Biology subjects

Gerstein, A.

Publications and source records attributed to Gerstein, A..

8 recordsLinked to original sources

bioRxiv2026

The influence of drug dosing regimen on extinction and adaptation during experimental evolution in Candida albicans

The antifungal azole drug fluconazole (FLC) is frequently prescribed for treating Candida albicans infections. Azole drugs are typically fungistatic; they inhibit cell growth rather than kill susceptible cells within a population. Therefore, fungal populations can adapt to fluconazole through increased drug resistance or increased drug tolerance (the ability of a subpopulation of susceptible cells to grow slowly at inhibitory drug concentrations). To our knowledge, no prior experiments have examined how different factors influence extinction propensity, although previous in vitro studies show that both drug concentration and strain background can influence adaptation. To determine how the drug dosing regimen, namely fluconazole concentration and transfer duration (i.e., the amount of time between transfer into fresh medium), influenced extinction and adaptation in Candida albicans, we conducted a fully factorial series of short in vitro evolution experiments. Dosing regimen significantly influenced extinction: all replicates evolved at all drug concentrations survived the 48 h and 72 h transfer experiments, yet the extinction rate increased sharply from low to high drug concentration with 24 h transfers. Increases in drug tolerance were frequent regardless of the dosing regimen. In contrast, drug resistance and growth rate both tended to decrease following evolution at higher drug concentrations. Fluconazole-evolved replicates exhibited frequent increases in cross-tolerance and decreases in cross-resistance in a panel of other azole drugs. Genome size changes were common in some strain backgrounds evolved to higher fluconazole concentrations, regardless of transfer times. Combined, our results indicate that many experimental factors can influence the trajectory of adaptation, and show that extinction in a fungistatic drug occurs readily at high drug concentrations with rapid transfers.

microbiology↗

bioRxiv2026

oidyPeaks: R package for automated flow cytometry cell cycle analysis and visualization in fungal microbes

Karyotypic variation in fungal populations from ecological, clinical, and industrial environments and is also a hallmark of many cancers. The evolutionary dynamics of karyotypic vriants (i.e., changes in the number of chromosomes, either as complete sets or single chromosomes) are the same as any de novo mutation. To quantify karyotypic subpopulations within evolving populations, an unbiased computational method is required to identify subpopulations and quantify their abundance. Flow cytometry is the standard method to measure the genome size from large populations of individual cells. Cell populations typically include cells in all stages of the cell cycle (G0/G1 before DNA replication, S phase during replication, and G2/M after DNA replication but before cell division). Existing mathematical models can be fit to genome-size distributions to determine the base ploidy of a population, but existing algorithms assume a single base ploidy. Mixed-karyotype populations therefore require manual gating to subdivide the flow cytometry data before analysis, which introduces bias, as it is not always clear where or when gates should be drawn. To overcome this, we developed a new method that identifies and quantifies karyotypic subpopulations from flow cytometry data. The open-source R package ploidyPeaks builds on existing Bioconductor tools to import and manipulate standard .fcs flow cytometry files. Using reference populations of known ploidy, we established thresholds to distinguish single-genome size populations and identify candidate mixed-karyotype samples. We then developed a peak-detection algorithm to identify G0/G1 and G2/M peaks, resolve karyotypic subpopulations within mixed populations, and apply nonlinear least-squares fitting of the Dean-Jett-Fox cell-cycle model to each subpopulation, providing a confidence measure for model fit. Compared with existing methods, our approach quantifies genome size in mixed-karyotype populations without manual intervention while providing an objective measure of model fit. We demonstrate the utility of this method on 240 replicate populations initiated from 20 Candida albicans genetic backgrounds evolving for 100 generations to the antifungal drug fluconazole.

microbiology↗

bioRxiv2025

Pooled CRISPRi screening reveals fungal-specific vulnerabilities across environments and genetic backgrounds

The rising rate of drug-resistant fungal infections and the emergence of fungal pathogens with intrinsic resistance phenotypes are a growing concern. The close evolutionary distance between mammals and fungi complicates the design of new antifungals and increases the chances of toxic off-target effects. As such, antifungal drug development usually focuses on fungal-specific proteins when considering potential new targets. Ideal drug targets should mediate essential cell processes and be highly sensitive to inhibition. Targeted gene repression can serve as a model for drug-mediated inhibition and for determining the dosage-sensitivity profile of genes of interest. In the fungal pathogen Candida albicans, classical approaches for gene repression can be labour-intensive and limited to one genetic background due to low throughput. Here, we adapt pooled CRISPRi screening in C. albicans for the first time and exploit this technique for large-scale functional genomic analysis. Through pooled CRISPRi screening, we test the repression sensitivity of over a hundred essential genes conserved in fungi but absent in humans, and successfully identify highly dosage-sensitive genes across multiple cell components and pathways. By extending our analysis to ten diverse environmental conditions, we show how the environment influences dosage-sensitivity profiles. Finally, we extend our experiments to two clinical drug-resistant C. albicans strain backgrounds and demonstrate that many of the fitness defects we observed are conserved in resistant clinical isolates. Together, our results highlight a set of genes that are highly dosage-sensitive across different genetic and environmental contexts, making them attractive targets for further investigation. By facilitating rapid, efficient large-scale functional genomics assays across diverse genetic backgrounds, CRISPRi pooled screening will open new frontiers in C. albicans biology.

microbiology↗

bioRxiv2024

FungAMR: A comprehensive portrait of antimicrobial resistance mutations in fungi

Antimicrobial resistance (AMR) is a global threat. To optimize the use of our antifungal arsenal, we need rapid detection and monitoring tools that rely on high-quality AMR mutation data. Here, we performed a thorough manual curation of published AMR mutations in fungal pathogens to produce the FungAMR reference dataset. A total of 501 papers were curated, leading to 35,792 mutation entries all classified with the degree of evidence that supports their role in resistance. FungAMR covers 95 species, 246 genes and 208 drugs. We combined variant effect predictors with FungAMR resistance mutations and showed that these tools could be used to help predict the potential impact of mutations on AMR. Additionally, a comparative analysis among species revealed a high level of convergence in the molecular basis of resistance, highlighting some potentially universal resistance mutations. The analysis also showed that a significant number of resistance mutations lead to cross-resistance within antifungals of a class, as well as between classes for certain mutated genes. The acquisition of fungal resistance in the clinic and the field is an urging concern. Finally, we provide a computational tool, ChroQueTas, that leverages FungAMR to screen fungal genomes for AMR mutations. These resources are anticipated to have great utility for researchers in the fight against antifungal resistance.

microbiology↗

bioRxiv2024

Constraint on boric acid resistance and tolerance evolvability in Candida albicans

Boric acid is a broad-spectrum antimicrobial used to treat vulvovaginal candidiasis when patients relapse on the primary azole drug fluconazole. Candida albicans is the most common cause of vulvovaginal candidiasis, colloquially referred to as a "vaginal yeast infection". Little is known about the propensity of C. albicans to develop BA resistance or tolerance (the ability of a subpopulation to grow slowly in high levels of drug). We evolved 96 replicates from eight diverse C. albicans strains to increasing BA concentrations to test the evolvability of BA resistance and tolerance. Replicate growth was individually assessed daily, with replicates passaged when they had reached an optical density consistent with exponential growth. Many replicates went extinct quickly. Although some replicates could grow in much higher levels of BA than the ancestral strains, evolved populations isolated from the highest terminal BA levels (after 11 weeks of passages) surprisingly showed only modest growth improvements and only at low levels of BA. No large increases in resistance or tolerance were observed in the evolved replicates. Overall, our findings illustrate that there may be evolutionary constraints limiting the emergence of BA resistance and tolerance, which could explain why it remains an effective treatment for recurrent yeast infections.

evolutionary biology↗

bioRxiv2022

Acquisition of cross-azole tolerance and aneuploidy in Candida albicans strains evolved to posaconazole

A number of in vitro studies have examined the acquisition of drug resistance to the triazole fluconazole, a first-line treatment for many Candida infections. Much less is known about posaconazole, a newer triazole. We conducted the first in vitro experimental evolution of replicates from eight diverse strains of C. albicans in a high level of the fungistatic drug posaconazole. Approximately half of the 132 evolved replicates survived 50 generations of evolution, biased towards some of the strain backgrounds. We found that although increases in drug resistance were rare, increases in drug tolerance (the slow growth of a subpopulation of cells in a level of drug above the resistance level) were common across strains. We also found that adaptation to posaconazole resulted in widespread cross-tolerance to other azole drugs. Widespread aneuploidy variation was also observed in evolved replicates from some strain backgrounds. Trisomy of chromosomes 3, 6, and R was identified in 11 of 12 whole-genome sequenced evolved SC5314 replicates. These findings document rampant evolved cross-tolerance among triazoles and highlight that increases in drug tolerance can evolve independently of drug resistance in a diversity of C. albicans strain backgrounds.

evolutionary biology↗

bioRxiv2021

Evolving a mitigation of the stress response pathway to change the basic chemistry of life

Billions of years of evolution have produced only slight variations in the standard genetic code, and the number and identity of proteinogenic amino acids have remained mostly consistent throughout all three domains of life. These observations suggest a certain rigidity of the genetic code and prompt musings as to the origin and evolution of the code. Here we conducted an adaptive laboratory evolution (ALE) to push the limits of the code restriction, by evolving Escherichia coli to fully replace tryptophan, thought to be the latest addition to the genetic code, with the analog L-{beta}-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa). We identified an overshooting of the stress response system to be the main inhibiting factor for limiting ancestral growth upon exposure to {beta}-(thieno[3,2-b]pyrrole ([3,2]Tp), a metabolic precursor of [3,2]Tpa, and Trp limitation. During the ALE, E. coli was able to "calm down" its stress response machinery, thereby restoring growth. In particular, the inactivation of RpoS itself, the master regulon of the general stress response, was a key event during the adaptation. Knocking out the rpoS gene in the ancestral background independent of other changes conferred growth on [3,2]Tp. Our results add additional evidence that frozen regulatory constraints rather than a rigid protein translation apparatus are Lifes gatekeepers of the canonical amino acid repertoire. This information will not only enable us to design enhanced synthetic amino acid incorporation systems but may also shed light on a general biological mechanism trapping organismal configurations in a status quo. SIGNIFICANCE STATEMENTThe (apparent) rigidity of the genetic code, as well as its universality, have long since ushered explorations into expanding the code with synthetic, new-to-nature building blocks and testing its boundaries. While nowadays even proteome-wide incorporation of synthetic amino acids has been reported on several occasions1-3, little is known about the underlying mechanisms. We here report ALE with auxotrophic E. coli that yielded successful proteome-wide replacement of Trp by its synthetic analog [3,2]Tpa accompanied with the selection for loss of RpoS4 function. Such laboratory domestication of bacteria by the acquisition of rpoS mitigation mutations is beneficial not only to overcome the stress of nutrient (Trp) starvation but also to evolve the paths to use environmental xenobiotics (e.g. [3,2]Tp) as essential nutrients for growth. We pose that regulatory constraints rather than a rigid and conserved protein translation apparatus are Lifes gatekeepers of the canonical amino acid repertoire (at least where close structural analogs are concerned). Our findings contribute a step towards understanding possible environmental causes of genetic changes and their relationship to evolution. Our evolved strain affords a platform for homogenous protein labeling with [3,2]Tpa as well as for the production of biomolecules5, which are challenging to synthesize chemically. Top-down synthetic biology will also benefit greatly from breaking through the boundaries of the frozen bacterial genetic code, as this will enable us to begin creating synthetic cells capable to utilize an expanded range of substrates essential for life.

synthetic biology↗

bioRxiv2016

Ploidy tug-of-war: evolutionary and genetic environments influence the rate of ploidy drive in a human fungal pathogen

Variation in baseline ploidy is seen throughout the tree of life, yet the factors that determine why one ploidy level is selected over another remain poorly understood. Experimental evolution studies using asexual fungal microbes with manipulated ploidy levels intriguingly reveals a propensity to return to the historical baseline ploidy, a phenomenon that we term ploidy drive. We evolved haploid, diploid, and polyploid strains of the human fungal pathogen Candida albicans under three different nutrient limitation environments to test whether these conditions, hypothesized to select for low ploidy levels, could counteract ploidy drive. Strains generally maintained or acquired smaller genome sizes in minimal medium and under phosphorus depletion compared to in a complete medium, while mostly maintained or acquired increased genome sizes under nitrogen depletion. Surprisingly, improvements in fitness often ran counter to changes in total nuclear genome size; in a number of scenarios lines that maintained their original genome size often increased in fitness more than lines that converged towards diploidy. Combined, this work demonstrates a role for both the environment and genotype in determination of the rate of ploidy drive, and highlights questions that remain about the force(s) that cause genome size variation.

evolutionary biology↗