to a lack of suitable targets. The most commonly used antifungals target the same molecule in the cell membrane and, while efficacious, are either extremely toxic or susceptible to resistance. This article examines elongation factor-3, which is unique to fungi and essential for fungal cell survival and, thus, an attractive antifungal target. A search for inhibitors of this 'perfect target' led to identification of compounds (sordarins) which inhibited elongation factor-2, a protein with a mammalian homologue. Molecular analysis demonstrated why sordarins can specifically act against fungal elongation factor-2. This data questions the validity of pursuing genes as targets only if they are unique to fungi. Proteins that are homologous to elongation factor-3 are also discussed. The advances in molecular techniques and bioinformatics will allow the re-evaluation of targets previously thought to be unattractive. In addition, molecular genetics provides new and novel information on cellular processes that can potentially introduce new targets.

The Candida albicans 14-3-3 gene, BMH1, is essential for growth.

The 14-3-3 proteins are a family of conserved small acidic proteins that have been implicated in playing major roles in a wide variety of signalling cascades. In Saccharomyces cerevisiae, the 14-3-3 genes (BMH1 and BMH2) are essential for normal pseudohyphal induction and normal bud cell development. The Bmh proteins function in the cAMP-dependent RAS/MAPK and rapamycin-sensitive signalling cascades. Deletion of only one BMH gene demonstrates no phenotypic differences under normal growth conditions. Strains deleted of both BMH1 and BMH2 are either non-viable or demonstrate sensitivity to environmental stresses. In Schizosaccharomyces pombe, the BMH homologues (RAD24 and RAD25) are essential for cell cycle control after DNA damage and deletion of both genes renders the cell inviable. The 14-3-3 gene in Candida albicans (BMH1) was identified using a novel adherence assay and differential display RT-PCR. Unlike other yeasts, C. albicans has only one 14-3-3 gene (BMH1). It was not possible to construct double knockouts by routine methods. These results suggested that the C. albicans BMH1 gene is essential. The essentiality of C. albicans BMH1 was confirmed by a PCR disruption technique. The C. albicans bmh1 Delta/BMH1 heterozygotes exhibit growth and morphogenetic defects. Therefore, the BMH1 gene in C. albicans (Accession No. AF038154) is an excellent candidate to improve our understanding of the coordinate regulation of cell cycle and morphogenesis.

Disruption studies of a Candida albicans gene, ELF1: a member of the ATP-binding cassette family.

A 3.6 kb gene (ELF1) with homology to the ATP-binding cassette (ABC) gene family has been isolated from genomic libraries of Candida albicans. Members of this gene family include both membrane transport proteins which confer a drug-resistance phenotype, and proteins whose functions are associated with protein translation. ELF1 (Elongation Like Factor) showed greatest homology with a Saccharomyces cerevisiae ORF (YPL226W), whose function is unknown, and lower homology with fungal elongation factor 3 (EF-3) genes. In comparison, homology with a gene conferring a drug-resistant phenotype (CDR1) was low. To understand the function of ELF1 in C. albicans, gene-knockout experiments were conducted using the hisG-URA3-hisG disruption cassette. Both single-copy (heterozygote) and double-disrupted strains in ELF1 were isolated. Phenotypically, the disrupted strains grew more slowly than wild-type and produced a mixture of large, irregular cells and apparently normal cells.