Faiza Tebbji

Global Gene Deletion Analysis Exploring Yeast Filamentous Growth

Infectious Phenotype The pathogenic yeast Candida albicans needs to adopt a filamentous form to invade tissues. The distantly related yeast species Saccharomyces cerevisiae also takes on a filamentous form for nutrient foraging. Comparing genome-wide deletion libraries between the two species, Ryan et al. (p. 1353) identified genes involved in three different filamentous yeast phenotypes and found unique genes for each of these phenotypes. However, in addition, core genes, including a previously unknown conserved regulator, appear to have homologous roles in regulating filamentous growth in these distantly related yeast species. Yeast genes involved in the dimorphic switch between cell budding and filamentous growth types are identified. The dimorphic switch from a single-cell budding yeast to a filamentous form enables Saccharomyces cerevisiae to forage for nutrients and the opportunistic pathogen Candida albicans to invade human tissues and evade the immune system. We constructed a genome-wide set of targeted deletion alleles and introduced them into a filamentous S. cerevisiae strain, Σ1278b. We identified genes involved in morphologically distinct forms of filamentation: haploid invasive growth, biofilm formation, and diploid pseudohyphal growth. Unique genes appear to underlie each program, but we also found core genes with general roles in filamentous growth, including MFG1 (YDL233w), whose product binds two morphogenetic transcription factors, Flo8 and Mss11, and functions as a critical transcriptional regulator of filamentous growth in both S. cerevisiae and C. albicans.

A novel role for the transcription factor Cwt1p as a negative regulator of nitrosative stress in Candida albicans.

The ability of Candida albicans to survive in the presence of nitrosative stress during the initial contact with the host immune system is crucial for its ability to colonize mammalian hosts. Thus, this fungus must activate robust mechanisms to neutralize and repair nitrosative-induced damage. Until now, very little was known regarding the regulatory circuits associated with reactive nitrogen species detoxification in fungi. To gain insight into the transcriptional regulatory networks controlling nitrosative stress response (NRS) in C. albicans a compilation of transcriptional regulator-defective mutants were screened. This led to the identification of Cwt1p as a negative regulator of NSR. By combining genome-wide location and expression analyses, we have characterized the Cwt1p regulon and demonstrated that Cwt1p is directly required for proper repression of the flavohemoglobin Yhb1p, a key NO-detoxification enzyme. Furthermore, Cwt1p operates both by activating and repressing genes of specific functions solicited upon NSR. Additionally, we used Gene Set Enrichment Analysis to reinvestigate the C. albicans NSR-transcriptome and demonstrate a significant similarity with the transcriptional profiles of C. albicans interacting with phagocytic host-cells. In summary, we have characterized a novel negative regulator of NSR and bring new insights into the transcriptional regulatory network governing fungal NSR.

A functional portrait of Med7 and the mediator complex in Candida albicans.

Mediator is a multi-subunit protein complex that regulates gene expression in eukaryotes by integrating physiological and developmental signals and transmitting them to the general RNA polymerase II machinery. We examined, in the fungal pathogen Candida albicans, a set of conditional alleles of genes encoding Mediator subunits of the head, middle, and tail modules that were found to be essential in the related ascomycete Saccharomyces cerevisiae. Intriguingly, while the Med4, 8, 10, 11, 14, 17, 21 and 22 subunits were essential in both fungi, the structurally highly conserved Med7 subunit was apparently non-essential in C. albicans. While loss of CaMed7 did not lead to loss of viability under normal growth conditions, it dramatically influenced the pathogens ability to grow in different carbon sources, to form hyphae and biofilms, and to colonize the gastrointestinal tracts of mice. We used epitope tagging and location profiling of the Med7 subunit to examine the distribution of the DNA sites bound by Mediator during growth in either the yeast or the hyphal form, two distinct morphologies characterized by different transcription profiles. We observed a core set of 200 genes bound by Med7 under both conditions; this core set is expanded moderately during yeast growth, but is expanded considerably during hyphal growth, supporting the idea that Mediator binding correlates with changes in transcriptional activity and that this binding is condition specific. Med7 bound not only in the promoter regions of active genes but also within coding regions and at the 3′ ends of genes. By combining genome-wide location profiling, expression analyses and phenotyping, we have identified different Med7p-influenced regulons including genes related to glycolysis and the Filamentous Growth Regulator family. In the absence of Med7, the ribosomal regulon is de-repressed, suggesting Med7 is involved in central aspects of growth control.

The Monoterpene Carvacrol Generates Endoplasmic Reticulum Stress in the Pathogenic Fungus Candida albicans

ABSTRACT The monoterpene carvacrol, the major component of oregano and thyme oils, is known to exert potent antifungal activity against the pathogenic yeast Candida albicans. This monoterpene has been the subject of a considerable number of investigations that uncovered extensive pharmacological properties, including antifungal and antibacterial effects. However, its mechanism of action remains elusive. Here, we used integrative chemogenomic approaches, including genome-scale chemical-genetic and transcriptional profiling, to uncover the mechanism of action of carvacrol associated with its antifungal property. Our results clearly demonstrated that fungal cells require the unfolded protein response (UPR) signaling pathway to resist carvacrol. The mutants most sensitive to carvacrol in our genome-wide competitive fitness assay in the yeast Saccharomyces cerevisiae expressed mutations of the transcription factor Hac1 and the endonuclease Ire1, which is required for Hac1 activation by removing a nonconventional intron from the 3′ region of HAC1 mRNA. Confocal fluorescence live-cell imaging revealed that carvacrol affects the morphology and the integrity of the endoplasmic reticulum (ER). Transcriptional profiling of pathogenic yeast C. albicans cells treated with carvacrol demonstrated a bona fide UPR transcriptional signature. Ire1 activity detected by the splicing of HAC1 mRNA in C. albicans was activated by carvacrol. Furthermore, carvacrol was found to potentiate antifungal activity of the echinocandin antifungal caspofungin and UPR inducers dithiothreitol and tunicamycin against C. albicans. This comprehensive chemogenomic investigation demonstrated that carvacrol exerts its antifungal activity by altering ER integrity, leading to ER stress and the activation of the UPR to restore protein-folding homeostasis.

Tuning Hsf1 levels drives distinct fungal morphogenetic programs with depletion impairing Hsp90 function and overexpression expanding the target space

The capacity to respond to temperature fluctuations is critical for microorganisms to survive within mammalian hosts, and temperature modulates virulence traits of diverse pathogens. One key temperature-dependent virulence trait of the fungal pathogen Candida albicans is its ability to transition from yeast to filamentous growth, which is induced by environmental cues at host physiological temperature. A key regulator of temperature-dependent morphogenesis is the molecular chaperone Hsp90, which has complex functional relationships with the transcription factor Hsf1. Although Hsf1 controls global transcriptional remodeling in response to heat shock, its impact on morphogenesis remains unknown. Here, we establish an intriguing paradigm whereby overexpression or depletion of C. albicans HSF1 induces morphogenesis in the absence of external cues. HSF1 depletion compromises Hsp90 function, thereby driving filamentation. HSF1 overexpression does not impact Hsp90 function, but rather induces a dose-dependent expansion of Hsf1 direct targets that drives overexpression of positive regulators of filamentation, including Brg1 and Ume6, thereby bypassing the requirement for elevated temperature during morphogenesis. This work provides new insight into Hsf1-mediated environmentally contingent transcriptional control, implicates Hsf1 in regulation of a key virulence trait, and highlights fascinating biology whereby either overexpression or depletion of a single cellular regulator induces a profound developmental transition.

The Human Gut Microbial Metabolome Modulates Fungal Growth via the TOR Signaling Pathway

Candida albicans is a natural component of the human microbiota but also an opportunistic pathogen that causes life-threatening infections. The human gastrointestinal tract is the main reservoir of C. albicans, from where systemic infections originate as a consequence of the disruption of the intestinal mucosal barrier. Recent studies provided convincing evidence that overgrowth of C. albicans and other related species in the gut is predominantly associated with chronic intestinal inflammatory bowel diseases. Here, we showed, for the first time, the antagonistic interkingdom interactions between C. albicans and common intestinal commensal bacteria. From a therapeutic perspective, administering a defined bacterial community, such as the one described here with anti-Candida activity, could provide potential therapeutic protection against gastrointestinal inflammatory diseases. ABSTRACT Candida albicans is well known as a major human fungal pathogen, but it is also a permanent resident of healthy gastrointestinal tracts. Recent studies have shown that the human gut microbial metabolome represents an interesting source of bioactive molecules with a significant degree of chemical diversity. Some of these bioactive molecules may have useful antivirulence activities. For instance, intestinal bacterial species belonging to the Lachnospiraceae family were found to secrete molecules that attenuate Salmonella pathogenicity and repress the expression of virulence genes. Here, we have investigated whether the microbial gut metabolome (GM) contains molecules that might promote the commensal lifestyle and/or inhibit the expression of virulence of C. albicans in the intestine. We found that metabolites from human feces inhibited the growth of C. albicans and other opportunistic yeasts. A genetic screen in C. albicans suggested that TOR is the molecular target of the antifungal molecule(s) of the GM. In addition, we found that the GM metabolites inhibit both C. albicans hyphal growth and the invasion of human enterocytes. The antigrowth and antivirulence activities were partially recapitulated by secretions from Roseburia spp. and Bacteroides ovatus strains, respectively. This study demonstrates that the antimicrobial activity of the GM can be extended to a eukaryotic pathogen, C. albicans, illuminating the antagonistic interkingdom interactions between a fungus and intestinal commensal bacteria. IMPORTANCE Candida albicans is a natural component of the human microbiota but also an opportunistic pathogen that causes life-threatening infections. The human gastrointestinal tract is the main reservoir of C. albicans, from where systemic infections originate as a consequence of the disruption of the intestinal mucosal barrier. Recent studies provided convincing evidence that overgrowth of C. albicans and other related species in the gut is predominantly associated with chronic intestinal inflammatory bowel diseases. Here, we showed, for the first time, the antagonistic interkingdom interactions between C. albicans and common intestinal commensal bacteria. From a therapeutic perspective, administering a defined bacterial community, such as the one described here with anti-Candida activity, could provide potential therapeutic protection against gastrointestinal inflammatory diseases.

The Genomic Landscape of the Fungus-Specific SWI/SNF Complex Subunit, Snf6, in Candida albicans

Candida albicans is a natural component of the human microbiota but also an opportunistic pathogen that causes life-threatening infections in immunosuppressed patients. Current therapeutics include a limited number of molecules that suffer from limitations, including growing clinical resistance and toxicity. New molecules are being clinically investigated; however, the majority of these potential antifungals target the same processes as do the standard antifungals and might confront the same problems of toxicity and loss of efficiency due to the common resistance mechanisms. Here, we characterized the role of Snf6, a fungus-specific subunit of the chromatin-remodeling complex SWI/SNF. Our genomic and phenotypic data demonstrated a central role of Snf6 in biological processes that are critical for a fungal pathogen to colonize its host and cause disease, suggesting Snf6 as a possible antifungal target. ABSTRACT SWI/SNF is an ATP-dependent chromatin-remodeling complex that is required for the regulation of gene expression in eukaryotes. While most of the fungal SWI/SNF components are evolutionarily conserved with those of the metazoan SWI/SNF, subunits such as Snf6 are specific to certain fungi and thus represent potential antifungal targets. We have characterized the role of the Snf6 protein in Candida albicans. Our data showed that although there was low conservation of its protein sequence with other fungal orthologs, Snf6 was copurified with bona fide SWI/SNF complex subunits. The role of Snf6 in C. albicans was investigated by determining its genome-wide occupancy using chromatin immunoprecipitation coupled to tiling arrays in addition to transcriptional profiling of the snf6 conditional mutant. Snf6 directs targets that were enriched in functions related to carbohydrate and amino acid metabolic circuits, to cellular transport, and to heat stress responses. Under hypha-promoting conditions, Snf6 expanded its set of targets to include promoters of genes related to respiration, ribosome biogenesis, mating, and vesicle transport. In accordance with the genomic occupancy data, an snf6 doxycycline-repressible mutant exhibited growth defects in response to heat stress and also when grown in the presence of different fermentable and nonfermentable carbon sources. Snf6 was also required to differentiate invasive hyphae in response to different cues. This study represents the first comprehensive characterization, at the genomic level, of the role of SWI/SNF in the pathogenic yeast C. albicans and uncovers functions that are essential for fungal morphogenesis and metabolic flexibility. IMPORTANCE Candida albicans is a natural component of the human microbiota but also an opportunistic pathogen that causes life-threatening infections in immunosuppressed patients. Current therapeutics include a limited number of molecules that suffer from limitations, including growing clinical resistance and toxicity. New molecules are being clinically investigated; however, the majority of these potential antifungals target the same processes as do the standard antifungals and might confront the same problems of toxicity and loss of efficiency due to the common resistance mechanisms. Here, we characterized the role of Snf6, a fungus-specific subunit of the chromatin-remodeling complex SWI/SNF. Our genomic and phenotypic data demonstrated a central role of Snf6 in biological processes that are critical for a fungal pathogen to colonize its host and cause disease, suggesting Snf6 as a possible antifungal target.

pH-Dependant Antifungal Activity of Valproic Acid against the Human Fungal Pathogen Candida albicans

Current antifungal drugs suffer from limitations including toxicity, the emergence of resistance and decreased efficacy at low pH that are typical of human vaginal surfaces. Here, we have shown that the antipsychotic drug valproic acid (VPA) exhibited a strong antifungal activity against both sensitive and resistant Candida albicans in pH condition similar to that encountered in vagina. VPA exerted a strong anti-biofilm activity and attenuated damage of vaginal epithelial cells caused by C. albicans. We also showed that VPA synergizes with the allylamine antifungal, Terbinafine. We undertook a chemogenetic screen to delineate biological processes that underlies VPA-sensitivity in C. albicans and found that vacuole-related genes were required to tolerate VPA. Confocal fluorescence live-cell imaging revealed that VPA alters vacuole integrity and support a model where alteration of vacuoles contributes to the antifungal activity. Taken together, this study suggests that VPA could be used as an effective antifungal against vulvovaginal candidiasis.