Stef Jacobs

Echinocandins are the first-line treatment for invasive fungal infections and work by inhibiting the fungal β-1,3-glucan synthase Fks1. Recently, a first-in-class triterpenoid Fks1 inhibitor, ibrexafungerp, was approved for clinical use. The extent to which the difference between these drug classes alters the landscape of resistance mutations in Fks1 is unknown. We used deep mutational scanning (DMS) to quantify the effects of all single-amino-acid variants spanning 159 residues across the three canonical Fks1 resistance hotspots and flanking regions in Saccharomyces cerevisiae. Drug selections revealed widespread position-specific cross-resistance and differential resistance, including an ibrexafungerp-specific resistance cluster downstream of hotspot 2 that defines a novel triterpenoid-specific resistance hotspot. We defined this as hotspot 4 and redefined the boundaries of the remaining three canonical echinocandin hotspots, based on the mutational landscape of our DMS analyses. Susceptibility testing of individual FKS1 variants of S. cerevisiae and Candidozyma auris strains confirmed conserved class-specific and cross-resistance patterns. Structure-guided ligand placement, molecular dynamics-based simulated annealing, and DMS-informed pose selection linked drug-specific phenotypes to distinct interaction networks, providing a molecular rationale for the observed differential resistance patterns. These data provide a framework for predicting antifungal resistance, informing treatment strategies and future drug design.

Candidozyma auris is an emerging multidrug-resistant fungal pathogen that poses a major public-health challenge owing to high mortality and the limited efficacy of current therapies. Echinocandins, which inhibit β-glucan synthesis, are first-line therapy for invasive C. auris infection; however, resistance to this class is rising, underscoring the urgent need for new antifungal targets. Here we show that enzymes in the trehalose-biosynthetic pathway regulate stress responses, antifungal resistance/tolerance and virulence in C. auris. The tps2Δ strain displays heightened susceptibility to echinocandins, whereas tps1Δ and tps1Δ tps2Δ strains show resistance and tolerance comparable to wild type (WT). Mechanistically, the tps2Δ strain accumulates trehalose 6-phosphate (T6P), which inhibits hexokinase activity and reduces the flux of glucose 6-phosphate (G6P) into the chitin biosynthesis pathway, leading to substantially decreased cell wall chitin. During echinocandin exposure, the tps2Δ strain fails to compensate for reduced β-glucan with increased chitin, thereby rendering it highly susceptible to these drugs. In a systemic mouse infection model, deletion of the TPS2 gene results in lower tissue fungal burdens after treatment with caspofungin. Together, these findings identify Tps2 as a potential therapeutic target that can potentiate echinocandin efficacy in C. auris via a distinct mechanism of action.

The rise of multidrug-resistant fungal pathogens, like Candida auris, poses a significant public health challenge due to high mortality rates and the limited effectiveness of current treatment options. Echinocandins, targeting β-glucan synthesis, are the first-line therapy for invasive C. auris infections. However, the resistance to this drug class is increasing, underscoring the urgent need for new antifungal targets. This study investigates the role of trehalose biosynthesis in C. auris by either blocking biosynthesis of the intermediate molecule trehalose-6-phosphate (T6P), by disrupting the trehalose-phosphate synthase (Tps1), or blocking biosynthesis of trehalose, by disrupting the trehalose-6-phosphate phosphatase (Tps2). The tps2Δ strain demonstrated heightened susceptibility to echinocandins, while the tps1Δ and tps1Δ tps2Δ strains maintained resistance and tolerance levels comparable to the wild type (WT) strain. Subsequent analysis revealed a link between chitin biosynthesis and T6P levels. In the absence of T6P (in the tps1∆ or tps1∆tps2∆ strains) there was a strong increase in hexokinase activity and accelerated glycolysis, with no significant impact on chitin biosynthesis. However, the tps2Δ strain accumulated high levels of T6P, resulting in the inhibition of hexokinase and a reduced flux of glucose 6-phosphate into the chitin biosynthesis pathway, thereby significantly decreasing cell wall chitin content. The inability to compensate the reduction in β-glucan levels with increased chitin production during echinocandin treatment in the tps2Δ strain, rendered this strain highly susceptible to these drugs. Furthermore, an in vivo systemic infection model demonstrated that the tps2Δ strain exhibited a reduced fungal burden in tissues, with infected mice showing marked improvement during caspofungin treatment. This suggests that Tps2 is a putative target for improving echinocandin treatment and reducing virulence in C. auris.

Echinocandins, which target the fungal β-1,3-glucan synthase (Fks), are essential for treating invasive fungal infections, yet resistance is increasingly reported. While resistance typically arises through mutations in Fks hotspots, emerging evidence suggests a contributing role of changes in membrane sterol composition due to ERG3 mutations. Here, we present a clinical case of Nakaseomyces glabratus ( Candida glabrata ) in which combined mutations in ERG3 and FKS2 , but not FKS2 alone, appear to confer echinocandin resistance. Integrated analyses reveal a recurrent association between Erg3 loss-of-function and echinocandin resistance mediated by Fks variation across Candida species, but exclude ERG3 loss-of-function as an independent resistance mechanism. Advances in Fks structural biology and insights into echinocandin-Fks interactions support a model of epistatic crosstalk between membrane sterols and Fks function. Understanding this interaction is crucial, as it may underlie not only acquired echinocandin resistance but also the broader development of multidrug resistance across major antifungal drug classes.

Exploring the dynamics and molecular mechanisms of antimicrobial drug resistance provides critical insights for developing effective strategies to combat it. This review highlights the potential of experimental evolution methods to study resistance in pathogenic fungi, drawing on insights from bacteriology and innovative approaches in mycology. We emphasize the versatility of experimental evolution in replicating clinical and environmental scenarios and propose that incorporating evolutionary modelling can enhance our understanding of antifungal resistance evolution. We advocate for a broader application of experimental evolution in medical mycology to improve our still limited understanding of drug resistance in fungi.

Candida auris is a growing concern due to its resistance to antifungal drugs, particularly amphotericin B (AMB), detected in 30 to 60% of clinical isolates. However, the mechanisms of AMB resistance remain poorly understood. Here we investigated 441 in vitro- and in vivo-evolved C. auris lineages from 4 AMB-susceptible clinical strains of different clades. Genetic and sterol analyses revealed four major types of sterol alterations as a result of clinically rare variations in sterol biosynthesis genes ERG6, NCP1, ERG11, ERG3, HMG1, ERG10 and ERG12. In addition, aneuploidies in chromosomes 4 and 6 emerged during resistance evolution. Fitness trade-off phenotyping and mathematical modelling identified diverse strain- and mechanism-dependent fitness trade-offs. Variation in CDC25 rescued fitness trade-offs, thereby increasing the infection capacity. This possibly contributed to therapy-induced acquired AMB resistance in the clinic. Our findings highlight sterol-modulating mechanisms and fitness trade-off compensation as risks for AMB treatment failure in clinical settings.

Antifungal drug resistance represents a serious global health threat, necessitating new treatment strategies. Here we investigated collateral sensitivity (CS), in which resistance to one drug increases sensitivity to another, and cross-resistance (XR), in which one drug resistance mechanism reduces susceptibility to multiple drugs, since CS and XR dynamics can guide treatment design to impede resistance development, but have not been systematically explored in pathogenic fungi. We used experimental evolution and mathematical modelling of Candida auris population dynamics during cyclic and combined drug exposures and found that especially CS-based drug cycling can effectively prevent the emergence of drug resistance. In addition, we found that a CS-based treatment switch can actively select against or eradicate resistant sub-populations, highlighting the potential to consider CS in therapeutic decision-making upon resistance detection. Furthermore, we show that some CS trends are robust among different strains and resistance mechanisms. Overall, these findings provide a promising direction for improved antifungal treatment approaches.

The increased prevalence of antifungal drug resistance and emergence of multidrug-resistant species such as Candida auris represent a global public health threat. By mapping drug susceptibility responses of experimentally evolved C. auris across diverse antifungals, we found trends of cross-resistance (XR) and collateral sensitivity (CS) and show that CS can reduce resistance development through drug cycling and combination therapy, providing a promising direction for future research.