Regulation of copper uptake by the SWI/SNF chromatin remodeling complex in Candida albicans affects susceptibility to antifungal and oxidative stresses under hypoxia.

Candida albicans is a human colonizer and also an opportunistic yeast occupying different niches that are mostly hypoxic. While hypoxia is the prevalent condition within the host, the machinery that integrates oxygen status to tune the fitness of fungal pathogens remains poorly characterized. Here, we uncovered that Snf5, a subunit of the chromatin remodeling complex SWI/SNF, is required to tolerate antifungal stress particularly under hypoxia. RNA-seq profiling of snf5 mutant exposed to amphotericin B and fluconazole under hypoxic conditions uncovered a signature that is reminiscent of copper (Cu) starvation. We found that under hypoxic and Cu-starved environments, Snf5 is critical for preserving Cu homeostasis and the transcriptional modulation of the Cu regulon. Furthermore, snf5 exhibits elevated levels of reactive oxygen species and an increased sensitivity to oxidative stress principally under hypoxia. Supplementing growth medium with Cu or increasing gene dosage of the Cu transporter CTR1 alleviated snf5 growth defect and attenuated reactive oxygen species levels in response to antifungal challenge. Genetic interaction analysis suggests that Snf5 and the bona fide Cu homeostasis regulator Mac1 function in separate pathways. Together, our data underlined a unique role of SWI/SNF complex as a potent regulator of Cu metabolism and antifungal stress under hypoxia.

A novel genetic circuitry governing hypoxic metabolic flexibility, commensalism and virulence in the fungal pathogen Candida albicans.

Inside the human host, the pathogenic yeast Candida albicans colonizes predominantly oxygen-poor niches such as the gastrointestinal and vaginal tracts, but also oxygen-rich environments such as cutaneous epithelial cells and oral mucosa. This suppleness requires an effective mechanism to reversibly reprogram the primary metabolism in response to oxygen variation. Here, we have uncovered that Snf5, a subunit of SWI/SNF chromatin remodeling complex, is a major transcriptional regulator that links oxygen status to the metabolic capacity of C. albicans. Snf5 and other subunits of SWI/SNF complex were required to activate genes of carbon utilization and other carbohydrates related process specifically under hypoxia. snf5 mutant exhibited an altered metabolome reflecting that SWI/SNF plays an essential role in maintaining metabolic homeostasis and carbon flux in C. albicans under hypoxia. Snf5 was necessary to activate the transcriptional program linked to both commensal and invasive growth. Accordingly, snf5 was unable to maintain its growth in the stomach, the cecum and the colon of mice. snf5 was also avirulent as it was unable to invade Galleria larvae or to cause damage to human enterocytes and murine macrophages. Among candidates of signaling pathways in which Snf5 might operate, phenotypic analysis revealed that mutants of Ras1-cAMP-PKA pathway, as well as mutants of Yak1 and Yck2 kinases exhibited a similar carbon flexibility phenotype as did snf5 under hypoxia. Genetic interaction analysis indicated that the adenylate cyclase Cyr1, a key component of the Ras1-cAMP pathway interacted genetically with Snf5. Our study yielded new insight into the oxygen-sensitive regulatory circuit that control metabolic flexibility, stress, commensalism and virulence in C. albicans.

Repurposing the anthelminthic salicylanilide oxyclozanide against susceptible and clinical resistant Candida albicans strains.

Current antifungal drugs suffer from limitations including toxicity, adverse interactions with other commonly prescribed drugs, and the emergence of resistant strains. Here, we repurposed the anthelmintic oxyclozanide as a potent antifungal agent against both sensitive and resistant clinical isolates of Candida albicans, as well as other human opportunistic fungi. Antifungal activity of oxyclozanide was enhanced when C. albicans grew in nonfermentable carbon sources. Our data support a mechanism of action where oxyclozanide uncoupled the mitochondrial electron transport from oxidative phosphorylation and perturbed the mitochondrial membrane potential.

Transcriptional Control of Hypoxic Hyphal Growth in the Fungal Pathogen Candida albicans.

The ability of Candida albicans, an important human fungal pathogen, to develop filamentous forms is a crucial determinant for host invasion and virulence. While hypoxia is one of the predominant host cues that promote C. albicans filamentous growth, the regulatory circuits that link oxygen availability to filamentation remain poorly characterized. We have undertaken a genetic screen and identified the two transcription factors Ahr1 and Tye7 as central regulators of the hypoxic filamentation. Both ahr1 and tye7 mutants exhibited a hyperfilamentous phenotype specifically under an oxygen-depleted environment suggesting that these transcription factors act as negative regulators of hypoxic filamentation. By combining microarray and ChIP-chip analyses, we have characterized the set of genes that are directly modulated by Ahr1 and Tye7. We found that both Ahr1 and Tye7 modulate a distinct set of genes and biological processes. Our genetic epistasis analysis supports our genomic finding and suggests that Ahr1 and Tye7 act independently to modulate hyphal growth in response to hypoxia. Furthermore, our genetic interaction experiments uncovered that Ahr1 and Tye7 repress the hypoxic filamentation via the Efg1 and Ras1/Cyr1 pathways, respectively. This study yielded a new and an unprecedented insight into the oxygen-sensitive regulatory circuit that control morphogenesis in a fungal pathogen.

Metabolic Reprogramming in the Opportunistic Yeast Candida albicans in Response to Hypoxia.

Hypoxia is the predominant condition that the human opportunistic fungus Candida albicans encounters in the majority of the colonized niches within the host. So far, the impact of such a condition on the overall metabolism of this important human-pathogenic yeast has not been investigated. Here, we have undertaken a time-resolved metabolomics analysis to uncover the metabolic landscape of fungal cells experiencing hypoxia. Our data showed a dynamic reprogramming of many fundamental metabolic pathways, such as glycolysis, the pentose phosphate pathway, and different metabolic routes related to fungal cell wall biogenesis. The C. albicans lipidome was highly affected by oxygen depletion, with an increased level of free fatty acids and biochemical intermediates of membrane lipids, including phospholipids, lysophospholipids, sphingolipids, and mevalonate. The depletion of oxygen-dependent lipids such as ergosterol or phosphatidylcholine with longer and polyunsaturated lateral fatty acid chains was observed only at the later hypoxic time point (180 min). Transcriptomics data supported the main metabolic response to hypoxia when matched to our metabolomic profiles. The hypoxic metabolome reflected different physiological alterations of the cell wall and plasma membrane of C. albicans under an oxygen-limiting environment that were confirmed by different approaches. This study provided a framework for future in vivo investigations to examine relevant hypoxic metabolic trajectories in fungal virulence and fitness within the host.IMPORTANCE A critical aspect of cell fitness is the ability to sense and adapt to variations in oxygen levels in their local environment. Candida albicans is an opportunistic yeast that is the most prevalent human fungal pathogen. While hypoxia is the predominant condition that C. albicans encounters in most of its niches, its impact on fungal metabolism remains unexplored so far. Here, we provided a detailed landscape of the C. albicans metabolome that emphasized the importance of many metabolic routes for the adaptation of this yeast to oxygen depletion. The fungal hypoxic metabolome identified in this work provides a framework for future investigations to assess the contribution of relevant metabolic pathways in the fitness of C. albicans and other human eukaryotic pathogens with similar colonized human niches. As hypoxia is present at most of the fungal infection foci in the host, hypoxic metabolic pathways are thus an attractive target for antifungal therapy.

A phenotypic small-molecule screen identifies halogenated salicylanilides as inhibitors of fungal morphogenesis, biofilm formation and host cell invasion.

A poorly exploited paradigm in the antimicrobial therapy field is to target virulence traits for drug development. In contrast to target-focused approaches, antivirulence phenotypic screens enable identification of bioactive molecules that induce a desirable biological readout without making a priori assumption about the cellular target. Here, we screened a chemical library of 678 small molecules against the invasive hyphal growth of the human opportunistic yeast Candida albicans. We found that a halogenated salicylanilide (N1-(3,5-dichlorophenyl)-5-chloro-2-hydroxybenzamide) and one of its analogs, Niclosamide, an FDA-approved anthelmintic in humans, exhibited both antifilamentation and antibiofilm activities against C. albicans and the multi-resistant yeast C. auris. The antivirulence activity of halogenated salicylanilides were also expanded to C. albicans resistant strains with different resistance mechanisms. We also found that Niclosamide protected the intestinal epithelial cells against invasion by C. albicans. Transcriptional profiling of C. albicans challenged with Niclosamide exhibited a signature that is characteristic of the mitochondria-to-nucleus retrograde response. Our chemogenomic analysis showed that halogenated salicylanilides compromise the potential-dependant mitochondrial protein translocon machinery. Given the fact that the safety of Niclosamide is well established in humans, this molecule could represent the first clinically approved antivirulence agent against a pathogenic fungus.

Validation of a predictive model for the growth of chalk yeasts on bread.

The present study focused on the effects of temperature, T, and water activity, aw, on the growth of Hyphopichia burtonii, Pichia anomala, and Saccharomycopsis fibuligera on Sabouraud Agar Medium. Cardinal values were estimated by means of cardinal models with inflection. All the yeasts were xerophilic, and they exhibited growth at 0.85 aw. The combined effects of T, aw, and pH on the growth of these species were described by the gamma-concept and validated on bread in the range of 15-25 °C, 0.91-0.97 aw, and pH 4.6-6.8. The optimum growth rates on bread were 2.88, 0.259, and 1.06 mm/day for H. burtonii, P. anomala, and S. fibuligera, respectively. The optimal growth rate of S. fibuligera on bread was about 2 fold that obtained on Sabouraud. Due to reproduction by budding, P. anomala exhibited low growth on Sabouraud and bread. However, this species is of major concern in the baker's industry because of the production of ethyl acetate in bread.

A model for the effect of pH on the growth of chalk yeasts.

Hyphopichia burtonii, Pichia anomala, and Saccharomycopsis fibuligera were isolated from spoiled packaged sliced bread. These chalk yeasts were characterized by a wide range of pH for which growth was almost optimum. Thus, the curve growth vs pH exhibited plateau and sharp profiles close to the minimum and the maximum pH. This study described a chalk yeast model (CYM) for the effect of pH derived from a new germination model for fungi (Dantigny, P., Nanguy, S., P.-M., Judet-Correia, D., and Bensoussan, M. 2011, International Journal of Food Microbiology, 146, 176-181). The CYM is asymmetric, versatile, based on parameters with biological significance, and compatible with the gamma concept. The CYM was compared to the cardinal pH model (CPM) which is widely used to describe the effect of pH on microbial growth. The CYM exhibited RMSE values two fold less than those obtained with the CPM for H. burtonii, and S. fibuligera for which plateaus were clearly observed. For P. anomala, the plateau was less obvious, but the RMSE value obtained with the CYM was similar to that found with the CPM. The CYM could extend its use to represent the effect of pH on mold growth.

Effect of inoculum size and water activity on the time to visible growth of Penicillium chrysogenum colony.

In order to assess the effect of the inoculum size on the time to visible growth for Penicillium chrysogenum, the correlation described by González et al. (González, H.H.L., Resnik, S.L., Vaamonde, G., 1987. Influence of inoculum size on growth rate and lag phase of fungi isolate from Argentine corn. International Journal of Food Microbiology 4, 111-117) was compared to the model introduced by Gougouli et al. (Gougouli, M., Kalantzi, K., Beletsiotis, E., Koutsoumanis, K.P., 2011. Development and application of predictive models for fungal growth as tools to improve quality control in yogurt production. Food Microbiology 28, 1453-1462). Based on the regression coefficient, the latter model performed better than the former one to fit the data obtained for P. chrysogenum grown on Potato Dextrose Agar at 25 °C. Inoculum sizes in the range 10(1)-10(5) spores were tested at 0.930, 0.950, 0.970, and 0.995 aw. By extrapolation of the straight line, the model of Gougouli et al. (2011) provided accurate estimations of the time to visible growth for a single spore inoculum, tvg (N=1). In order to avoid experiments at reduced water activities, the influence of water activity on the model parameters, and on the ratio tvg (N=1) over the germination time was assessed.