Identification of Recessive Lethal Alleles in the Diploid Genome of aCandida albicansLaboratory Strain Unveils a Potential Role of Repetitive Sequences in Buffering Their Deleterious Impact

ABSTRACTThe heterozygous diploid genome ofCandida albicansis highly plastic, with frequent loss of heterozygosity (LOH) events. In the SC5314 laboratory strain, while LOH events are ubiquitous, a chromosome homozygosis bias is observed for certain chromosomes, whereby only one of the two homologs can occur in the homozygous state. This suggests the occurrence of recessive lethal allele(s) (RLA) preventing large-scale LOH events on these chromosomes from being stably maintained. To verify the presence of an RLA on chromosome 7 (Chr7), we utilized a system that allows (i) DNA double-strand break (DSB) induction on Chr7 by the I-SceI endonuclease and (ii) detection of the resulting long-range homozygosis. I-SceI successfully induced a DNA DSB on both Chr7 homologs, generally repaired by gene conversion. Notably, cells homozygous for the right arm of Chr7B were not recovered, confirming the presence of RLA(s) in this region. Genome data mining for RLA candidates identified a premature nonsense-generating single nucleotide polymorphism (SNP) within the HapB allele of C7_03400c whoseSaccharomycescerevisiaeortholog encodes the essential Mtr4 RNA helicase. Complementation with a wild-type copy ofMTR4rescued cells homozygous for the right arm of Chr7B, demonstrating that themtr4K880*RLA is responsible for the Chr7 homozygosis bias in strain SC5314. Furthermore, we observed that the major repeat sequences (MRS) on Chr7 acted as hot spots for interhomolog recombination. Such recombination events provideC. albicanswith increased opportunities to survive DNA DSBs whose repair can lead to homozygosis of recessive lethal or deleterious alleles. This might explain the maintenance of MRS in this species.IMPORTANCECandida albicansis a major fungal pathogen, whose mode of reproduction is mainly clonal. Its genome is highly tolerant to rearrangements, in particular loss of heterozygosity events, known to unmask recessive lethal and deleterious alleles in heterozygous diploid organisms such asC. albicans. By combining a site-specific DSB-inducing system and mining genome sequencing data of 182 C. albicansisolates, we were able to ascribe the chromosome 7 homozygosis bias of theC. albicanslaboratory strain SC5314 to an heterozygous SNP introducing a premature STOP codon in theMTR4gene. We have also proposed genome-wide candidates for new recessive lethal alleles. We additionally observed that the major repeat sequences (MRS) on chromosome 7 acted as hot spots for interhomolog recombination. Maintaining MRS inC. albicanscould favor haplotype exchange, of vital importance to LOH events, leading to homozygosis of recessive lethal or deleterious alleles that inevitably accumulate upon clonality.

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Analysis of Repair Mechanisms following an Induced Double-Strand Break Uncovers Recessive Deleterious Alleles in theCandida albicansDiploid Genome

mBio ◽

10.1128/mbio.01109-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 19

Author(s):

Adeline Feri ◽

Raphaël Loll-Krippleber ◽

Pierre-Henri Commere ◽

Corinne Maufrais ◽

Natacha Sertour ◽

...

Keyword(s):

Candida Albicans ◽

Genome Sequencing ◽

Strand Break ◽

Double Strand Break ◽

Clonal Reproduction ◽

Strong Increase ◽

Recessive Lethal ◽

Independent Events ◽

Deleterious Alleles ◽

Strain Sc5314

ABSTRACTThe diploid genome of the yeastCandida albicansis highly plastic, exhibiting frequent loss-of-heterozygosity (LOH) events. To provide a deeper understanding of the mechanisms leading to LOH, we investigated the repair of a unique DNA double-strand break (DSB) in the laboratoryC. albicansSC5314 strain using the I-SceI meganuclease. Upon I-SceI induction, we detected a strong increase in the frequency of LOH events at an I-SceI target locus positioned on chromosome 4 (Chr4), including events spreading from this locus to the proximal telomere. Characterization of the repair events by single nucleotide polymorphism (SNP) typing and whole-genome sequencing revealed a predominance of gene conversions, but we also observed mitotic crossover or break-induced replication events, as well as combinations of independent events. Importantly, progeny that had undergone homozygosis of part or all of Chr4 haplotype B (Chr4B) were inviable. Mining of genome sequencing data for 155C. albicansisolates allowed the identification of a recessive lethal allele in theGPI16gene on Chr4B unique toC. albicansstrain SC5314 which is responsible for this inviability. Additional recessive lethal or deleterious alleles were identified in the genomes of strain SC5314 and two clinical isolates. Our results demonstrate that recessive lethal alleles in the genomes ofC. albicansisolates prevent the occurrence of specific extended LOH events. While these and other recessive lethal and deleterious alleles are likely to accumulate inC. albicansdue to clonal reproduction, their occurrence may in turn promote the maintenance of corresponding nondeleterious alleles and, consequently, heterozygosity in theC. albicansspecies.IMPORTANCERecessive lethal alleles impose significant constraints on the biology of diploid organisms. Using a combination of an I-SceI meganuclease-mediated DNA DSB, a fluorescence-activated cell sorter (FACS)-optimized reporter of LOH, and a compendium of 155 genome sequences, we were able to unmask and identify recessive lethal and deleterious alleles in isolates ofCandida albicans, a diploid yeast and the major fungal pathogen of humans. Accumulation of recessive deleterious mutations upon clonal reproduction ofC. albicanscould contribute to the maintenance of heterozygosity despite the high frequency of LOH events in this species.

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Genome Sequence of a Staphylococcus epidermidis Strain (GTH12) Associated with Candida albicans SC5314 Cultured under Hypoxia at 37°C in Glycerol for 12 Weeks

Genome Announcements ◽

10.1128/genomea.00533-18 ◽

2018 ◽

Vol 6 (25) ◽

Author(s):

Danielle do Carmo Ferreira Bruno ◽

Thais Fernanda Bartelli ◽

Marcelo R. S. Briones

Keyword(s):

Candida Albicans ◽

Patient Outcomes ◽

Genome Sequence ◽

Staphylococcus Epidermidis ◽

Whole Genome Sequence ◽

Mixed Species ◽

Content Type ◽

Important Health ◽

Strain Sc5314 ◽

Polymicrobial Infections

Polymicrobial infections with mixed-species biofilms are important health problems because of increased antimicrobial resistance and worse patient outcomes than with monomicrobial infections. Here, we present the whole-genome sequence of Staphylococcus epidermidis strain GTH12, which was cocultured with the yeast Candida albicans SC5314 (generating C. albicans strain SC5314 GTH12), thus providing genomic information on polymicrobial infections.

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Identification of a Cell Death Pathway in Candida albicans during the Response to Pheromone

Eukaryotic Cell ◽

10.1128/ec.00155-10 ◽

2010 ◽

Vol 9 (11) ◽

pp. 1690-1701 ◽

Cited By ~ 12

Author(s):

Kevin Alby ◽

Dana Schaefer ◽

Racquel Kim Sherwood ◽

Stephen K. Jones ◽

Richard J. Bennett

Keyword(s):

Cell Death ◽

Candida Albicans ◽

Laboratory Strain ◽

Opportunistic Pathogen ◽

Cell Types ◽

Yeast Cells ◽

Phenotypic Switching ◽

Morphological Response ◽

Content Type ◽

Cell Death Pathway

ABSTRACT Mating in hemiascomycete yeasts involves the secretion of pheromones that induce sexual differentiation in cells of the opposite mating type. Studies in Saccharomyces cerevisiae have revealed that a subpopulation of cells experiences cell death during exposure to pheromone. In this work, we tested whether the phenomenon of pheromone-induced death (PID) also occurs in the opportunistic pathogen Candida albicans. Mating in C. albicans is uniquely regulated by white-opaque phenotypic switching; both cell types respond to pheromone, but only opaque cells undergo the morphological transition and cell conjugation. We show that approximately 20% of opaque cells, but not white cells, of laboratory strain SC5314 experience pheromone-induced death. Furthermore, analysis of mutant strains revealed that PID was significantly reduced in strains lacking Fig1 or Fus1 transmembrane proteins that are induced during the mating process and, we now show, are necessary for efficient mating in C. albicans. The level of PID was also Ca2+ dependent, as chelation of Ca2+ ions increased cell death to almost 50% of the population. However, in contrast to S. cerevisiae PID, pheromone-induced killing of C. albicans cells was largely independent of signaling via the Ca2+-dependent protein phosphatase calcineurin, even when combined with the loss of Cmk1 and Cmk2 proteins. Finally, we demonstrate that levels of PID vary widely between clinical isolates of C. albicans, with some strains experiencing close to 70% cell death. We discuss these findings in light of the role of prodeath and prosurvival pathways operating in yeast cells undergoing the morphological response to pheromone.

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Clinical Candida albicans Vaginal Isolates and a Laboratory Strain Show Divergent Behaviors during Macrophage Interactions

mSphere ◽

10.1128/msphere.00393-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Franziska Gerwien ◽

Christine Dunker ◽

Philipp Brandt ◽

Enrico Garbe ◽

Ilse D. Jacobsen ◽

...

Keyword(s):

Candida Albicans ◽

Fungal Infections ◽

Laboratory Strain ◽

Infection Process ◽

Vulvovaginal Candidiasis ◽

Host Immune System ◽

Host Pathogen Interactions ◽

Content Type ◽

Host Pathogen

ABSTRACT Typically, established lab strains are widely used to study host-pathogen interactions. However, to better reflect the infection process, the experimental use of clinical isolates has come more into focus. Here, we analyzed the interaction of multiple vaginal isolates of the opportunistic fungal pathogen Candida albicans, the most common cause of vulvovaginal candidiasis in women, with key players of the host immune system: macrophages. We tested several strains isolated from asymptomatic or symptomatic women with acute and recurrent infections. While all clinical strains showed a response similar to the commonly used lab strain SC5314 in various in vitro assays, they displayed remarkable differences during interaction with macrophages. This coincided with significantly reduced β-glucan exposure on the cell surface, which appeared to be a shared property among the tested vaginal strains for yeast extract/peptone/dextrose-grown cells, which is partly lost when the isolates faced vaginal niche-like nutrient conditions. However, macrophage damage, survival of phagocytosis, and filamentation capacities were highly strain-specific. These results highlight the high heterogeneity of C. albicans strains in host-pathogen interactions, which have to be taken into account to bridge the gap between laboratory-gained data and disease-related outcomes in an actual patient. IMPORTANCE Vulvovaginal candidiasis is one of the most common fungal infections in humans with Candida albicans as the major causative agent. This study is the first to compare clinical vaginal isolates of defined patient groups in their interaction with macrophages, highlighting the vastly different outcomes in comparison to a laboratory strain using commonly applied virulence-determining assays.

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Sphingolipids Mediate Differential Echinocandin Susceptibility in Candida albicans and Aspergillus nidulans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04667-14 ◽

2015 ◽

Vol 59 (6) ◽

pp. 3377-3384 ◽

Cited By ~ 9

Author(s):

Kelley R. Healey ◽

Krishna K. Challa ◽

Thomas D. Edlind ◽

Santosh K. Katiyar

Keyword(s):

Candida Albicans ◽

Aspergillus Nidulans ◽

Laboratory Strain ◽

Wild Type ◽

Enoyl Reductase ◽

Content Type ◽

Long Chain Base ◽

Membrane Target ◽

Sphingolipid Biosynthesis ◽

Long Chain

ABSTRACTThe cell wall synthesis-inhibiting echinocandins, including caspofungin and micafungin, play important roles in the treatment of candidiasis and aspergillosis. Previous studies revealed that, in the haploid yeastCandida glabrata, sphingolipid biosynthesis pathway mutations confer caspofungin reduced susceptibility (CRS) but micafungin increased susceptibility (MIS). Here, we describe oneCandida albicansstrain (of 10 tested) that similarly yields CRS-MIS mutants at relatively high frequency. Mutants demonstrated increased levels of long-chain bases (sphingolipid pathway intermediates) and, unique to this strain, loss of His104/Pro104 heterozygosity in theTSC13-encoded enoyl reductase. CRS-MIS was similarly observed in aC. albicanshomozygousfen1Δfen12Δ laboratory strain and in diverse wild-type strains following exogenous long-chain-base treatment. Analogous to these results, CRS-MIS was demonstrated in anAspergillus nidulansbasAmutant encoding defective sphingolipid C4-hydroxylase and in its wild-type parent exposed to long-chain bases. Sphingolipids likely modulate echinocandin interaction with their Fks membrane target in all susceptible fungi, with potential implications for optimizing therapy with existing antifungals and the development of novel agents.

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Tolerance to Caspofungin in Candida albicans Is Associated with at Least Three Distinctive Mechanisms That Govern Expression of FKS Genes and Cell Wall Remodeling

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00071-17 ◽

2017 ◽

Vol 61 (5) ◽

Cited By ~ 23

Author(s):

Feng Yang ◽

Lulu Zhang ◽

Hironao Wakabayashi ◽

Jason Myers ◽

Yuanying Jiang ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Fungal Infections ◽

Direct Selection ◽

Resistance Development ◽

Content Type ◽

Cellular Factors ◽

Echinocandin Resistance ◽

Diploid Level ◽

The Right

ABSTRACT Expanding echinocandin use to prevent or treat invasive fungal infections has led to an increase in the number of breakthrough infections due to resistant Candida species. Although it is uncommon, echinocandin resistance is well documented for Candida albicans, which is among the most prevalent bloodstream organisms. A better understanding is needed to assess the cellular factors that promote tolerance and predispose infecting cells to clinical breakthrough. We previously showed that some mutants that were adapted to growth in the presence of toxic sorbose due to loss of one chromosome 5 (Ch5) also became more tolerant to caspofungin. We found here, following direct selection of mutants on caspofungin, that tolerance can be conferred by at least three mechanisms: (i) monosomy of Ch5, (ii) combined monosomy of the left arm and trisomy of the right arm of Ch5, and (iii) an aneuploidy-independent mechanism. Tolerant mutants possessed cell walls with elevated chitin and showed downregulation of genes involved in cell wall biosynthesis, namely, FKS, located outside Ch5, and CHT2, located on Ch5, irrespective of Ch5 ploidy. Also irrespective of Ch5 ploidy, the CNB1 and MID1 genes on Ch5, which are involved in the calcineurin signaling pathway, were expressed at the diploid level. Thus, multiple mechanisms can affect the relative expression of the aforementioned genes, controlling them in similar ways. Although breakthrough mutations in two specific regions of FKS1 have previously been associated with caspofungin resistance, we found mechanisms of caspofungin tolerance that are independent of FKS1 and thus represent an earlier event in resistance development.

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Inducible and Constitutive Activation of Two Polymorphic Promoter Alleles of the Candida albicans Multidrug Efflux PumpMDR1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00264-12 ◽

2012 ◽

Vol 56 (8) ◽

pp. 4490-4494 ◽

Cited By ~ 11

Author(s):

Christoph Sasse ◽

Rebecca Schillig ◽

Alexandra Reimund ◽

Julia Merk ◽

Joachim Morschhäuser

Keyword(s):

Candida Albicans ◽

Transcription Factors ◽

Loss Of Heterozygosity ◽

Efflux Pump ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Fluconazole Resistance ◽

Content Type ◽

Constitutive Activation ◽

Strain Sc5314

ABSTRACTOverexpression of the multidrug efflux pumpMDR1confers resistance to the antifungal drug fluconazole onCandida albicans. It has been reported that two types ofMDR1promoters exist inC. albicansand that homozygosity for the allele with higher activity may promote fluconazole resistance. We found that the twoMDR1promoter alleles in strain SC5314 were equally well activated by inducing chemicals or hyperactive forms of the transcription factors Mrr1 and Cap1, which controlMDR1expression. In addition, no loss of heterozygosity at theMDR1locus was observed inMDR1-overexpressing clinicalC. albicansstrains that developed fluconazole resistance during therapy.

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The White Cell Response to Pheromone Is a General Characteristic of Candida albicans Strains

Eukaryotic Cell ◽

10.1128/ec.00320-08 ◽

2008 ◽

Vol 8 (2) ◽

pp. 251-256 ◽

Cited By ~ 23

Author(s):

Nidhi Sahni ◽

Song Yi ◽

Claude Pujol ◽

David R. Soll

Keyword(s):

Candida Albicans ◽

Cell Response ◽

Expression Patterns ◽

Synthetic Medium ◽

Laboratory Strain ◽

General Characteristic ◽

White Cell ◽

Yeast Extract Peptone Dextrose ◽

Strain Sc5314 ◽

Α Pheromone

ABSTRACT For Candida albicans, evidence has suggested that the mating pheromones activate not only the mating response in mating-competent opaque cells but also a unique response in mating-incompetent white cells that includes increased cohesion and adhesion, enhanced biofilm formation, and expression of select mating-related and white cell-specific genes. On the basis of a recent microarray analysis comparing changes in the global expression patterns of white cells in two strains in response to α-pheromone, however, skepticism concerning the validity and generality of the white cell response has been voiced. Here, we present evidence that the response occurs in all tested media (Lee's, RPMI, SpiderM, yeast extract-peptone-dextrose, and a synthetic medium) and in all of the 27 tested strains, including a/a and α/α strains, derivatives of the common laboratory strain SC5314, and representatives from all of the five major clades. The white cell response to pheromone is therefore a general characteristic of MTL-homozygous strains of C. albicans.

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Susceptibility to Medium-Chain Fatty Acids Is Associated with Trisomy of Chromosome 7 in Candida albicans

mSphere ◽

10.1128/msphere.00402-19 ◽

2019 ◽

Vol 4 (3) ◽

Cited By ~ 1

Author(s):

Qinxi Ma ◽

Mihaela Ola ◽

Elise Iracane ◽

Geraldine Butler

Keyword(s):

Fatty Acids ◽

Candida Albicans ◽

Chromosome Number ◽

Antifungal Drugs ◽

Chromosome 7 ◽

Chromosome 2 ◽

Adaptation To Stress ◽

Pathogenic Yeast ◽

Gene Deletions ◽

Content Type

Aneuploidy (changes in chromosome number) and loss of heterozygosity (LOH) occur frequently in the human-pathogenic yeastCandida albicansand are associated with adaptation to stress and to antifungal drugs. Aneuploidy and LOH can also be induced during laboratory manipulations, such as during genetic transformation. We find thatC. albicansstrain SN152, commonly used to generate gene deletions, has undergone a major LOH event on chromosome 2. One deletion strain generated in this background has acquired extra copies of chromosomes 5 and 7. We find that trisomy (three copies) of chromosome 7 is associated with sensitivity to fatty acids.

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Natural Variation in Clinical Isolates of Candida albicans Modulates Neutrophil Responses

mSphere ◽

10.1128/msphere.00501-20 ◽

2020 ◽

Vol 5 (4) ◽

Cited By ~ 1

Author(s):

Madhu Shankar ◽

Tricia L. Lo ◽

Ana Traven

Keyword(s):

Reactive Oxygen Species ◽

Candida Albicans ◽

Reactive Oxygen ◽

Neutrophil Extracellular Traps ◽

Prototype Strain ◽

Human Neutrophils ◽

Oxygen Species ◽

Content Type ◽

Extracellular Traps ◽

Strain Sc5314

ABSTRACT Neutropenia predisposes patients to life-threatening infection with Candida albicans, a commensal and opportunistic fungal pathogen. How phenotypic variation in C. albicans isolates dictates neutrophil responses is poorly understood. By using a panel of clinical C. albicans strains, here we report that the prototype strain SC5314 induces the most potent accumulation of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) by human neutrophils of all tested isolates. ROS and NET accumulation positively correlated with the degree of hyphal formation by the isolates, the hypha being the fungal morphotype that promotes pathogenesis. However, there was no correlation of ROS and NET accumulation with fungal killing by neutrophils. Fungal killing was also not correlated with phagocytosis levels or oxidative stress susceptibility of the isolates. The bloodstream isolate P94015 cannot make hyphae and was previously shown to be hyperfit in the murine gut commensalism model. Our results show that P94015 displays poor phagocytosis by neutrophils, the least ROS and NET accumulation of all tested isolates, and resistance to neutrophil-mediated killing. Our data suggest that reduced susceptibility to neutrophils is likely to be independent from a previously described genetic mutation in P94015 that promotes commensalism. Reduced clearance by neutrophils could benefit commensal fitness of C. albicans and could also have promoted the virulence of P94015 in the human patient in the absence of hyphal morphogenesis. Collectively, our study provides new insights into neutrophil interactions with C. albicans and suggests that studying diverse isolates informs knowledge of the relevant aspects of this key immune interaction. IMPORTANCE Neutrophils are the key immune cell type for host defenses against infections with Candida albicans. C. albicans strains isolated from patients display large phenotypic diversity, but how this diversity impacts host-pathogen interactions with neutrophils is incompletely defined. Here, we show that important neutrophil responses, such as accumulation of reactive oxygen species and neutrophil extracellular traps, as well as the levels of phagocytosis and killing of the pathogen, differ when comparing diverse C. albicans isolates. A bloodstream patient isolate previously described as more suited to commensalism than pathogenesis in animal models is relatively “silent” to neutrophils and resistant to killing. Our findings illuminate the relationships between fungal morphogenesis, neutrophil responses, and C. albicans survival. Our findings suggest that host phenotypes of a commensally adapted strain could be driven by resistance to immune clearance and indicate that we should extend our studies beyond the “prototype” strain SC5314 for deeper understanding of Candida-neutrophil interactions.

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