Biology subjects

Selmecki, A.

Publications and source records attributed to Selmecki, A..

9 recordsLinked to original sources

bioRxiv2026

CRISPR-activation reveals key resistance genes and vulnerabilities of copy number variants in Candida albicans

Changes in gene copy number are an extremely important source of variation and are frequently observed during the acquisition of drug resistance. The opportunistic human fungal pathogen Candida albicans frequently adapts to antifungal drugs via large copy number variations (CNVs) that amplify hundreds of genes simultaneously. Despite the recurrent amplification of CNVs across diverse clinical isolates, the genes that contribute to drug resistance are not known. Additionally, by amplifying many genes, CNVs might result in cross-adaptation or fitness trade-offs to multiple environments, which has major implications for how CNVs are expected to contribute to adaptation in complex environments like a mammalian host. We use CRISPR-activation to systematically assay the fitness effects of individually overexpressing [~]800 genes in four genetically diverse isolates across eight physiologically relevant environments. We identify 198 genes with significant fitness effects in at least one environment in one or more genetic backgrounds. We identify novel genes with positive fitness effects in two different classes of antifungal drug and observe frequent gene-by-environment interactions for the fitness effects of gene overexpression. Additive fitness effects of individual gene overexpression are a significant predictor of the fitness of multiple isolates with CNVs and can explain fitness trade-offs observed between classes of antifungal drug for the CNV isolates. These findings identify genes that increase fitness in drug and those that create vulnerabilities in CNV isolates and can help inform treatment of isolates adapting to antifungal drug via CNVs.

microbiology↗

bioRxiv2026

Micafungin exposure drives multidrug resistance in Clavispora lusitaniae

Fungal infections are an escalating global health concern, with rare Candida species posing an urgent threat due to emerging multidrug resistance. Clavispora (Candida) lusitaniae is an uncommon pathogen in which multidrug resistance has been documented during antifungal therapy, yet the selective forces driving this phenotype remain unclear. Here, we show that exposure to the echinocandin micafungin (MCF) alone can select for multidrug resistance in C. lusitaniae. Through controlled evolution experiments we identified individual point mutations in genes encoding ergosterol biosynthesis enzymes (ERGs), sterol trafficking proteins (OSH2), and the echinocandin drug target (FKS1) that confer a significant fitness benefit to one or more classes of antifungals. We find that ERG3 loss-of-function is the primary and independent driver of pan-antifungal resistance to echinocandins, azoles and polyenes. The ERG3 mutants have <1% ergosterol, increased levels of non-toxic sterol intermediates, and increased chitin content, consistent with both cell membrane and cell wall remodeling that enables the fungal pathogen to evade all three drug classes. The convergence of sterol reprogramming and compensatory cell wall remodeling that occurs during adaptation to echinocandin monotherapy can evolve through a single point mutation and parallels our recent case study of acquired multidrug resistance. IMPORTANCEMultidrug resistance in Candida species severely limits treatment options and increases mortality, particularly in immunocompromised patients. Despite increasing reports of multidrug resistance, the molecular mechanisms driving multidrug resistance remain poorly understood. We find that in vitro MCF exposure alone can drive multidrug resistance in C. lusitaniae via acquisition of de novo point mutations in ERG3, an observation that parallels our recent patient case study. By identifying causative mutations and associated physiological changes, we provide mechanistic insight into the emergence of multidrug resistance and highlight the need for surveillance strategies that account for resistance evolution under echinocandin monotherapy.

microbiology↗

bioRxiv2025

Aneuploidy, polyploidy, and loss of heterozygosity distinguish serial bloodstream isolates of Candida albicans

BackgroundThe opportunistic pathogen Candida albicans is the leading species causing invasive Candida infections worldwide. Genomic variation is widespread in clinical isolates and complicates identification of genetic variants underlying antifungal drug resistance and tolerance. Our understanding of genomic and phenotypic diversity during invasive infections is limited and studies of serial isolates from individual patients are uncommon. We performed comparative analyses of 101 C. albicans bloodstream isolates from 55 patients in the Minnesota Minneapolis-Saint Paul region, including serial isolates from 19 patients. We analyzed the phylogenetic relationships of these isolates relative to 199 globally-collected public C. albicans genomes. ResultsThis studys regional isolates span the phylogenetic diversity of C. albicans; 6 isolates represent novel outliers to known clades. Serial isolates from individual patients were separated by limited single nucleotide polymorphisms. Nevertheless, we identified extensive large-scale genomic variation between serial isolates including polyploidy, aneuploidy, copy number variation, loss of heterozygosity, and chromosomal rearrangements. We demonstrated how a heterozygous ERG251 loss of function variant drives azole tolerance in a clinical isolate from a patient with a history of recurrent infections. Using serial isolates, we demonstrated that polyploidy provides an adaptive advantage in the presence of fluconazole despite the absence of overt antifungal drug resistance. ConclusionsOur analysis of serial isolates reveals the genomic plasticity of C. albicans during invasive infections and identifies variation driving antifungal drug tolerance. Our findings reveal limitations in current antifungal susceptibility testing and highlight the need to account for genomic and phenotypic variation during invasive Candida infections.

microbiology↗

bioRxiv2025

Heterogeneity of Candida bloodstream isolates in an academic medical center and affiliated hospitals

Invasive Candida bloodstream infections (candidemia) are a deadly global health threat. Rare Candida species are increasingly important causes of candidemia and phenotypic data, including patterns of antifungal drug resistance, is limited. There is geographic variation in the distribution of Candida species and frequency of antifungal drug resistance, which means that collecting and reporting regional data can have significant clinical value. Here, we report the first survey of species distribution, frequency of antifungal drug resistance, and phenotypic variability of Candida bloodstream isolates from an academic medical center and 5 affiliated hospitals in the Minneapolis-Saint Paul region of Minnesota, collected during an 18-month period from 2019 to 2021. We collected 288 isolates spanning 11 species from 119 patients. C. albicans was the most frequently recovered species, followed by C. glabrata and C. parapsilosis, with 10% of cases representing additional, rare species. We performed antifungal drug susceptibility for the three major drug classes and, concerningly, we identified fluconazole, micafungin and multidrug resistance rates in C. glabrata that were [~] 2 times higher than that reported in other regions of the United States. We report some of the first phenotypic data in rare non-albicans Candida species. Through analysis of serial isolates from individual patients, we identified clinically relevant within-patient differences of MIC values in multiple drug classes. Our results provide valuable clinical data relevant to antifungal stewardship efforts and highlight important areas of future research, including within-patient dynamics of infection and the mechanisms of drug resistance in rare Candida species.

microbiology↗

bioRxiv2024

Erg251 has complex and pleiotropic effects on azole susceptibility, filamentation, and stress response phenotypes

Ergosterol is essential for fungal cell membrane integrity and growth, and numerous antifungal drugs target ergosterol. Inactivation or modification of ergosterol biosynthetic genes can lead to changes in antifungal drug susceptibility, filamentation and stress response. Here, we found that the ergosterol biosynthesis gene ERG251 is a hotspot for point mutations during adaptation to antifungal drug stress within two distinct genetic backgrounds of Candida albicans. Heterozygous point mutations led to single allele dysfunction of ERG251 and resulted in azole tolerance in both genetic backgrounds. This is the first known example of point mutations causing azole tolerance in C. albicans. Importantly, single allele dysfunction of ERG251 in combination with recurrent chromosome aneuploidies resulted in bona fide azole resistance. Homozygous deletions of ERG251 caused increased fitness in low concentrations of fluconazole and decreased fitness in rich medium, especially at low initial cell density. Dysfunction of ERG251 resulted in transcriptional upregulation of the alternate sterol biosynthesis pathway and ZRT2, a Zinc transporter. Notably, we determined that overexpression of ZRT2 is sufficient to increase azole tolerance in C. albicans. Our combined transcriptional and phenotypic analyses revealed the pleiotropic effects of ERG251 on stress responses including cell wall, osmotic and oxidative stress. Interestingly, while loss of either allele of ERG251 resulted in similar antifungal drug responses, we observed functional divergence in filamentation regulation between the two alleles of ERG251 (ERG251-A and ERG251-B) with ERG251-A exhibiting a dominant role in the SC5314 genetic background. Finally, in a murine model of systemic infection, homozygous deletion of ERG251 resulted in decreased virulence while the heterozygous deletion mutants maintain their pathogenicity. Overall, this study provides extensive genetic, transcriptional and phenotypic analysis for the effects of ERG251 on drug susceptibility, fitness, filamentation and stress responses. AUTHOR SUMMARYInvasive infections caused by the fungal pathogen Candida albicans have high mortality rates (20-60%), even with antifungal drug treatment. Numerous mechanisms contributing to drug resistance have been characterized, but treatment failure remains a problem indicating that there are many facets that are not yet understood. The azole class of antifungals targets production of ergosterol, an essential component of fungal cell membranes. Here, we provide insights into the contributions of ERG251, a component of the ergosterol biosynthesis pathway, to increased growth in azoles as well as broad scale effects on stress responses filamentation and pathogenicity. One of the most striking results from our study is that even a single nucleotide change in one allele of ERG251 in diploid C. albicans can lead to azole tolerance. Tolerance, a distinct phenotype from resistance, is the ability of fungal cells to grow above the minimum inhibitory concentration in a drug concentration-independent manner. Tolerance frequently goes undetected in the clinic because it is not observable in standard assays. Strikingly, azole tolerance strains lacking one allele of ERG251 remained virulent in a mouse model of infection highlighting the potential for mutations in ERG251 to arise and contribute to treatment failure in patients.

microbiology↗

bioRxiv2024

Step-wise evolution of azole resistance through copy number variation followed by KSR1 loss of heterozygosity in Candida albicans.

Antimicrobial drug resistance poses a global health threat, requiring a deeper understanding of the evolutionary processes that lead to its emergence in pathogens. Complex evolutionary dynamics involve multiple mutations that can result in cooperative or competitive (clonal interference) effects. Candida albicans, a major fungal pathogen, displays high rates of copy number variation (CNV) and loss of heterozygosity (LOH). CNV and LOH events involve large numbers of genes and could synergize during evolutionary adaptation. Understanding the contributions of CNV and LOH to antifungal drug adaptation is challenging, especially in the context of whole-population genome sequencing. Here, we document the sequential evolution of fluconazole tolerance and then resistance in a C. albicans isolate involving an initial CNV on chromosome 4, followed by an LOH on chromosome R that involves KSR1. Similar LOH events involving KSR1, which encodes a reductase involved in sphingolipid biosynthesis, were also detected in independently evolved fluconazole resistant isolates. We dissect the specific KSR1 codons that affect fluconazole resistance and tolerance. The combination of the chromosome 4 CNV and KSR1 LOH results in a >500-fold increase in azole resistance, illustrating a compelling example of rapid, yet step-wise, interplay between CNV and LOH in drug resistance evolution.

microbiology↗

bioRxiv2022

Genomic diversity across Candida auris clinical isolates shapes rapid development of antifungal resistance in vitro and in vivo

Antifungal drug resistance and tolerance poses a serious threat to global public health. In the human fungal pathogen, Candida auris, resistance to triazole, polyene, and echinocandin antifungals is rising, resulting in multidrug resistant isolates. Here, we use genome analysis and in vitro evolution of seventeen new clinical isolates of C. auris from clades I and IV to determine how quickly resistance mutations arise, the stability of resistance in the absence of drug, and the impact of genetic background on evolutionary trajectories. We evolved each isolate in the absence of drug as well as in low and high concentrations of fluconazole. In just three passages, we observed genomic and phenotypic changes including karyotype alterations, aneuploidy, acquisition of point mutations, and increases in MIC values within the populations. Fluconazole resistance was stable in the absence of drug, indicating little to no fitness cost associated with resistance. Importantly, two isolates substantially increased fluconazole resistance to [≥]256{micro}g/ml fluconazole. Multiple evolutionary pathways and mechanisms to increase fluconazole resistance occurred simultaneously within the same population. Strikingly, the sub-telomeric regions of C. auris were highly dynamic as deletion of multiple genes near the sub-telomeres occurred during the three passages in several populations. Finally, we discovered a mutator phenotype in a clinical isolate of C. auris. This isolate had elevated mutation rates compared to other isolates and acquired substantial resistance during evolution in vitro and in vivo supporting that the genetic background of clinical isolates can have a significant effect on evolutionary potential. ImportanceDrug resistant Candida auris infections are recognized by the CDC as an urgent threat. Here, we obtained and characterized a set of clinical isolates of C. auris including multiple isolates from the same patient. To understand how drug resistance arises, we evolved these isolates and found that resistance to fluconazole, the most commonly prescribed antifungal, can occur rapidly and that there are multiple pathways to resistance. During our experiment, resistance was gained, but it was not lost, even in the absence of drug. We also found that some C. auris isolates have higher mutation rates than others and are primed to acquire antifungal resistance mutations. Furthermore, we found that multidrug resistance can evolve within a single patient. Overall, our results highlight the high stability and high rates of acquisition of antifungal resistance of C. auris that allow evolution of pan-resistant, transmissible isolates in the clinic.

microbiology↗

bioRxiv2021

The SUMO protease Ulp2 regulates genome stability and drug resistance in the human fungal pathogen Candida albicans

Stress-induced genome instability in microbial organisms is emerging as a critical regulatory mechanism for driving rapid and reversible adaption to drastic environmental changes. In Candida albicans, a human fungal pathogen that causes life-threatening infections, genome plasticity confers increased virulence and antifungal drug resistance. Discovering the mechanisms regulating C. albicans genome plasticity is a priority to understand how this and other microbial pathogens establish life-threatening infections and develop resistance to antifungal drugs. We identified the SUMO protease Ulp2 as a critical regulator of C. albicans genome integrity through genetic screening. Deletion of ULP2 leads to hypersensitivity to genotoxic agents and increased genome instability. This increased genome diversity causes reduced fitness under standard laboratory growth conditions but enhances adaptation to stress, making ulp2{Delta}/{Delta} cells more likely to thrive in the presence of antifungal drugs. Whole-genome sequencing indicates that ulp2{Delta}/{Delta} cells counteract antifungal drug-induced stress by developing segmental aneuploidies of chromosome R and chromosome I. We demonstrate that intrachromosomal repetitive elements drive the formation of complex novel genotypes with adaptive power.

microbiology↗

bioRxiv2019

Selection of Candida albicans Trisomy during Oropharyngeal Infection Results in a Commensal-Like Phenotype

When the fungus Candida albicans proliferates in the oropharyngeal cavity during experimental oropharyngeal candidiasis (OPC), it undergoes large-scale genome changes at a much higher frequency than when it grows in vitro. Previously, we identified a specific whole chromosome amplification, trisomy of Chr 6 (Chr6x3), that was highly overrepresented among strains recovered from the tongues of mice with OPC. To determine the functional significance of this trisomy, we assessed the virulence of two Chr6 trisomic strains and a Chr5 trisomic strain in the mouse model of OPC. We also analyzed the expression of virulence-associated traits in vitro. All three trisomic strains exhibited characteristics of a commensal during OPC in mice. They achieved the same oral fungal burden as the diploid progenitor strain but caused significantly less weight loss and elicited a significantly lower inflammatory host response. In vitro, all three trisomic strains had reduced capacity to adhere to and invade oral epithelial cells and increased susceptibility to neutrophil killing. Whole genome sequencing of pre- and post-infection isolates found that the trisomies were usually maintained. Most post-infection isolates also contained de novo point mutations, but these were not conserved. While in vitro growth assays did not reveal phenotypes specific to de novo point mutations, they did reveal novel phenotypes specific to each lineage. These data reveal that during OPC, clones that are trisomic for Chr5 or Chr6 are selected and they facilitate a commensal-like phenotype.

genomics↗