Design and Generation of a CRISPR Interference System for Genetic Repression and Essential Gene Analysis in the Fungal Pathogen Candida albicans

ABSTRACT Fungal pathogens are emerging as an important cause of human disease, and Candida albicans is among the most common causative agents of fungal infections. Studying this fungal pathogen is of the utmost importance and necessitates the development of molecular technologies to perform comprehensive genetic and functional genomic analysis. Here, we designed and developed a novel clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for targeted genetic repression in C. albicans. We engineered a nuclease-dead Cas9 (dCas9) construct that, paired with a guide RNA targeted to the promoter of an endogenous gene, is capable of targeting that gene for transcriptional repression. We further optimized a favorable promoter locus to achieve repression and demonstrated that fusion of dCas9 to an Mxi1 repressor domain was able to further enhance transcriptional repression. Finally, we demonstrated the application of this CRISPRi system through genetic repression of the essential molecular chaperone HSP90. This is the first demonstration of a functional CRISPRi repression system in C. albicans, and this valuable technology will enable many future applications in this critical fungal pathogen. IMPORTANCE Fungal pathogens are an increasingly important cause of human disease and mortality, and Candida albicans is among the most common causes of fungal disease. Studying this important fungal pathogen requires a comprehensive genetic toolkit to establish how different genetic factors play roles in the biology and virulence of this pathogen. Here, we developed a CRISPR-based genetic regulation platform to achieve targeted repression of C. albicans genes. This CRISPR interference (CRISPRi) technology exploits a nuclease-dead Cas9 protein (dCas9) fused to transcriptional repressors. The dCas9 fusion proteins pair with a guide RNA to target genetic promoter regions and to repress expression from these genes. We demonstrated the functionality of this system for repression in C. albicans and show that we can apply this technology to repress essential genes. Taking the results together, this work presents a new technology for efficient genetic repression in C. albicans, with important applications for genetic analysis in this fungal pathogen.

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Faculty Opinions recommendation of A novel role for the NLRC4 inflammasome in mucosal defenses against the fungal pathogen Candida albicans.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13490963.14868063 ◽

2012 ◽

Author(s):

Philip Murphy ◽

Michail Lionakis

Keyword(s):

Candida Albicans ◽

Fungal Pathogen

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pH-Dependant Antifungal Activity of Valproic Acid against the Human Fungal Pathogen Candida albicans

Frontiers in Microbiology ◽

10.3389/fmicb.2017.01956 ◽

2017 ◽

Vol 8 ◽

Cited By ~ 9

Author(s):

Julien Chaillot ◽

Faiza Tebbji ◽

Carlos García ◽

Hugo Wurtele ◽

René Pelletier ◽

...

Keyword(s):

Valproic Acid ◽

Candida Albicans ◽

Antifungal Activity ◽

Fungal Pathogen ◽

Human Fungal Pathogen

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An improved transformation protocol for the human fungal pathogen Candida albicans

Current Genetics ◽

10.1007/s00294-002-0349-0 ◽

2003 ◽

Vol 42 (6) ◽

pp. 339-343 ◽

Cited By ~ 143

Author(s):

Andrea Walther ◽

Jürgen Wendland

Keyword(s):

Candida Albicans ◽

Fungal Pathogen ◽

Transformation Protocol ◽

Human Fungal Pathogen

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An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans

mSphere ◽

10.1128/mspheredirect.00149-17 ◽

2017 ◽

Vol 2 (2) ◽

Cited By ~ 33

Author(s):

Namkha Nguyen ◽

Morgan M. F. Quail ◽

Aaron D. Hernday

Keyword(s):

Candida Albicans ◽

Genome Editing ◽

Fungal Pathogen ◽

Gene Knockout ◽

Strain Engineering ◽

Molecular Genetic Analysis ◽

Content Type ◽

Highly Efficient ◽

Discovery Research ◽

Gene Knockouts

ABSTRACT Candida albicans is the most common fungal pathogen of humans. Historically, molecular genetic analysis of this important pathogen has been hampered by the lack of stable plasmids or meiotic cell division, limited selectable markers, and inefficient methods for generating gene knockouts. The recent development of clustered regularly interspaced short palindromic repeat(s) (CRISPR)-based tools for use with C. albicans has opened the door to more efficient genome editing; however, previously reported systems have specific limitations. We report the development of an optimized CRISPR-based genome editing system for use with C. albicans. Our system is highly efficient, does not require molecular cloning, does not leave permanent markers in the genome, and supports rapid, precise genome editing in C. albicans. We also demonstrate the utility of our system for generating two independent homozygous gene knockouts in a single transformation and present a method for generating homozygous wild-type gene addbacks at the native locus. Furthermore, each step of our protocol is compatible with high-throughput strain engineering approaches, thus opening the door to the generation of a complete C. albicans gene knockout library. IMPORTANCE Candida albicans is the major fungal pathogen of humans and is the subject of intense biomedical and discovery research. Until recently, the pace of research in this field has been hampered by the lack of efficient methods for genome editing. We report the development of a highly efficient and flexible genome editing system for use with C. albicans. This system improves upon previously published C. albicans CRISPR systems and enables rapid, precise genome editing without the use of permanent markers. This new tool kit promises to expedite the pace of research on this important fungal pathogen.

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The Cek1 and Hog1 Mitogen-Activated Protein Kinases Play Complementary Roles in Cell Wall Biogenesis and Chlamydospore Formation in the Fungal Pathogen Candida albicans

Eukaryotic Cell ◽

10.1128/ec.5.2.347-358.2006 ◽

2006 ◽

Vol 5 (2) ◽

pp. 347-358 ◽

Cited By ~ 109

Author(s):

B. Eisman ◽

R. Alonso-Monge ◽

E. Román ◽

D. Arana ◽

C. Nombela ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Map Kinase ◽

Fungal Pathogen ◽

Hog Pathway ◽

Cell Wall Biogenesis ◽

Chlamydospore Formation ◽

Mitogen Activated Protein ◽

Morphological Transitions ◽

Relationship Of

ABSTRACT The Hog1 mitogen-activated protein (MAP) kinase mediates an adaptive response to both osmotic and oxidative stress in the fungal pathogen Candida albicans. This protein also participates in two distinct morphogenetic processes, namely the yeast-to-hypha transition (as a repressor) and chlamydospore formation (as an inducer). We show here that repression of filamentous growth occurs both under serum limitation and under other partially inducing conditions, such as low temperature, low pH, or nitrogen starvation. To understand the relationship of the HOG pathway to other MAP kinase cascades that also play a role in morphological transitions, we have constructed and characterized a set of double mutants in which we deleted both the HOG1 gene and other signaling elements (the CST20, CLA4, and HST7 kinases, the CPH1 and EFG1 transcription factors, and the CPP1 protein phosphatase). We also show that Hog1 prevents the yeast-to-hypha switch independent of all the elements analyzed and that the inability of the hog1 mutants to form chlamydospores is suppressed when additional elements of the CEK1 pathway (CST20 or HST7) are altered. Finally, we report that Hog1 represses the activation of the Cek1 MAP kinase under basal conditions and that Cek1 activation correlates with resistance to certain cell wall inhibitors (such as Congo red), demonstrating a role for this pathway in cell wall biogenesis.

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Pmt-Mediated O Mannosylation Stabilizes an Essential Component of the Secretory Apparatus, Sec20p, in Candida albicans

Eukaryotic Cell ◽

10.1128/ec.3.5.1164-1168.2004 ◽

2004 ◽

Vol 3 (5) ◽

pp. 1164-1168 ◽

Cited By ~ 22

Author(s):

Yvonne Weber ◽

Stephan K.-H. Prill ◽

Joachim F. Ernst

Keyword(s):

Endoplasmic Reticulum ◽

Candida Albicans ◽

Fungal Pathogen ◽

Wild Type ◽

Rapid Degradation ◽

Vesicle Traffic ◽

Human Fungal Pathogen ◽

Low Levels ◽

Lumenal Domain ◽

Secretory Apparatus

ABSTRACT Sec20p is an essential endoplasmic reticulum (ER) membrane protein in yeasts, functioning as a tSNARE component in retrograde vesicle traffic. We show that Sec20p in the human fungal pathogen Candida albicans is extensively O mannosylated by protein mannosyltransferases (Pmt proteins). Surprisingly, Sec20p occurs at wild-type levels in a pmt6 mutant but at very low levels in pmt1 and pmt4 mutants and also after replacement of specific Ser/Thr residues in the lumenal domain of Sec20p. Pulse-chase experiments revealed rapid degradation of unmodified Sec20p (38.6 kDa) following its biosynthesis, while the stable O-glycosylated form (50 kDa) was not formed in a pmt1 mutant. These results suggest a novel function of O mannosylation in eukaryotes, in that modification by specific Pmt proteins will prevent degradation of ER-resident membrane proteins via ER-associated degradation or a proteasome-independent pathway.

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Nanoscale adhesion forces between the fungal pathogen Candida albicans and macrophages

Nanoscale Horizons ◽

10.1039/c5nh00049a ◽

2016 ◽

Vol 1 (1) ◽

pp. 69-74 ◽

Cited By ~ 15

Author(s):

Sofiane El-Kirat-Chatel ◽

Yves F. Dufrêne

Keyword(s):

Atomic Force Microscopy ◽

Candida Albicans ◽

Immune Cells ◽

Fungal Pathogen ◽

Fungal Pathogens ◽

Adhesion Forces ◽

Force Microscopy ◽

Atomic Force

We establish atomic force microscopy as a new nanoscopy platform for quantifying the forces between fungal pathogens and immune cells.

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In Fungal Intracellular Pathogenesis, Form Determines Fate

mBio ◽

10.1128/mbio.02092-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 4

Author(s):

Robin C. May ◽

Arturo Casadevall

Keyword(s):

Candida Albicans ◽

Fungal Pathogen ◽

Membrane Integrity ◽

Pathogenic Fungi ◽

Morphological Transition ◽

Physical Damage ◽

Fungal Cell ◽

Morphological Transitions ◽

Pathogenic Microbes ◽

Phagosomal Membrane

ABSTRACT For pathogenic microbes to survive ingestion by macrophages, they must subvert powerful microbicidal mechanisms within the phagolysosome. After ingestion, Candida albicans undergoes a morphological transition producing hyphae, while the surrounding phagosome exhibits a loss of phagosomal acidity. However, how these two events are related has remained enigmatic. Now Westman et al. (mBio 9:e01226-18, 2018, https://doi.org/10.1128/mBio.01226-18) report that phagosomal neutralization results from disruption of phagosomal membrane integrity by the enlarging hyphae, directly implicating the morphological transition in physical damage that promotes intracellular survival. The C. albicans intracellular strategy shows parallels with another fungal pathogen, Cryptococcus neoformans, where a morphological changed involving capsular enlargement intracellularly is associated with loss of membrane integrity and death of the host cell. These similarities among distantly related pathogenic fungi suggest that morphological transitions that are common in fungi directly affect the outcome of the fungal cell-macrophage interaction. For this class of organisms, form determines fate in the intracellular environment.

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