Candidiasis can be present as a cutaneous, mucosal or deep-seated organ infection, which is caused by more than 20 types of Candida sp., with C. albicans being the most common. These are pathogenic yeast and are usually present in the normal microbiome. High-risk individuals are patients of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), organ transplant, and diabetes. During infection, pathogens can adhere to complement receptors and various extracellular matrix proteins in the oral and vaginal cavity. Oral and vaginal Candidiasis results from the overgrowth of Candida sp. in the hosts, causing penetration of the oral and vaginal tissues. Symptoms include white patches in the mouth, tongue, throat, and itchiness or burning of genitalia. Diagnosis involves visual examination, microscopic analysis, or culturing. These infections are treated with a variety of antifungals that target different biosynthetic pathways of the pathogen. For example, echinochandins target cell wall biosynthesis, while allylamines, azoles, and morpholines target ergosterol biosynthesis, and 5-Flucytosine (5FC) targets nucleic acid biosynthesis. Azoles are commonly used in therapeutics, however, because of its fungistatic nature, Candida sp. evolve azole resistance. Besides azoles, Candida sp. also acquire resistance to polyenes, echinochandins, and 5FC. This review discusses, in detail, the drug resistance mechanisms adapted by Candida sp.

Ergosterol (ERG) is a critical sterol in the cell membranes of fungi, and its biosynthesis is tightly regulated by 25 known enzymes along the ERG production pathway. The effects of changes in expression of each ERG biosynthesis enzyme in Saccharomyces cerevisiae were analyzed by the use of gene deletion or plasmid-borne overexpression constructs. The strains overexpressing the ERG pathway genes were examined for changes in doubling time and responses to a variety of stress agents. In addition, ERG gene overexpression strains and ERG gene deletion strains were tested for alterations in antifungal drug susceptibility. The data show that disruptions in ergosterol biosynthesis regulation can affect a diverse set of cellular processes and can cause numerous phenotypic effects. Some of the phenotypes observed include dramatic increases in doubling times, respiratory deficiencies on glycerol media, cell wall insufficiencies on Congo red media, and disrupted ion homeostasis under iron or calcium starvation conditions. Overexpression or deletion of specific enzymes in the ERG pathway causes altered susceptibilities to a variety of classes of antifungal ergosterol inhibitors, including fluconazole, fenpropimorph, lovastatin, nystatin, amphotericin B, and terbinafine. This analysis of the effect of perturbations to the ERG pathway caused by systematic overexpression of each of the ERG pathway genes contributes significantly to the understanding of the ergosterol biosynthetic pathway and its relationship to stress response and basic biological processes. The data indicate that precise regulation of ERG genes is essential for cellular homeostasis and identify several ERG genes that could be exploited in future antifungal development efforts.

Candida auris is an emerging multi-drug resistant yeast that causes systemic infections. Here we show that C. auris undergoes replicative aging (RA) that results from asymmetric cell division and causes phenotypic differences between mother and daughter cells similar to other pathogenic yeasts. Importantly, older C. auris cells (10 generations) exhibited higher tolerance to fluconazole (FLC), micafungin, 5- flucytosine and amphotericin B compared to younger (0–3 generation) cells. Increased FLC tolerance was associated with increased Rhodamine 6G (R6G) efflux and therapeutic failure of FLC in a Galleria infection model. The higher efflux in the older cells correlated with overexpression of the efflux pump encoding gene CDR1 (4-fold). In addition, 8-fold upregulation of the azole target encoding gene ERG11 was noted in the older cells. Analysis of genomic DNA from older cells by qPCR indicates that transient gene duplication of CDR1 and ERG11 causes the observed age-dependent enhanced FLC tolerance in C. auris strains. Furthermore, older cells exhibited a thickened cell wall, decreased neutrophil killing (24% vs 50%), increased epithelial cell adhesion (31.6% vs 17.8%) and upregulation of adhesin protein Als5p. Thus, this study demonstrates that transient gene duplication can occur during RA, causing increased FLC tolerance in old C. auris cells.

To evaluate the pump activity associated with overexpression, an assay that combined data from two separate fluorescent assays using rhodamine 6G and alanine ␤-naphthylamide was developed. The qRT-PCR results and activity assay results were in good agreement. This combination of two fluorescent assays can be used to study efflux pumps as resistance mechanisms in clinical isolates. These results demonstrate that efflux pumps are a significant resistance mechanism in vaginal C. albicans isolates.

As Cryptococcus neoformans mother cells generationally age, their cell walls become thicker and cell-wall associated virulence factors are upregulated. Antiphagocytic protein 1 (App1), and laccase enzymes (Lac1 and Lac2) are virulence factors known to contribute to virulence of C. neoformans during infection through inhibition of phagocytic uptake and melanization. Here we show that these cell-wall-associated proteins are not only significantly upregulated in old C. neoformans cells, but also that their upregulation likely contributes to the increased resistance to antifungal and host-mediated killing during infection and to the subsequent accumulation of old cells. We found that old cells melanize to a greater extent than younger cells and as a consequence, old melanized cells are more resistant to killing by amphotericin B compared to young melanized cells. A decrease in melanization of old lacΔ mutants lead to a decrease in old-cell resilience, indicating that age-related melanization is contributing to the overall resilience of older cells and is being mediated by laccase genes. Additionally, we found that older cells are more resistant to macrophage phagocytosis, but this resistance is lost when APP1 is knocked out, indicating that upregulation of APP1 in older cells is in part responsible for their increased resistance to phagocytosis by macrophages. Finally, infections with old cells in the Galleria mellonella model support our conclusions, as loss of the APP1, LAC1, and LAC2 gene ablates the enhanced virulence of old cells, indicating their importance in age-dependent resilience.

Cryptococcus neoformans (Cn) is a deadly fungal pathogen responsible for ~ 180,000 deaths per year and despite effective antifungals, treatment failure and resistance to antifungals are increasingly problematic. Aging and age-related phenotypes are prominent virulence traits that contribute to the resilience of Cn to host responses and antifungals. Traditional methods to study aging in Cn are expensive, inefficient and in need of improvement. Here, we demonstrate the development and use of a High-Throughput Yeast Aging Analysis for Cryptococcus (HYAAC) microfluidic device to better study aging and age-associated genes in Cn . Compared to traditional methods, the HYAAC is superior in its efficiency to isolate, manipulate and observe old cells for analysis. It allows for the trapping and tracking of individual cells over the course of their lifespan, allowing for more precise measurements of lifespan, tracking of age-related phenotypes with age, and a more high-throughput ability to investigate genes associated with aging.

We investigated the effect of replicative aging on antifungal resistance in Our studies demonstrate significantly increased transcription of ABC transporters and efflux pump activity in old versus young cells of a fluconazole-sensitive and -resistant strain. In addition, higher tolerance to killing by micafungin and amphotericin B was noted and is associated with higher transcription of glucan synthase gene and lower ergosterol content in older cells.

Candida albicans, Candida auris, Candida glabrata, and Cryptococcus neoformans are pathogenic yeasts which can cause systemic infections in immune-compromised as well as immune-competent individuals. These yeasts undergo replicative aging analogous to a process first described in the nonpathogenic yeast Saccharomyces cerevisiae. The hallmark of replicative aging is the asymmetric cell division of mother yeast cells that leads to the production of a phenotypically distinct daughter cell. Several techniques to study aging that have been pioneered in S. cerevisiae have been adapted to study aging in other pathogenic yeasts. The studies indicate that aging is relevant for virulence in pathogenic fungi. As the mother yeast cell progressively ages, every ensuing asymmetric cell division leads to striking phenotypic changes, which results in increased antifungal and antiphagocytic resistance. This review summarizes the various techniques that are used to study replicative aging in pathogenic fungi along with their limitations. Additionally, the review summarizes some key phenotypic variations that have been identified and are associated with changes in virulence or resistance and thus promote persistence of older cells.