1887

Abstract

is closely related to important pathogenic species, especially and but it has been rarely isolated from humans. For this reason, through comparative studies, it could be a powerful model to understand the genetic underpinnings of the pathogenicity of species. Here, we generated a cohesive assembly of the genome and developed genetic engineering tools that will facilitate studying this species at a molecular level. We used a combination of short and long-read sequencing to build a polished genomic draft composed of 14 Mbp, 45 contigs and close to 5700 genes. This assembly represents a substantial improvement from the currently available sequences that are composed of thousands of contigs. Genomic comparison with and revealed a substantial reduction in the total number of genes in . However, gene loss seems not to be associated to the avirulence of this species given that most genes that have been previously associated with pathogenicity were also present in . To be able to edit the genome of we generated a set of triple auxotrophic strains so that gene deletions can be performed similarly to what has been routinely done in pathogenic species. As a proof of concept, we generated gene knockouts of a gene that encodes a transcription factor that is essential for filamentation and biofilm formation in and . Characterization of these mutants showed that Efg1 also plays a role in biofilm formation and filamentous growth in , but it seems to be a repressor of filamentation in this species. The genome assembly and auxotrophic mutants developed here are a key step forward to start using for comparative and evolutionary studies at a molecular level.

Funding
This study was supported by the:
  • Consejo Nacional de Humanidades, Ciencias y Tecnologías (Award 2019-000037-02NACF-26396)
    • Principle Award Recipient: MarcoChávez-Tinoco
  • Consejo Nacional de Humanidades, Ciencias y Tecnologías (Award 4133922)
    • Principle Award Recipient: LuisF. García-Ortega
  • Consejo Nacional de Humanidades, Ciencias y Tecnologías (Award FORDECYT-PRONACES/103000/2020)
    • Principle Award Recipient: EugenioMancera
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001447
2024-03-08
2024-04-22
Loading full text...

Full text loading...

/deliver/fulltext/micro/170/3/mic001447.html?itemId=/content/journal/micro/10.1099/mic.0.001447&mimeType=html&fmt=ahah

References

  1. Krassowski T, Coughlan AY, Shen XX, Zhou X, Kominek J et al. Evolutionary instability of CUG-Leu in the genetic code of budding yeasts. Nat Commun 2018; 9:1887 [View Article] [PubMed]
    [Google Scholar]
  2. Butler G, Rasmussen MD, Lin MF, Santos MAS, Sakthikumar S et al. Evolution of pathogenicity and sexual reproduction in eight Candida genomes. Nature 2009; 459:657–662 [View Article] [PubMed]
    [Google Scholar]
  3. Jackson AP, Gamble JA, Yeomans T, Moran GP, Saunders D et al. Comparative genomics of the fungal pathogens Candida dubliniensis and Candida albicans. Genome Res 2009; 19:2231–2244 [View Article] [PubMed]
    [Google Scholar]
  4. Bongomin F, Gago S, Oladele RO, Denning DW. Global and multi-national prevalence of fungal diseases-estimate precision. J Fungi 2017; 3:57 [View Article] [PubMed]
    [Google Scholar]
  5. Parums DV. The World Health Organization (WHO) fungal priority pathogens list in response to emerging fungal pathogens during the COVID-19 pandemic. Med Sci Monit 2022; 28:e939088 [View Article] [PubMed]
    [Google Scholar]
  6. Maciel NOP, Piló FB, Freitas LFD, Gomes FCO, Johann S et al. The diversity and antifungal susceptibility of the yeasts isolated from coconut water and reconstituted fruit juices in Brazil. Int J Food Microbiol 2013; 160:201–205 [View Article] [PubMed]
    [Google Scholar]
  7. Mauersberger S, Ohkuma M, Schunck W. Candida maltosa. In Nonconventional Yeasts in Biotechnology 1996 pp 412–580 [View Article]
    [Google Scholar]
  8. Vadkertiová R, Sláviková E. Metal tolerance of yeasts isolated from water, soil and plant environments. J Basic Microbiol 2006; 46:145–152 [View Article] [PubMed]
    [Google Scholar]
  9. Zhai YC, Huang LN, Xi ZW, Chai CY, Hui FL. Candida yunnanensis sp. nov. and Candida parablackwelliae sp. nov., two yeast species in the Candida albicans/Lodderomyces clade. Int J Syst Evol Microbiol 2019; 69:2775–2780 [View Article]
    [Google Scholar]
  10. Gabaldón T, Naranjo-Ortíz MA, Marcet-Houben M. Evolutionary genomics of yeast pathogens in the Saccharomycotina. FEMS Yeast Res 2016; 16:fow064 [View Article] [PubMed]
    [Google Scholar]
  11. Schikora-Tamarit , Gabaldón T. Using genomics to understand the mechanisms of virulence and drug resistance in fungal pathogens. Biochem Soc Trans 2022; 50:1259–1268 [View Article] [PubMed]
    [Google Scholar]
  12. Muñoz JF, Gade L, Chow NA, Loparev VN, Juieng P et al. Genomic insights into multidrug-resistance, mating and virulence in Candida auris and related emerging species. Nat Commun 2018; 9:5346 [View Article] [PubMed]
    [Google Scholar]
  13. Flanagan PR, Fletcher J, Boyle H, Sulea R, Moran GP et al. Expansion of the TLO gene family enhances the virulence of Candida species. PLoS ONE 2018; 13:e0200852 [View Article] [PubMed]
    [Google Scholar]
  14. Moran GP, Coleman DC, Sullivan DJ. Candida albicans versus Candida dubliniensis: why is C. albicans more pathogenic?. Int J Microbiol 2012205921 [View Article] [PubMed]
    [Google Scholar]
  15. Turner SA, Butler G. The Candida pathogenic species complex. Cold Spring Harb Perspect Med 2014; 4:a019778 [View Article] [PubMed]
    [Google Scholar]
  16. Komagata K, Katsuya N. Assimilation of hydrocarbons by yeasts II. determination of hydrocarbon-assimilating yeasts. J Gen Appl Microbiol 1964; 10: [View Article]
    [Google Scholar]
  17. Liu N-N, Zhao X, Tan J-C, Liu S, Li B-W et al. Mycobiome dysbiosis in women with intrauterine adhesions. Microbiol Spectr 2022; 10:e0132422 [View Article] [PubMed]
    [Google Scholar]
  18. Gacho C, Coronado F, Tansengco M, Barcelo J, Borromeo C et al. Isolation, identification and heavy metal biosorption assessment of yeast isolates indigenous to abandoned mine sites of Itogon Benguet, Philippines. JESAM 2019; 22:109–121 [View Article]
    [Google Scholar]
  19. El-Latif Hesham A, Gupta VK, Singh BP. Use of PCR-denaturing gradient gel electrophoresis for the discrimination of Candida species isolated from natural habitats. Microb Pathog 2018; 120:19–22 [View Article] [PubMed]
    [Google Scholar]
  20. Kawai S, Hikiji T, Murao S, Takagi M, Yano K. Isolation and sequencing of a gene, C-ADE1, and its use for a host-vector system in Candida maltosa with two genetic markers. Agric Biol Chem 1991; 55:59–65 [PubMed]
    [Google Scholar]
  21. Gradova NB, Kovalskii IU. Production of fodder yeast cultures on media containing hydrocarbons (Russ) google Académico. Mikrobiologiia 1978; 47:259–264
    [Google Scholar]
  22. Becher D, Schulze S, Kasüske A, Schulze H, Samsonova IA et al. Molecular analysis of a leu2-mutant of Candida maltosa demonstrates the presence of multiple alleles. Curr Genet 1994; 26:208–216 [View Article] [PubMed]
    [Google Scholar]
  23. Nakazawa T, Motoyama T, Horiuchi H, Ohta A, Takagi M. Evidence that part of a centromeric DNA region induces pseudohyphal growth in a dimorphic yeast, Candida maltosa. J Bacteriol 1997; 179:5030–5036 [View Article] [PubMed]
    [Google Scholar]
  24. Takaku H, Mutoh E, Horiuchi H, Ohta A, Takagi M. Ray38p, a homolog of a purine motif triple-helical DNA-binding protein, Stm1p, is a ribosome-associated protein and dissociated from ribosomes prior to the induction of cycloheximide resistance in Candida maltosa. Biochem Biophys Res Commun 2001; 284:194–202 [View Article] [PubMed]
    [Google Scholar]
  25. Ake AHJ, Hafidi M, Ouhdouch Y, Jemo M, Aziz S et al. Microorganisms from tannery wastewater: isolation and screening for potential chromium removal. Envir Technol Inn 2023; 31:103167 [View Article]
    [Google Scholar]
  26. Defosse TA, Le Govic Y, Courdavault V, Clastre M, Vandeputte P et al. Yeasts from the CTG clade (Candida clade): biology, impact in human health, and biotechnological applications. J Mycol Med 2018; 28:257–268 [View Article] [PubMed]
    [Google Scholar]
  27. Lin Y, He P, Wang Q, Lu D, Li Z et al. The alcohol dehydrogenase system in the xylose-fermenting yeast Candida maltosa. PLoS ONE 2010; 5:e11752 [View Article] [PubMed]
    [Google Scholar]
  28. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 2018; 34:i884–i890 [View Article] [PubMed]
    [Google Scholar]
  29. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH et al. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res 2017; 27:722–736 [View Article] [PubMed]
    [Google Scholar]
  30. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE 2014; 9:e112963 [View Article] [PubMed]
    [Google Scholar]
  31. Simão FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 2015; 31:3210–3212 [View Article] [PubMed]
    [Google Scholar]
  32. Smit A, Hubley R, Green P. RepeatMasker 4.0; 2013 http://www.repeatmasker.org/RMDownload.html
  33. Campbell MS, Holt C, Moore B, Yandell M. Genome annotation and curation using MAKER and MAKER-P. Curr Protoc Bioinformatics 2014; 48:4 [View Article] [PubMed]
    [Google Scholar]
  34. Korf I. Gene finding in novel genomes. BMC Bioinformatics 2004; 5:1–9 [View Article] [PubMed]
    [Google Scholar]
  35. Stanke M, Keller O, Gunduz I, Hayes A, Waack S et al. AUGUSTUS: ab initio prediction of alternative transcripts. Nucleic Acids Res 2006; 34:W435–9 [View Article] [PubMed]
    [Google Scholar]
  36. Jones P, Binns D, Chang HY, Fraser M, Li W et al. InterProScan 5: genome-scale protein function classification. Bioinformatics 2014; 30:1236–1240 [View Article] [PubMed]
    [Google Scholar]
  37. Paril J, Zare T, Fournier-Level A. Compare_genomes: a comparative genomics workflow to streamline the analysis of evolutionary divergence across eukaryotic genomes. Curr Protoc 2023; 3:e876 [View Article] [PubMed]
    [Google Scholar]
  38. Emms DM, Kelly S. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol 2019; 20:238 [View Article] [PubMed]
    [Google Scholar]
  39. Mistry J, Finn RD, Eddy SR, Bateman A, Punta M. Challenges in homology search: HMMER3 and convergent evolution of coiled-coil regions. Nucleic Acids Res 2013; 41:e121 [View Article] [PubMed]
    [Google Scholar]
  40. Thomas PD, Ebert D, Muruganujan A, Mushayahama T, Albou L-P et al. PANTHER: making genome-scale phylogenetics accessible to all. Protein Sci 2022; 31:8–22 [View Article] [PubMed]
    [Google Scholar]
  41. De Bie T, Cristianini N, Demuth JP, Hahn MW. CAFE: a computational tool for the study of gene family evolution. Bioinformatics 2006; 22:1269–1271 [View Article]
    [Google Scholar]
  42. Edgar RC. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 2004; 5:113 [View Article] [PubMed]
    [Google Scholar]
  43. Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 2009; 25:1972–1973 [View Article] [PubMed]
    [Google Scholar]
  44. Miller MA, Schwartz T, Hoover P, Yoshimoto K, Sivagnanam S et al. The CIPRES workbench: a flexible framework for creating science gateways. ACM Int Con Proc Series 2015 [View Article]
    [Google Scholar]
  45. Wang Y, Tang H, Debarry JD, Tan X, Li J et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res 2012; 40:2–14 [View Article] [PubMed]
    [Google Scholar]
  46. Carlson M, Herves P. AnnotationForge: Tools for building SQLite-based annotation data packages DOI: 10.18129/B9.bioc.AnnotationForge R package version 1.44.0,. Bioconductor Package Maintainer; 2023 https://bioconductor.org/packages/release/bioc/html/AnnotationForge.html
  47. Yu G, Wang L-G, Han Y, He Q-Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 2012; 16:284–287 [View Article] [PubMed]
    [Google Scholar]
  48. Gómez-Gaviria M, Martínez-Álvarez JA, Chávez-Santiago JO, Mora-Montes HM. Candida haemulonii complex and Candida auris: biology, virulence factors, immune response, and multidrug resistance. Infect Drug Resist 2023; 16:1455–1470 [View Article] [PubMed]
    [Google Scholar]
  49. Noble SM, French S, Kohn LA, Chen V, Johnson AD. Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity. Nat Genet 2010; 42:590–598 [View Article] [PubMed]
    [Google Scholar]
  50. Oh S-H, Smith B, Miller AN, Staker B, Fields C et al. Agglutinin-Like Sequence (ALS) genes in the Candida parapsilosis species complex: blurring the boundaries between gene families that encode cell-wall proteins. Front Microbiol 2019; 10:781 [View Article] [PubMed]
    [Google Scholar]
  51. Zhang Z, Carriero N, Zheng D, Karro J, Harrison PM et al. PseudoPipe: an automated pseudogene identification pipeline. Bioinformatics 2006; 22:1437–1439 [View Article] [PubMed]
    [Google Scholar]
  52. Mancera E, Frazer C, Porman AM, Ruiz-Castro S, Johnson AD et al. Genetic modification of closely related Candida species. Front Microbiol 2019; 10:357 [View Article]
    [Google Scholar]
  53. Noble SM, Johnson AD. Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans. Eukaryot Cell 2005; 4:298–309 [View Article] [PubMed]
    [Google Scholar]
  54. Lackey E, Vipulanandan G, Childers DS, Kadosh D. Comparative evolution of morphological regulatory functions in Candida species. Eukaryot Cell 2013; 12:1356–1368 [View Article] [PubMed]
    [Google Scholar]
  55. Lee KL, Buckley HR, Campbell CC. An amino acid liquid synthetic medium for the development of mycelial and yeast forms of Candida albicans. Sabouraudia 1975; 13:148–153 [View Article] [PubMed]
    [Google Scholar]
  56. Zhang Q, Tao L, Guan G, Yue H, Liang W et al. Regulation of filamentation in the human fungal pathogen Candida tropicalis. Mol Microbiol 2016; 99:528–545 [View Article] [PubMed]
    [Google Scholar]
  57. Naseem S, Douglas LM, Konopka JB. Candida albicans agar invasion assays. Bio-Protocol 2020; 10:16 [View Article]
    [Google Scholar]
  58. Mancera E, Porman AM, Cuomo CA, Bennett RJ, Johnson AD. Finding a missing gene: EFG1 regulates morphogenesis in Candida tropicalis. G3 2015; 5:849–856 [View Article] [PubMed]
    [Google Scholar]
  59. Ramos L, Borghi A, Ramadán S, Lopez C. The use of calcofluor white for the detection of fungi which cause subcutaneous and systemic mycoses. Bol Micol 1999; 14:9 [View Article]
    [Google Scholar]
  60. Mancera E, Nocedal I, Hammel S, Gulati M, Mitchell KF et al. Evolution of the complex transcription network controlling biofilm formation in Candida species. Elife 2021; 10:e64682 [View Article] [PubMed]
    [Google Scholar]
  61. Nobile CJ, Fox EP, Nett JE, Sorrells TR, Mitrovich QM et al. A recently evolved transcriptional network controls biofilm development in Candida albicans. Cell 2012; 148:126–138 [View Article] [PubMed]
    [Google Scholar]
  62. Gupta V, Abhisheik K, Balasundari S, Devendra NK, Shadab K et al. Identification of Candida albicans using different culture media and its association in leukoplakia and oral squamous cell carcinoma. J Oral Maxillofac Pathol 2019; 23:28–35 [View Article] [PubMed]
    [Google Scholar]
  63. Takagi M, Yano K. Separation of chromosomal DNA molecules of Candida maltosa on agarose gels using the OFAGE technique. Agri Biol Chem 1987; 51:3161–3163 [View Article]
    [Google Scholar]
  64. Mba IE, Nweze EI, Eze EA, Anyaegbunam ZKG. Genome plasticity in Candida albicans: a cutting-edge strategy for evolution, adaptation, and survival. Infect Genet Evol 2022; 99:105256 [View Article] [PubMed]
    [Google Scholar]
  65. Xu J. Is natural population of Candida tropicalis sexual, parasexual, and/or asexual?. Front Cell Infect Microbiol 2021; 11:751676 [View Article] [PubMed]
    [Google Scholar]
  66. Guin K, Chen Y, Mishra R, Muzaki SRB, Thimmappa BC et al. Spatial inter-centromeric interactions facilitated the emergence of evolutionary new centromeres. Elife 2020; 9:e58556 [View Article] [PubMed]
    [Google Scholar]
  67. Mishra PK, Baum M, Carbon J. Centromere size and position in Candida albicans are evolutionarily conserved independent of DNA sequence heterogeneity. Mol Genet Genomics 2007; 278:455–465 [View Article] [PubMed]
    [Google Scholar]
  68. Proux-Wéra E, Armisén D, Byrne KP, Wolfe KH. A pipeline for automated annotation of yeast genome sequences by a conserved-synteny approach. BMC Bioinformatics 2012; 13:1–12 [View Article] [PubMed]
    [Google Scholar]
  69. Haran J, Boyle H, Hokamp K, Yeomans T, Liu Z et al. Telomeric ORFs (TLOs) in Candida spp. Encode mediator subunits that regulate distinct virulence traits. PLoS Genet 2014; 10:e1004658 [View Article] [PubMed]
    [Google Scholar]
  70. Oh S-H, Isenhower A, Rodriguez-Bobadilla R, Smith B, Jones J et al. Pursuing advances in DNA sequencing technology to solve a complex genomic jigsaw puzzle: the agglutinin-like sequence (ALS) genes of Candida tropicalis. Front Microbiol 2020; 11:594531 [View Article] [PubMed]
    [Google Scholar]
  71. Parra-Ortega B, Cruz-Torres H, Villa-Tanaca L, Hernández-Rodríguez C. Phylogeny and evolution of the aspartyl protease family from clinically relevant Candida species. Mem Inst Oswaldo Cruz 2009; 104:505–512 [View Article] [PubMed]
    [Google Scholar]
  72. Taylor BN, Staib P, Binder A, Biesemeier A, Sehnal M et al. Profile of Candida albicans-secreted aspartic proteinase elicited during vaginal infection. Infect Immun 2005; 73:1828–1835 [View Article] [PubMed]
    [Google Scholar]
  73. Holland LM, Schröder MS, Turner SA, Taff H, Andes D et al. Comparative phenotypic analysis of the major fungal pathogens Candida parapsilosis and Candida albicans. PLoS Pathog 2014; 10:e1004365 [View Article] [PubMed]
    [Google Scholar]
  74. Nguyen N, Quail MMF, Hernday AD. An efficient, rapid, and recyclable system for CRISPR-mediated genome editing in Candida albicans. mSphere 2017; 2:e00149-17 [View Article] [PubMed]
    [Google Scholar]
  75. Hirakawa MP, Martinez DA, Sakthikumar S, Anderson MZ, Berlin A et al. Genetic and phenotypic intra-species variation in Candida albicans. Genome Res 2015; 25:413–425 [View Article] [PubMed]
    [Google Scholar]
  76. Montelongo-Jauregui D, Saville SP, Lopez-Ribot JL. Contributions of Candida albicans dimorphism, adhesive interactions, and extracellular matrix to the formation of dual-species biofilms with Streptococcus gordonii. mBio 2019; 10:e01179-19 [View Article] [PubMed]
    [Google Scholar]
  77. Ramage G, VandeWalle K. The filamentation pathway controlled by the Efg1 regulator protein is required for normal biofilm formation and development in Candida albicans. FEMS Microbiol Lett 2002; 214:95–100 [View Article]
    [Google Scholar]
  78. Sonneborn A, Bockmühl DP, Ernst JF. Chlamydospore formation in Candida albicans requires the Efg1p morphogenetic regulator. Infect Immun 1999; 67:5514–5517 [View Article] [PubMed]
    [Google Scholar]
  79. Takaku H, Horiuchi H, Takagi M, Ohta A. Pseudohyphal growth in a dimorphic yeast, Candida maltosa, after disruption of the C-GCN4 gene, a homolog of Saccharomyces cerevisiae GCN4. Biosci Biotechnol Biochem 2002; 66:1936–1939 [View Article] [PubMed]
    [Google Scholar]
  80. Ramírez-Ramírez R, Calvo-Méndez C, Avila-Rodríguez M, Lappe P, Ulloa M et al. Cr(VI) reduction in a chromate-resistant strain of Candida maltosa isolated from the leather industry. Antonie van Leeuwenhoek 2004; 85:63–68 [View Article] [PubMed]
    [Google Scholar]
  81. Connolly LA, Riccombeni A, Grózer Z, Holland LM, Lynch DB et al. The APSES transcription factor Efg1 is a global regulator that controls morphogenesis and biofilm formation in Candida parapsilosis. Mol Microbiol 2013; 90:36–53 [View Article] [PubMed]
    [Google Scholar]
  82. Robinson D, Vanacloig-Pedros E, Cai R, Place M, Hose J et al. Gene-by-environment interactions influence the fitness cost of gene copy-number variation in yeast. G3 2023; 13:jkad159 [View Article] [PubMed]
    [Google Scholar]
  83. Steenwyk JL, Rokas A. Copy number variation in fungi and its implications for wine yeast genetic diversity and adaptation. Front Microbiol 2018; 9:288 [View Article] [PubMed]
    [Google Scholar]
  84. Kiss RD, Stephanopoulos G. Culture instability of auxotrophic amino acid producers. Biotechnol Bioeng 1992; 40:75–85 [View Article] [PubMed]
    [Google Scholar]
  85. Huang MY, Woolford CA, May G, McManus CJ, Mitchell AP. Circuit diversification in a biofilm regulatory network. PLoS Pathog 2019; 15:e1007787 [View Article] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.001447
Loading
/content/journal/micro/10.1099/mic.0.001447
Loading

Data & Media loading...

Supplements

Supplementary material 1

EXCEL
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error