Recurrent vulvovaginal candidiasis (RVVC) affects women worldwide and has far-reaching implications for a patient's quality of life. For decades, maintenance treatment using the azole antifungal fluconazole was the preferred treatment. Although efficient in controlling the symptoms, the development of azole resistance and high rates of recurrence after therapy cessation have emerged as significant limitations. Nevertheless, persistent efforts have delivered novel treatment options. Oteseconazole (VT-1161), marketed as VIVJOA, is an oral, tetrazole antifungal with unprecedented specificity toward the fungal lanosterol 14α-demethylase. We reviewed literature data on oteseconazole with a focus on the management of RVVC. Therapeutic options for RVVC are limited, and novel, innovative approaches are needed to treat this debilitating condition. These therapies need to be well-tolerated and prevent RVVC recurrence. The available clinical data show excellent safety and efficacy, with an unprecedentedly low recurrence rate. However, we believe health-care providers should be mindful to monitor for the development of resistance, as this may result in treatment failure. Further, the availability and cost may, like for most novel drugs, affect the widespread clinical implementation of VIVJOA. Altogether, we are convinced that VIVJOA is a significant advance in RVVC management.

The incidence of non- albicans Candida infections has witnessed a substantial rise in recent decades. Candida glabrata (Nakaseomyces glabratus ), an opportunistic human fungal pathogen, is accountable for both superficial mucosal and life-threatening bloodstream infections, particularly in immunocompromised individuals. Distinguished by its remarkable resilience to environmental stressors, C. glabrata exhibits intrinsic tolerance to azoles and a high propensity to swiftly develop azole resistance during treatment. The molecular mechanism for the high tolerance is not fully understood. In this work, we investigated the possible role of trehalose in this tolerance. We generated mutants in the C. glabrata TPS1 , TPS2 , and NTH1 genes, encoding trehalose 6-phosphate synthase (Tps1), trehalose 6-phosphate phosphatase (Tps2), and neutral trehalase (Nth1), respectively. As expected, the tps1∆ strain cannot grow on glucose. The tps2 ∆ strain demonstrated diminished trehalose accumulation and very high levels of trehalose 6-phosphate (T6P), the biosynthetic intermediate, in comparison to the wild-type (WT) strain. Whereas these higher T6P levels did not affect growth, the lower trehalose levels clearly resulted in lower environmental stress tolerance and a lower susceptibility to fluconazole. More interestingly, the tps2∆ strain completely lost tolerance to fluconazole, characterized by the absence of slow growth at supra-MIC concentrations of this drug. All these phenotypes are reversed in the nth1 ∆ strain, which accumulates high levels of trehalose. Our findings underscore the role of trehalose in enabling tolerance toward fluconazole in C. glabrata . We further show that the change in tolerance is a result of the effect that trehalose has on the sterol pattern in the cell.

Understanding the spatial and temporal organization of proteins of interest is key to unraveling cellular processes and identifying novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans , and resistance mechanisms against these therapeutic agents are rapidly acquired.

The human fungal pathogen, Candida albicans , is very proficient at several classical virulence factors such as morphogenesis, adhesion, biofilm formation and immune evasion through β-glucan masking. The protein kinase A (PKA) pathway is involved in both morphogenesis and β-glucan masking. Several signals converge onto the PKA pathway, but it contains only a single upstream G-protein coupled receptor, Gpr1. We identified specific residues within the N-terminal tail of Gpr1 that are required for methionine-induced morphogenesis through Tpk2. Furthermore, we observe that Gpr1-Gpa2 has an active role in exposing glucans. Even though Gpr1 is required for survival when C. albicans is challenged with macrophages, specifically disrupting morphogenesis did not attenuate this survival. Additionally, constitutive β-glucan masking did not improve C. albicans survival rates in the macrophage assay. Taken together, this indicates that Gpr1 may regulate additional mechanisms, possibly through glutamine 461, which are crucial in a macrophage context. Significance Statement: Candida albicans is a human fungal pathogen mostly present as a commensal in the gastrointestinal tract. It can rapidly adapt to its everchanging environment through continuous monitoring of extracellular signals. These extracellular signals include methionine and lactate which induce respectively morphogenesis and β-glucan masking through the G-protein coupled receptor, Gpr1. Through a mutagenic approach we different amino acids of the receptor sense methionine and/or lactate but we show that Gpr1 may have an additional ligand that affect its survival in macrophages.

Fungal infections are combatted using three main classes of antifungals, of which the azoles, considered to be fungistatic, are the most widely used. Slow growth of Candida albicans at supra-minimal inhibitory concentrations (MIC) of fluconazole (FLC), termed tolerance, is routinely observed. A combination therapy resulting in the eradication of this fungistatic character would be a valid therapeutic strategy, and indeed, the synergistic combination of the antibiotic doxycycline and FLC has such an effect. We hypothesized that iron-requiring mitochondrial functions may be the targets of the synergistic combination. The proteome enriched for mitochondria obtained from FLC + Fe-treated cells hinted that iron alleviated the FLC stress and that intracellular iron homeostasis, more specifically the vacuolar iron exporter Smf3, might be a key factor during FLC treatment, as its expression was induced. Moreover, a ROS assay revealed that a smf3Δ/Δ strain treated with FLC accumulated ROS to a similar extent as that displayed by the WT undergoing a FLC+DOX combination treatment. Thus, deletion of SMF3 mimics the addition of doxycycline in wild-type cells. The ROS accumulation can be attenuated through overexpression of the mitochondrial superoxide dismutase SOD2, and this restored the synergy between DOX and FLC in the smf3Δ/Δ background. ROS accumulation, in part through altered iron availability from the vacuolar storage pool, is thus the molecular mechanism underlying the synergy between doxycycline and FLC. Furthermore, no effect on either cidality or tolerance was observed in the smf3Δ/Δ strain, highlighting that synergy is not necessarily an indication of cidal therapies. IMPORTANCE: Azoles are widely used against Candida albicans , yet many cells survive above the minimal inhibitory concentrations (MIC) by growing slowly, which can prolong infection and foster resistance. We show that intracellular iron homeostasis alters the fluconazole characteristics by affecting ROS accumulation in mitochondria, and that this is the molecular mechanism underlying the combination therapy of fluconazole and doxycycline. These results place iron release from the vacuole at the center of azole responses, suggesting novel ways to boost azole efficacy.

Fluorescent proteins (FPs) have always been a crucial part of molecular research in life sciences, including the research into the human fungal pathogen Candida albicans, but have obvious shortcomings such as their relatively large size and long maturation time. However, the next generation of FPs overcome these issues and rely on the binding of a fluorogen for the protein to become fluorescently active. This generation of FPs includes the improved version of Fluorescence activating and Absorption Shifting Tag (iFAST). The binding between the fluorogen and the iFAST protein is reversible, thus resulting in reversible fluorescence. The fluorogens of iFAST are analogues of 4-hydroxylbenzylidene-rhodanine (HBR). These HBR analogues differ in spectral properties depending on functional group substitutions, which gives the iFAST system flexibility in terms of absorbance and emission maxima. In this work we describe and illustrate the application of iFAST as a protein tag and its reversible multi-colour characteristics in C. albicans.