Jane Usher

Candida ( Candidozyma ) auris is a critical priority fungal pathogen that emerged two decades ago near simultaneously on multiple continents. Since emergence, C. auris resistance to all four classes of antifungal drugs has been described, including pan-drug resistant isolates, sometimes evolving within patients. Here, we confirm the first isolation of C. auris from Antarctica and show cold-adapted phenotypes and an affinity for binding to nylon. We also provide evidence to suggest mutator phenotypes contribute to the rapid evolution of C. auris and are responsible for the emergence of multiple, distinct genetic clades worldwide. Isolates in clades I, III and IV with a mutator phenotype displayed elevated mutation rates compared to non- auris Candida species. This phenotype had a complex genetic basis and was associated with drug resistance mutations. We postulate that the mutator phenotype has a significant effect on evolutionary potential and is responsible for the emergence and rapid spread of drug-resistance C. auris and novel genetic clades.

Candida glabrata is an opportunistic fungal pathogen and a leading cause of invasive candidiasis, particularly in immunocompromised patients, where treatment is increasingly compromised by intrinsic and acquired antifungal resistance. Despite lacking morphological plasticity, C. glabrata employs distinct virulence strategies, including adhesin-mediated host colonization, intracellular survival within phagocytes, stress tolerance, iron acquisition and biofilm formation. This review synthesizes current knowledge of C. glabrata virulence and antifungal resistance mechanisms, with a particular focus on azole and echinocandin resistance driven by efflux pump regulation and FKS mutations. We critically evaluate the greater wax moth larva, Galleria mellonella , as a non-mammalian in vivo model for studying C. glabrata pathogenesis, host–pathogen interactions and antifungal efficacy. Evidence demonstrating concordance between G. mellonella , murine models and clinical outcomes is discussed, alongside the model’s limitations, including the absence of adaptive immunity. Collectively, this review highlights G. mellonella as a robust, cost-effective platform for dissecting C. glabrata virulence traits and for preclinical screening of antifungal therapies, supporting its growing role in translational fungal research.

Fungal infections affect over 6.55 million individuals annually, with Candida glabrata (recently reclassified as Nakaseomyces glabratus ) being the second most common cause of invasive candidiasis and candidemia, conditions that carry a high mortality rate of approximately 63.6%. C. glabrata exhibits significant genomic plasticity, enabling rapid adaptation to antifungal treatments. This ability is largely driven by gain-of-function (GOF) mutations in the CgPDR1 gene, which enhance the activity of efflux pumps and confer azole resistance. Over 80 unique CgPDR1 mutations have been identified, leading to diverse resistance profiles, from single-azole resistance to pan-azole resistance. These mutations not only increase resistance but also enhance the virulence of C. glabrata . To combat multi-drug-resistant (MDR) strains, which show resistance to azoles, echinocandins, and polyenes, novel multi-target therapeutic approaches are essential. This study employs synthetic genetic array (SGA) screening to explore the genetic interactions underlying CgPDR1 ⁺ -mediated azole resistance, using the clinical allele R592S. By generating genome-wide double mutant strains, we identified gene deletions that cause synthetic sick (SS) and synthetic lethal (SL) interactions, providing potential therapeutic targets. In silico screening identified inhibitors of these targets, and one was validated in vitro . Our findings support the potential of SGA screening to identify antifungal adjuvants that can slow the emergence of azole resistance in C. glabrata . IMPORTANCE: Candida glabrata is a significant cause of life-threatening fungal infections, especially concerning due to its rapid development of resistance to commonly used antifungal drugs like azoles. The growing prevalence of multi-drug-resistant C. glabrata strains, coupled with their high mortality rate, underscores the urgency of identifying new treatment strategies. This study investigates the genetic mechanisms underlying drug resistance, specifically focusing on mutations in the CgPDR1 gene, which increase azole resistance. By leveraging a novel genetic screening method, this work identifies genes that interact with drug-resistant mutations, offering potential new therapeutic targets. Additionally, an inhibitor identified through in silico screening shows promise in delaying azole resistance when tested in vitro . The findings highlight the potential of multi-target therapies to combat drug resistance, paving the way for more effective treatment options for C. glabrata infections and addressing an urgent clinical need.

"Metabolic generalism" refers to the ability of an organism to utilise a wide range of nutrients and flexibly adapt to dynamic environmental conditions. For Candida spp., this versatility is crucial for their survival and virulence in diverse host niches [1], allowing them to thrive. In contrast, metabolic specialists follow a strict hierarchy of carbon source utilisation. While this hierarchy does not preclude specialists from potentially utilising a wide range of nutrients, it could delay switching to alternative carbon sources during transitions between host niches. In this Pearl, we will explore major metabolic themes supporting Candida species pathogenesis and highlight how the metabolic specialist, Candida glabrata, provides insights into what metabolic determinants really matter for survival in the host.

Antifungal drugs have had a tremendous impact on human health and the yields of crops. However, in recent years, due to usage both in a health setting and in agriculture, there has been a rapid emergence of antifungal drug resistance that has outpaced novel compound discovery. It is now globally recognized that new strategies to tackle fungal infection are urgently needed, with such approaches requiring the cooperation of both sectors and the development of robust antifungal stewardship rationales. In this review, we examine the current antifungal regimes in clinical and agricultural settings, focusing on two pathogens of importance, Candida auris and Aspergillus fumigatus, examining their drivers of antifungal resistance, the impact of dual-use azoles and the impact agricultural practices have on driving the emergence of resistance. Finally, we postulate that a One Health approach could offer a viable alternative to prolonging the efficacy of current antifungal agents.

Candida glabrata (Nakaseomyces glabratus) is an opportunistic fungal pathogen that has become a significant concern in clinical settings due to its increasing resistance to antifungal treatments. Understanding the genetic basis of its pathogenicity and resistance mechanisms is crucial for developing new therapeutic strategies. One powerful method of studying gene function is through targeted gene deletion. This paper outlines a comprehensive protocol for the deletion of genes in C. glabrata, encompassing primer design, preparation of electrocompetent cells, transformation, and finally confirmation of the gene deletion. The protocol begins with the identification and design of primers necessary for generating deletion constructs, involving the precise targeting of up‐ and downstream regions flanking the gene of interest to ensure high specificity and efficiency of homologous recombination. Followed is the preparation of electrocompetent cells, a critical step for successful transformation. Transformation of the competent cells is achieved through electroporation, facilitating the introduction of exogenous DNA into the cells. This is followed by the selection and confirmation of successfully transformed colonies. Confirmation involves the use of colony PCR to verify the correct integration of the NAT resistance cassette and deletion of the target gene. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Primer design for gene deletion in C. glabrataBasic Protocol 2: Preparing competent C. glabrata cellsBasic Protocol 3: Transforming C. glabrata using electroporationBasic Protocol 4: Confirming deletion strains with colony PCR

Candida causes an estimated half-billion cases of vulvovaginal candidiasis (VVC) every year. VVC is most commonly caused by Candida albicans , which, in this setting, triggers nonprotective neutrophil infiltration, aggressive local inflammation, and symptomatic disease. Despite its prevalence, little is known about the molecular mechanisms underpinning the immunopathology of this fungal infection. In this study, we describe the molecular determinant of VVC immunopathology and a potentially straightforward way to prevent disease. In response to zinc limitation, C. albicans releases a trace mineral binding molecule called Pra1 (pH-regulated antigen). Here, we show that the PRA1 gene is strongly up-regulated during vaginal infections and that its expression positively correlated with proinflammatory cytokine concentrations in women. Genetic deletion of PRA1 prevented vaginal inflammation in mice, and application of a zinc solution down-regulated expression of the gene and also blocked immunopathology. We also show that treatment of women suffering from recurrent VVC with a zinc gel prevented reinfections. We have therefore identified a key mediator of symptomatic VVC, giving us an opportunity to develop a range of preventative measures for combatting this disease.

IntroductionAU : Pleaseconfirmthatallheadinglevelsarerepresentedcorrectly: Candida glabrata (Nakoseomyces glabratus) is a haploid, budding yeast that causes opportunistic nosocomial infections and is garnering increasing attention in line with its changing epidemiological importance. It is a commensal of the human mucosa, particularly oral, gastrointestinal, and vaginal epithelia, which predisposes to infection. The physical disruption of the epithelial layer is a major risk factor for systemic C. glabrata infection, which generally occurs in immunocompromised individuals. C. glabrata appears to lack the brute force traits of other Candida species such as filament formation leading to tissue damage and immune cell lysis but nevertheless leads the non-Candida albicansAU : PleasenotethatasperPLOSstyle; donotusespec species in the number of patients it infects. Infections caused by C. glabrata are of great concern due to their propensity for drug resistance and limited treatment options. This article discusses the intrinsic and acquired factors contributing to C. glabrata's increasing resistance to pharmaceutical intervention.

Purpose of Review: Human fungal pathogens are rapidly increasing in incidence and readily able to evade the host immune responses. Our ability to study the genetic behind this has been limited due to the apparent lack of a sexual cycle and forward genetic tools. In this review, we discuss the evolution of mating, meiosis, and pathogenesis and if these processes are advantageous to pathogens. Recent Findings: This review summarises what is currently known about the sexual cycles of two important human fungal pathogens, Candida albicans and Candida glabrata. This includes the identification of parasexual cycle in C. albicans and the observed low levels of recombination in C. glabrata populations. Summary: In this review, we present what is currently known about the mating types and mating/sexual cycles of two clinically important human fungal pathogens, Candida albicans and Candida glabrata. We discuss the evolution of meiosis using the knowledge that has been amassed from the decades of studying Saccharomyces cerevisiae and how this can be applied to fungal pathogens. We further discuss how the evolution of pathogenesis has played a role in influencing mating processes in human fungal pathogens and compare sexual cycles between C. albicans and C. glabrata, highlighting knowledge gaps and suggesting how these two fungi have evolved distinct mating niches to allow the development of disease in a human host.