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Nrg1 is a repressor of hypha formation and hypha-associated gene expression in the fungal pathogen Candida albicans. It has been well studied in the genetic background of the type strain SC5314. Here, we tested Nrg1 function in four other diverse clinical isolates through an analysis of nrg1 Δ/Δ mutants, with SC5314 included as a control. In three strains, nrg1 Δ/Δ mutants unexpectedly produced aberrant hyphae under inducing conditions, as assayed by microscopic observation and endothelial cell damage. The nrg1 Δ/Δ mutant of strain P57055 had the most severe defect. We examined gene expression features under hypha-inducing conditions by RNA-sequencing (RNA-Seq) for the SC5314 and P57055 backgrounds. The SC5314 nrg1 Δ/Δ mutant expressed six hypha-associated genes at reduced levels compared with wild-type SC5314. The P57055 nrg1 Δ/Δ mutant expressed 17 hypha-associated genes at reduced levels compared with wild-type P57055, including IRF1 , RAS2 , and ECE1 . These findings indicate that Nrg1 has a positive role in hypha-associated gene expression and that this role is magnified in strain P57055. Remarkably, the same hypha-associated genes affected by the nrg1 Δ/Δ mutation in strain P57055 were also naturally expressed at lower levels in wild-type P57055 than those in wild-type SC5314. Our results suggest that strain P57055 is defective in a pathway that acts in parallel with Nrg1 to upregulate the expression of several hypha-associated genes. IMPORTANCE Hypha formation is a central virulence trait of the fungal pathogen Candida albicans. Control of hypha formation has been studied in detail in the type strain but not in other diverse C. albicans clinical isolates. Here, we show that the hyphal repressor Nrg1 has an unexpected positive role in hypha formation and hypha-associated gene expression, as revealed by the sensitized P57055 strain background. Our findings indicate that reliance on a single type strain limits understanding of gene function and illustrate that strain diversity is a valuable resource for C. albicans molecular genetic analysis.

The use of antibiotics can disrupt the body's natural balance and increase the susteptibility of patients towards fungal infections. Candida albicans is a dimorphic opportunistic fungal pathogen with niches similar to those of bacteria. Our aim was to study the interaction between this pathogen and bacteria to facilitate the control of C. albicans infection. Alpha-hemolysin (Hla), a protein secreted from Staphylococcus aureus, causes cell wall damage and impedes the yeast-hyphae transition in C. albicans. Mechanistically, Hla stimulation triggered the formation of reactive oxygen species that damaged the cell wall and mitochondria of C. albicans. The cell cycle was arrested in the G0/G1 phase, CDC42 was downregulated, and Ywp1 was upregulated, disrupting yeast hyphae switching. Subsequently, hyphae development was inhibited. In mouse models, C. albicans pretreated with Hla reduced the C. albicans burden in skin and vaginal mucosal infections, suggesting that S. aureus Hla can inhibit hyphal development and reduce the pathogenicity of candidiasis in vivo., (© 2023. The Author(s), under exclusive licence to Microbiological Society of Korea.)

Engineered conditional gene expression is used in appraisal of gene function and pathway relationships. For pathogens like the fungus Candida albicans, conditional expression systems are most useful if they are active in the infection environment and if they can be utilized in multiple clinical isolates. Here, we describe such a system. It employs the RBT5 promoter and can be implemented with a few PCRs. We validated the system with RBT5 promoter fusions to two genes that promote filamentation and polarized growth, UME6 and HGC1 , and with efg1 Δ/Δ mutants, which are defective in an activator of filamentous growth. An RBT5 promoter fusion to either gene enabled filamentous growth of an efg1 Δ/Δ mutant of strain SC5314 in iron-limited media, including RPMI with serum and yeast extract-peptone-dextrose with bathophenanthrolinedisulfonic acid. The RBT5-UME6 fusion promoted filamentation of efg1 Δ/Δ mutants in RPMI with serum of four other clinical C. albicans isolates as well. In a mouse model of disseminated candidiasis, the RBT5-UME6 fusion promoted filamentation of the SC5314 efg1 Δ/Δ mutant in kidney tissue, an indication that the RBT5 promoter is active in the iron-limited host environment. The RBT5 promoter expands the conditional expression toolkit for C. albicans genetics. IMPORTANCE Genetic strategies have been vital for mechanistic analysis of biological processes. Here, we describe a genetic tool for the fungal pathogen Candida albicans.

Efficient assimilation of alternative carbon sources in glucose-limited host niches is critical for colonization of Candida albicans, a commensal yeast that frequently causes opportunistic infection in human. C. albicans evolved mechanistically to regulate alternative carbon assimilation for the promotion of fungal growth and commensalism in mammalian hosts. However, this highly adaptive mechanism that C. albicans employs to cope with alternative carbon assimilation has yet to be clearly understood. Here we identified a novel role of C. albicans mitochondrial complex I (CI) in regulating assimilation of alternative carbon sources such as mannitol. Our data demonstrate that CI dysfunction by deleting the subunit Nuo2 decreases the level of NAD+, downregulates the NAD+-dependent mannitol dehydrogenase activity, and consequently inhibits hyphal growth and biofilm formation in conditions when the carbon source is mannitol, but not fermentative sugars like glucose. Mannitol-dependent morphogenesis is controlled by a ROS-induced signaling pathway involving Hog1 activation and Brg1 repression. In vivo studies show that nuo2Δ/Δ mutant cells are severely compromised in gastrointestinal colonization and the defect can be rescued by a glucose-rich diet. Thus, our findings unravel a mechanism by which C. albicans regulates carbon flexibility and commensalism. Alternative carbon assimilation might represent a fitness advantage for commensal fungi in successful colonization of host niches.