Genetic analysis of sirtuin deacetylases in hyphal growth of Candida albicans

Candida albicans is a major human fungal pathogen that encounters varied host environments during infection. In response to environmental cues, C. albicans switches between ovoid yeast and elongated hyphal growth forms, and this morphological plasticity contributes to virulence. Environmental changes that alter the cell’s metabolic state could be sensed by sirtuins, which are NAD+-dependent deacetylases. Here we studied the roles of three sirtuin deacetylases, Sir2, Hst1, and Hst2, in hyphal growth of C. albicans. We made single, double, and triple sirtuin knockout strains and tested their ability to switch from yeast to hyphae. We found that true hyphae formation was significantly reduced by the deletion of SIR2 but not HST1 or HST2. Moreover, the expression of hyphal-specific genes HWP1, ALS3, and ECE1 decreased in the sir2Δ/Δ mutant compared to wild-type. This regulation of hyphae formation was dependent on the deacetylase activity of Sir2, as a point mutant lacking deacetylase activity had a similar defect in hyphae formation as the sir2Δ/Δ mutant. Finally, we found that Sir2 and Hst1 were localized to the nucleus, with Sir2 specifically focused in the nucleolus. This nuclear localization suggests a role for Sir2 and Hst1 in regulating gene expression. In contrast, Hst2 was localized to the cytoplasm. In conclusion, our results suggest that Sir2 plays a critical and non-redundant role in hyphal growth of C. albicans.

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Farnesol and Cyclic AMP Signaling Effects on the Hypha-to-Yeast Transition in Candida albicans

Eukaryotic Cell ◽

10.1128/ec.00144-12 ◽

2012 ◽

Vol 11 (10) ◽

pp. 1219-1225 ◽

Cited By ~ 61

Author(s):

Allia K. Lindsay ◽

Aurélie Deveau ◽

Amy E. Piispanen ◽

Deborah A. Hogan

Keyword(s):

Candida Albicans ◽

Cyclic Amp ◽

Fungal Pathogen ◽

Hyphal Growth ◽

Morphological Plasticity ◽

Camp Signaling ◽

Environmental Cues ◽

Transcriptional Repressors ◽

Content Type

ABSTRACTCandida albicans, a fungal pathogen of humans, regulates its morphology in response to many environmental cues and this morphological plasticity contributes to virulence. Farnesol, an autoregulatory molecule produced byC. albicans, inhibits the induction of hyphal growth by inhibiting adenylate cyclase (Cyr1). The role of farnesol and Cyr1 in controlling the maintenance of hyphal growth has been less clear. Here, we demonstrate that preformed hyphae transition to growth as yeast in response to farnesol and that strains with increased cyclic AMP (cAMP) signaling exhibit more resistance to farnesol. Exogenous farnesol did not induce the hypha-to-yeast transition in mutants lacking the Tup1 or Nrg1 transcriptional repressors in embedded conditions. Although body temperature is not required for embedded hyphal growth, we found that the effect of farnesol on the hypha-to-yeast transition varies inversely with temperature. Our model of Cyr1 activity being required for filamentation is also supported by our liquid assay data, which show increased yeast formation when preformed filaments are treated with farnesol. Together, these data suggest that farnesol can modulate morphology in preformed hyphal cells and that the repression of hyphal growth maintenance likely occurs through the inhibition of cAMP signaling.

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Genetic Analysis of Sirtuin Deacetylases in Hyphal Growth of Candida albicans

mSphere ◽

10.1128/msphere.00053-21 ◽

2021 ◽

Vol 6 (3) ◽

Author(s):

Guolei Zhao ◽

Laura N. Rusche

Keyword(s):

Candida Albicans ◽

Environmental Changes ◽

Fungal Infections ◽

Hyphal Growth ◽

Morphological Plasticity ◽

The United States ◽

Environmental Cues ◽

Growth Forms ◽

Content Type ◽

Hypha Formation

ABSTRACT Candida albicans is a major human fungal pathogen that encounters varied host environments during infection. In response to environmental cues, C. albicans switches between ovoid yeast and elongated hyphal growth forms, and this morphological plasticity contributes to virulence. Environmental changes that alter the cell’s metabolic state could be sensed by sirtuins, which are NAD+-dependent deacetylases. Here, we studied the roles of three sirtuin deacetylases—Sir2, Hst1, and Hst2—in the hyphal growth of C. albicans. We made single, double, and triple sirtuin knockout strains and tested their ability to switch from yeast to hyphae. We found that true hypha formation was significantly reduced by the deletion of SIR2 but not HST1 or HST2. Moreover, the expression of hypha-specific genes HWP1, ALS3, and ECE1 decreased in the sir2Δ/Δ mutant compared to the wild type. This regulation of hypha formation was likely dependent on the deacetylase activity of Sir2, as a similar defect in hypha formation was observed when an asparagine known to be required for deacetylation was mutated. Finally, we found that Sir2 and Hst1 were localized to the nucleus, with Sir2 specifically focused in the nucleolus. This nuclear localization suggests a role for Sir2 and Hst1 in regulating gene expression. In contrast, Hst2 was localized to the cytoplasm. In conclusion, our results suggest that Sir2 plays a critical and nonredundant role in hyphal growth of C. albicans. IMPORTANCE Candida albicans is one of the most common causes of hospital-acquired systemic fungal infections in the United States. It can switch between ovoid yeast and elongated hyphal growth forms in response to environmental cues. This morphological transition is essential for its survival in the host. Thus, identifying regulators involved in this process can lead to new therapies. In this study, we examined the contribution of three regulators called sirtuins (Sir2, Hst1, and Hst2) to the yeast-to-hypha transition of C. albicans. We found that loss of Sir2 but not Hst1 or Hst2 hampered hypha formation. Moreover, the defect was caused by the loss of the catalytic activity of Sir2. Our study may lay the groundwork for discovering novel targets for antifungal therapies.

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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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Recent advances in understanding Candida albicans hyphal growth

F1000Research ◽

10.12688/f1000research.18546.1 ◽

2019 ◽

Vol 8 ◽

pp. 700 ◽

Cited By ~ 2

Author(s):

Robert A. Arkowitz ◽

Martine Bassilana

Keyword(s):

Candida Albicans ◽

Medical Research ◽

Filamentous Fungi ◽

Fungal Pathogen ◽

Fungal Pathogens ◽

Morphological Changes ◽

Hyphal Growth ◽

Yeast Form ◽

Intensive Research ◽

Human Fungal Pathogen

Morphological changes are critical for the virulence of a range of plant and human fungal pathogens. Candida albicans is a major human fungal pathogen whose ability to switch between different morphological states is associated with its adaptability and pathogenicity. In particular, C. albicans can switch from an oval yeast form to a filamentous hyphal form, which is characteristic of filamentous fungi. What mechanisms underlie hyphal growth and how are they affected by environmental stimuli from the host or resident microbiota? These questions are the focus of intensive research, as understanding C. albicans hyphal growth has broad implications for cell biological and medical research.

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Deletion of the CaBIG1 Gene Reduces β-1,6-Glucan Synthesis, Filamentation, Adhesion, and Virulence in Candida albicans

Infection and Immunity ◽

10.1128/iai.74.4.2373-2381.2006 ◽

2006 ◽

Vol 74 (4) ◽

pp. 2373-2381 ◽

Cited By ~ 30

Author(s):

Takashi Umeyama ◽

Aki Kaneko ◽

Hiroshi Watanabe ◽

Asuka Hirai ◽

Yoshimasa Uehara ◽

...

Keyword(s):

Candida Albicans ◽

Hyphal Growth ◽

Wild Type ◽

Environmental Signals ◽

Human Fungal Pathogen ◽

Functional Homolog ◽

Solid Agar ◽

Adhesion Failure ◽

Specific Localization ◽

Glucan Synthesis

ABSTRACT The human fungal pathogen Candida albicans is able to change its shape in response to various environmental signals. We analyzed the C. albicans BIG1 homolog, which might be involved in β-1,6-glucan biosynthesis in Saccharomyces cerevisiae. C. albicans BIG1 is a functional homolog of an S. cerevisiae BIG1 gene, because the slow growth of an S. cerevisiae big1 mutant was restored by introduction of C. albicans BIG1. CaBig1p was expressed constitutively in both the yeast and hyphal forms. A specific localization of CaBig1p at the endoplasmic reticulum or plasma membrane similar to the subcellular localization of S. cerevisiae Big1p was observed in yeast form. The content of β-1,6-glucan in the cell wall was decreased in the Cabig1Δ strain in comparison with the wild-type or reconstituted strain. The C. albicans BIG1 disruptant showed reduced filamentation on a solid agar medium and in a liquid medium. The Cabig1Δ mutant showed markedly attenuated virulence in a mouse model of systemic candidiasis. Adherence to human epithelial HeLa cells and fungal burden in kidneys of infected mice were reduced in the Cabig1Δ mutant. Deletion of CaBIG1 abolished hyphal growth and invasiveness in the kidneys of infected mice. Our results indicate that adhesion failure and morphological abnormality contribute to the attenuated virulence of the Cabig1Δ mutant.

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Strains and Strategies for Large-Scale Gene Deletion Studies of the Diploid Human Fungal Pathogen Candida albicans

Eukaryotic Cell ◽

10.1128/ec.4.2.298-309.2005 ◽

2005 ◽

Vol 4 (2) ◽

pp. 298-309 ◽

Cited By ~ 389

Author(s):

Suzanne M. Noble ◽

Alexander D. Johnson

Keyword(s):

Candida Albicans ◽

Fungal Pathogen ◽

Large Scale ◽

Gene Deletion ◽

Selectable Marker ◽

Wild Type ◽

Position Effects ◽

Human Fungal Pathogen ◽

Chromosome Position ◽

Knockout Mutants

ABSTRACT Candida albicans is the most common human fungal pathogen and causes significant morbidity and mortality worldwide. Nevertheless, the basic principles of C. albicans pathogenesis remain poorly understood. Of central importance to the study of this organism is the ability to generate homozygous knockout mutants and to analyze them in a mammalian model of pathogenesis. C. albicans is diploid, and current strategies for gene deletion typically involve repeated use of the URA3 selectable marker. These procedures are often time-consuming and inefficient. Moreover, URA3 expression levels—which are susceptible to chromosome position effects—can themselves affect virulence, thereby complicating analysis of strains constructed with URA3 as a selectable marker. Here, we describe a set of newly developed reference strains (leu2Δ/leu2Δ, his1Δ/his1Δ; arg4Δ/arg4Δ, his1Δ/his1Δ; and arg4Δ/arg4Δ, leu2Δ/leu2Δ, his1Δ/his1Δ) that exhibit wild-type or nearly wild-type virulence in a mouse model. We also describe new disruption marker cassettes and a fusion PCR protocol that permit rapid and highly efficient generation of homozygous knockout mutations in the new C. albicans strains. We demonstrate these procedures for two well-studied genes, TUP1 and EFG1, as well as a novel gene, RBD1. These tools should permit large-scale genetic analysis of this important human pathogen.

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SpRY Cas9 Can Utilize a Variety of Protospacer Adjacent Motif Site Sequences To Edit the Candida albicans Genome

mSphere ◽

10.1128/msphere.00303-21 ◽

2021 ◽

Vol 6 (3) ◽

Author(s):

Ben A. Evans ◽

Douglas A. Bernstein

Keyword(s):

Candida Albicans ◽

Genome Editing ◽

Fungal Pathogen ◽

Wild Type ◽

Content Type ◽

Human Fungal Pathogen ◽

Protospacer Adjacent Motif ◽

Motif Site ◽

Life Threatening ◽

Genomic Locations

ABSTRACT Candida albicans is a human fungal pathogen capable of causing life-threatening infections. The ability to edit the C. albicans genome using CRISPR/Cas9 is an important tool investigators can leverage in their search for novel therapeutic targets. However, wild-type Cas9 requires an NGG protospacer adjacent motif (PAM), leaving many AT-rich regions of DNA inaccessible. A recently described near-PAMless CRISPR system that utilizes the SpRY Cas9 variant can target non-NGG PAM sequences. Using this system as a model, we developed C. albicans CRISPR/SpRY. We tested our system by mutating C. albicans ADE2 and show that CRISPR/SpRY can utilize non-NGG PAM sequences in C. albicans. Our CRISPR/SpRY system will allow researchers to efficiently modify C. albicans DNA that lacks NGG PAM sequences. IMPORTANCE Genetic modification of the human fungal pathogen Candida albicans allows us to better understand how fungi differ from humans at the molecular level and play essential roles in the development of therapeutics. CRISPR/Cas9-mediated genome editing systems can be used to introduce site-specific mutations to C. albicans. However, wild-type Cas9 is limited by the requirement of an NGG PAM site. CRISPR/SpRY targets a variety of different PAM sequences. We modified the C. albicans CRISPR/Cas9 system using the CRISPR/SpRY as a guide. We tested CRISPR/SpRY on C. albicans ADE2 and show that our SpRY system can facilitate genome editing independent of an NGG PAM sequence, thus allowing the investigator to target AT-rich sequences. Our system will potentially enable mutation of the 125 C. albicans genes which have been previously untargetable with CRISPR/Cas9. Additionally, our system will allow for precise targeting of many genomic locations that lack NGG PAM sites.

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Alternative oxidase induction protects Candida albicans from respiratory stress and promotes hyphal growth

10.1101/405670 ◽

2018 ◽

Cited By ~ 1

Author(s):

Lucian Duvenage ◽

Louise A. Walker ◽

Aleksandra Bojarczuk ◽

Simon A. Johnston ◽

Donna M. McCallum ◽

...

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Candida Albicans ◽

Fungal Pathogen ◽

Alternative Oxidase ◽

Hyphal Growth ◽

Infection Model ◽

Human Fungal Pathogen ◽

Zebrafish Infection

AbstractThe human fungal pathogenCandida albicanspossesses two genes expressing a cyanide-insensitive Alternative Oxidase (Aox) enzymes in addition to classical and parallel electron transfer chains (ETC). In this study, we examine the role of Aox inC.albicansunder conditions of respiratory stress, which may be inflicted during its interaction with the human host or co-colonising bacteria. We find that the level of Aox expression is sufficient to modulate resistance to classical ETC inhibition under respiratory stress and are linked to gene expression changes that can promote both survival and pathogenicity. For example we demonstrate that Aox function is important for the regulation of filamentation inC.albicansand observe that cells lacking Aox function lose virulence in a zebrafish infection model. Our investigations also identify that pyocyanin, a phenazine produced by the co-colonising bacteriumPseudomonas aeruginosa, inhibits Aox-based respiration inC.albicans. These results suggest that Aox plays important roles within respiratory stress response pathways whichC.albicansmay utilise both as a commensal organism and as a pathogen.

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