A human-curated annotation of the Candida albicans genome.
ABSTRACT: Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications.
Project description:We used transcriptional profiling to investigate the response of the fungal pathogen Candida albicans to temperature and osmotic and oxidative stresses under conditions that permitted >60% survival of the challenged cells. Each stress generated the transient induction of a specific set of genes including classic markers observed in the stress responses of other organisms. We noted that the classical hallmarks of the general stress response observed in Saccharomyces cerevisiae are absent from C. albicans; no C. albicans genes were significantly induced in a common response to the three stresses. This observation is supported by our inability to detect stress cross-protection in C. albicans. Similarly, in C. albicans there is essentially no induction of carbohydrate reserves like glycogen and trehalose in response to a mild stress, unlike the situation in S. cerevisiae. Thus C. albicans lacks the strong general stress response exhibited by S. cerevisiae.
Project description:A dominant selectable marker for Candida albicans and other Candida species, which confers resistance to nourseothricin, was characterized. In a heterologous promoter system and a recyclable cassette, the marker efficiently permitted deletion and complementation of C. albicans genes. Neither growth nor filamentous development was affected in strains expressing this marker.
Project description:Early childhood caries (ECC), which can lead to rampant tooth-decay that is painful and costly to treat, is one of the most prevalent infectious diseases affecting children worldwide. Previous studies support that interactions between Streptococcus mutans and Candida albicans are associated with the pathogenesis of ECC. The presence of Candida enhances S. mutans growth, fitness and accumulation within biofilms in vitro, although the molecular basis for these behaviors is undefined. Using an established co-cultivation biofilm model and RNA-Seq, we investigated how C. albicans influences the transcriptome of S. mutans. The presence of C. albicans dramatically altered gene expression in S. mutans in the dual-species biofilm, resulting in 393 genes differentially expressed, compared to mono-species biofilms of S. mutans. By Gene Ontology analysis, the majority of up-regulated genes were related to carbohydrate transport and metabolic/catabolic processes. KEGG pathway impact analysis showed elevated pyruvate and galactose metabolism, suggesting that co-cultivation with C. albicans influences carbohydrate utilization by S. mutans. Analysis of metabolites confirmed the increases in carbohydrate metabolism, with elevated amounts of formate in the culture medium of co-cultured biofilms. Moreover, co-cultivation with C. albicans altered transcription of S. mutans signal transduction (comC and ciaRH) genes associated with fitness and virulence. Interestingly, the expression of genes for mutacins (bacteriocins) and CRISPR were down-regulated. Collectively, the data provide a comprehensive insight into S. mutans transcriptomic changes induced by C. albicans, and offer novel insights into how bacterial-fungal interactions may enhance the severity of dental caries.
Project description:The first barrier against infection by Candida albicans involves fungal recognition and destruction by phagocytic cells of the innate immune system. It is well established that interactions between different phagocyte receptors and components of the fungal cell wall trigger phagocytosis and subsequent immune responses, but the fungal ligands mediating the initial stage of recognition have not been identified. Here, we describe a novel assay for fungal recognition and uptake by macrophages which monitors this early recognition step independently of other downstream events of phagocytosis. To analyze infection in live macrophages, we validated the neutrality of a codon-optimized red fluorescent protein (yEmRFP) biomarker in C. albicans; growth, hyphal formation, and virulence in infected mice and macrophages were unaffected by yEmRFP production. This permitted a new approach for studying phagocytosis by carrying out competition assays between red and green fluorescent yeast cells to measure the efficiency of yeast uptake by murine macrophages as a function of dimorphism or cell wall defects. These competition experiments demonstrate that, given a choice, macrophages display strong preferences for phagocytosis based on genus, species, and morphology. Candida glabrata and Saccharomyces cerevisiae are taken up by J774 macrophage cells more rapidly than C. albicans, and C. albicans yeast cells are favored over hyphal cells. Significantly, these preferences are mannan dependent. Mutations that affect mannan, but not those that affect glucan or chitin, reduce the uptake of yeast challenged with wild-type competitors by both J774 and primary murine macrophages. These results suggest that mannose side chains or mannosylated proteins are the ligands recognized by murine macrophages prior to fungal uptake.
Project description:Candida albicans is an opportunistic fungal pathogen that resides in the human body as a commensal and can turn pathogenic when the host is immunocompromised. Adaptation of C. albicans to host niche-specific conditions is important for the establishment of pathogenicity, where the ability of C. albicans to utilize multiple carbon sources provides additional flexibility. One alternative sugar is N-acetylglucosamine (GlcNAc), which is now established as an important carbon source for many pathogens and can also act as a signaling molecule. Although GlcNAc catabolism has been well studied in many pathogens, the importance of several enzymes involved in the formation of metabolic intermediates still remains elusive. In this context, microarray analysis was carried out to investigate the transcriptional responses induced by GlcNAc under different conditions. A novel gene that was highly upregulated immediately following the GlcNAc catabolic genes was identified and was named GIG2 (GlcNAc-induced gene 2). This gene is regulated in a manner distinct from that of the GlcNAc-induced genes described previously in that GlcNAc metabolism is essential for its induction. Furthermore, this gene is involved in the metabolism of N-acetylneuraminate (sialic acid), a molecule equally important for initial host-pathogen recognition. Mutant cells showed a considerable decrease in fungal burden in mouse kidneys and were hypersensitive to oxidative stress conditions. Since GIG2 is also present in many other fungal and enterobacterial genomes, targeted inhibition of its activity would offer insight into the treatment of candidiasis and other fungal or enterobacterial infections.
Project description:Lipid mediators, derived from arachidonic acid metabolism, play an important role in immune regulation. The functions of bioactive eicosanoids range from modulating cytokine signaling and inflammasome formation to anti-inflammatory and pro-resolving activities. Human pathogenic fungi such as Candida albicans, Candida parapsilosis, Cryptococcus neoformans and Aspergillus fumigatus have been shown to produce such lipid mediators, associated with their virulence. To date, investigations into the molecular mechanisms of fungal eicosanoid biosynthesis in different species have revealed that several genes are associated with prostaglandin production. However, these routes remain uncharacterized in C. parapsilosis with early results suggesting it uses pathways distinct from those found in C. albicans. Therefore, we aimed to identify and characterize C. parapsilosis genes involved in eicosanoid biosynthesis. Following arachidonic acid treatment of C. parapsilosis cells, we identified several genes interfering with prostaglandin production. Out of the identified genes, homologues of a multi copper oxidase (FET3), an Acyl-CoA thiolase (POT1) and an Acyl-CoA oxidase (POX1-3) were found to play a significant role in prostaglandin synthesis. Furthermore, all three genes were confirmed to enhance C. parapsilosis pathogenicity, as the corresponding deletion mutants were cleared more efficiently by human macrophages and induced higher levels of pro-inflammatory cytokines. In addition, the mutants were less virulent than the wild-type strain in a mouse model of systemic infection. Taken together, we identified three genes that regulate eicosanoid biosynthesis in C. parapsilosis and impact the fungus' virulence.
Project description:Candidiasis affects a wide variety of immunocompromised and medically compromised patients. Candida albicans, a major human fungal pathogen, accounts for about 50% of all cases, while the remainder are caused by the less pathogenic non-albicans Candida species (NACS). These species are believed to be less pathogenic, in part, because they do not filament as readily or robustly as C. albicans, although definitive evidence is lacking. To address this question, we used strains for two NACS, Candida tropicalis and Candida parapsilosis, which were genetically engineered to constitutively express the key transcriptional regulator UME6 and drive strong filamentation both in vitro and during infection in vivo Unexpectedly, both strains showed a dramatic reduction in organ fungal burden in response to UME6 expression. Consistent with these findings, we observed that a C. tropicalis hyperfilamentous mutant was significantly reduced and a filamentation-defective mutant was slightly increased for organ fungal burden. Comprehensive immune profiling generally did not reveal any significant changes in the host response to UME6 expression in the NACS that could explain the increased clearance of infection. Interestingly, whole-genome transcriptional profiling indicated that while genes important for filamentation were induced by UME6 expression in C. tropicalis and C. parapsilosis, other genes involved in a variety of processes important for pathogenesis were strongly downregulated. These findings suggest that there are fundamental evolutionary differences in the relationship between morphology and pathogenicity among Candida species and that NACS do not necessarily possess the same virulence properties as C. albicans IMPORTANCE Many immunocompromised individuals, including HIV/AIDS and cancer patients, are susceptible to candidiasis. About half of all cases are caused by the major fungal pathogen Candida albicans, whereas the remainder are due to less pathogenic non-albicans Candida species (NACS). Generation of filamentous cells represents a major virulence property of C. albicans, and the NACS are believed to be less pathogenic, in part, because they do not filament as well as C. albicans does. To address this question, we determined the pathogenicity of two NACS strains that have been genetically engineered to promote filamentation during infection. Surprisingly, these strains showed a dramatic reduction in pathogenicity. The host immune response did not appear to be affected. However, unlike C. albicans, filamentation of the NACS was associated with downregulation of several genes important for pathogenicity processes. Our results suggest that there are fundamental evolutionary differences in the relationship between filamentation and pathogenesis in NACS compared to C. albicans.
Project description:Since current antifungal drugs have not kept pace with the escalating medical demands of fungal infections, new, effective medications are required. However, antifungal drug discovery is hindered by the evolutionary similarity of mammalian and fungal cells, which results in fungal drug targets having human homologs and drug non-selectivity. The group III hybrid histidine kinases (HHKs) are an attractive drug target since they are conserved in fungi and absent in mammals. We used a Saccharomyces cerevisiae reporter strain that conditionally expresses HHK to establish a high-throughput bioassay to screen microbial extracts natural products for antifungals. We identified macrotetrolides, a group of related ionophores thought to exhibit restricted antifungal activity. In addition to confirming the use of this bioassay for the discovery of antifungal natural products, we demonstrated broader, more potent fungistatic activity of the macrotetrolides against multiple Candida spp., Cryptococcus spp., and Candida albicans in biofilms. Macrotetrolides were also active in an animal model of C. albicans biofilm, but were found to have inconsistent activity against fluconazole-resistant C. albicans, with most isolates resistant to this natural product. The macrotetrolides do not directly target HHKs, but their selective activity against S. cerevisiae grown in galactose (regardless of Drk1 expression) revealed potential new insight into the role of ion transport in the mode of action of these promising antifungal compounds. Thus, this simple, high-throughput bioassay permitted us to screen microbial extracts, identify natural products as antifungal drugs, and expand our understanding of the activity of macrotetrolides.
Project description:UNLABELLED:Single-celled organisms have different strategies to sense and utilize nutrients in their ever-changing environments. The opportunistic fungal pathogen Candida albicans is a common member of the human microbiota, especially that of the gastrointestinal (GI) tract. An important question concerns how C. albicans gained a competitive advantage over other microbes to become a successful commensal and opportunistic pathogen. Here, we report that C. albicans uses N-acetylglucosamine (GlcNAc), an abundant carbon source present in the GI tract, as a signal for nutrient availability. When placed in water, C. albicans cells normally enter the G0 phase and remain viable for weeks. However, they quickly lose viability when cultured in water containing only GlcNAc. We term this phenomenon GlcNAc-induced cell death (GICD). GlcNAc triggers the upregulation of ribosomal biogenesis genes, alterations of mitochondrial metabolism, and the accumulation of reactive oxygen species (ROS), followed by rapid cell death via both apoptotic and necrotic mechanisms. Multiple pathways, including the conserved cyclic AMP (cAMP) signaling and GlcNAc catabolic pathways, are involved in GICD. GlcNAc acts as a signaling molecule to regulate multiple cellular programs in a coordinated manner and therefore maximizes the efficiency of nutrient use. This adaptive behavior allows C. albicans' more efficient colonization of the gut. IMPORTANCE:The ability to rapidly and appropriately respond to nutrients in the environment is crucial to free-living microorganisms. To maximize the use of available nutrients, microorganisms often use a limiting nutritional component as a signal to coordinate multiple biological processes. The human fungal pathogen Candida albicans uses N-acetylglucosamine (GlcNAc) as a signal for the availability of external nutrient resources. GlcNAc induces rapid cell death in C. albicans due to the constitutive activation of oxidative metabolism and accumulation of reactive oxygen species (ROS), and multiple pathways are involved in its regulation. This study sheds light on the mechanisms of niche specialization of pathogenic fungi and raises the possibility that this cell death pathway could be an unexplored therapeutic target.
Project description:Candida albicans, a common fungal pathogen which diverged from the baker's yeast Saccharomyces cerevisiae has the unique ability to utilise N-acetylglucosamine, an amino sugar and exhibits phenotypic differences. It has acquired intricate regulatory mechanisms at different levels in accordance with its life style. N-acetylglucosamine kinase, a component of the N-acetylglucosamine catabolic cascade is an understudied gene since Saccharomyces cerevisiae lacks it. We report HXK1 to act as both positive and negative regulator of transcription of genes involved in maintaining cellular homeostasis. It is involved in repression of hyphal specific genes in addition to metabolic genes. Its regulation of filamentation and GlcNAc metabolism is independent of the known classical regulators like EFG1, CPH1, RAS1, TPK2 or TUP1. Moreover, Hxk1-GFP is localised to cytoplasm, nucleus and mitochondria in a condition specific manner. By employing two-step affinity purification, we report the interaction of HXK1 with SIR2 under filamentation inducing conditions. Our work highlights a novel regulatory mechanism involved in filamentation repression and attempts to decipher the GlcNAc catabolic regulatory cascade in eukaryotes.