Biology subjects

Gerstein, A. C.

Publications and source records attributed to Gerstein, A. C..

8 recordsLinked to original sources

bioRxiv2026

Chromosome-scale CRISPR screening reveals secretory pathway genes as drivers of aneuploidy-mediated antifungal tolerance

The gain or loss of chromosomes in eukaryotes often drives aberrant phenotypes by altering the expression levels of hundreds or thousands of genes. In the case of beneficial aneuploidies, the genetic basis of fitness improvement has rarely been pinpointed, and identifying the causal genes remains a major challenge in engineering biology. The leading cause of human fungal infections, Candida albicans, frequently acquires extra copies of chromosome R (ChrR) following exposure to azole antifungal drugs, resulting in heightened antifungal tolerance. Here, we combine RNA-seq with parallel chromosome-wide CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) screens to systematically profile the ChrR genes contributing to azole tolerance. Using multiplexed CRISPR-dCas12a tools, we further characterize the combinatorial effects of candidate genes and uncover a central role for post-Golgi secretory trafficking in antifungal tolerance. Specifically, we demonstrate that the secretory pathway regulators SEC4 and YPT31 are both necessary and sufficient for ChrR-mediated azole tolerance. By leveraging a large-scale CRISPRa screen in a fungal pathogen, our work functionally dissects one of the most common aneuploidies observed in C. albicans, provides mechanistic insight into the molecular basis of antifungal tolerance, and establishes a generalizable framework for studying aneuploidy-mediated phenotypes across eukaryotic organisms.

microbiology↗

bioRxiv2026

Global whole-genome phylogenomics of Nakaseomyces glabratus reveals admixture and refines sequence type-based classification

Nakaseomyces glabratus is a globally distributed opportunistic fungal pathogen. An ongoing discussion in studies of N. glabratus population structure has been whether genetic clusters are best defined using multilocus sequence typing (MLST) or short-read whole-genome sequencing (WGS). To assess the concordance between MLST- and WGS-based phylogenies, we analyzed a dataset of 548 N. glabratus WGS sequences from 12 countries. Clusters identified from WGS largely recapitulated the MLST-defined sequence type (ST) groups: fourteen WGS clusters were composed of a single MLST ST, and the remaining contained STs with very closely related MLST profiles. We thus propose a pragmatic naming convention, consistent with the system used in other microbial species, which specifies WGS cluster labels based on the primary ST. From the large WGS isolate dataset, we determined the prevalence of admixture and genomic variants. Interestingly, seven of the nine singleton isolates were admixed, in addition to 58 isolates from six different clusters. Aneuploidy was detected in 4% of isolates, most commonly in chrE, which contains ERG11, the gene encoding the enzyme targeted by azole antifungals. Aneuploid chromosomes did not exhibit elevated heterozygosity relative to the sequencing error rate, consistent with instability of extra chromosome copies. Copy number variants were found in 3% of the isolates; some of the CNVs co-occurred with aneuploidies, and were primarily identified on chrD, chrE, chrI, and chrM. Our findings demonstrate that deep splits between clusters preserve the utility of MLST ST designations for clade-level designation, yet underscore the utility of WGS for high-resolution genomic analyses. Article SummaryThere is an ongoing debate in studies on Nakaseomyces glabratus about whether traditional MLST analysis is sufficient to determine population structure, or whether the precision of whole genome sequencing (WGS) is necessary. We analyzed WGS data from 548 isolates from around the world. We found a very strong agreement between the two methods. We propose a hybrid naming system, where cluster names are based on the dominant MLST group. We used the WGS data to show that admixed isolates, and those with extra chromosomes or CNVs are rare (<7% of isolates in each class) and are distributed throughout the phylogeny.

evolutionary biology↗

bioRxiv2023

Genotypic and phenotypic homogeneity of vaginal and rectal yeast isolates from recurrent vulvovaginal candidiasis

Vulvovaginal candidiasis is one of the most common fungal infections. Most are successfully treated with antifungal drugs, yet [~]8% lead to recurrent vulvovaginal candidiasis ("RVVC"). Previous research found closely-related isolates within vaginal and rectal populations. However, their methods preclude assessing fine-scale relationships among closely related isolates and measuring genetic variation, a fundamental property with evolutionary potential implications. To address this gap, we isolated 12 vaginal and 12 rectal yeast isolates during symptomatic relapse from four individuals with a history of RVVC. Three had Candida albicans infections, while the fourth had Nakaseomyces glabratus. All isolates were whole-genome sequenced and phenotyped. The isolates were placed into global phylogenies built from short-read WGS data, including an updated N. glabratus tree with over 500 isolates. Genotypic and phenotypic analyses were consistent with migration between sites. There was little phenotypic diversity for drug response and no consistent difference between isolates from different sites for invasive growth. Although there are few comparables, C. albicans nucleotide diversity was similar to most commensal oral and rectal populations, while N. glabratus was similar to some bloodstream infections (though higher than others). Single nucleotide changes drove diversity; no aneuploidies were found, and only a single loss-of-heterozygosity tract on chr1L varied among isolates from one participant. This study provides baseline measurements and describes techniques to quantify within-population diversity in fungal microbes. We highlight a need for comparable studies that use the same sampling effort and analysis methods to understand the interplay in shaping fungal microbial communities in important contexts. Author SummaryRecurrent vaginal yeast infections are relatively common, and we do not understand why some people experience these chronic infections when many others have a single infection that is successfully treated and cleared. Many open questions remain about the basic biology of the yeast populations involved. We quantified diversity using modern sequencing technology within vaginal and rectal yeast populations from four individuals with a history of recurrent yeast infections experiencing symptoms. Three participants had a Candida albicans infection (the most common causative species), while the fourth had a Nakaseomyces glabratus infection (the second most common and increasingly implicated). We found that vaginal and rectal isolates were closely related, indicating the same population is present at the two sites. Surprisingly, we found that diversity was similar to the yeast populations found at other body sites in healthy people. Our study highlights a critical need for additional studies following the same methods in different contexts to better understand the fungal microbial populations in our bodies.

microbiology↗

bioRxiv2022

Differential response of Candida species morphologies and isolates to fluconazole and boric acid

Candida albicans is the most prevalent cause of vulvovaginal candidiasis ( yeast infection) and recurrent vulvovaginal candidiasis, though the incidence of non-albicans yeast species is increasing. The azole fluconazole is the primary antifungal drug used to treat R/VVC yet isolates from some species have intrinsic resistance to fluconazole, and recurrent infection can occur even with fluconazole-susceptible populations. The second-line broad-spectrum antimicrobial drug, boric acid, is an alternative treatment that has been found to successfully treat complicated VVC infections. Far less is known about how boric acid inhibits growth of yeast isolates in different morphologies compared to fluconazole. We found significant differences in drug resistance and drug tolerance (the ability of a subpopulation to grow slowly in high levels of drug) between C. albicans, C. glabrata, and C. parapsilosis isolates, with the specific relationships dependent on both drug and phenotype. Population-level variation for both susceptibility and tolerance was broader for fluconazole than boric acid in all species. Unlike fluconazole, which neither prevented hyphal formation nor disrupted mature biofilms, boric acid inhibited C. albicans hyphal formation and reduced mature biofilm biomass and metabolic activity in all isolates in a dose-dependent manner. Variation in planktonic response did not generally predict biofilm phenotypes. Overall, our findings illustrate that boric acid is broadly effective at inhibiting growth across many isolates and morphologies, which could explain why it is an effective treatment for R/VVC.

microbiology↗

bioRxiv2019

Identification of pathogen genomic differences that impact human immune response and disease during Cryptococcus neoformans infection

Patient outcomes during infection are due to a complex interplay between the quality of medical care, host immunity factors, and the infecting pathogens characteristics. To probe the influence of pathogen genotype on human immune response and disease, we examined Cryptococcus neoformans isolates collected during the Cryptococcal Optimal ART Timing (COAT) trial in Uganda. We measured human participants immunologic phenotypes, meningitis disease parameters, and survival. We compared this clinical data to whole genome sequences from 38 C. neoformans isolates of the most frequently observed sequence type (ST) ST93 in our Ugandan participant population, and an additional 18 strains from 9 other sequence types representing the known genetic diversity within the Ugandan Cryptococcus clinical isolates. We focused our analyses on 652 polymorphisms that: were variable among the ST93 genomes, were not in centromeres or extreme telomeres, and were predicted to have a fitness effect. Logistic regression and principal component analyses identified 40 candidate Cryptococcus genes and 3 hypothetical RNAs associated with human immunologic response or clinical parameters. We infected mice with 17 available KN99 gene deletion strains for these candidate genes and found that 35% (6/17) directly influenced murine survival. Four of the six gene deletions that impacted murine survival were novel. Such bedside-to-bench translational research provides important candidate genes for future studies on virulence-associated traits in human Cryptococcus infections.\n\nAuthor SummaryEven with the best available care, mortality rates in cryptococcal meningitis range from 20-60%. Disease is often due to infection by the fungus Cryptococcus neoformans and involves a complex interaction between the human host and the fungal pathogen. Although previous studies have suggested genetic differences in the pathogen impact human disease, it has proven quite difficult to identify the specific C. neoformans genes that impact the outcome of the human infection. Here, we take advantage of a Ugandan patient cohort infected with closely related C. neoformans strains to examine to role of pathogen genetic variants on several human disease characteristics. Using a pathogen whole genome sequencing approach, we showed that 40 C. neoformans genes are associated with human disease. Surprisingly, many of these genes are specific to Cryptococcus and have unknown functions. We also show deletion of these genes alters disease in a mouse model of infection, confirming their role in disease. These findings are particularly important because they are the first to identify C. neoformans genes associated with human cryptococcal meningitis and lay the foundation for future studies that may lead to new treatment strategies aimed at reducing patient mortality.

genetics↗

bioRxiv2018

Ancestral fitness and genetic background influence diversity acquired during adaptation to low drug concentration in Candida albicans

The importance of within-species diversity in determining the evolutionary potential of a population to evolve drug resistance or tolerance is not well understood, including in eukaryotic pathogens. To examine the influence of genetic background, we evolved replicates of twenty different clinical isolates of Candida albicans, a human fungal pathogen, in fluconazole, the commonly used antifungal drug. The isolates hailed from the major C. albicans clades and had different initial levels of drug resistance and tolerance to the drug. The majority of replicates rapidly increased in fitness in the evolutionary environment, with the degree of improvement inversely correlated with ancestral strain fitness in the drug. Improvement was largely restricted to up to the evolutionary level of drug: only 4% of the evolved replicates increased resistance (MIC) above the evolutionary level of drug. Prevalent changes were altered levels of drug tolerance (slow growth of a subpopulation of cells at drug concentrations above the MIC) and increased diversity of genome size. The prevalence and predominant direction of these changes differed in a strain-specific manner but neither correlated directly with ancestral fitness or improvement in fitness. Rather, low ancestral strain fitness was correlated with high levels of heterogeneity in fitness, tolerance, and genome size among evolved replicates. Thus, ancestral strain background is an important determinant in mean improvement to the evolutionary environment as well as the diversity of evolved phenotypes, and the range of possible responses of a pathogen to an antimicrobial drug cannot be captured by in-depth study of a single strain background. ImportanceAntimicrobial resistance is an evolutionary phenomenon with clinical implications. We tested how replicates from diverse strains of Candida albicans, a prevalent human fungal pathogen, evolve in the commonly-prescribed antifungal drug fluconazole. Replicates on average increased in fitness in the level of drug they were evolved to, with the least fit ancestral strains improving the most. Very few replicates increased resistance above the drug level they were evolved in. Notably, many replicates increased in genome size and changed in drug tolerance (a drug response where a subpopulation of cells grow slowly in high levels of drug) and variability among replicates in fitness, tolerance and genome size was higher in strains that initially were more sensitive to the drug. Genetic background influenced the average degree of adaptation and the evolved variability of many phenotypes, highlighting that different strains from the same species may respond and adapt very differently during adaptation.

evolutionary biology↗

bioRxiv2018

Quantifying how constraints limit the diversity of viable routes to adaptation

Convergent adaptation can occur at the genome scale when independently evolving lineages use the same genes to respond to similar selection pressures. These patterns provide insights into the factors that facilitate or constrain the diversity of genetic responses that contribute to adaptive evolution. A first step in studying such factors is to quantify the observed amount of repeatability relative to expectations under a null hypothesis. Here, we formulate a novel metric to quantify the constraints driving the observed amount of repeated adaptation in pairwise contrasts based on the hypergeometric distribution, and then generalize this for simultaneous analysis of multiple lineages. This metric is explicitly based on the probability of observing a given amount of repeatability by chance under an arbitrary null hypothesis, and is readily compared among different species and types of trait. We also formulate a metric to quantify the effective proportion of genes in the genome that have the potential to contribute to adaptation. As an example of how these metrics can be used to draw inferences, we assess the amount of repeatability observed in existing datasets on adaptation to antibiotics in yeast and climate in conifers. This approach provides a method to test a wide range of hypotheses about how different kinds of factors can facilitate or constrain the diversity of genetic responses observed during adaptive evolution.

evolutionary biology↗

bioRxiv2018

Exposure to the oral host niche yields rapid phenotypic and genotypic diversification in Candida albicans

In vitro studies suggest that stress may generate random standing variation, and that different cellular and ploidy states may evolve more rapidly under stress. Yet this idea has not been tested with pathogenic fungi growing within their host niche in vivo. Here, we analyzed the generation of both genotypic and phenotypic diversity during exposure of Candida albicans to the mouse oral cavity. Ploidy, aneuploidy, loss of heterozygosity (LOH) and recombination were determined using flow cytometry and ddRADseq. Colony phenotypic changes (CPs) in size and filamentous growth were evident without selection, and were enriched among colonies selected for LOH of the GAL1 marker. Aneuploidy and LOH occurred on all chromosomes (Chrs), with aneuploidy more frequent for smaller Chrs and whole Chr LOH more frequent for larger Chrs. Large genome shifts in ploidy to haploidy often maintained one or more heterozygous disomic Chrs, consistent with random Chr missegregation events. Most isolates displayed several different types of genomic changes, suggesting that the oral environment rapidly generates diversity de novo. In sharp contrast, following in vitro propagation isolates were not enriched for multiple LOH events, except in those that underwent haploidization and/or had high levels of Chr loss. The frequency of events was overall 100 times higher for C. albicans populations following in vivo passage compared to in vitro. These hyperdiverse in vivo isolates likely provide C. albicans with the ability to adapt rapidly to the diversity of stress environments it encounters inside the host.\n\nAuthor summaryAdaption is a continuous dynamic process that requires genotypic and phenotypic variation. Here we studied the effects of a single passage in a mouse oropharyngeal model of infection on the appearance of diversity in C. albicans, a common commensal of the human oral cavity and GI tract. We found that variation could be rapidly detected following oral colonization, with the frequency of genome change being considerably higher with pre-selection for recombination and colony phenotypic changes. Importantly, one third of all isolates had multiple genome changes, significantly higher than expected by chance alone. We suggest that some cells in the population are naturally hypervariable and that they are a major source of diversity upon which selection can act in stressful conditions in vivo and in vitro.

genetics↗