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.

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" or VVC) and recurrent vulvovaginal candidiasis (RVVC), although the incidence of non-albicans yeast species is increasing. The azole fluconazole is the primary antifungal drug used to treat RVVC, 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, Candida glabrata, and Candida 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 for either drug. 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 RVVC.

The Canadian Fungal Research Network: current challenges and future opportunities.

Fungi critically impact the health and function of global ecosystems and economies. In Canada, fungal researchers often work within silos defined by subdiscipline and institutional type, complicating the collaborations necessary to understand the impacts fungi have on the environment, economy, and plant and animal health. Here, we announce the establishment of the Canadian Fungal Research Network (CanFunNet, https://fungalresearch.ca), whose mission is to strengthen and promote fungal research in Canada by facilitating dialogue among scientists. We summarize the challenges and opportunities for Canadian fungal research that were discussed at CanFunNet's inaugural meeting in 2019, and identify 4 priorities for our community: (i) increasing collaboration among scientists, (ii) studying diversity in the context of ecological disturbance, (iii) preserving culture collections in the absence of sustained funding, and (iv) leveraging diverse expertise to attract trainees. We have gathered additional information to support our recommendations, including a survey identifying underrepresentation of fungal-related courses at Canadian universities, a list of Canadian fungaria and culture collections, and a case study of a human fungal pathogen outbreak. We anticipate that these discussions will help prioritize fungal research in Canada, and we welcome all researchers to join this nationwide effort to enhance knowledge dissemination and funding advocacy.

Quantifying how constraints limit the diversity of viable routes to adaptation.

Convergent adaptation occurs at the genome scale when independently evolving lineages use the same genes to respond to similar selection pressures. These patterns of genetic repeatability 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 index 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 index is explicitly based on the probability of observing a given amount of repeatability by chance under a given null hypothesis and is readily compared among different species and types of trait. We also formulate an index to quantify the effective proportion of genes in the genome that have the potential to contribute to adaptation. As an example of how these indices can be used to draw inferences, we assess the amount of repeatability observed in existing datasets on adaptation to stress 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.

Widespread Genetic Incompatibilities between First-Step Mutations during Parallel Adaptation of Saccharomyces cerevisiae to a Common Environment.

Independently evolving populations may adapt to similar selection pressures via different genetic changes. The interactions between such changes, such as in a hybrid individual, can inform us about what course adaptation may follow and allow us to determine whether gene flow would be facilitated or hampered following secondary contact. We used Saccharomyces cerevisiae to measure the genetic interactions between first-step mutations that independently evolved in the same biosynthetic pathway following exposure to the fungicide nystatin. We found that genetic interactions are prevalent and predominantly negative, with the majority of mutations causing lower growth when combined in a double mutant than when alone as a single mutant (sign epistasis). The prevalence of sign epistasis is surprising given the small number of mutations tested and runs counter to expectations for mutations arising in a single biosynthetic pathway in the face of a simple selective pressure. Furthermore, in one third of pairwise interactions, the double mutant grew less well than either single mutant (reciprocal sign epistasis). The observation of reciprocal sign epistasis among these first adaptive mutations arising in the same genetic background indicates that partial postzygotic reproductive isolation could evolve rapidly between populations under similar selective pressures, even with only a single genetic change in each. The nature of the epistatic relationships was sensitive, however, to the level of drug stress in the assay conditions, as many double mutants became fitter than the single mutants at higher concentrations of nystatin. We discuss the implications of these results both for our understanding of epistatic interactions among beneficial mutations in the same biochemical pathway and for speciation.

Variable gene transcription underlies phenotypic convergence of hypoxia tolerance in sculpins.

BACKGROUND: The degree by which mechanisms underlying phenotypic convergence are similar among taxa depends on the number of evolutionary paths available for selection to act upon. Likelihood of convergence will be influenced by an interplay of factors such as genetic architecture, phylogenetic history and population demography. To determine if there is convergence or divergence in mechanisms underlying phenotypic similarity, we assessed whether gene transcription patterns differed among species with similar levels of hypoxia tolerance. RESULTS: Three species of marine fish from the superfamily Cottoidea (smoothhead sculpin [Artedius lateralis], sailfin sculpin [Nautichthys oculofasciatus] and Pacific staghorn sculpin [Leptocottus armatus]), all of which have previously been shown to share the same level of hypoxia tolerance, were exposed to short-(8 h) and longer-term (72 h) hypoxia and mRNA transcripts were assessed using a custom microarray. We examined hypoxia-induced transcription patterns in metabolic and protein production pathways and found that a high proportion of genes associated with these biological processes showed significant differences among the species. Specifically, the data suggest that the smoothhead sculpin, unlike the sailfin sculpin and the Pacific staghorn sculpin, relied on amino acid degradation rather than glycolysis or fatty acid oxidation to generate ATP during hypoxia exposure. There was also variation across the species in the transcription of genes involved in protein production (e.g. mRNA processing and protein translation), such that it increased in the smoothhead sculpin, decreased in the sailfin sculpin and was variable in the Pacific staghorn sculpin. CONCLUSIONS: Changes in metabolic and protein production pathways are part of the key responses of fishes to exposures to environmental hypoxia. Yet, species with similar overall hypoxia tolerance exhibited different transcriptional responses in these pathways, indicating flexibility and complexity of interactions in the evolution of the mechanisms underlying the hypoxia tolerance phenotype. The variation in the hypoxia-induced transcription of genes across species with similar hypoxia tolerance suggests that similar whole-animal phenotypes can emerge from divergent evolutionary paths that may affect metabolically important functions.

It's not all about us: evolution and maintenance of Cryptococcus virulence requires selection outside the human host.

Cryptococcus is predominantly an AIDS-related pathogen that causes significant morbidity and mortality in immunocompromised patients. Research studies have historically focused on understanding how the organism causes human disease through the use of in vivo and in vitro model systems to identify virulence factors. Cryptococcus is not an obligate pathogen, however, as human-human transmission is either absent or rare. Selection in the environment must thus be invoked to shape the evolution of this taxa, and directly influences genotypic and trait diversity. Importantly, the evolution and maintenance of pathogenicity must also stem directly from environmental selection. To that end, here we examine abiotic and biotic stresses in the environment, and discuss how they could shape the factors that are commonly identified as important virulence traits. We identify a number of important unanswered questions about Cryptococcus diversity and evolution that are critical for understanding this deadly pathogen, and discuss how implementation of modern sampling and genomic tools could be utilized to answer these questions. Copyright © 2016 John Wiley & Sons, Ltd.

Silencing is noisy: population and cell level noise in telomere-adjacent genes is dependent on telomere position and sir2.

Cell-to-cell gene expression noise is thought to be an important mechanism for generating phenotypic diversity. Furthermore, telomeric regions are major sites for gene amplification, which is thought to drive genetic diversity. Here we found that individual subtelomeric TLO genes exhibit increased variation in transcript and protein levels at both the cell-to-cell level as well as at the population-level. The cell-to-cell variation, termed Telomere-Adjacent Gene Expression Noise (TAGEN) was largely intrinsic noise and was dependent upon genome position: noise was reduced when a TLO gene was expressed at an ectopic internal locus and noise was elevated when a non-telomeric gene was expressed at a telomere-adjacent locus. This position-dependent TAGEN also was dependent on Sir2p, an NAD+-dependent histone deacetylase. Finally, we found that telomere silencing and TAGEN are tightly linked and regulated in cis: selection for either silencing or activation of a TLO-adjacent URA3 gene resulted in reduced noise at the neighboring TLO but not at other TLO genes. This provides experimental support to computational predictions that the ability to shift between silent and active chromatin states has a major effect on cell-to-cell noise. Furthermore, it demonstrates that these shifts affect the degree of expression variation at each telomere individually.

diskImageR: quantification of resistance and tolerance to antimicrobial drugs using disk diffusion assays.

Microbial pathogens represent an increasing threat to human health. Although many infections can be successfully treated and cleared, drug resistance is a widespread problem. The existence of subpopulations of 'tolerant' cells (where a fraction of the population is able to grow above the population resistance level) may increase the rate of treatment failure; yet, existing methods to measure subpopulation effects are cumbersome. Here we describe diskImageR, a computational pipeline that analyses photographs of disk diffusion assays to determine the degree of drug susceptibility [the radius of inhibition, (RAD)], and two aspects of subpopulation growth [the fraction of growth (FoG) within the zone of inhibition, (ZOI), and the rate of change in growth from no drug to inhibitory drug concentrations, (SLOPE)]. diskImageR was used to examine the response of the human fungal pathogen Candida albicans to the antifungal drug fluconazole across different strain backgrounds and growth conditions. Disk diffusion assays performed under Clinical and Laboratory Standards Institute (CLSI) conditions led to more susceptibility and less tolerance than assays performed using rich medium conditions. We also used diskImageR to quantify the effects of three drugs in combination with fluconazole, finding that all three combinations affected tolerance, with the effect of one drug (doxycycline) being very strain dependent. The three drugs had different effects on susceptibility, with doxycycline generally having no effect, chloroquine generally increasing susceptibility and pyrvinium pamoate generally reducing susceptibility. The ability to simultaneously quantitate different aspects of microbial drug responses will facilitate the study of mechanisms of subpopulation responses in the presence of antimicrobial drugs.

Mutational effects depend on ploidy level: all else is not equal.

Ploidy is predicted to influence adaptation directly, yet whether single mutations behave the same in different ploidy backgrounds has not been well studied. It has often been assumed theoretically that aside from dominance, selective parameters do not differ between cells of varying ploidy. Using the budding yeast Saccharomyces cerevisiae, I compared the effect size of 20 adaptive mutations in haploids and homozygous diploids and found, surprisingly, that the same mutations often had a much larger effect in haploids than homozygous diploids. This empirical result demonstrates that it cannot be assumed that mutations will have the same effect in haploids and homozygous diploids.

mSphere of Influence: Deterministic and Stochastic Processes Drive Microbial Evolution.

Aleeza C. Gerstein works in the field of microbial evolutionary genetics in human fungal pathogens. In this mSphere of Influence article, she reflects on how the papers "Experimental tests of the roles of adaptation, chance, and history in evolution" by Travisano et al. (1995) and "Pervasive genetic hitchhiking and clonal interference in forty evolving yeast populations" by Lang et al. (2013) provided her with a framework for thinking about the competing stochastic and deterministic evolutionary forces that act on microbial populations at the genotypic and phenotypic levels to cause both parallelism and divergence under different scenarios.

Powering up antifungal treatment: using small molecules to unlock the potential of existing therapies.

Fungal pathogens are increasingly appreciated as a significant infectious disease challenge. Compared to bacteria, fungal cells are more closely related to human cells, and few classes of antifungal drugs are available. Combination therapy offers a potential solution to reduce the likelihood of resistance acquisition and extend the lifespan of existing antifungals. There has been recent interest in combining first-line drugs with small-molecule adjuvants. In a recent article, Alabi et al. identified 1,4-benzodiazepines as promising molecules to enhance azole activity in pathogenic Candida spp. (P. E. Alabi, C. Gautier, T. P. Murphy, X. Gu, M. Lepas, V. Aimanianda, J. K. Sello, I. V. Ene, 2023, mBio https://doi.org/10.1128/mbio.00479-23). These molecules have no antifungal activity on their own but exhibited significant potentiation of fluconazole in azole-susceptible and -resistant isolates. Additionally, the 1,4-benzodiazepines increased the fungicidal activity of azoles that are typically fungistatic to Candida spp., inhibited filamentation (a virulence-associated trait), and accordingly increased host survival in Galleria mellonella. This research thus provides another encouraging step on the critical pathway toward reducing mortality due to antimicrobial resistance.

Experimental evolution of drug resistance in human fungal pathogens.

Experimental evolution in vitro is a powerful tool to uncover the factors that contribute to resistance evolution and understand the genetic basis of adaptation. Here, we discuss recent experimental evolution studies from human fungal pathogens. We synthesize the results to highlight the common threads that influence resistance acquisition. The picture that emerges is that drug resistance consistently appears readily and rapidly. Mutations are often found in an overlapping set of genes and genetic pathways known to be involved in drug resistance, including whole or partial chromosomal aneuploidy. The likelihood of acquiring resistance and cross-resistance between drugs seems to be influenced by the specific drug (not just drug class), level of drug, and strain genetic background. We discuss open questions, such as the potential for increases in drug tolerance to evolve in static drugs. We highlight opportunities to use this framework to probe how different factors influence the rate and nature of adaptation to antifungal drugs in fungal microbes through a call for increased reporting on all replicates that were evolved, not just those that acquired resistance.

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 8 diverse strains of Candida albicans in a high level of the fungistatic drug posaconazole. Approximately half of the 132 evolved replicates survived 50 generations of evolution, biased toward 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 was observed in evolved replicates from some strain backgrounds. Trisomy of at least one 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.

The future of fungi: threats and opportunities.

The fungal kingdom represents an extraordinary diversity of organisms with profound impacts across animal, plant, and ecosystem health. Fungi simultaneously support life, by forming beneficial symbioses with plants and producing life-saving medicines, and bring death, by causing devastating diseases in humans, plants, and animals. With climate change, increased antimicrobial resistance, global trade, environmental degradation, and novel viruses altering the impact of fungi on health and disease, developing new approaches is now more crucial than ever to combat the threats posed by fungi and to harness their extraordinary potential for applications in human health, food supply, and environmental remediation. To address this aim, the Canadian Institute for Advanced Research (CIFAR) and the Burroughs Wellcome Fund convened a workshop to unite leading experts on fungal biology from academia and industry to strategize innovative solutions to global challenges and fungal threats. This report provides recommendations to accelerate fungal research and highlights the major research advances and ideas discussed at the meeting pertaining to 5 major topics: (1) Connections between fungi and climate change and ways to avert climate catastrophe; (2) Fungal threats to humans and ways to mitigate them; (3) Fungal threats to agriculture and food security and approaches to ensure a robust global food supply; (4) Fungal threats to animals and approaches to avoid species collapse and extinction; and (5) Opportunities presented by the fungal kingdom, including novel medicines and enzymes.

Small is the new big: assessing the population structure of microorganisms.

Microorganisms are a tremendously large and diverse group spanning multiple kingdoms, yet they have been considerably under-studied by ecologists and evolutionary biologists compared to their larger relatives. Although a few microbial species have become the stars of laboratory experiments, relatively few studies have examined microbial species in their natural habitats. As such, the question of whether microbial diversity parallels that of larger bodied species is contentious (Lachance 2004; Fenchel & Finlay 2004). It has been suggested that large population sizes, high dispersal potential and low extinction rates lead to genetically homogeneous populations of microbial species over large geographical scales­arguments that bring to mind discussions about speciation and population structure in the marine environment. In this issue of Molecular Ecology, Herrera et al. (2011) add to this debate by examining 91 isolates of the flower-living yeast Metschnikowia gruessii from southeastern Spain. Their AFLP results support both spatial structuring of genetic diversity across the region, as well as microsite-dependent diversifying selection within single flowers. This study adds to a growing body of literature suggesting that although microbes have much larger population sizes and many differ in their principal mode of reproduction (primarily clonal rather than sexual), patterns of genetic diversity and phylogenetic structure for some microbial species may be similar to that of larger species. This study highlights the need for vastly more research that specifically examines biogeographic structure in this under-utilized group of organisms.

High-Throughput Computational Analysis of Biofilm Formation from Time-Lapse Microscopy.

Candida albicans biofilm formation in the presence of drugs can be examined through time-lapse microscopy. In many cases, the images are used qualitatively, which limits their utility for hypothesis testing. We employed a machine-learning algorithm implemented in the Orbit Image Analysis program to detect the percent area covered by cells from each image. This is combined with custom R scripts to determine the growth rate, growth asymptote, and time to reach the asymptote as quantitative proxies for biofilm formation. We describe step-by-step protocols that go from sample preparation for time-lapse microscopy through image analysis parameterization and visualization of the model fit. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation Basic Protocol 2: Time-lapse microscopy: Evos protocol Basic Protocol 3: Batch file renaming Basic Protocol 4: Machine learning analysis of Evos images with Orbit Basic Protocol 5: Parametrization of Orbit output in R Basic Protocol 6: Visualization of logistic fits in R.

The population genetics of ploidy change in unicellular fungi.

Changes in ploidy are a significant type of genetic variation, describing the number of chromosome sets per cell. Ploidy evolves in natural populations, clinical populations, and lab experiments, particularly in unicellular fungi. Predicting how ploidy will evolve has proven difficult, despite a long history of theoretical work on this topic, as it is often unclear why one ploidy state outperforms another. Here, we review what is known about contemporary ploidy evolution in diverse fungal species through the lens of population genetics. As with typical genetic variants, ploidy evolution depends on the rate that new ploidy states arise by mutation, natural selection on alternative ploidy states, and random genetic drift. However, ploidy variation also has unique impacts on evolution, with the potential to alter chromosomal stability, the rate and patterns of point mutation, and the nature of selection on all loci in the genome. We discuss how ploidy evolution depends on these general and unique factors and highlight areas where additional experimental evidence is required to comprehensively explain the ploidy transitions observed in the field, the clinic, and the lab.

Candida albicans Genetic Background Influences Mean and Heterogeneity of Drug Responses and Genome Stability during Evolution in Fluconazole.

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 20 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 parental 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 parental fitness or improvement in fitness. Rather, low parental strain fitness was correlated with high levels of heterogeneity in fitness, tolerance, and genome size among evolved replicates. Thus, parental 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.IMPORTANCE Antimicrobial 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 parental 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.

Ploidy and the causes of genomic evolution.

Genomes vary dramatically in size and in content. This variation is driven in part by numerous polyploidization events that have happened over the course of eukaryotic evolution. Experimental evolution studies, primarily using the yeast Saccharomyces cerevisiae, provide insights into the immediate fitness effects of ploidy mutations, the ability of organisms of different ploidy levels to mask deleterious mutations, the impact of ploidy on rates of adaptation, and the relative roles of selection versus drift in shaping ploidy evolution. We review these experimental evolution studies and present new data on differences in maximal growth rate for cells of different ploidy levels.