Generating genomic platforms to study<i>Candida albicans</i>pathogenesis

Legrand, Mélanie; Bachellier‐Bassi, Sophie; Lee, Keunsook K.; Chaudhari, Yogesh; Tournu, Hélène; Arbogast, Laurence; Boyer, Hélène; Chauvel, Murielle; Cabral, Vitor; Maufrais, Corinne; Nesseir, Audrey; Maslanka, Irena; Permal, Emmanuelle; Rossignol, Tristan; Walker, Larry P.; Zeidler, Ute; Znaidi, Sadri; Schoeters, Floris; Majgier, Charlotte; Julien, Renaud A; Ma, Laurence; Tichit, Magali; Bouchier, Christiane; Dijck, Patrick Van; Munro, Carol A.; d’Enfert, Christophe

doi:10.1101/261628

Cited by 3 publications

(4 citation statements)

References 62 publications

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“…The resulting strain was transformed with plasmid CIp10 to generate the prototrophic strain CEC4425 (Table S11) that we used as a BFP‐labelled control for our 1:1 competitive assays (Figure c,d). Strains overexpressing CRZ2 from the constitutive TDH3 promoter were generated by transferring the CRZ2 ORF from the respective pDONR207 derivative to the Gateway‐compatible CIp10‐P TDH3 ‐GTW plasmid (Legrand et al ) followed by transformation of strain BWP17AH (CEC161; Chauvel et al, ) to generate three independent P TDH3 ‐driven CRZ2 ‐overexpression clones (C26, C27, and C28; Table S11). The CEC161 strain was also transformed with CIp10‐P TDH3 ‐GFP, generating strain CEC4267 (Table S11), to serve as a control for phenotypic assays.…”

Section: Methodsmentioning

confidence: 99%

“…To create a TAP epitope‐tagged version of CRZ2 , the CRZ2 ORF was transferred from the corresponding pDONR207 to the Gateway‐compatible CIp10‐P TET ‐TAP‐GTW that allows P TET ‐driven expression of N‐terminally‐tagged ORFs (Legrand et al, , in press). The ura3 auxotrophic derivative of the crz2 Δ/ crz2 Δ mutants was first transformed with plasmid pNIMX then with CIp10‐P TET ‐TAP‐ CRZ2 or CIp10‐P TET ‐ CRZ2 (untagged control) to generate strains C251, C252 (two independent TAP‐tagged clones) and C255, C257, C258 (three independent untagged control clones), respectively (Table S11).…”

Section: Methodsmentioning

confidence: 99%

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Systematic gene overexpression inCandida albicansidentifies a regulator of early adaptation to the mammalian gut

Znaidi

Wijlick

Hernández‐Cervantes

et al. 2018

Cellular Microbiology

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Candida albicans is part of the human gastrointestinal (GI) microbiota. To better understand how C. albicans efficiently establishes GI colonisation, we competitively challenged growth of 572 signature‐tagged strains (~10% genome coverage), each conditionally overexpressing a single gene, in the murine gut. We identified CRZ2, a transcription factor whose overexpression and deletion respectively increased and decreased early GI colonisation. Using clues from genome‐wide expression and gene‐set enrichment analyses, we found that the optimal activity of Crz2p occurs under hypoxia at 37°C, as evidenced by both phenotypic and transcriptomic analyses following CRZ2 genetic perturbation. Consistent with early colonisation of the GI tract, we show that CRZ2 overexpression confers resistance to acidic pH and bile salts, suggesting an adaptation to the upper sections of the gut. Genome‐wide location analyses revealed that Crz2p directly modulates the expression of many mannosyltransferase‐ and cell‐wall protein‐encoding genes, suggesting a link with cell‐wall function. We show that CRZ2 overexpression alters cell‐wall phosphomannan abundance and increases sensitivity to tunicamycin, suggesting a role in protein glycosylation. Our study reflects the powerful use of gene overexpression as a complementary approach to gene deletion to identify relevant biological pathways involved in C. albicans interaction with the host environment.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Systematic gene overexpression inCandida albicansidentifies a regulator of early adaptation to the mammalian gut

Znaidi

Wijlick

Hernández‐Cervantes

et al. 2018

Cellular Microbiology

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“…Overexpression screens can also help dissect molecular pathways (53)(54)(55). In C. albicans, several techniques exist for genetic overexpression (39), including promoter replacement strategies (55)(56)(57)(58), the generation of ORFeome collection strains (59,60), and CRISPR-based methods (27). While these these tools have made important contributions to understanding overexpression in C. albicans, there remains a need for simple and rapid methods to efficiently target fungal genes of interest for overexpression, and to do so in diverse strain backgrounds.…”

Section: Introductionmentioning

confidence: 99%

Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans

Gervais

Bella

Wensing

et al. 2022

Preprint

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For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness genetic tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans. Our CRISPRa system exploits a nuclease-dead dCas9 fused to the tripartite activator complex VP64-p65-Rta (VPR), and a Golden Gate site for efficient guide RNA cloning to overexpress genes of interest. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust overexpression. We further demonstrate the application of CRISPRa to overexpress genes involved in fungal pathogenesis and drug resistance and detect corresponding phenotypic alterations in these traits. Together, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.

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“…These resources have been generated for several model organisms, including humans, Escherichia coli, Caenorhabditis elegans , and Arabidopsis thaliana (18–23). ORFeomes have also been developed for the fungal kingdom, including fission yeast (18), budding yeast (24) and, most recently, the human pathogenic yeast Candida albicans (8). Usually, ORFeomes are compatible with the Gateway ® cloning technology (Invitrogen), which enables rapid and high throughput recombinase-based transfer of an ORF coding sequence to generate expression vectors (25, 26).…”

Section: Introductionmentioning

confidence: 99%

The Zymoseptoria tritici ORFeome: a functional genomics community resource

Chaudhari

Cairns

Sidhu

et al. 2019

Preprint

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Libraries of protein-encoding sequences can be generated by identification of open reading frames (ORFs) from a genome of choice that are then assembled into collections of plasmids termed ORFeome libraries. These represent powerful resources to facilitate functional genomic characterization of genes and their encoded products. Here, we report the generation of an ORFeome for Zymoseptoria tritici, which causes the most serious disease of wheat in temperate regions of the world. We screened the genome of strain IP0323 for high confidence gene models, identifying 4075 candidates from 10,933 predicted genes. These were amplified from genomic DNA, cloned into the Gateway® Entry Vector pDONR207, and sequenced, providing a total of 3022 quality-controlled plasmids. The ORFeome includes genes predicted to encode effectors (n = 410) and secondary metabolite biosynthetic proteins (n = 171), in addition to genes residing at dispensable chromosomes (n= 122), or those that are preferentially expressed during plant infection (n = 527). The ORFeome plasmid library is compatible with our previously developed suite of Gateway® Destination vectors, which have various combinations of promoters, selection markers, and epitope tags. The Z. tritici ORFeome constitutes a powerful resource for functional genomics, and offers unparalleled opportunities to understand the biology of Z. tritici.

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Generating genomic platforms to studyCandida albicanspathogenesis

Cited by 3 publications

References 62 publications

Systematic gene overexpression inCandida albicansidentifies a regulator of early adaptation to the mammalian gut

Systematic gene overexpression inCandida albicansidentifies a regulator of early adaptation to the mammalian gut

Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans

The Zymoseptoria tritici ORFeome: a functional genomics community resource

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