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Cited by 150Fungal and yeast genetics researchAntifungal resistance and susceptibilityBacterial Genetics and Biotechnology

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Gene Essentiality Analyzed by In Vivo Transposon Mutagenesis and Machine Learning in a Stable Haploid Isolate of Candida albicans

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Segal, Ella Shtifman, Gritsenko, Vladimir, Levitan, Anton, Yadav, Bhawna, Dror, Naama, Steenwyk, Jacob L., Silberberg, Yael, Mielich, Kevin, Rokas, Antonis, Gow, Neil A. R., Kunze, Reinhard, Sharan, Roded, Berman, Judith·2018·mBio
DOI 10.1128/mbio.02048-18PMC PMC6212825
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Abstract

Comprehensive understanding of an organism requires that we understand the contributions of most, if not all, of its genes. Classical genetic approaches to this issue have involved systematic deletion of each gene in the genome, with comprehensive sets of mutants available only for very-well-studied model organisms. We took a different approach, harnessing the power of in vivo transposition coupled with deep sequencing to identify >500,000 different mutations, one per cell, in the prevalent human fungal pathogen Candida albicans and to map their positions across the genome. The transposition approach is efficient and less labor-intensive than classic approaches. Here, we describe the production and analysis (aided by machine learning) of a large collection of mutants and the comprehensive identification of 1,610 C. albicans genes that are essential for growth under standard laboratory conditions. Among these C. albicans essential genes, we identify those that are also essential in two distantly related model yeasts as well as those that are conserved in all four major human fungal pathogens and that are not conserved in the human genome. This list of genes with functions important for the survival of the pathogen provides a good starting point for the development of new antifungal drugs, which are greatly needed because of the emergence of fungal pathogens with elevated resistance and/or tolerance of the currently limited set of available antifungal drugs.

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Figures

Fig 1 Strain and Tn library construction. (A) Growth curves for strains are indicated as follows: blue, diploid (SC5314); red, haploid MATa parent (YJB-T900); yellow, +Ac (YJB-T 1792); pink, +Ac+Ds(1) (YJB-T1081) (growth of YJB-T1082 [also +Ac+Ds] was indistinguishable from that of YJB-T1081); orange, haploid I (9); green, haploid XI (9). All growth curve analyses were performed in triplicate, with standard deviations ranging from 0 to 0.05. OD, optical density. (B) Summary of DNA content for multiple isolates for each strain (n = 24) [+Ds (YJB-T1794); MATα haploid parent YJB-T257; +Ac+Ds(3); YJB-T2743]. (C and D) Flow cytometric DNA content for strain YJB-T1081 before (C) and after (D) excision of Ds-NAT1. (E) Schematic of transposon mutant library preparation process.
Fig 1 Strain and Tn library construction. (A) Growth curves for strains are indicated as follows: blue, diploid (SC5314); red, haploid MATa parent (YJB-T900); yellow, +Ac (YJB-T 1792); pink, +Ac+Ds(1) (YJB-T1081) (growth of YJB-T1082 [also +Ac+Ds] was indistinguishable from that of YJB-T1081); orange, haploid I (9); green, haploid XI (9). All growth curve analyses were performed in triplicate, with standard deviations ranging from 0 to 0.05. OD, optical density. (B) Summary of DNA content for multiple isolates for each strain (n = 24) [+Ds (YJB-T1794); MATα haploid parent YJB-T257; +Ac+Ds(3); YJB-T2743]. (C and D) Flow cytometric DNA content for strain YJB-T1081 before (C) and after (D) excision of Ds-NAT1. (E) Schematic of transposon mutant library preparation process.CC BY · PubMed Central
Fig 2 Maps of transposon insertions at different scales. (A) Distribution of insertion sites (hits) for the pooled library used in this study (libraries 3, 7, and 11). In the top two rows, the 8 C. albicans chromosomes and Chr7 are in 10,000-bp bins, the y axis scale is log10, and the ADE2 gene is indicated by a red bar. In the lower two rows, 100-kb and 10-kb sections of Chr7 show three tracks of hit data in black, representing libraries 3, 7, and 11 (from top to bottom). Blue rectangles, predicted ORFs; black rectangles, recognized domains from protein information in CGD (38). The arrow points to the 3′ end of the gene. (B) Distribution of predicted essential and nonessential genes across the chromosomes. Essential genes are indicated with tall bars; nonessential genes are indicated with short bars; bars above and below the central axis represent genes transcribed on the Watson and Crick strands, respectively. (C) Example of low-hit region of CR_00880W gene.
Fig 2 Maps of transposon insertions at different scales. (A) Distribution of insertion sites (hits) for the pooled library used in this study (libraries 3, 7, and 11). In the top two rows, the 8 C. albicans chromosomes and Chr7 are in 10,000-bp bins, the y axis scale is log10, and the ADE2 gene is indicated by a red bar. In the lower two rows, 100-kb and 10-kb sections of Chr7 show three tracks of hit data in black, representing libraries 3, 7, and 11 (from top to bottom). Blue rectangles, predicted ORFs; black rectangles, recognized domains from protein information in CGD (38). The arrow points to the 3′ end of the gene. (B) Distribution of predicted essential and nonessential genes across the chromosomes. Essential genes are indicated with tall bars; nonessential genes are indicated with short bars; bars above and below the central axis represent genes transcribed on the Watson and Crick strands, respectively. (C) Example of low-hit region of CR_00880W gene.CC BY · PubMed Central
Fig 3 (A) Examples of genes with hits in the C-terminal portion of the coding sequence for C. albicans and S. cerevisiae orthologs. Note that patterns are similar although S. cerevisiae genes sometimes have insertions in the extreme N-terminal coding sequence that are not evident in C. albicans orthologs. Yellow bars below the genes indicate introns. Similar maps for the other libraries are found in Fig. S2A and maps of all ORFs are available in Dataset 9 at https://doi.org/10.6084/m9.figshare.c.4251182. (B) Relative feature importance contributions to the random forest classifiers for all three model yeasts.
Fig 3 (A) Examples of genes with hits in the C-terminal portion of the coding sequence for C. albicans and S. cerevisiae orthologs. Note that patterns are similar although S. cerevisiae genes sometimes have insertions in the extreme N-terminal coding sequence that are not evident in C. albicans orthologs. Yellow bars below the genes indicate introns. Similar maps for the other libraries are found in Fig. S2A and maps of all ORFs are available in Dataset 9 at https://doi.org/10.6084/m9.figshare.c.4251182. (B) Relative feature importance contributions to the random forest classifiers for all three model yeasts.CC BY · PubMed Central
Fig 4 (A) Examples of genes with misannotated start codon positions. Symbols are as described in the Fig. 3 legend. Green histograms illustrate RNAseq expression levels (48). (B) Proportions of essential and nonessential genes compared to prior information are indicated. CaTn-Ess and CaTn-NE genes, as indicated. Red, genes with prior data indicating essentiality; blue, genes that were not previously tested for essentiality; green, genes with contradictory data. Dark green, genes with contradictory data prior to this study; light green, genes for which CaTn predictions contradicted at least one other report. (C) ScTn-Ess and ScTn-NE genes, as indicated. In yellow are genes that had conflicting annotations in SGD. (D) SpTn-Ess and SpTn-NE genes, as indicated.
Fig 4 (A) Examples of genes with misannotated start codon positions. Symbols are as described in the Fig. 3 legend. Green histograms illustrate RNAseq expression levels (48). (B) Proportions of essential and nonessential genes compared to prior information are indicated. CaTn-Ess and CaTn-NE genes, as indicated. Red, genes with prior data indicating essentiality; blue, genes that were not previously tested for essentiality; green, genes with contradictory data. Dark green, genes with contradictory data prior to this study; light green, genes for which CaTn predictions contradicted at least one other report. (C) ScTn-Ess and ScTn-NE genes, as indicated. In yellow are genes that had conflicting annotations in SGD. (D) SpTn-Ess and SpTn-NE genes, as indicated.CC BY · PubMed Central
Fig 5 Venn diagrams of the intersections of (A) Core5 essential genes; (B) Core5 nonessential genes; (C) S. cerevisiae and S. pombe paralogs with CaTnEss, ScSpNE group; and (D) CaTnEss versus ScEss and SpEss.
Fig 5 Venn diagrams of the intersections of (A) Core5 essential genes; (B) Core5 nonessential genes; (C) S. cerevisiae and S. pombe paralogs with CaTnEss, ScSpNE group; and (D) CaTnEss versus ScEss and SpEss.CC BY · PubMed Central
Fig 6 Genetic interaction degree and number of essential genes conserved in pathogenic fungi and not in humans. (A) Genetic interaction degree, a measure of interactivity of C. albicans genes in experiments performed using SGA software (see Materials and Methods) plotted for essential (Ess) and nonessential (NE) genes/orthologs in C. albicans (CaTn) and S. cerevisiae (ScΔ and ScTn) and from S. pombe analyses (SpΔ and SpTn) as well as from the Core5 analyses (CaTn, ScΔ, ScTn, SpΔ, and SpTn). The y axis data represent the number of gene interactions normalized by the number of observations (GIPD) (46). The statistical significance of results of comparisons between Ess and NE genes was obtained using the Wilcoxon rank sum test (***, P < 0.001). (B) Four groups of essential genes of CaTn-Ess and Core5-Ess as described in the Fig. 5 legend as well as of genes of Ca-ECS and Core5-ECS-Ess (after filtering was performed using an essentiality confidence score) were then filtered for those without human homologs and then for those with homologs in the other three major pathogenic yeasts (A. fumigatus, C. neoformans, and H. capsulatum).
Fig 6 Genetic interaction degree and number of essential genes conserved in pathogenic fungi and not in humans. (A) Genetic interaction degree, a measure of interactivity of C. albicans genes in experiments performed using SGA software (see Materials and Methods) plotted for essential (Ess) and nonessential (NE) genes/orthologs in C. albicans (CaTn) and S. cerevisiae (ScΔ and ScTn) and from S. pombe analyses (SpΔ and SpTn) as well as from the Core5 analyses (CaTn, ScΔ, ScTn, SpΔ, and SpTn). The y axis data represent the number of gene interactions normalized by the number of observations (GIPD) (46). The statistical significance of results of comparisons between Ess and NE genes was obtained using the Wilcoxon rank sum test (***, P < 0.001). (B) Four groups of essential genes of CaTn-Ess and Core5-Ess as described in the Fig. 5 legend as well as of genes of Ca-ECS and Core5-ECS-Ess (after filtering was performed using an essentiality confidence score) were then filtered for those without human homologs and then for those with homologs in the other three major pathogenic yeasts (A. fumigatus, C. neoformans, and H. capsulatum).CC BY · PubMed Central

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Metadata

Authors
Segal, E. S., Gritsenko, V., Levitan, A., Yadav, B., Dror, N., Steenwyk, J. L., Silberberg, Y., Mielich, K., Rokas, A., Gow, N. A. R., Kunze, R., Sharan, R., & Berman, J.
Affiliations
  • Tel Aviv University (IL)
  • University of Aberdeen (GB)
  • Vanderbilt University (US)
  • Freie Universität Berlin (DE)
Corresponding author
Berman, Judith
ORCID
Venue
mBio
Publisher
American Society for Microbiology
Published
2018-11-07
Volume
9
Issue
5
ISSN
  • 2150-7511 (ISSN-L)
  • 2161-2129
DOI
10.1128/mbio.02048-18
PMC
PMC6212825
OpenAlex
W2898801056
Cited by
150
References
105
Citations by year
2026: 11 · 2025: 20 · 2024: 18 · 2023: 9 · 2022: 21 · 2021: 20 · 2020: 26 · 2019: 24 · 2018: 1
FWCI
6.31
Type
journal article
Language
en
Open access
Licence
Locations
  • mBio (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Aberdeen University Research Archive (Aberdeen University) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Refubium (Universitätsbibliothek der Freien Universität Berlin) (submittedVersion, cc-by)
  • Universitätsbibliothek der FU Berlin Hochschulschriftenstelle u. Dokumentenserver (cc-by)
Funders
  • Wellcome Trust (086827 075470 101873 200208)
  • MRC Center for Medical Mycology (N006364/1)
  • European Research Council (340087 RAPLODAPT)
  • Dahlem Center of Plant Sciences (DCPS)
  • Israel Science Foundation (715/18)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Health Sciences › Medicine › Infectious Diseases › Antifungal resistance and susceptibility
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
gene essentiality, Candida albicans, transposon mutagenesis, essential genes, fungal pathogens, antifungal drug targets, machine learning, deep sequencing
MeSH terms
Machine Learning, Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, DNA Transposable Elements, Genes, Fungal, Genetics, Microbial, Haploidy, Histoplasma, Mutagenesis, Insertional, Genes, Essential

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