Microorganisms have a remarkable capacity to evolve resistance to antimicrobial agents, threatening the efficacy of the limited arsenal of antimicrobials and becoming a dire public health crisis. This is of particular concern for fungal pathogens, which cause devastating invasive infections with treatment options limited to only three major classes of antifungal drugs. The paucity of antifungals with clinical utility is in part due to close evolutionary relationships between these eukaryotic pathogens and their human hosts, which limits the unique targets to be exploited therapeutically. This review highlights the mechanisms by which fungal pathogens of humans evolve resistance to antifungal drugs, which provide crucial insights to enable development of novel therapeutic strategies to thwart drug resistance and combat fungal infectious disease.

Cryptococcus spp., in particular Cryptococcus neoformans and Cryptococcus gattii , have an enormous impact on human health worldwide. The global burden of cryptococcal meningitis is almost a quarter of a million cases and 181,000 deaths annually, with mortality rates of 100% if infections remain untreated. Despite these alarming statistics, treatment options for cryptococcosis remain limited, with only three major classes of drugs approved for clinical use. Exacerbating the public health burden is the fact that the only new class of antifungal drugs developed in decades, the echinocandins, displays negligible antifungal activity against Cryptococcus spp., and the efficacy of the remaining therapeutics is hampered by host toxicity and pathogen resistance. Here, we describe the current arsenal of antifungal agents and the treatment strategies employed to manage cryptococcal disease. We further elaborate on the recent advances in our understanding of the intrinsic and adaptive resistance mechanisms that are utilized by Cryptococcus spp. to evade therapeutic treatments. Finally, we review potential therapeutic strategies, including combination therapy, the targeting of virulence traits, impairing stress response pathways and modulating host immunity, to effectively treat infections caused by Cryptococcus spp. Overall, understanding of the mechanisms that regulate anti-cryptococcal drug resistance, coupled with advances in genomics technologies and high-throughput screening methodologies, will catalyse innovation and accelerate antifungal drug discovery.

The fungus Candida albicans is an opportunistic pathogen that can exploit imbalances in microbiome composition to invade its human host, causing pathologies ranging from vaginal candidiasis to fungal sepsis. Bacteria of the genus Lactobacillus are colonizers of human mucosa and can produce compounds with bioactivity against C. albicans. Here, we show that some Lactobacillus species produce a small molecule under laboratory conditions that blocks the C. albicans yeast-to-filament transition, an important virulence trait. It remains unexplored whether the compound is produced in the context of the human host. Bioassay-guided fractionation of Lactobacillus-conditioned medium linked this activity to 1-acetyl-β-carboline (1-ABC). We use genetic approaches to show that filamentation inhibition by 1-ABC requires Yak1, a DYRK1-family kinase. Additional biochemical characterization of structurally related 1-ethoxycarbonyl-β-carboline confirms that it inhibits Yak1 and blocks C. albicans biofilm formation. Thus, our findings reveal Lactobacillus-produced 1-ABC can prevent the yeast-to-filament transition in C. albicans through inhibition of Yak1.

Invasive fungal infections caused by the pathogen Candida albicans have transitioned from a rare curiosity to a major cause of human mortality. This is in part due to the emergence of resistance to the limited number of antifungals available to treat fungal infections. Azoles function by targeting the biosynthesis of ergosterol, a key component of the fungal cell membrane. Loss-of-function mutations in the ergosterol biosynthetic gene ERG3 mitigate azole toxicity and enable resistance that depends upon fungal stress responses. Here, we performed a genome-wide synthetic genetic array screen in Saccharomyces cerevisiae to map ERG3 genetic interactors and uncover novel circuitry important for azole resistance. We identified nine genes that enabled erg3-mediated azole resistance in the model yeast and found that only two of these genes had a conserved impact on resistance in C. albicans. Further, we screened a C. albicans homozygous deletion mutant library and identified 13 genes for which deletion enhances azole susceptibility. Two of the genes, RGD1 and PEP8, were also important for azole resistance acquired by diverse mechanisms. We discovered that loss of function of retrograde transport protein Pep8 overwhelms the functional capacity of the stress response regulator calcineurin, thereby abrogating azole resistance. To identify the mechanism through which the GTPase activator protein Rgd1 enables azole resistance, we selected for mutations that restore resistance in strains lacking Rgd1. Whole genome sequencing uncovered parallel adaptive mechanisms involving amplification of both chromosome 7 and a large segment of chromosome 3. Overexpression of a transporter gene on the right portion of chromosome 3, NPR2, was sufficient to enable azole resistance in the absence of Rgd1. Thus, we establish a novel mechanism of adaptation to drug-induced stress, define genetic circuitry underpinning azole resistance, and illustrate divergence in resistance circuitry over evolutionary time.

Fungal pathogens pose a global threat to human health, with Candida albicans among the leading killers. Systematic analysis of essential genes provides a powerful strategy to discover potential antifungal targets. Here, we build a machine learning model to generate genome-wide gene essentiality predictions for C. albicans and expand the largest functional genomics resource in this pathogen (the GRACE collection) by 866 genes. Using this model and chemogenomic analyses, we define the function of three uncharacterized essential genes with roles in kinetochore function, mitochondrial integrity, and translation, and identify the glutaminyl-tRNA synthetase Gln4 as the target of N-pyrimidinyl-β-thiophenylacrylamide (NP-BTA), an antifungal compound.