The human fungal pathogen Candida albicans poses a significant burden on global health, causing high rates of mortality and antifungal drug resistance. C. albicans is a heterozygous diploid organism that reproduces asexually. Structural variants (SVs) are an important source of genomic rearrangement, particularly in species that lack sexual recombination. To comprehensively investigate SVs across clinical isolates of C. albicans, we conducted long-read sequencing and genome-wide SV analysis in three distantly related clinical isolates. Our work includes a new, comprehensive analysis of transposable element (TE) composition, location, and diversity. SVs and TEs are frequently close to coding sequences and many SVs are heterozygous, suggesting that SVs might impact gene and allele-specific expression. Most SVs are uniquely present in only one clinical isolate, indicating that SVs represent a significant source of intraspecies genetic variation. We identify multiple, distinct SVs at the centromeres of Chromosome 4 and Chromosome 5, including inversions and transposon polymorphisms. These two chromosomes are often aneuploid in drug-resistant clinical isolates and can form isochromosome structures with breakpoints near the centromere. Further screening of 100 clinical isolates confirms the widespread presence of centromeric SVs in C. albicans, often appearing in a heterozygous state, indicating that SVs are contributing to centromere evolution in C. albicans. Together, these findings highlight that SVs and TEs are common across diverse clinical isolates of C. albicans and that the centromeres of this organism are important sites of genome rearrangement.

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.

Abstract Invasive fungal infections are a leading global cause of human mortality. Only three major classes of antifungal drugs are widely used, and resistance to all three classes can arise rapidly. The most widely prescribed antifungal drug, fluconazole, disseminates rapidly and reaches a wide range of concentrations throughout the body. The impact of drug concentration on the spectrum and effect of mutations acquired during adaptation is not known for any fungal pathogen, and how the specific level of a given stress influences the distribution of beneficial mutations has been poorly explored in general. We evolved 144 lineages from three genetically distinct clinical isolates of Candida albicans to four concentrations of fluconazole (0, 1, 8, and 64 μg/ml) and performed comprehensive phenotypic and genomic comparisons of ancestral and evolved populations. Adaptation to different fluconazole concentrations resulted in distinct adaptive trajectories. In general, lineages evolved to drug concentrations close to their MIC50 (the level of drug that reduces growth by 50% in the ancestor) tended to rapidly evolve an increased MIC50 and acquired distinct segmental aneuploidies and copy number variations. By contrast, lineages evolved to drug concentrations above their ancestral MIC50 tended to acquire a different suite of mutational changes and increased in drug tolerance (the ability of a subpopulation of cells to grow slowly above their MIC50). This is the first evidence that different concentrations of drug can select for different genotypic and phenotypic outcomes in vitro and may explain observed in vivo drug response variation.

Whole genome sequencing of human fungal pathogens has revolutionized the speed and accuracy in which sequence variants that cause antifungal resistance can be identified. Genome rearrangements resulting in copy number variation (CNV) are a significant source of acquired antifungal drug resistance across diverse fungal species. Some CNVs are transient in nature, while other CNVs are stable and well tolerated even in the absence of antifungal drugs. By visualizing whole genome sequencing read depth as a function of genomic location, CNVs and CNV breakpoints (genomic positions where the copy number changes occur relative to the rest of the genome) are rapidly identified. A similar analysis can be used to visualize allele ratio changes that occur across the genomes of heterozygous fungal species, both in the presence and absence of CNVs. This protocol walks through the bioinformatic analysis of CNVs and allele ratios utilizing free, open-source visualization tools. We provide code to use with an example dataset (matched antifungal drug-sensitive and drug-resistant Candida albicans isolates) and notes on how to expand this protocol to other fungal genomes.

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 ≥256µg/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. Importance Drug 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.

Abstract Background Candidemia has significant clinical implications, due to high rates of mortality and increasing resistance to antifungal drugs. Candida auris shows person-to-person transmission and survival on fomites. We aimed to determine if similar hospital transmission of Candida species, other than C. auris, is occurring. We analyzed candidemia infections for species, geographical, and temporal clusters, and clonality. We also aimed to study resistance patterns in individual patients on antifungal treatment. Here we present our current data from December 2019 – March 2021. Methods Patients with candidemia were identified with the help of the clinical microbiology lab serving an urban health system. Isolates were stored prospectively as glycerol stocks at -80 C. Data were collected by retrospective chart review and described in terms of frequency distributions and percentages. Patient locations within the hospital setting were traced by Infection Prevention. A cluster was defined as the same species being isolated from ≥ 2 patients in the same unit within a 90-day period. Genomic DNA was isolated and whole genome sequencing was performed. Genomic data were visualized using the Yeast Mapping Analysis Pipeline. The Minimum Inhibitory Concentration (MIC) was determined using broth microdilution. Results 105 patients were identified from six hospitals (Table 1). Seven clusters of candidemia were identified (Table 2). Genome sequencing supported that all isolates from an individual patient were genetically related. No clonality was observed for isolates from different patients, including those representing two of the seven clusters (Figure 1). Loss of heterozygosity was detected in isolates collected 15 minutes apart in the same patient, indicating distinct populations. MICs remained the same at 0.5 ug/ml over 6 days in Patients l and m (Table 1). For Patient n, MICs remained at 0.5-1 ug/ml Day 1-8, but increased to 4 ug/ml on Day 9. No copy number variation was observed in these isolates. Table 1. Species and resistance patterns of candidemia Table 2. Clusters of candidemia Figure 1. Yeast Mapping Analysis Pipeline for Clusters 3 and 4 – Candida albicans Conclusion We have not found evidence of hospital transmission of candida isolates in our investigations to date. We plan to evaluate clonality in the remaining 5 clusters. Future single nucleotide polymorphism analysis will determine if acquisition of point mutations is causing the increased MIC in Patient n. Disclosures All Authors: No reported disclosures

Candida albicans is a prevalent human fungal pathogen. Rapid genomic change, due to aneuploidy, is a common mechanism that facilitates survival from multiple types of stresses including the few classes of available antifungal drugs. The stress survival of aneuploids occurs despite the fitness costs attributed to most aneuploids growing under idealized lab conditions. Systematic study of the aneuploid state in C. albicans has been hindered by the lack of a comprehensive collection of aneuploid strains. Here, we describe a collection of diploid C. albicans aneuploid strains, each carrying one extra copy of each chromosome, all from the same genetic background. We tested the fitness of this collection under several physiological conditions including shifts in pH, low glucose, oxidative stress, temperature, high osmolarity, membrane stress and cell wall stress. We found that, most aneuploids, under most conditions, were less fit than their euploid parent, yet there were specific conditions under which specific aneuploid isolates provided a fitness benefit relative to the euploid parent strain. Importantly, this fitness benefit was attributable to the change in the copy number of specific chromosomes. Thus, C. albicans can tolerate aneuploidy of each chromosome and some aneuploids confer improved growth under conditions that the yeast encounters in its host niches.

We utilize the PageRank vector to generalize the $k$-means clustering algorithm to directed and undirected graphs. We demonstrate that PageRank and other centrality measures can be used in our setting to robustly compute centrality of nodes in a given graph. Furthermore, we show how our method can be generalized to metric spaces and apply it to other domains such as point clouds and triangulated meshes

Previously, we identified long repeat sequences that are frequently associated with genome rearrangements, including copy number variation (CNV), in many diverse isolates of the human fungal pathogen Candida albicans (Todd et al., 2019). Here, we describe the rapid acquisition of novel, high copy number CNVs during adaptation to azole antifungal drugs. Single-cell karyotype analysis indicates that these CNVs appear to arise via a dicentric chromosome intermediate and breakage-fusion-bridge cycles that are repaired using multiple distinct long inverted repeat sequences. Subsequent removal of the antifungal drug can lead to a dramatic loss of the CNV and reversion to the progenitor genotype and drug susceptibility phenotype. These findings support a novel mechanism for the rapid acquisition of antifungal drug resistance and provide genomic evidence for the heterogeneity frequently observed in clinical settings.

Genome rearrangements resulting in copy number variation (CNV) and loss of heterozygosity (LOH) are frequently observed during the somatic evolution of cancer and promote rapid adaptation of fungi to novel environments. In the human fungal pathogen Candida albicans, CNV and LOH confer increased virulence and antifungal drug resistance, yet the mechanisms driving these rearrangements are not completely understood. Here, we unveil an extensive array of long repeat sequences (65–6499 bp) that are associated with CNV, LOH, and chromosomal inversions. Many of these long repeat sequences are uncharacterized and encompass one or more coding sequences that are actively transcribed. Repeats associated with genome rearrangements are predominantly inverted and separated by up to ~1.6 Mb, an extraordinary distance for homology-based DNA repair/recombination in yeast. These repeat sequences are a significant source of genome plasticity across diverse strain backgrounds including clinical, environmental, and experimentally evolved isolates, and represent previously uncharacterized variation in the reference genome.

Morphogenetic transitions are prevalent in the fungal kingdom. For a leading human fungal pathogen, Candida albicans, the capacity to transition between yeast and filaments is key for virulence. For the model yeast Saccharomyces cerevisiae, filamentation enables nutrient acquisition. A recent functional genomic screen in S. cerevisiae identified Mfg1 as a regulator of morphogenesis that acts in complex with Flo8 and Mss11 to mediate transcriptional responses crucial for filamentation. In C. albicans, Mfg1 also interacts physically with Flo8 and Mss11 and is critical for filamentation in response to diverse cues, but the mechanisms through which it regulates morphogenesis remained elusive. Here, we explored the consequences of perturbation of Mfg1, Flo8, and Mss11 on C. albicans morphogenesis, and identified functional divergence of complex members. We observed that C. albicans Mss11 was dispensable for filamentation, and that overexpression of FLO8 caused constitutive filamentation even in the absence of Mfg1. Harnessing transcriptional profiling and chromatin immunoprecipitation coupled to microarray analysis, we identified divergence between transcriptional targets of Flo8 and Mfg1 in C. albicans. We also established that Flo8 and Mfg1 cooperatively bind to promoters of key regulators of filamentation, including TEC1, for which overexpression was sufficient to restore filamentation in the absence of Flo8 or Mfg1. To further explore the circuitry through which Mfg1 regulates morphogenesis, we employed a novel strategy to select for mutations that restore filamentation in the absence of Mfg1. Whole genome sequencing of filamentation-competent mutants revealed chromosome 6 amplification as a conserved adaptive mechanism. A key determinant of the chromosome 6 amplification is FLO8, as deletion of one allele blocked morphogenesis, and chromosome 6 was not amplified in evolved lineages for which FLO8 was re-located to a different chromosome. Thus, this work highlights rewiring of key morphogenetic regulators over evolutionary time and aneuploidy as an adaptive mechanism driving fungal morphogenesis.

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.

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.

Ploidy, the number of sets of homologous chromosomes in a cell, can alter cellular physiology, gene regulation, and the spectrum of acquired mutations. Advances in single-cell flow cytometry have greatly improved the understanding of how genome size contributes to diverse biological processes including speciation, adaptation, pathogenesis, and tumorigenesis. For example, fungal pathogens can undergo whole genome duplications during infection of the human host and during acquisition of antifungal drug resistance. Quantification of ploidy is dramatically affected by the nucleic acid staining technique and the flow cytometry analysis of single cells. Ploidy in fungi is also impacted by samples that are heterogeneous for both ploidy and morphology, and control strains with known ploidy must be included in every flow cytometry experiment. To detect ploidy changes within fungal strains, the following protocol was developed to accurately and dependably interrogate single-cell ploidy. © 2018 by John Wiley & Sons, Inc.

Gemmata obscuriglobus is a Gram-negative bacterium with several intriguing biological features. Here, we present a complete, de novo whole genome assembly for G. obscuriglobus which consists of a single, circular 9 Mb chromosome, with no plasmids detected. The genome was annotated using the NCBI Prokaryotic Genome Annotation pipeline to generate common gene annotations. Analysis of the rRNA genes revealed three interesting features for a bacterium. First, linked G. obscuriglobus rrn operons have a unique gene order, 23S–5S–16S, compared to typical prokaryotic rrn operons (16S–23S–5S). Second, G. obscuriglobus rrn operons can either be linked or unlinked (a 16S gene is in a separate genomic location from a 23S and 5S gene pair). Third, all of the 23S genes (5 in total) have unique polymorphisms. Genome analysis of a different Gemmata species (SH-PL17), revealed a similar 23S–5S–16S gene order in all of its linked rrn operons and the presence of an unlinked operon. Together, our findings show that unique and rare features in Gemmata rrn operons among prokaryotes provide a means to better define the evolutionary relatedness of Gemmata species and the divergence time for different Gemmata species. Additionally, these rrn operon differences provide important insights into the rrn operon architecture of common ancestors of the planctomycetes.

The ability of an organism to replicate and segregate its genome with high fidelity is vital to its survival and for the production of future generations. Errors in either of these steps (replication or segregation) can lead to a change in ploidy or chromosome number. While these drastic genome changes can be detrimental to the organism, resulting in decreased fitness, they can also provide increased fitness during periods of stress. A change in ploidy or chromosome number can fundamentally change how a cell senses and responds to its environment. Here, we discuss current ideas in fungal biology that illuminate how eukaryotic genome size variation can impact the organism at a cellular and evolutionary level. One of the most fascinating observations from the past 2 decades of research is that some fungi have evolved the ability to tolerate large genome size changes and generate vast genomic heterogeneity without undergoing canonical meiosis.

The deconvolution of biological dielectric spectra can be difficult enough with artefact-free spectra but is more problematic when machine baseline artefacts and electrode polarisation are present as well. In addition, these two sources of anomalies can be responsible for significant interference with dielectric biomass measurements made using one- or two-spot frequencies. The aim of this paper is to develop mathematical models of baseline artefacts and electrode polarisation which can be used to remove these anomalies from dielectric spectra in a way that can be easily implemented on-line and in real-time on the Biomass Monitor (BM). We show that both artefacts can be successfully removed in solutions of organic and inorganic ions; in animal cell and microbial culture media; and in yeast suspensions of varying biomass. The high quality of the compensations achieved were independent of whether gold and platinum electrodes were used; the electrode geometry; electrode fouling; current density; the type of BM; and of whether electrolytic cleaning pulses had been applied. In addition, the calibration experiments required could be done off-line using a simple aqueous KCl dilution series with the calibration constants being automatically calculated by a computer without the need for user intervention. The calibration values remained valid for a minimum of 3 months for the baseline model and indefinitely for the electrode polarisation one. Importantly, application of baseline correction prior to polarisation correction allowed the latter's application to the whole conductance range of the BM. These techniques are therefore exceptionally convenient to use under practical conditions.