Candida Albicans Transcription Research Articles

Host-derived reactive oxygen species trigger activation of the Candida albicans transcription regulator Rtg1/3.

The signals that denote mammalian host environments and dictate the activation of signaling pathways in human-associated microorganisms are often unknown. The transcription regulator Rtg1/3 in the human fungal pathogen Candida albicans is a crucial determinant of host colonization and pathogenicity. Rtg1/3's activity is controlled, in part, by shuttling the regulator between the cytoplasm and nucleus of the fungus. The host signal(s) that Rtg1/3 respond(s) to, however, have remained unclear. Here we report that neutrophil-derived reactive oxygen species (ROS) direct the subcellular localization of this C. albicans transcription regulator. Upon engulfment of Candida cells by human or mouse neutrophils, the regulator shuttles to the fungal nucleus. Using genetic and chemical approaches to disrupt the neutrophils' oxidative burst, we establish that the oxidants produced by the NOX2 complex-but not the oxidants generated by myeloperoxidase-trigger Rtg1/3's migration to the nucleus. Furthermore, screening a collection of C. albicans kinase deletion mutants, we implicate the MKC1 signaling pathway in the ROS-dependent regulation of Rtg1/3 in this fungus. Finally, we show that Rtg1/3 contributes to C. albicans virulence in the nematode Caenorhabditis elegans in an ROS-dependent manner as the rtg1 and rtg3 mutants display virulence defects in wild-type but not in ROS deficient worms. Our findings establish NOX2-derived ROS as a key signal that directs the activity of the pleiotropic fungal regulator Rtg1/3.

Construction of Double Heterozygous Deletion Strains for Complex Haploinsufficiency-Based Genetic Analysis in Candida albicans.

Complex haploinsufficiency refers to the genetic interaction that occurs in strains with heterozygous mutations at two different loci (a double heterozygous deletion mutant). Double heterozygous deletion mutants can be used to identify gene partners that act within the same pathway or to determine expression-dependent genetic interactions that result in phenotypic changes outside of what would be expected based on the phenotypes of the single heterozygous deletion mutants. The approach outlined here uses a lithium acetate transformation method on a parental "query" strain to introduce a transcription factor deletion DNA construct that is derived from the Homann et al. Candida albicans transcription factor deletion library (Homann et al. PLoS Genet 5(12):e1000783, 2009). We also outline the steps to confirming the genotype of the resulting transformants as well as an example of the use of double heterozygous deletion mutants for complex haploinsufficiency analysis of biofilm formation.

Understanding the Uptake Mechanism and Interaction Potential of the Designed Peptide and Preparation of Composite Fiber Matrix for Fungal Keratitis.

The conventional use of antibiotics for the treatment of infectious keratitis currently faces two major challenges: poor drug penetration and the emergence of antibiotic resistance in microbial strains. Cell-penetrating peptides (CPPs) with antimicrobial properties have the potential to address these challenges. However, their mode of action, mechanism of uptake, and interaction potential have not been explored in detail. In this study, we probed the mechanism of uptake and interaction potential of our previously designed peptides (VRF005 and VRF007). Our results showed that VRF005 undergoes direct translocation and induces a rough membrane surface, whereas VRF007 undergoes clathrin-mediated endocytic uptake. The gel shift assay showed that VRF005 is bound to genomic DNA, whereas VRF007 is bound to chitin and β-d-glucan. Gene expression studies revealed the effect of peptide VRF005 on Candida albicans transcription. Molecular docking and simulations showed that VRF005 forms noncovalent interactions (such as H-bonding and water bridges) with natamycin. It exhibited synergistic antifungal activity in the colony-forming assay. VRF005, functionalized in the polycaprolactone fiber matrix, showed sustained delivery and antifungal activity.

2887. Identifying Candida albicans Transcription Factors (TFs) That Regulate Pathogenesis of Intra-abdominal Candidiasis (IAC) by Screening a Deletion Mutant Library

BackgroundIAC is a common manifestation of invasive candidiasis, but its pathogenesis is poorly understood. We developed a mouse model of C. albicans IAC, in which disease progresses from peritonitis to abscesses (IAA) in a manner that recapitulates human infection. Our goal was to use the model to identify C. albicans TFs that regulate virulence during IAC.MethodsWe screened a signature-tagged library (48 unique oligonucleotide markers) of homozygous deletion mutants for 165 C. albicans TF genes, created in duplicate in strain SC5314 (S. Noble). Mice were infected intra-peritoneally in triplicate with pools of 24 mutants and wild-type, and strains harvested at 72 hours in IAA.ResultsTwenty-one TF mutants were significantly attenuated for virulence in both libraries, and 2 TF mutants were significantly more virulent in both libraries, as measured by tissue burdens (figure). Biologic processes over-represented among attenuated mutants were regulation of pH responses, biofilm, hyphal formation, echinocandin responses, and copper metabolism. pH responses are likely to be crucial to pathogenesis of IAC, as C. albicans transitions from pH 8 during peritonitis to pH 6.8 within IAA. 9 pH response regulators contributing to virulence included RIM101, STP2 (alkaline), ASH1, SFL1, SFL2 (neutral), MNL1, SKO1, PHO4 (weak acid), and CSR1 (acid). We created rim101 null mutant and reconstitution strains, and demonstrated that the gene was essential for complete virulence during peritonitis and IAA. Transcriptional profiling of strains by RT-PCR during peritonitis and in vitro showed both conserved and rewired Rim101 targets. SAP5, which encodes an aspartyl protease, is a major Rim101 target in vivo and in vitro; over-expression of SAP5 in rim101 restored virulence during IAA at 3, 7, and 10 days, but not during peritonitis. Other pH regulatory TF genes are currently being validated as virulence determinants, and pathway relationships between MNL1, SKO1, and PHO4 during IAA formation are being explored through epistasis approaches.ConclusionScreening of a C. albicans TF mutant library identified pH responses and other biologic processes as important during pathogenesis of IAC. Rim101, an alkaline pH response regulator, contributes to both peritonitis and IAA, the latter at least in part through its effects on Sap5. Disclosures All Authors: No reported Disclosures.

The Role of Candida albicans Transcription Factor RLM1 in Response to Carbon Adaptation.

Candida albicans is the main causative agent of candidiasis and one of the most frequent causes of nosocomial infections worldwide. In order to establish an infection, this pathogen supports effective stress responses to counter host defenses and adapts to changes in the availability of important nutrients, such as alternative carbon sources. These stress responses have clear implications on the composition and structure of Candida cell wall. Therefore, we studied the impact of lactate, a physiologically relevant carbon source, on the activity of C. albicans RLM1 transcriptional factor. RLM1 is involved in the cell wall integrity pathway and plays an important role in regulating the flow of carbohydrates into cell wall biosynthesis pathways. The role of C. albicans RLM1 in response to lactate adaptation was assessed in respect to several virulence factors, such as the ability to grow under cell wall damaging agents, filament, adhere or form biofilm, as well as to immune recognition. The data showed that growth of C. albicans cells in the presence of lactate induces the secretion of tartaric acid, which has the potential to modulate the TCA cycle on both the yeast and the host cells. In addition, we found that adaptation of C. albicans cells to lactate reduces their internalization by immune cells and consequent % of killing, which could be correlated with a lower exposure of the cell wall β-glucans. In addition, absence of RLM1 has a minor impact on internalization, compared with the wild-type and complemented strains, but it reduces the higher efficiency of lactate grown cells at damaging phagocytic cells and induces a high amount of IL-10, rendering these cells more tolerable to the immune system. The data suggests that RLM1 mediates cell wall remodeling during carbon adaptation, impacting their interaction with immune cells.

The Candida albicans transcription factor Cas5 couples stress responses, drug resistance and cell cycle regulation

The capacity to coordinate environmental sensing with initiation of cellular responses underpins microbial survival and is crucial for virulence and stress responses in microbial pathogens. Here we define circuitry that enables the fungal pathogen Candida albicans to couple cell cycle dynamics with responses to cell wall stress induced by echinocandins, a front-line class of antifungal drugs. We discover that the C. albicans transcription factor Cas5 is crucial for proper cell cycle dynamics and responses to echinocandins, which inhibit β-1,3-glucan synthesis. Cas5 has distinct transcriptional targets under basal and stress conditions, is activated by the phosphatase Glc7, and can regulate the expression of target genes in concert with the transcriptional regulators Swi4 and Swi6. Thus, we illuminate a mechanism of transcriptional control that couples cell wall integrity with cell cycle regulation, and uncover circuitry governing antifungal drug resistance.

Candida albicans Transcriptional Profiling Within Biliary Fluid From a Patient With Cholangitis, Before and After Antifungal Treatment and Surgical Drainage.

We used ribonucleic acid sequencing to profile Candida albicans transcription within biliary fluid from a patient with cholangitis; samples were collected before and after treatment with fluconazole and drainage. Candida albicans transcriptomes at the infection site distinguished treated from untreated cholangitis. After treatment, 1131 C. albicans genes were differentially expressed in biliary fluid. Up-regulated genes were enriched in hyphal growth, cell wall organization, adhesion, oxidation reduction, biofilm, and fatty acid and ergosterol biosynthesis. This is the first study to define Candida global gene expression during deep-seated human infection. Successful treatment of cholangitis induced C. albicans genes involved in fluconazole responses and pathogenesis.

Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models.

The aim of the present study was to identify Candida albicans transcription factors (TFs) involved in virulence. Although mice are considered the gold-standard model to study fungal virulence, mini-host infection models have been increasingly used. Here, barcoded TF mutants were first screened in mice by pools of strains and fungal burdens (FBs) quantified in kidneys. Mutants of unannotated genes which generated a kidney FB significantly different from that of wild-type were selected and individually examined in Galleria mellonella. In addition, mutants that could not be detected in mice were also tested in G. mellonella. Only 25% of these mutants displayed matching phenotypes in both hosts, highlighting a significant discrepancy between the two models. To address the basis of this difference (pool or host effects), a set of 19 mutants tested in G. mellonella were also injected individually into mice. Matching FB phenotypes were observed in 50% of the cases, highlighting the bias due to host effects. In contrast, 33.4% concordance was observed between pool and single strain infections in mice, thereby highlighting the bias introduced by the “pool effect.” After filtering the results obtained from the two infection models, mutants for MBF1 and ZCF6 were selected. Independent marker-free mutants were subsequently tested in both hosts to validate previous results. The MBF1 mutant showed impaired infection in both models, while the ZCF6 mutant was only significant in mice infections. The two mutants showed no obvious in vitro phenotypes compared with the wild-type, indicating that these genes might be specifically involved in in vivo adapt

How duplicated transcription regulators can diversify to govern the expression of nonoverlapping sets of genes.

The duplication of transcription regulators can elicit major regulatory network rearrangements over evolutionary timescales. However, few examples of duplications resulting in gene network expansions are understood in molecular detail. Here we show that four Candida albicans transcription regulators that arose by successive duplications have differentiated from one another by acquiring different intrinsic DNA-binding specificities, different preferences for half-site spacing, and different associations with cofactors. The combination of these three mechanisms resulted in each of the four regulators controlling a distinct set of target genes, which likely contributed to the adaption of this fungus to its human host. Our results illustrate how successive duplications and diversification of an ancestral transcription regulator can underlie major changes in an organism's regulatory circuitry.

Regulation of theCandida albicansCell Wall Damage Response by Transcription Factor Sko1 and PAS Kinase Psk1

The environmental niche of each fungus places distinct functional demands on the cell wall. Hence cell wall regulatory pathways may be highly divergent. We have pursued this hypothesis through analysis of Candida albicans transcription factor mutants that are hypersensitive to caspofungin, an inhibitor of beta-1,3-glucan synthase. We report here that mutations in SKO1 cause this phenotype. C. albicans Sko1 undergoes Hog1-dependent phosphorylation after osmotic stress, like its Saccharomyces cerevisiae orthologues, thus arguing that this Hog1-Sko1 relationship is conserved. However, Sko1 has a distinct role in the response to cell wall inhibition because 1) sko1 mutants are much more sensitive to caspofungin than hog1 mutants; 2) Sko1 does not undergo detectable phosphorylation in response to caspofungin; 3) SKO1 transcript levels are induced by caspofungin in both wild-type and hog1 mutant strains; and 4) sko1 mutants are defective in expression of caspofungin-inducible genes that are not induced by osmotic stress. Upstream Sko1 regulators were identified from a panel of caspofungin-hypersensitive protein kinase-defective mutants. Our results show that protein kinase Psk1 is required for expression of SKO1 and of Sko1-dependent genes in response to caspofungin. Thus Psk1 and Sko1 lie in a newly described signal transduction pathway.

Candida albicans transcription factor Ace2 regulates metabolism and is required for filamentation in hypoxic conditions.

Ace2 transcription factor family genes are found in many fungal genomes and are required for regulation of expression of genes involved in cell separation. We used transcriptional profiling to identify the targets of Ace2 in Candida albicans, and we show that these include several cell wall components, such as glucanases and glycosylphosphatidylinositol-anchored proteins. Expression is downregulated in ace2 deletion mutants in both yeast and hyphal cells. In addition, deleting ace2 results in dramatic changes in expression of metabolic pathways. Expression of glycolytic enzymes is reduced, while expression of respiratory genes (including those involved in the tricarboxylic acid cycle, oxidative phosphorylation, and ATP synthesis) is increased. Similar changes occur in both yeast and hyphal cells. In contrast, genes required for acetyl-coenzyme A and lipid metabolism are upregulated in an ace2 deletion mutant grown predominantly as yeast cells but are downregulated in hyphae. These results suggest that in wild-type strains, Ace2 acts to increase glycolysis and reduce respiration. This is supported by the observation that deleting ace2 results in increased resistance to antimycin A, a drug that inhibits respiration. We also show that Ace2 is required for filamentation in response to low oxygen concentrations (hypoxia). We suggest that filamentation is induced in wild-type cells by reducing respiration (using low oxygen or respiratory drugs) and that mutants with increased respiratory activity fail to undergo filamentation under these conditions.

A Mutation in Tac1p, a Transcription Factor Regulating CDR1 and CDR2, Is Coupled With Loss of Heterozygosity at Chromosome 5 to Mediate Antifungal Resistance in Candida albicans

TAC1, a Candida albicans transcription factor situated near the mating-type locus on chromosome 5, is necessary for the upregulation of the ABC-transporter genes CDR1 and CDR2, which mediate azole resistance. We showed previously the existence of both wild-type and hyperactive TAC1 alleles. Wild-type alleles mediate upregulation of CDR1 and CDR2 upon exposure to inducers such as fluphenazine, while hyperactive alleles result in constitutive high expression of CDR1 and CDR2. Here we recovered TAC1 alleles from two pairs of matched azole-susceptible (DSY294; FH1: heterozygous at mating-type locus) and azole-resistant isolates (DSY296; FH3: homozygous at mating-type locus). Two different TAC1 wild-type alleles were recovered from DSY294 (TAC1-3 and TAC1-4) while a single hyperactive allele (TAC1-5) was isolated from DSY296. A single amino acid (aa) difference between TAC1-4 and TAC1-5 (Asn977 to Asp or N977D) was observed in a region corresponding to the predicted activation domain of Tac1p. Two TAC1 alleles were recovered from FH1 (TAC1-6 and TAC1-7) and a single hyperactive allele (TAC1-7) was recovered from FH3. The N977D change was seen in TAC1-7 in addition to several other aa differences. The importance of N977D in conferring hyperactivity to TAC1 was confirmed by site-directed mutagenesis. Both hyperactive alleles TAC1-5 and TAC1-7 were codominant with wild-type alleles and conferred hyperactive phenotypes only when homozygous. The mechanisms by which hyperactive alleles become homozygous was addressed by comparative genome hybridization and single nucleotide polymorphism arrays and indicated that loss of TAC1 heterozygosity can occur by recombination between portions of chromosome 5 or by chromosome 5 duplication.

Comparative Gene Expression Analysis by a Differential Clustering Approach: Application to the Candida albicans Transcription Program

Differences in gene expression underlie many of the phenotypic variations between related organisms, yet approaches to characterize such differences on a genome-wide scale are not well developed. Here, we introduce the “differential clustering algorithm” for revealing conserved and diverged co-expression patterns. Our approach is applied at different levels of organization, ranging from pair-wise correlations within specific groups of functionally linked genes, to higher-order correlations between such groups. Using the differential clustering algorithm, we systematically compared the transcription program of the fungal pathogen Candida albicans with that of the model organism Saccharomyces cerevisiae. Many of the identified differences are related to the differential requirement for mitochondrial function in the two yeasts. Distinct regulation patterns of cell cycle genes and of amino acid metabolic genes were also revealed and, in some cases, could be linked to the differential appearance of cis-regulatory elements in the gene promoter regions. Our study provides a comprehensive framework for comparative gene expression analysis and a rich source of hypotheses for uncharacterized open reading frames and putative cis-regulatory elements in C. albicans.