A comprehensive functional portrait of two heat shock factor-type transcriptional regulators involved in Candida albicans morphogenesis and virulence.

International audienceSfl1p and Sfl2p are two homologous heat shock factor-type transcriptional regulators that antagonistically control morphogenesis in Candida albicans, while being required for full pathogenesis and virulence. To understand how Sfl1p and Sfl2p exert their function, we combined genome-wide location and expression analyses to reveal their transcriptional targets in vivo together with the associated changes of the C. albicans transcriptome. We show that Sfl1p and Sfl2p bind to the promoter of at least 113 common targets through divergent binding motifs and modulate directly the expression of key transcriptional regulators of C. albicans morphogenesis and/or virulence. Surprisingly, we found that Sfl2p additionally binds to the promoter of 75 specific targets, including a high proportion of hyphal-specific genes (HSGs; HWP1, HYR1, ECE1, others), revealing a direct link between Sfl2p and hyphal development. Data mining pointed to a regulatory network in which Sfl1p and Sfl2p act as both transcriptional activators and repressors. Sfl1p directly represses the expression of positive regulators of hyphal growth (BRG1, UME6, TEC1, SFL2), while upregulating both yeast form-associated genes (RME1, RHD1, YWP1) and repressors of morphogenesis (SSN6, NRG1). On the other hand, Sfl2p directly upregulates HSGs and activators of hyphal growth (UME6, TEC1), while downregulating yeast form-associated genes and repressors of morphogenesis (NRG1, RFG1, SFL1). Using genetic interaction analyses, we provide further evidences that Sfl1p and Sfl2p antagonistically control C. albicans morphogenesis through direct modulation of the expression of important regulators of hyphal growth. Bioinformatic analyses suggest that binding of Sfl1p and Sfl2p to their targets occurs with the co-binding of Efg1p and/or Ndt80p. We show, indeed, that Sfl1p and Sfl2p targets are bound by Efg1p and that both Sfl1p and Sfl2p associate in vivo with Efg1p. Taken together, our data suggest that Sfl1p and Sfl2p act as central "switch on/off" proteins to coordinate the regulation of C. albicans morphogenesis

Contribution of the glycolytic flux and hypoxia adaptation to efficient biofilm formation by Candida albicans.

International audienceThe fungal pathogen Candida albicans forms therapeutically challenging biofilms on biomedical implants. Using a transcript profiling approach genes whose expression is favoured upon biofilm growth compared with planktonic growth have been previously identified. Knock-out mutants for 38 of these genes were constructed, six of which showed a specific defect in biofilm formation. Among these genes, TYE7 that encodes a transcriptional activator of glycolytic genes in planktonic and biofilm growth conditions was identified as being required for the cohesiveness of biofilms. Biofilms formed by the tye7Δ knock-out mutant showed a hyperfilamentous morphology, and growth of this mutant on solid medium under hypoxia was also associated with the production of hyphae. Similar to TYE7 inactivation, inhibition of glycolysis or ATP synthesis using oxalate or an uncoupler, respectively, triggered morphogenesis when a wild-type strain was grown under hypoxia. These treatments also induced the formation of weakly cohesive, hyper-filamentous biofilms by a wild-type strain. Our data indicate that a hypoxic environment is generated within C. albicans biofilms and that continued biofilm development requires a Tye7p-dependent upregulation of glycolytic genes necessary to adapt to hypoxia and prevent uncontrolled hyphal formation. Thus, adaptation to hypoxia is an integral component of biofilm formation in C. albicans

Factors that influence bidirectional long-tract homozygosis due to double-strand break repair in Candida albicans

International audienceGenomic rearrangements have been associated with the acquisition of adaptive phenotypes, allowing organisms to efficiently generate new favorable genetic combinations. The diploid genome of Candida albicans is highly plastic, displaying numerous genomic rearrangements that are often the by-product of the repair of DNA breaks. For example, DNA double-strand breaks (DSB) repair using homologous-recombination pathways are a major source of loss-of-heterozygosity (LOH), observed ubiquitously in both clinical and laboratory strains of C. albicans. Mechanisms such as break-induced replication (BIR) or mitotic crossover (MCO) can result in long tracts of LOH, spanning hundreds of kilobases until the telomere. Analysis of I-SceI-induced BIR/MCO tracts in C. albicans revealed that the homozygosis tracts can ascend several kilobases toward the centromere, displaying homozygosis from the break site toward the centromere. We sought to investigate the molecular mechanisms that could contribute to this phenotype by characterizing a series of C. albicans DNA repair mutants, including pol32-/-, msh2-/-, mph1-/-, and mus81-/-. The impact of deleting these genes on genome stability revealed functional differences between Saccharomyces cerevisiae (a model DNA repair organism) and C. albicans. In addition, we demonstrated that ascending LOH tracts toward the centromere are associated with intrinsic features of BIR and potentially involve the mismatch repair pathway which acts upon natural heterozygous positions. Overall, this mechanistic approach to study LOH deepens our limited characterization of DNA repair pathways in C. albicans and brings forth the notion that centromere proximal alleles from DNA break sites are not guarded from undergoing LOH

Transcript profiling reveals the role of PDB1, a subunit of the pyruvate dehydrogenase complex, in Candida albicans biofilm formation

International audienceCandida albicans, the most prevalent fungal pathogen in the human microbiota can form biofilms on implanted medical devices. These biofilms are tolerant to conventional antifungal drugs and the host immune system as compared to the free-floating planktonic cells. Several in vitro models of biofilm formation have been used to determine the C. albicans biofilm-forming process, regulatory networks, and their properties. Here, we performed a genome-wide transcript profiling with C. albicans cells grown in YPD medium both in planktonic and biofilm condition. Transcript profiling of YPD-grown biofilms was further compared with published Spider medium-grown biofilm transcriptome data. This comparative analysis highlighted the differentially expressed genes and the pathways altered during biofilm formation. In addition, we demonstrated that overexpression of the PDB1 gene encoding a subunit of the pyruvate dehydrogenase resulted in defective biofilm formation. Altogether, this comparative analysis of transcript profiles from two different studies provides a robust reading on biofilm-altered genes and pathways during C. albicans biofilm development

Vancomycin-Dependent Enterococcus faecalis Clinical Isolates and Revertant Mutants

Three vancomycin-dependent clinical isolates of Enterococcus faecalis of the VanB type were studied by determining (i) the sequence of the ddl gene encoding the host d-Ala:d-Ala ligase and the vanS(B)-vanR(B) genes specifying the two-component regulatory system that activates transcription of the vanB operon, (ii) the level of expression of resistance genes by using dd-dipeptidase activity as a reporter, and (iii) the proportions of the peptidoglycan precursors synthesized. Each strain had a mutation in ddl leading to an amino acid substitution (D295 to V; T316 to I) or deletion (DAK251-253 to E) at invariant positions in d-Ala:d-Ala, d-Ala:d-Lac, and d-Ala:d-Ser ligases. These mutations resulted in impaired host d-Ala:d-Ala ligases since only precursors terminating in d-Ala-d-Lac were synthesized under vancomycin-inducing conditions. Two types of vancomycin-independent revertants of one isolate were obtained in vitro after growth in the absence of vancomycin: (i) vancomycin-resistant, teicoplanin-susceptible mutants had a 6-bp insertion in the host ddl gene, causing the E251-to-EYK change that restored d-Ala:d-Ala ligase activity, (ii) constitutive vancomycin-resistant, teicoplanin-resistant mutants had substitutions (S232 to F or E247 to K) in the vicinity of the autophosphorylation site of the VanS(B) sensor and produced exclusively precursors ending in d-Ala-d-Lac. Vancomycin- and teicoplanin-dependent mutants obtained by growth in the presence of teicoplanin had an 18-bp deletion in VanS(B), affecting residues 402 to 407 and overlapping the G2 ATP binding domain. The rapid emergence of vancomycin-independent revertants in vitro suggests that interruption of vancomycin therapy may not be sufficient to cure patients infected with vancomycin-dependent enterococci

Overexpression approaches to advance understanding of Candida albicans

International audienceCandida albicans is an opportunistic fungal pathogen that is responsible for infections linked to high mortality. Loss-of-function approaches, taking advantage of gene knockouts or inducible down-regulation, have been successfully used in this species in order to understand gene function. However, overexpression of a gene provides an alternative, powerful tool to elucidate gene function and identify novel phenotypes. Notably, overexpression can identify pathway components that might remain undetected using loss-of-function approaches. Several repressible or inducible promoters have been developed which allow to shut off or turn on the expression of a gene in C. albicans upon growth in the presence of a repressor or inducer. In this review, we summarize recent overexpression approaches used to study different aspects of C. albicans biology, including morphogenesis, biofilm formation, drug tolerance, and commensalism

A phylogenetically-restricted essential cell cycle progression factor in the human pathogen Candida albicans

International audienceAbstract Chromosomal instability caused by cell division errors is associated with antifungal drug resistance in fungal pathogens. Here, we identify potential mechanisms underlying such instability by conducting an overexpression screen monitoring chromosomal stability in the human fungal pathogen Candida albicans . Analysis of ~1000 genes uncovers six c hromosomal s t a bility ( CSA ) genes, five of which are related to cell division genes of other organisms. The sixth gene, CSA6 , appears to be present only in species belonging to the CUG-Ser clade, which includes C. albicans and other human fungal pathogens. The protein encoded by CSA6 localizes to the spindle pole bodies, is required for exit from mitosis, and induces a checkpoint-dependent metaphase arrest upon overexpression. Thus, Csa6 is an essential cell cycle progression factor that is restricted to the CUG-Ser fungal clade, and could therefore be explored as a potential antifungal target

Genome-wide location of <i>Candida albicans</i> Sfl1p and Sfl2p, <i>in vivo</i>, at a single-nucleotide resolution.

<p>(<b>A</b>) Venn diagram of the overlap between Sfl1p and Sfl2p binding targets. All 113 Sfl1p targets are also bound by Sfl2p, while 75 target promoters are Sfl2p-specific. The total number of Sfl1p or Sfl2p target promoters are indicated between parentheses. Target promoters include those that are clearly associated with given ORFs as well as those that are shared by two ORFs in opposite orientations. (<b>B</b>) A single-nucleotide resolution of Sfl1p and Sfl2p binding at selected <i>C. albicans</i> genomic regions <i>in vivo</i>. Plotted are read-count signal intensities of HA₃-tagged <i>SFL1</i>- (<i>sfl1</i>-CaEXP-<i>SFL1-HA₃</i>) or <i>SFL2</i>- (<i>sfl2</i>-CaEXP-<i>SFL2-HA₃</i>) coimmunoprecipitated DNA and the corresponding empty-vector control signals (<i>sfl1</i>-CaEXP, <i>sfl2</i>-CaEXP, respectively) from merged BAM files of two independent biological replicates. Some read-count signals extend beyond the maximum graduation (not shown) that ranges between 0–500 reads for Sfl1 data (<i>sfl1</i>-CaEXP and <i>sfl1</i>-CaEXP-<i>SFL1-HA₃</i>) and 0–1000 reads for Sfl2 data (<i>sfl2</i>-CaEXP and <i>sfl2</i>-CaEXP-<i>SFL2-HA₃</i>). The position of each signal in selected <i>C. albicans</i> genomic regions from assembly 21 is shown on the <i>x</i>-axis. The location of each selected region from the corresponding chromosome (Chr) is indicated at the top of each panel (limits are shown between parentheses in base pairs). The orientation of each ORF is depicted by the arrowed black rectangle. (<b>C</b>) Enrichment scores of the Gene Ontology (GO) terms to which are assigned Sfl1p and Sfl2p common (shaded area) or Sfl2p-specific (unshaded area) binding targets. GO term enrichment scores are calculated as the negative value of the log₁₀-transformed <i>P</i>-value. The number of genes of each category is shown at the right of each horizontal bar.</p

Strains used in this study.

<p>Strains used in this study.</p