Biology subjects

Berman, J.

Publications and source records attributed to Berman, J..

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bioRxiv2019

Autonomously replicating linear plasmids facilitate the analysis of replication origin function in Candida albicans

The ability to generate autonomously replicating plasmids has been elusive in Candida albicans, a prevalent human fungal commensal and pathogen. Instead, plasmids generally integrate into the genome. Here, we assessed plasmid and transformant properties, including plasmid geometry, transformant colony size, four selectable markers, and potential origins of replication for their ability to drive autonomous plasmid maintenance. Importantly, linear plasmids with terminal telomere repeats yielded many more autonomous transformants than circular plasmids with the identical sequences. Furthermore, we could distinguish by colony size, transient, autonomously replicating and chromosomally integrated transformants (tiny, medium and large, respectively). Candida albicans URA3 and a heterologous marker, ARG4, yielded many transient transformants indicative of weak origin activity; replication of plasmid carrying heterologous LEU2 marker was highly dependent upon the addition of a bona fide origin sequence. Several bona fide chromosomal origins, with an origin fragment of ~100 bp as well as a heterologous origin, panARS, from Kluyveromyces lactis drove autonomous replication, yielding moderate transformation efficiency and plasmid stability. Thus, C. albicans maintains linear plasmids that yield high transformation efficiency and are maintained autonomously in an origin-dependent manner. ImportanceCircular plasmids are important tools for molecular manipulation in model fungi such as bakers yeast, yet, in Candida albicans, an important yeast pathogen of humans, prior studies were not able to generate circular plasmids that were autonomous (duplicated without inserting themselves into the chromosome). Here, we found that linearizing circular plasmids with sequences from telomeres, the chromosome ends, allows the plasmids to duplicate and segregate in C. albicans. We used this system to identify chromosomal sequences that facilitate the initiation of plasmid replication (origins) and to show that a ~100 bp fragment of a C. albicans origin, as well as an origin sequence from a distantly related yeast, can both function as origins in C. albicans. Thus, the requirements for plasmid geometry, but not necessarily for origin sequences, differ between C. albicans and bakers yeast.

molecular biology↗

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↗

bioRxiv2018

Chromatin profiling of the repetitive and non-repetitive genome of the human fungal pathogen Candida albicans

BackgroundEukaryotic genomes are packaged into chromatin structures with pivotal roles in regulating all DNA-associated processes. Post-translational modifications of histone proteins modulate chromatin structure leading to rapid, reversible regulation of gene expression and genome stability which are key steps in environmental adaptation. Candida albicans is the leading fungal pathogen in humans, and can rapidly adapt and thrive in diverse host niches. The contribution of chromatin to C. albicans biology is largely unexplored.\n\nResultsHere, we harnessed genome-wide sequencing approaches to generate the first comprehensive chromatin profiling of histone modifications (H3K4me3, H3K9Ac, H4K16Ac and {gamma}-H2A) across the C. albicans genome and relate it to gene expression. We demonstrate that gene-rich non-repetitive regions are packaged in canonical euchromatin associated with histone modifications that mirror their transcriptional activity. In contrast, repetitive regions are assembled into distinct chromatin states: subtelomeric regions and the rDNA locus are assembled into canonical heterochromatin, while Major Repeat Sequences and transposons are packaged in chromatin bearing features of euchromatin and heterochromatin. Genome-wide mapping of {gamma}H2A, a marker of genome instability, allowed the identification of potential recombination-prone genomic sites. Finally, we present the first quantitative chromatin profiling in C. albicans to delineate the role of the chromatin modifiers Sir2 and Set1 in controlling chromatin structure and gene expression.\n\nConclusionsThis study presents the first genome-wide chromatin profiling of histone modifications associated with the C. albicans genome. These epigenomic maps provide an invaluable resource to understand the contribution of chromatin to C. albicans biology.

genomics↗

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

Candida albicans dispersed cells signify a developmental state distinct from biofilm and planktonic phases

Candida albicans surface-attached biofilms are sites of amplification of an infection through continuous discharge of cells capable of initiating new infectious foci. Yeast cells released from biofilms on intravenous catheters have direct access to the bloodstream. We previously reported that dispersed cells are largely lateral yeast cells that originate from the hyphal layers of the biofilm. Compared to their planktonic counterparts, these biofilm-dispersed yeast cells displayed enhanced virulence-associated gene expression and drug resistance. Little is known about the molecular properties of dispersed cells. We found that the inducer of dispersal, PES1, genetically interacts with the repressor of filamentation, NRG1, in a manner that supports a genetic definition of dispersed cells as yeast. We combined a flow biofilm model with RNA sequencing technology, to identify transcriptomic characteristics of freshly dispersed yeast cells versus biofilms or age-matched planktonic yeast cells growing in glucose-rich medium. Dispersed cells largely inherited a biofilm-like mRNA profile but with one stark difference: dispersed cells were transcriptionally reprogrammed to metabolize alternative carbon sources, while their sessile parents expressed glycolytic genes, despite exposure to the same nutritional signals. Our studies hence define dispersal cell production as an intrinsic step of biofilm development which generates propagules capable of colonizing distant host sites. This developmental step anticipates the need for virulence-associated gene expression before experiencing the associated external signals.

microbiology↗

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↗

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↗