Christophe d’Enfert

Stage-specific gene expression of Candida albicans in human blood

The pathogenic fungus Candida albicans commonly causes mucosal surface infections. In immunocompromised patients, C. albicans may penetrate into deeper tissue, enter the bloodstream and disseminate within the host causing life‐threatening systemic infections. In order to elucidate how C. albicans responds to the challenge of a blood environment, we analysed the transcription profile of C. albicans cells exposed to human blood using genomic arrays and a cDNA subtraction protocol. By combining data obtained with these two methods, we were able to identify unique sets of different fungal genes specifically expressed at different stages of this model that mimics bloodstream infections. By removing host cells and incubation in plasma, we were also able to identify several genes in which the expression level was significantly influenced by the presence of these cells. Differentially expressed genes included those that are involved in the general stress response, antioxidative response, glyoxylate cycle as well as putative virulence attributes. These data point to possible mechanisms by which C. albicans ensures survival in the hostile environment of the blood and how the fungus may escape the bloodstream as an essential step in its systemic dissemination.

cAMP and ras signalling independently control spore germination in the filamentous fungus Aspergillus nidulans

The role of cAMP signalling during germination of asexual spores (conidia) of the filamentous fungus Aspergillus nidulans was investigated. A. nidulans strains defective for adenylate cyclase (CyaA) or for the functionally overlapping cAMP‐dependent protein kinase (PkaA) and newly characterized SchA protein kinase, homologous to Saccharomyces cerevisiae Sch9, show altered trehalose mobilization and kinetics of germ tube outgrowth, in addition to other defects in colony formation. cAMP‐dependent trehalose breakdown is triggered by the addition of a carbon source independently of further catabolism, suggesting that cAMP signalling controls early events of conidial germination in response to carbon source sensing. Additional results suggest that cAMP has targets other than PkaA and SchA and that PkaA retains activity in the absence of cAMP. Conversely, PkaA regulates cAMP levels in A. nidulans because these are elevated by ≈ 250‐fold in a strain that lacks PkaA. Furthermore, analysis of mutant strains impaired in both adenylate cyclase and RasA GTPase previously implicated in the control of A. nidulans spore germination suggested that RasA and cAMP signalling proceed independently during germination in A. nidulans.

The Yak1p kinase controls expression of adhesins and biofilm formation in Candida glabrata in a Sir4p‐dependent pathway

Biofilm is the predominant type of microbial development in natural environments, and potentially represents a major form of resistance or source of recurrence during host infection. Although a large number of studies have focussed on the genetics of bacterial biofilm formation, very little is known about the genes involved in this type of growth in fungi. A genetic screen for Candida glabrata Biofilm mutants was performed using a 96‐well plate model of biofilm formation. Study of the isolated mutant strains allowed the identification of four genes involved in biofilm formation (RIF1, SIR4, EPA6 and YAK1). Epa6p is a newly identified adhesin required for biofilm formation in this pathogenic yeast. EPA6 and its close paralogue EPA7 are located in subtelomeric regions and their transcription is regulated by Sir4p and Rif1p, two proteins involved in subtelomeric silencing. Biofilm growth conditions induce the transcription of EPA6 and EPA7: this is dependent on the presence of an intact subtelomeric silencing machinery and is independent of the Mpk1p signalling pathway. Finally, the kinase Yak1p is required for expression of both adhesin genes and acts through a subtelomeric silencing machinery‐dependent pathway.

Candida albicans biofilms: building a heterogeneous, drug-tolerant environment.

Fungi are able to form biofilms on medical implants, causing serious infections. A better understanding of fungal biofilm formation is necessary to develop tools for detection or prevention and to identify new antifungal strategies. This review explores recent advances in the characterization at the molecular level of fungal biofilms, especially those formed by the yeast Candida albicans: the identification of complex transcriptional networks that control their formation; the pivotal role of the extracellular matrix in biofilm antifungal tolerance; and the knowledge gained on the physiology of biofilm cells and heterogeneity within these communities. These findings may help develop new, targeted therapeutic strategies.

Glycerol dehydrogenase, encoded by gldB is essential for osmotolerance in Aspergillus nidulans

We have characterized the Aspergillus nidulans gldB gene encoding a NADP+‐dependent glycerol dehydrogenase. A basal expression level was observed for gldB, which increased significantly under conditions of hyper‐osmotic shock (1 M NaCl). Growth of strains in which gldB was disrupted was severely reduced on plates containing 1% glucose and 1 M NaCl, but these strains were able to grow on plates containing 1 M NaCl and 1% glycerol, arabitol, mannitol or erythritol. Uptake of these polyols compensated for the inability of the gldB disruptants to produce glycerol. Presence of 1% glucose in these plates prevented growth restoration by all the polyols tested with the exemption of glycerol, indicating that uptake of mannitol, arabitol and erythritol is subject to glucose repression, whereas uptake of glycerol is significantly less or not repressed. No intracellular glycerol dehydrogenase activity could be detected in the gldB disruption strains. Intracellular glycerol levels in these strains were strongly decreased compared to wild type, whereas intracellular mannitol, erythritol and arabitol levels were increased. Conidia of the gldB disruption strain did not accumulate glycerol upon germination in glucose media with or without 1 M NaCl and germ tube emergence was significantly delayed in this strain in the presence of 1 M NaCl in comparison to the wild type. These data indicate that gldB is essential for osmotolerance in A. nidulans and that the pathways for glycerol biosynthesis under osmotic stress differ between yeast and filamentous fungi.

Hidden killers: persistence of opportunistic fungal pathogens in the human host

Opportunistic fungal pathogens are responsible for life-threatening systemic infections in immunocompromized individuals. Yet, they are also able to persist in immunocompetent individuals through different strategies. This review explores recent advances in our understanding of several survival strategies: the establishment of a commensal relationship between yeast of the genus Candida and the host; the formation of biofilms that allow microbes in these communities to be protected from chemical and cellular attacks; and the persistence of airborne pathogens within macrophages following primary infection. While research has concentrated on deciphering virulence factors of pathogenic fungi, additional understanding of how fungal pathogens persist in healthy hosts might help us design new strategies to prevent the transition from harmless interactions to devastating infections.

Loss of heterozygosity in commensal isolates of the asexual diploid yeast Candida albicans.

Candida albicans is a commensal and the most frequent fungal pathogen of humans. One mechanism of genetic variation in this diploid asexual yeast involves loss of heterozygosity (LOH). LOH events occur upon infection and contribute to the acquisition of antifungal resistance in patients. In contrast, little is known about the nature and extent of LOH events during commensalism. Using a combination of single nucleotide polymorphism typing, positional transcript profiling and karyotyping, we have characterized related C. albicans commensal isolates that differ by LOH events. Most of these LOH events encompassed the entirety of the chromosome or a large region extending to the telomere, suggesting chromosome loss or mitotic recombination/break-induced replication events, respectively. They were frequently accompanied by karyotype alterations such as chromosome length polymorphism and copy number variations at other chromosomes. These results demonstrate the high plasticity of the C. albicans genome during commensalism.

A Versatile Overexpression Strategy in the Pathogenic Yeast Candida albicans: Identification of Regulators of Morphogenesis and Fitness

Candida albicans is the most frequently encountered human fungal pathogen, causing both superficial infections and life-threatening systemic diseases. Functional genomic studies performed in this organism have mainly used knock-out mutants and extensive collections of overexpression mutants are still lacking. Here, we report the development of a first generation C. albicans ORFeome, the improvement of overexpression systems and the construction of two new libraries of C. albicans strains overexpressing genes for components of signaling networks, in particular protein kinases, protein phosphatases and transcription factors. As a proof of concept, we screened these collections for genes whose overexpression impacts morphogenesis or growth rates in C. albicans. Our screens identified genes previously described for their role in these biological processes, demonstrating the functionality of our strategy, as well as genes that have not been previously associated to these processes. This article emphasizes the potential of systematic overexpression strategies to improve our knowledge of regulatory networks in C. albicans. The C. albicans plasmid and strain collections described here are available at the Fungal Genetics Stock Center. Their extension to a genome-wide scale will represent important resources for the C. albicans community.

Gymnemic Acids Inhibit Hyphal Growth and Virulence in Candida albicans

Candida albicans is an opportunistic and polymorphic fungal pathogen that causes mucosal, disseminated and invasive infections in humans. Transition from the yeast form to the hyphal form is one of the key virulence factors in C. albicans contributing to macrophage evasion, tissue invasion and biofilm formation. Nontoxic small molecules that inhibit C. albicans yeast-to-hypha conversion and hyphal growth could represent a valuable source for understanding pathogenic fungal morphogenesis, identifying drug targets and serving as templates for the development of novel antifungal agents. Here, we have identified the triterpenoid saponin family of gymnemic acids (GAs) as inhibitor of C. albicans morphogenesis. GAs were isolated and purified from Gymnema sylvestre leaves, the Ayurvedic traditional medicinal plant used to treat diabetes. Purified GAs had no effect on the growth and viability of C. albicans yeast cells but inhibited its yeast-to-hypha conversion under several hypha-inducing conditions, including the presence of serum. Moreover, GAs promoted the conversion of C. albicans hyphae into yeast cells under hypha inducing conditions. They also inhibited conidial germination and hyphal growth of Aspergillus sp. Finally, GAs inhibited the formation of invasive hyphae from C. albicans-infected Caenorhabditis elegans worms and rescued them from killing by C. albicans. Hence, GAs could be useful for various antifungal applications due to their traditional use in herbal medicine.

Ultraviolet-C Light for Treatment of Candida albicans Burn Infection in Mice

Burn patients are at high risk of invasive fungal infections, which are a leading cause of morbidity, mortality, and related expense exacerbated by the emergence of drug resistant fungal strains. In this study, we investigated the use of UVC light (254 nm) for the treatment of yeast Candida albicans infection in mouse third degree burns. In vitro studies demonstrated that UVC could selectively kill the pathogenic C. albicans compared with a normal mouse keratinocyte cell line in a light exposure dependent manner. A mouse model of chronic C. albicans infection in non‐lethal third degree burns was developed. The C. albicans strain was stably transformed with a version of the Gaussia princeps luciferase gene that allowed real‐time bioluminescence imaging of the progression of C. albicans infection. UVC treatment with a single exposure carried out on day 0 (30 min postinfection) gave an average 2.16‐log10‐unit (99.2%) loss of fungal luminescence when 2.92 J cm−2 UVC had been delivered, while UVC 24 h postinfection gave 1.94‐log10‐unit (95.8%) reduction of fungal luminescence after 6.48 J cm−2. Statistical analysis demonstrated that UVC treatment carried out on both day 0 and day 1 significantly reduced the fungal bioburden of infected burns. UVC was found to be superior to a topical antifungal drug, nystatin cream. UVC was tested on normal mouse skin and no gross damage was observed 24 h after 6.48 J cm−2. DNA lesions (cyclobutane pyrimidine dimers) were observed by immunofluorescence in normal mouse skin immediately after a 6.48 J cm−2 UVC exposure, but the lesions were extensively repaired at 24 h after UVC exposure.