Marker Recycling in Candida albicans through CRISPR-Cas9-Induced Marker Excision

Huang, Manning Y.; Mitchell, Aaron P.

doi:10.1128/msphere.00050-17

Cited by 52 publications

(59 citation statements)

References 27 publications

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“…The approach also requires that both CAS9 and the sgRNA be integrated into the genome, which can be slow and makes sequential editing of several genes difficult. Later, improved versions of CRISPR-Cas9-based genome-editing methods were further developed by exploiting recent observations of C. albicans genetic properties (141)(142)(143)(144)(145)(146). These methods now allow single-or double-gene deletions, tagging, as well as open reading frame (ORF) reconstitutions.…”

Section: Genetic Toolboxmentioning

confidence: 99%

Candida parapsilosis: from Genes to the Bedside

et al. 2019

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SUMMARY Patients with suppressed immunity are at the highest risk for hospital-acquired infections. Among these, invasive candidiasis is the most prevalent systemic fungal nosocomial infection. Over recent decades, the combined prevalence of non-albicans Candida species outranked Candida albicans infections in several geographical regions worldwide, highlighting the need to understand their pathobiology in order to develop effective treatment and to prevent future outbreaks. Candida parapsilosis is the second or third most frequently isolated Candida species from patients. Besides being highly prevalent, its biology differs markedly from that of C. albicans, which may be associated with C. parapsilosis’ increased incidence. Differences in virulence, regulatory and antifungal drug resistance mechanisms, and the patient groups at risk indicate that conclusions drawn from C. albicans pathobiology cannot be simply extrapolated to C. parapsilosis. Such species-specific characteristics may also influence their recognition and elimination by the host and the efficacy of antifungal drugs. Due to the availability of high-throughput, state-of-the-art experimental tools and molecular genetic methods adapted to C. parapsilosis, genome and transcriptome studies are now available that greatly contribute to our understanding of what makes this species a threat. In this review, we summarize 10 years of findings on C. parapsilosis pathogenesis, including the species’ genetic properties, transcriptome studies, host responses, and molecular mechanisms of virulence. Antifungal susceptibility studies and clinician perspectives are discussed. We also present regional incidence reports in order to provide an updated worldwide epidemiology summary.

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Section: Genetic Toolboxmentioning

confidence: 99%

Candida parapsilosis: from Genes to the Bedside

et al. 2019

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“…For general growth and propagation, C. albicans strains were grown in yeast extract-peptone-dextrose (YPD) medium (1% yeast extract, 2% peptone, 2% glucose, 2% agar for solid medium) at 30°C. YPD plus 200g/ml nourseothricin (Werner Bioagents, Jena, Germany) was used to select for deletion mutants, and YP maltose (YPM) induced flipping out of the nourseothricin cassette as described previously (69).…”

Section: Strains and Mediamentioning

confidence: 99%

Multiple Alternative Carbon Pathways Combine To Promote Candida albicans Stress Resistance, Immune Interactions, and Virulence

Williams

Lorenz

2020

mBio

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The phagocytic cells of the innate immune system are an essential first line of antimicrobial defense, and yet Candida albicans, one of the most problematic fungal pathogens, is capable of resisting the stresses imposed by the macrophage phagosome, eventually resulting in the destruction of the phagocyte. C. albicans rapidly adapts to the phagosome by upregulating multiple alternative carbon utilization pathways, particularly those for amino acids, carboxylic acids, and N-acetylglucosamine (GlcNAc). Here, we report that C. albicans recognizes these carbon sources both as crucial nutrients and as independent signals in its environment. Even in the presence of glucose, each carbon source promotes increased resistance to a unique profile of stressors; lactate promotes increased resistance to osmotic and cell wall stresses, amino acids increased resistance to oxidative and nitrosative stresses, and GlcNAc increased resistance to oxidative stress and caspofungin, while all three alternative carbon sources have been shown to induce resistance to fluconazole. Moreover, we show mutants incapable of utilizing these carbon sources, in particular, strains engineered to be defective in all three pathways, are significantly attenuated in both macrophage and mouse models, with additive effects observed as multiple carbon pathways are eliminated, suggesting that C. albicans simultaneously utilizes multiple carbon sources within the macrophage phagosome and during disseminated candidiasis. Taking the data together, we propose that, in addition to providing energy to the pathogen within host environments, alternative carbon sources serve as niche-specific priming signals that allow C. albicans to recognize microenvironments within the host and to prepare for stresses associated with that niche, thus promoting host adaptation and virulence. IMPORTANCE Candida albicans is a fungal pathogen and a significant cause of morbidity and mortality, particularly in people with defects, sometimes minor ones, in innate immunity. The phagocytes of the innate immune system, particularly macrophages and neutrophils, generally restrict this organism to its normal commensal niches, but C. albicans shows a robust and multifaceted response to these cell types. Inside macrophages, a key component of this response is the activation of multiple pathways for the utilization of alternative carbon sources, particularly amino acids, carboxylic acids, and N-acetylglucosamine. These carbon sources are key sources of energy and biomass but also independently promote stress resistance, induce cell wall alterations, and affect C. albicans interactions with macrophages. Engineered strains incapable of utilizing these alternative carbon pathways are attenuated in infection models. These data suggest that C. albicans recognizes nutrient composition as an indicator of specific host environments and tailors its responses accordingly.

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“…albicans. In a follow-up to the Min et al publication, Huang and Mitchell demonstrated a system that enables marker excision in subsequent transformations (8). Briefly, the marker used to delete target gene 1 can be excised during a subsequent transformation that simultaneously integrates a distinct marker in place of target gene 2.…”

Section: Discussionmentioning

confidence: 99%

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

Nguyen

Quail

Hernday

2017

mSphere

103

135

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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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Marker Recycling in Candida albicans through CRISPR-Cas9-Induced Marker Excision

Cited by 52 publications

References 27 publications

Candida parapsilosis: from Genes to the Bedside

Candida parapsilosis: from Genes to the Bedside

Multiple Alternative Carbon Pathways Combine To Promote Candida albicans Stress Resistance, Immune Interactions, and Virulence

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

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