New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans

Shahana, Shahida; Childers, Delma S.; Ballou, Elizabeth R.; Bohovych, Iryna; Odds, Frank C.; Gow, Neil A. R.; Brown, Alistair J. P.

doi:10.1371/journal.pone.0100390

Cited by 39 publications

(39 citation statements)

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“…Indeed, the adaptation of C. albicans gene deletion technologies for use in C. auris will enable future molecular investigations to understand gene regulatory networks and virulence in this emerging pathogen. Consequently, the C. albicans NAT1-Clox system has been successfully employed to disrupt C. auris HOG1 and demonstrate its conserved role in stress responses (Shahana et al, 2014;Day et al, 2018). Future pathfinding molecular studies should reveal the conservation and divergence of gene regulatory networks in C. albicans and C. auris and highlight new avenues for antifungal development.…”

Section: Epigenetics and Gene Regulation In C Aurismentioning

confidence: 99%

Nine Things Genomics Can Tell Us About Candida auris

2020

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Candida auris is a recently emerged multidrug-resistant fungal pathogen causing severe illness in hospitalized patients. C. auris is most closely related to a few environmental or rarely observed but cosmopolitan Candida species. However, C. auris is unique in the concern it is generating among public health agencies for its rapid emergence, difficulty to treat, and the likelihood for further and more extensive outbreaks and spread. To date, five geographically distributed and genetically divergent lineages have been identified, none of which includes isolates that were collected prior to 1996. Indeed, C. auris' ecological niche(s) and emergence remain enigmatic, although a number of hypotheses have been proposed. Recent genomic and transcriptomic work has also identified a variety of gene and chromosomal features that may have conferred C. auris with several important clinical phenotypes including its drug-resistance and growth at high temperatures. In this review we discuss nine major lines of enquiry into C. auris that big-data technologies and analytical approaches are beginning to answer.

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Section: Epigenetics and Gene Regulation In C Aurismentioning

confidence: 99%

Nine Things Genomics Can Tell Us About Candida auris

2020

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“…Thus, Hda2 and Hda3 physically interact with Hda1. To delineate the function of C. albicans Hda2 and Hda3, we generated homozygous deletions mutants for HDA2 ( hda2 Δ/Δ ) and HDA3 ( hda3 Δ/Δ ) genes using a recyclable Clox system (Fig 1D) (33). The Clox system allows for the generation of a homozygous deletion mutant lacking any marker genes and therefore genetically identical to the parental strain except for the deleted gene.…”

Section: Resultsmentioning

confidence: 99%

“…Deletions of HDA2 and HDA3 were generated in the SN152 or BWP17 background using the Clox system for gene disruption (33) using long-oligos PCR, the LAL (loxP-ARG4-loxP) and NAT1-Clox (loxP-NAT1-MET3p-cre-loxP) plasmids as templates. During all selections for Clox transformants media was supplemented with 2.5 mM methionine and 2.5 mM cysteine to repress the MET3 promoter and minimize Cre-loxP mediated recombination.…”

Section: Methodsmentioning

confidence: 99%

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The fungal-specific Hda2 and Hda3 proteins regulate morphological switches in the human fungal pathogenCandida albicans

Peterson

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Gourlay

et al. 2018

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20The human fungal pathogen Candida albicans is responsible for millions of infections 21 annually. Due to the few available anti-fungal drugs and the increasing incidence of 22 drug resistance, the number of C. albicans infections is dramatically increasing. 23 Morphological switches, such as the white-opaque switch and the yeast-hyphae 24 switch, are key for the development of C. albicans pathogenic traits. Lysine 25 deacetylases are emerging as important regulators of morphological switches. Yet, 26 targeting lysine deacetylases for drug development is problematic due to the high 27 homology between the fungal and human proteins that could result in toxicity. Here 28 we provide evidence that the fungal specific proteins Hda2 and Hda3 interact with 29 the lysine deacetylase Hda1. By combining phenotypic analyses with genome-wide 30 transcriptome analyses, we demonstrate that Hda2 and Hda3 control C. albicans 31 morphological switches. Under nutrient-rich conditions, deletion of HDA2 or HDA3 32 leads to moderate overexpression of the master regulator of white-opaque switching 33 WOR1 and increase switching frequency. Under hyphae inducing conditions, 34 deletion of HDA2 and HDA3 block hyphae development. However, deletion of HDA2 35and HDA3 does not affect hyphae-formation and virulence in vivo. We propose that 36 Hda2 and Hda3 are good targets for the development of anti-fungal drugs to be used 37 in combination therapy. 38 39 KEYWORDS 40 KDACs, Candida albicans, morphological switches, white and opaque switch, yeast 41 to hyphae switch 48 the principal causal agent of mycotic death (2). This is because, in immune-49 compromised patients, C. albicans can invade vital organs and cause serious, life-50threating systemic infections associated with a mortality rate up to 70 % (2). The 51 ability to transition between different morphological forms in response to changing 52 environments is a key virulence trait in C. albicans. 53For example, C. albicans cells can reversibly switch between white and opaque 54 forms (3). White and opaque cells are genetically identical, yet they differ in cellular 55 morphology, colony shape, gene expression profile and mating behaviour (4). In 56 addition, white cells are more virulent in a murine model of systemic infection (5, 6) 57 whereas opaque cells preferentially colonise the skin (7). White-opaque switching is 58 under the control of the master regulator Wor1, a transcription factor whose 59 expression is necessary and sufficient for opaque cell formation (8)(9)(10)(11). Stochastic 60 increases in Wor1 levels drives the transition from the white to the opaque phase. 61Furthermore, Wor1 expression produces a direct positive feedback loop by binding 62 its own promoter and turning on its own expression (8, 9, 11). Switching is also 63 regulated by the mating type locus as opaque formation occurs predominantly in a or 64  cells (12). 65C. albicans virulence also depends on its ability to convert between yeast and hyphal 66 morphology: yeast cells are critical for colonisa...

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“…Deletion of one of these genes disrupts glutathione biosynthesis in C . albicans leading to glutathione auxotrophy and to increase oxidative stress sensitivity [ 58 , 59 ]. To determine the sensitivity to H 2 O 2 of wild-type and hat1 Δ/Δ cells upon deletion of GSH2 or GCS1 , the strains were grown in the absence of glutathione and treated with H 2 O 2 for 2h.…”

Section: Resultsmentioning

confidence: 99%

The Candida albicans Histone Acetyltransferase Hat1 Regulates Stress Resistance and Virulence via Distinct Chromatin Assembly Pathways

et al. 2015

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Human fungal pathogens like Candida albicans respond to host immune surveillance by rapidly adapting their transcriptional programs. Chromatin assembly factors are involved in the regulation of stress genes by modulating the histone density at these loci. Here, we report a novel role for the chromatin assembly-associated histone acetyltransferase complex NuB4 in regulating oxidative stress resistance, antifungal drug tolerance and virulence in C. albicans. Strikingly, depletion of the NuB4 catalytic subunit, the histone acetyltransferase Hat1, markedly increases resistance to oxidative stress and tolerance to azole antifungals. Hydrogen peroxide resistance in cells lacking Hat1 results from higher induction rates of oxidative stress gene expression, accompanied by reduced histone density as well as subsequent increased RNA polymerase recruitment. Furthermore, hat1Δ/Δ cells, despite showing growth defects in vitro, display reduced susceptibility to reactive oxygen-mediated killing by innate immune cells. Thus, clearance from infected mice is delayed although cells lacking Hat1 are severely compromised in killing the host. Interestingly, increased oxidative stress resistance and azole tolerance are phenocopied by the loss of histone chaperone complexes CAF-1 and HIR, respectively, suggesting a central role for NuB4 in the delivery of histones destined for chromatin assembly via distinct pathways. Remarkably, the oxidative stress phenotype of hat1Δ/Δ cells is a species-specific trait only found in C. albicans and members of the CTG clade. The reduced azole susceptibility appears to be conserved in a wider range of fungi. Thus, our work demonstrates how highly conserved chromatin assembly pathways can acquire new functions in pathogenic fungi during coevolution with the host.

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New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans

Cited by 39 publications

References 64 publications

Nine Things Genomics Can Tell Us About Candida auris

Nine Things Genomics Can Tell Us About Candida auris

The fungal-specific Hda2 and Hda3 proteins regulate morphological switches in the human fungal pathogenCandida albicans

The Candida albicans Histone Acetyltransferase Hat1 Regulates Stress Resistance and Virulence via Distinct Chromatin Assembly Pathways

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