The SUMO protease Ulp2 regulates genome stability and drug resistance in the human fungal pathogen Candida albicans

AbstractStress-induced genome instability in microbial organisms is emerging as a critical regulatory mechanism for driving rapid and reversible adaption to drastic environmental changes. In Candida albicans, a human fungal pathogen that causes life-threatening infections, genome plasticity confers increased virulence and antifungal drug resistance. Discovering the mechanisms regulating C. albicans genome plasticity is a priority to understand how this and other microbial pathogens establish life-threatening infections and develop resistance to antifungal drugs. We identified the SUMO protease Ulp2 as a critical regulator of C. albicans genome integrity through genetic screening. Deletion of ULP2 leads to hypersensitivity to genotoxic agents and increased genome instability. This increased genome diversity causes reduced fitness under standard laboratory growth conditions but enhances adaptation to stress, making ulp2Δ/Δ cells more likely to thrive in the presence of antifungal drugs. Whole-genome sequencing indicates that ulp2Δ/Δ cells counteract antifungal drug-induced stress by developing segmental aneuploidies of chromosome R and chromosome I. We demonstrate that intrachromosomal repetitive elements drive the formation of complex novel genotypes with adaptive power.

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Candida albicansgenetic background influences mean and heterogeneity of drug responses and genome stability during evolution to fluconazole

10.1101/360347 ◽

2018 ◽

Author(s):

Aleeza C. Gerstein ◽

Judith Berman

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Genome Size ◽

Fungal Pathogen ◽

Genetic Background ◽

Antifungal Drug ◽

Human Fungal Pathogen ◽

Evolutionary Level ◽

Ancestral Strain ◽

Strain Background

AbstractThe 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 ofCandida albicans,a human fungal pathogen, in fluconazole, the commonly used antifungal drug. The isolates hailed from the majorC. albicansclades 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 ofCandida 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.

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Tolerance to fluconazole in Candida albicans is regulated by temperature and aneuploidy

Access Microbiology ◽

10.1099/acmi.cc2021.po0106 ◽

2021 ◽

Vol 3 (12) ◽

Author(s):

FENG YANG ◽

YUANYING JIANG ◽

JUDITH BERMAN

Keyword(s):

Candida Albicans ◽

High Temperature ◽

Fungal Pathogen ◽

Drug Tolerance ◽

Antifungal Drugs ◽

Clinical Isolates ◽

Human Fungal Pathogen ◽

Genes Encoding ◽

Aneuploid Chromosome ◽

Definition Of

Candida albicans is a prevalent human fungal pathogen. Azoles are the most widely used antifungal drugs. Drug tolerance in bacteria is well defined and thoroughly studied, but in fungi, the definition of drug tolerance and the mechanism that drive it are not well understood. Here, we found that a large proportion of clinical isolates were intrinsically tolerant to fluconazole, and/or could be induced by high temperature (37°C) to become tolerant (conditionally tolerant). When treated with inhibitory doses of fluconazole, non-tolerant strains became tolerant by forming aneuploids involving different chromosomes, with chromosome R duplication as the most recurrent mechanism. Tolerance determines the ability to grow in the presence of fluconazole and other azoles, in a manner independent of the MIC. Both temperature conditional tolerance and the associated aneuploidy were sensitive to FK506, an inhibitor of calcineurin. Intrinsic and conditional tolerance were also abolished by deletions of genes encoding the calcineurin (CMP1 and CNB1). However, the dependence of tolerance on calcineurin could be bypassed by a different aneuploid chromosome. Thus, fluconazole tolerance in C. albicans is regulated by temperature and by aneuploidy and is dependent upon aneuploidy, but this dependence can be bypassed by an additional aneuploidy.

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Systematic truncations of chromosome 4 and their responses to antifungals in Candida albicans

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00197-0 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Wasim Uddin ◽

Darshan Dhabalia ◽

S. M. Udaya Prakash ◽

M. Anaul Kabir

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Resistant Mutant ◽

Wild Type ◽

Human Fungal Pathogen ◽

Zones Of Inhibition ◽

Life Threatening ◽

Fluconazole Resistant ◽

Diffusion Assay ◽

Type Strains

Abstract Background Candida albicans is an opportunistic human fungal pathogen responsible for superficial and systemic life-threatening infections. Treating these infections is challenging as many clinical isolates show increased drug resistance to antifungals. Chromosome (Chr) 4 monosomy was implicated in a fluconazole-resistant mutant. However, exposure to fluconazole adversely affects Candida cells and can generate numerous mutations. Hence, the present study aimed to truncate Chr4 and challenge the generated Candida strains to antifungals and evaluate their role in drug response. Results Herein, Chr4 was truncated in C. albicans using the telomere-mediated chromosomal truncation method. The resulting eight Candida strains carrying one truncated homolog of Chr4 were tested for response to multiple antifungals. The minimal inhibitory concentration (MIC) for these strains was determined against three classes of antifungals. The MIC values against fluconazole, amphotericin B, and caspofungin were closer to that of the wild type strain. Microdilution assay against fluconazole showed that the mutants and wild type strains had similar sensitivity to fluconazole. The disc diffusion assay against five azoles and two polyenes revealed that the zones of inhibition for all the eight strains were similar to those of the wild type. Thus, none of the generated strains showed any significant resistance to the tested antifungals. However, spot assay exhibited a reasonably high tolerance of a few generated strains with increasing concentrations of fluconazole. Conclusion This analysis suggested that Chr4 aneuploidy might not underlie drug resistance but rather drug tolerance in Candida albicans.

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Pathogenesis and Antifungal Drug Resistance of the Human Fungal Pathogen Candida glabrata

Pharmaceuticals ◽

10.3390/ph4010169 ◽

2011 ◽

Vol 4 (1) ◽

pp. 169-186 ◽

Cited By ~ 35

Author(s):

Michael Tscherner ◽

Tobias Schwarzmüller ◽

Karl Kuchler

Keyword(s):

Drug Resistance ◽

Fungal Pathogen ◽

Candida Glabrata ◽

Antifungal Drug ◽

Antifungal Drug Resistance ◽

Human Fungal Pathogen

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Ploidy determines the consequences of antifungal-induced mutagenesis in Candida albicans, a human fungal pathogen

10.1101/686881 ◽

2019 ◽

Cited By ~ 1

Author(s):

Ognenka Avramovska ◽

Meleah A. Hickman

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Environmental Stress ◽

Genome Size ◽

Fungal Pathogen ◽

Fungal Pathogens ◽

Antifungal Drugs ◽

Relevant Model ◽

Human Fungal Pathogen ◽

Induced Mutagenesis

AbstractOrganismal ploidy state and environmental stress impact the mutational spectrum and the mutational rate. The human fungal pathogen Candida albicans, serves as a clinically relevant model for studying the interaction between eukaryotic ploidy and stress-induced mutagenesis. In this study, we compared the rates and types of genome perturbations in diploid and tetraploid C. albicans following exposure to two classes of antifungal drugs, azoles and echinocandins. We measured mutations at three different scales: point mutation, loss-of-heterozygosity (LOH), and genome size changes in cells treated with fluconazole and caspofungin. We find that caspofungin induced higher rates of mutation than fluconazole, likely an indirect result from the stress associated with cell wall perturbations rather than an inherent genotoxicity. Furthermore, we found disproportionately elevated rates of LOH and genome size changes in response to both antifungals in tetraploid C. albicans compared to diploid C. albicans, suggesting that the magnitude of stress-induced mutagenesis results from an interaction between ploidy state and the environment. These results have both clinical and evolutionary implications for how fungal pathogens generate mutations in response to antifungal drug stress, and may facilitate the emergence of antifungal resistance.

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Chromosome 1 trisomy overcomes the block of sphingolipid biosynthesis caused by aureobasidin A and confers pleiotropic effects on susceptibility to antifungals in Candida albicans

10.1101/2020.04.07.031112 ◽

2020 ◽

Author(s):

Yi Xu ◽

Feng Yang

Keyword(s):

Candida Albicans ◽

Fungal Pathogen ◽

Membrane Lipid ◽

Antifungal Drugs ◽

Pleiotropic Effects ◽

Chromosome 1 ◽

Eukaryotic Cells ◽

Human Fungal Pathogen ◽

Aureobasidin A ◽

Sphingolipid Biosynthesis

AbstractSphingolipids are important membrane lipid components of eukaryotic cells. In Candida albicans, chromosome 1 trisomy not only overcame the block of sphingolipid biosynthesis caused by aureobasidin A, but also altered tolerance to three of the four major classes of antifungal drugs. Two haploinsufficient genes on chromosome 1, PDR16 and IPT1, were associated with tolerance to aureobasidin A. This study illustrates an example of multi-drug tolerance caused by aneuploidy in the human fungal pathogen C. albicans.

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Antifungal effects of alantolactone on Candida albicans: an in vitro study

10.1101/2021.01.25.428193 ◽

2021 ◽

Author(s):

Xin Liu ◽

Lili Zhong ◽

Zhiming Ma ◽

Yujie Sui ◽

Jia’nan Xie ◽

...

Keyword(s):

Candida Albicans ◽

Medical Devices ◽

Fungal Pathogen ◽

Biofilm Formation ◽

In Vitro Study ◽

Antifungal Drugs ◽

Promising Candidate ◽

Human Fungal Pathogen ◽

Antifungal Effects

AbstractThe human fungal pathogen Candida albicans can cause many kinds of infections, including biofilm infections on medical devices, while the available antifungal drugs are limited to only a few. In this study, alantolactone (Ala) demonstrated antifungal activities against C. albicans, as well as other Candida species, with a MIC of 72 μg/mL. Ala could also inhibit the adhesion, yeast-to-hyphal transition, biofilm formation and development of C. albicans. The exopolysaccharide of biofilm matrix and extracellular phospholipase production could also be reduced by Ala treatment. Ala could increase permeability of C. albicans cell membrane and ROS contribute to the antifungal activity of Ala. Overall, the present study suggests that Ala may provide a promising candidate for developing antifungal drugs against C. albicans infections.

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Transcriptomic and Genomic Approaches for Unravelling Candida albicans Biofilm Formation and Drug Resistance—An Update

Genes ◽

10.3390/genes9110540 ◽

2018 ◽

Vol 9 (11) ◽

pp. 540 ◽

Cited By ~ 6

Author(s):

Pei Chong ◽

Voon Chin ◽

Won Wong ◽

Priya Madhavan ◽

Voon Yong ◽

...

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Fungal Pathogen ◽

Biofilm Formation ◽

Antifungal Drugs ◽

Yeast Cells ◽

Whole Genome ◽

Different Strains

Candida albicans is an opportunistic fungal pathogen, which causes a plethora of superficial, as well as invasive, infections in humans. The ability of this fungus in switching from commensalism to active infection is attributed to its many virulence traits. Biofilm formation is a key process, which allows the fungus to adhere to and proliferate on medically implanted devices as well as host tissue and cause serious life-threatening infections. Biofilms are complex communities of filamentous and yeast cells surrounded by an extracellular matrix that confers an enhanced degree of resistance to antifungal drugs. Moreover, the extensive plasticity of the C. albicans genome has given this versatile fungus the added advantage of microevolution and adaptation to thrive within the unique environmental niches within the host. To combat these challenges in dealing with C. albicans infections, it is imperative that we target specifically the molecular pathways involved in biofilm formation as well as drug resistance. With the advent of the -omics era and whole genome sequencing platforms, novel pathways and genes involved in the pathogenesis of the fungus have been unraveled. Researchers have used a myriad of strategies including transcriptome analysis for C. albicans cells grown in different environments, whole genome sequencing of different strains, functional genomics approaches to identify critical regulatory genes, as well as comparative genomics analysis between C. albicans and its closely related, much less virulent relative, C. dubliniensis, in the quest to increase our understanding of the mechanisms underlying the success of C. albicans as a major fungal pathogen. This review attempts to summarize the most recent advancements in the field of biofilm and antifungal resistance research and offers suggestions for future directions in therapeutics development.

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SpRY Cas9 Can Utilize a Variety of Protospacer Adjacent Motif Site Sequences To Edit the Candida albicans Genome

mSphere ◽

10.1128/msphere.00303-21 ◽

2021 ◽

Vol 6 (3) ◽

Author(s):

Ben A. Evans ◽

Douglas A. Bernstein

Keyword(s):

Candida Albicans ◽

Genome Editing ◽

Fungal Pathogen ◽

Wild Type ◽

Content Type ◽

Human Fungal Pathogen ◽

Protospacer Adjacent Motif ◽

Motif Site ◽

Life Threatening ◽

Genomic Locations

ABSTRACT Candida albicans is a human fungal pathogen capable of causing life-threatening infections. The ability to edit the C. albicans genome using CRISPR/Cas9 is an important tool investigators can leverage in their search for novel therapeutic targets. However, wild-type Cas9 requires an NGG protospacer adjacent motif (PAM), leaving many AT-rich regions of DNA inaccessible. A recently described near-PAMless CRISPR system that utilizes the SpRY Cas9 variant can target non-NGG PAM sequences. Using this system as a model, we developed C. albicans CRISPR/SpRY. We tested our system by mutating C. albicans ADE2 and show that CRISPR/SpRY can utilize non-NGG PAM sequences in C. albicans. Our CRISPR/SpRY system will allow researchers to efficiently modify C. albicans DNA that lacks NGG PAM sequences. IMPORTANCE Genetic modification of the human fungal pathogen Candida albicans allows us to better understand how fungi differ from humans at the molecular level and play essential roles in the development of therapeutics. CRISPR/Cas9-mediated genome editing systems can be used to introduce site-specific mutations to C. albicans. However, wild-type Cas9 is limited by the requirement of an NGG PAM site. CRISPR/SpRY targets a variety of different PAM sequences. We modified the C. albicans CRISPR/Cas9 system using the CRISPR/SpRY as a guide. We tested CRISPR/SpRY on C. albicans ADE2 and show that our SpRY system can facilitate genome editing independent of an NGG PAM sequence, thus allowing the investigator to target AT-rich sequences. Our system will potentially enable mutation of the 125 C. albicans genes which have been previously untargetable with CRISPR/Cas9. Additionally, our system will allow for precise targeting of many genomic locations that lack NGG PAM sites.

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Rampant transposition following RNAi loss causes hypermutation and antifungal drug resistance in clinical isolates of a human fungal pathogen

10.21203/rs.3.rs-804218/v1 ◽

2021 ◽

Author(s):

Shelby Priest ◽

Vikas Yadav ◽

Cullen Roth ◽

Tim Dahlmann ◽

Ulrich Kueck ◽

...

Keyword(s):

Drug Resistance ◽

Nonsense Mutation ◽

Fungal Pathogen ◽

Genetic Locus ◽

Antifungal Drugs ◽

Antifungal Drug ◽

Gene Encoding ◽

Antifungal Drug Resistance ◽

Trait Locus ◽

Almost All

Abstract Microorganisms survive and compete within their environmental niches and avoid evolutionary stagnation by stochastically acquiring mutations that enhance fitness. Although increased mutation rates are often deleterious in multicellular organisms, hypermutation can be beneficial for microbes in the context of strong selective pressures. To explore how hypermutation arises in nature and elucidate its consequences, we employed a collection of 387 sequenced clinical and environmental isolates of Cryptococcus neoformans. This fungal pathogen is responsible for ~ 15% of annual AIDS-related deaths and is associated with high mortality rates, attributable to a dearth of antifungal drugs and increasing drug resistance. Isolates were screened for the ability to rapidly acquire antifungal drug resistance, and two robust hypermutators were identified. Insertion of the non-LTR Cnl1 retrotransposon was found to be responsible for the majority of drug-resistant isolates. Long-read whole-genome sequencing revealed both hypermutator genomes have two unique features: 1) hundreds of Cnl1 copies organized in subtelomeric arrays on both ends of almost all chromosomes, and 2) a nonsense mutation in the first exon of ZNF3, a gene encoding an RNAi component involved in silencing transposons. Quantitative trait locus mapping identified a significant genetic locus associated with hypermutation that includes the mutant znf3 allele, and CRISPR-mediated genome editing of the znf3 single-base pair nonsense mutation abolished the hypermutation phenotype and restored siRNA production. In sum, hypermutation and drug resistance in these isolates results from loss of RNAi combined with subsequent accumulation of a large genomic burden of a novel transposable element in C. neoformans.

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