Gene Editing in Dimorphic Fungi Using CRISPR/Cas9

Kujoth, Gregory C.; Sullivan, Thomas D.; Klein, Bruce S.

doi:10.1002/cpmc.132

Cited by 5 publications

(4 citation statements)

References 61 publications

(79 reference statements)

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“…Previously Yps-3 RNAi knockdown had shown no yeast phenotype in a macrophage cell line [ 34 ]. Since RNAi may not be definitive, to further characterize the role of Yps-3 in macrophage infection we generated a CRISPR deletion mutant of YPS-3 ( S7A and S7B Fig ) [ 52 , 53 ]. We found that the yps-3Δ mutant has a partial lysis defect in BMDMs ( Fig 6D ) despite the presence of Cbp1 in the cytosol of macrophages infected with this mutant ( Fig 5A ).…”

Section: Resultsmentioning

confidence: 99%

Cbp1, a fungal virulence factor under positive selection, forms an effector complex that drives macrophage lysis

et al. 2022

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Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication precedes macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the extracellular fungal pathogen Blastomyces dermatitidis. We observe a high rate of fixation of non-synonymous substitutions in the Cbp1 coding sequences, indicating that Cbp1 is under positive selection. We determine the de novo structures of Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel “binocular” fold consisting of a helical dimer arrangement wherein two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type Emergomyces africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a phagosomal human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor that accesses the cytosol. Taken together, these data imply that Cbp1 is a fungal virulence factor under positive selection that localizes to the cytosol to trigger host cell lysis.

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Section: Resultsmentioning

confidence: 99%

Cbp1, a fungal virulence factor under positive selection, forms an effector complex that drives macrophage lysis

et al. 2022

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“…Since RNAi may not be definitive, to further characterize the role of Yps-3 in macrophage infection we generated a CRISPR deletion mutant of YPS-3 (Fig. S8A and B) (53,54). We found that the yps-3∆ mutant has a partial lysis defect in BMDMs (Fig.…”

Section: Hc G217b and Pb03 Cbp1 Enter The Cytosol During Macrophage Infectionmentioning

confidence: 99%

Cbp1, a rapidly evolving fungal virulence factor, forms an effector complex that drives macrophage lysis

Azimova

Herrera

Duvenage

et al. 2021

Preprint

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Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication precedes macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the extracellular fungal pathogen Blastomyces dermatitidis. We determine the de novo structures of Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel 'binocular' fold consisting of a helical dimer arrangement wherein two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type Emergomyces africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a phagosomal human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor that accesses the cytosol. Taken together, these data imply that Cbp1 is a rapidly evolving fungal virulence factor that localizes to the cytosol to trigger host cell lysis.

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“…To overcome the existing limitations of current technologies for loss-of-function studies in Histoplasma and related dimorphic fungi, there has been considerable interest in and early work to apply CRISPR/Cas genome editing ( 22 ). We describe an optimized system for the expression of gene targeting crRNAs by Pol(II)-based transcription of a single chimeric guide RNA (gRNA or sometimes referred to as sgRNA) consisting of the crRNA and tracrRNA in one molecule.…”

Section: Introductionmentioning

confidence: 99%

Targeted gene deletions in the dimorphic fungal pathogen Histoplasma using an optimized episomal CRISPR/Cas9 system

Rappleye

2023

mSphere

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The rapid development of CRISPR/CRISPR-associated (Cas) systems has revolutionized the ability to produce genetic mutations in a desired locus, particularly in organisms with low rates of homologous recombination. Histoplasma is an important respiratory and systemic fungal pathogen that has few reverse genetic options. We describe an optimized CRISPR/Cas system for the efficient generation of mutations in desired genes. The limited requirements for CRISPR/Cas, namely a gene-targeting guide RNA (gRNA) and expression of a Cas endonuclease, enabled both the gRNA and the Streptococcus pyogenes Cas9 gene to be expressed from a single episomal vector. The gRNAs are expressed from a strong Pol(II) promoter, a critical parameter for increasing the recovery of mutated genes, and processed into the mature gRNA by ribozymes in the mRNA. Expression of dual-tandem gRNAs facilitates the generation of gene deletions at a good frequency which can be detected by PCR-based screening of pooled isolates resulting in the isolation of marker-less deletion mutants. The CRISPR/Cas system is encoded on an episomal telomeric vector facilitating curing strains of the CRISPR/Cas vector upon generation of the mutant. We demonstrate the successful application of this CRISPR/Cas system in diverse Histoplasma species and applicable for multiple genes. The optimized system shows promise for accelerating reverse genetic studies in Histoplasma spp. IMPORTANCE The ability to eliminate gene product functions is central to understanding molecular mechanisms. In the fungal pathogen Histoplasma , methods to inactivate or deplete gene products are inefficient, which hampers progress in defining Histoplasma ’s virulence mechanisms. We describe an efficient CRISPR/Cas-based system for generating gene deletions in Histoplasma and show its validation on multiple genes with selectable and non-selectable phenotypes.

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Gene Editing in Dimorphic Fungi Using CRISPR/Cas9

Cited by 5 publications

References 61 publications

Cbp1, a fungal virulence factor under positive selection, forms an effector complex that drives macrophage lysis

Cbp1, a fungal virulence factor under positive selection, forms an effector complex that drives macrophage lysis

Cbp1, a rapidly evolving fungal virulence factor, forms an effector complex that drives macrophage lysis

Targeted gene deletions in the dimorphic fungal pathogen Histoplasma using an optimized episomal CRISPR/Cas9 system

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