Targeted Gene Deletion in Candida albicans Wild-Type Strains by MPAR Flipping

2005 ◽  
pp. 035-044
Author(s):  
Joachim Morschhäuser ◽  
Peter Staib ◽  
Gerwald Köhler
Keyword(s):  
Candida Albicans ◽  
Gene Deletion ◽  
Wild Type ◽  
Targeted Gene Deletion ◽  
Type Strains

scholarly journals Barrier Activity in Candida albicans Mediates Pheromone Degradation and Promotes Mating

2007 ◽  
Vol 6 (6) ◽  
pp. 907-918 ◽  
Author(s):  
Dana Schaefer ◽  
Pierre Côte ◽  
Malcolm Whiteway ◽  
Richard J. Bennett
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Cell Growth ◽  
Cell Fusion ◽  
Aspartyl Protease ◽  
Wild Type ◽  
Pheromone Activity ◽  
Important Regulator ◽  
Mutant Cells ◽  
Type Strains

ABSTRACT Mating in Candida albicans and Saccharomyces cerevisiae is regulated by the secretion of peptide pheromones that initiate the mating process. An important regulator of pheromone activity in S. cerevisiae is barrier activity, involving an extracellular aspartyl protease encoded by the BAR1 gene that degrades the alpha pheromone. We have characterized an equivalent barrier activity in C. albicans and demonstrate that the loss of C. albicans BAR1 activity results in opaque a cells exhibiting hypersensitivity to alpha pheromone. Hypersensitivity to pheromone is clearly seen in halo assays; in response to alpha pheromone, a lawn of C. albicans Δbar1 mutant cells produces a marked zone in which cell growth is inhibited, whereas wild-type strains fail to show halo formation. C. albicans mutants lacking BAR1 also exhibit a striking mating defect in a cells, but not in α cells, due to overstimulation of the response to alpha pheromone. The block to mating occurs prior to cell fusion, as very few mating zygotes were observed in mixes of Δbar1 a and α cells. Finally, in a barrier assay using a highly pheromone-sensitive strain, we were able to demonstrate that barrier activity in C. albicans is dependent on Bar1p. These studies reveal that a barrier activity to alpha pheromone exists in C. albicans and that the activity is analogous to that caused by Bar1p in S. cerevisiae.

scholarly journals Systematic truncations of chromosome 4 and their responses to antifungals in Candida albicans

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Wasim Uddin ◽  
Darshan Dhabalia ◽  
S. M. Udaya Prakash ◽  
M. Anaul Kabir
Keyword(s):  
Drug Resistance ◽  
Candida Albicans ◽  
Resistant Mutant ◽  
Wild Type ◽  
Human Fungal Pathogen ◽  
Zones Of Inhibition ◽  
Life Threatening ◽  
Fluconazole Resistant ◽  
Diffusion Assay ◽  
Type Strains

Abstract Background Candida albicans is an opportunistic human fungal pathogen responsible for superficial and systemic life-threatening infections. Treating these infections is challenging as many clinical isolates show increased drug resistance to antifungals. Chromosome (Chr) 4 monosomy was implicated in a fluconazole-resistant mutant. However, exposure to fluconazole adversely affects Candida cells and can generate numerous mutations. Hence, the present study aimed to truncate Chr4 and challenge the generated Candida strains to antifungals and evaluate their role in drug response. Results Herein, Chr4 was truncated in C. albicans using the telomere-mediated chromosomal truncation method. The resulting eight Candida strains carrying one truncated homolog of Chr4 were tested for response to multiple antifungals. The minimal inhibitory concentration (MIC) for these strains was determined against three classes of antifungals. The MIC values against fluconazole, amphotericin B, and caspofungin were closer to that of the wild type strain. Microdilution assay against fluconazole showed that the mutants and wild type strains had similar sensitivity to fluconazole. The disc diffusion assay against five azoles and two polyenes revealed that the zones of inhibition for all the eight strains were similar to those of the wild type. Thus, none of the generated strains showed any significant resistance to the tested antifungals. However, spot assay exhibited a reasonably high tolerance of a few generated strains with increasing concentrations of fluconazole. Conclusion This analysis suggested that Chr4 aneuploidy might not underlie drug resistance but rather drug tolerance in Candida albicans.

scholarly journals New insights into the construction of wild-type Saba pig-derived Escherichia coli irp2 gene deletion strains

3 Biotech ◽  
2021 ◽  
Vol 11 (9) ◽  
Author(s):  
Bo Zhang ◽  
Hongdan Wang ◽  
Weiwei Zhao ◽  
Chunlan Shan ◽  
Chaoying Liu ◽  
...  
Keyword(s):  
Iron Uptake ◽  
Gene Editing ◽  
Gene Deletion ◽  
Growth Characteristics ◽  
Wild Type ◽  
Knock Out ◽  
E Coli ◽  
Functional Studies ◽  
Key Points ◽  
Type Strains

AbstractTo construct wild-type E. coli irp2 gene deletion strains, CRISPR/Cas9 gene editing technology was used, and the difficulty and key points of gene editing of wild-type strains were analyzed. Based on the resistance of the CRISPR/Cas9 system expression vector, 4 strains of 41 E. coli strains isolated from Saba pigs were selected as the target strains for the deletion of the irp2 gene, which were sensitive to both ampicillin and kanamycin. Then, CRISPR/Cas9 technology was combined with homologous recombination technology to construct recombinant vectors containing Cas9, sgRNA and donor sequences to knock out the irp2 gene. Finally, the absence of the irp2 gene in E. coli was further verified by iron uptake assays, iron carrier production assays and growth curve measurements. The results showed that three of the selected strains showed single base mutations and deletions (Δirp2-1, Δirp2-2 and Δirp2-3). The deletion of the irp2 gene reduced the ability of E. coli to take up iron ions and produce iron carriers, but not affect the growth characteristics of E. coli. It is shown that the CRISPR/Cas9 knock-out system constructed in this study can successfully knock out the irp2 gene of the wild-type E. coli. Our results providing new insights into genome editing in wild-type strains, which enable further functional studies of the irp2 gene in wild-type E. coli.

scholarly journals Generation of Viable Candida albicans Mutants Lacking the “Essential” Protein Kinase Snf1 by Inducible Gene Deletion

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Austin Mottola ◽  
Sonja Schwanfelder ◽  
Joachim Morschhäuser
Keyword(s):  
Candida Albicans ◽  
Protein Kinase ◽  
Gene Deletion ◽  
Metabolic Adaptation ◽  
Optimal Conditions ◽  
Powerful Method ◽  
Wild Type ◽  
Pathogenic Yeast ◽  
Content Type ◽  
Inducible Gene

ABSTRACT The protein kinase Snf1, a member of the highly conserved AMP-activated protein kinase family, is a central regulator of metabolic adaptation. In the pathogenic yeast Candida albicans, Snf1 is considered to be essential, as previous attempts by different research groups to generate homozygous snf1Δ mutants were unsuccessful. We aimed to elucidate why Snf1 is required for viability in C. albicans by generating snf1Δ null mutants through forced, inducible gene deletion and observing the terminal phenotype before cell death. Unexpectedly, we found that snf1Δ mutants were viable and could grow, albeit very slowly, on rich media containing the preferred carbon source glucose. Growth was improved when the cells were incubated at 37°C instead of 30°C, and this phenotype enabled us to isolate homozygous snf1Δ mutants also by conventional, sequential deletion of both SNF1 alleles in a wild-type C. albicans strain. All snf1Δ mutants could grow slowly on glucose but were unable to utilize alternative carbon sources. Our results show that, under optimal conditions, C. albicans can live and grow without Snf1. Furthermore, they demonstrate that inducible gene deletion is a powerful method for assessing gene essentiality in C. albicans. IMPORTANCE Essential genes are those that are indispensable for the viability and growth of an organism. Previous studies indicated that the protein kinase Snf1, a central regulator of metabolic adaptation, is essential in the pathogenic yeast Candida albicans, because no homozygous snf1 deletion mutants of C. albicans wild-type strains could be obtained by standard approaches. In order to investigate the lethal consequences of SNF1 deletion, we generated conditional mutants in which SNF1 could be deleted by forced, inducible excision from the genome. Unexpectedly, we found that snf1 null mutants were viable and could grow slowly under optimal conditions. The growth phenotypes of the snf1Δ mutants explain why such mutants were not recovered in previous attempts. Our study demonstrates that inducible gene deletion is a powerful method for assessing gene essentiality in C. albicans.

scholarly journals Contribution of Candida albicans Cell Wall Components to Recognition by and Escape from Murine Macrophages

2010 ◽  
Vol 78 (4) ◽  
pp. 1650-1658 ◽  
Author(s):  
C. G. J. McKenzie ◽  
U. Koser ◽  
L. E. Lewis ◽  
J. M. Bain ◽  
H. M. Mora-Montes ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Cell Wall Protein ◽  
Host Immune System ◽  
Wild Type ◽  
Human Fungal Pathogen ◽  
Protein Mutants ◽  
Hyphal Formation ◽  
Type Strains ◽  
Macrophage Uptake

ABSTRACT The pathogenicity of the opportunistic human fungal pathogen Candida albicans depends on its ability to escape destruction by the host immune system. Using mutant strains that are defective in cell surface glycosylation, cell wall protein synthesis, and yeast-hypha morphogenesis, we have investigated three important aspects of C. albicans innate immune interactions: phagocytosis by primary macrophages and macrophage cell lines, hyphal formation within macrophage phagosomes, and the ability to escape from and kill macrophages. We show that cell wall glycosylation is critically important for the recognition and ingestion of C. albicans by macrophages. Phagocytosis was significantly reduced for mutants deficient in phosphomannan biosynthesis (mmn4Δ, pmr1Δ, and mnt3 mnt5Δ), whereas O- and N-linked mannan defects (mnt1Δ mnt2Δ and mns1Δ) were associated with increased ingestion, compared to the parent wild-type strains and genetically complemented controls. In contrast, macrophage uptake of mutants deficient in cell wall proteins such as adhesins (ece1Δ, hwp1Δ, and als3Δ) and yeast-locked mutants (clb2Δ, hgc1Δ, cph1Δ, efg1Δ, and efg1Δ cph1Δ), was similar to that observed for wild-type C. albicans. Killing of macrophages was abrogated in hypha-deficient strains, significantly reduced in all glycosylation mutants, and comparable to wild type in cell wall protein mutants. The diminished ability of glycosylation mutants to kill macrophages was not a consequence of impaired hyphal formation within macrophage phagosomes. Therefore, cell wall composition and the ability to undergo yeast-hypha morphogenesis are critical determinants of the macrophage's ability to ingest and process C. albicans.

One-step targeted gene deletion in Candida albicans haploids

2014 ◽  
Vol 9 (2) ◽  
pp. 464-473 ◽  
Author(s):  
Guisheng Zeng ◽  
Yan-Ming Wang ◽  
Fong Yee Chan ◽  
Yue Wang
Keyword(s):  
Candida Albicans ◽  
Gene Deletion ◽  
Targeted Gene Deletion ◽  
One Step

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

2005 ◽  
Vol 4 (2) ◽  
pp. 298-309 ◽  
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.

scholarly journals Distinct Antifungal Mechanisms: β-Defensins Require Candida albicans Ssa1 Protein, while Trk1p Mediates Activity of Cysteine-Free Cationic Peptides

2006 ◽  
Vol 50 (1) ◽  
pp. 324-331 ◽  
Author(s):  
Slavena Vylkova ◽  
Xuewei S. Li ◽  
Jennifer C. Berner ◽  
Mira Edgerton
Keyword(s):  
Candida Albicans ◽  
Membrane Permeability ◽  
Fungicidal Activity ◽  
Wild Type ◽  
Histatin 5 ◽  
Antifungal Peptides ◽  
Mutant Strains ◽  
Multistep Process ◽  
Type Strains

ABSTRACT Salivary histatin 5 (Hst 5) kills the fungal pathogen Candida albicans via a multistep process which includes binding to Ssa1/2 proteins on the cell surface and requires the TRK1 potassium transporter. Hst 5-induced membrane permeability to propidium iodide (PI) was nearly abolished in strain CaTK1 (TRK1/trk1), suggesting that Hst 5-induced influx of PI is via Trk1p. To explore the functional role of Trk1p in the mechanism of other antifungal peptides, we evaluated candidacidal activity and PI uptake in wild-type strain CaTK2 (TRK1/TRK1) and strain CaTK1 following treatment with lactoferricin 11 (LFcn 11), bactenecin 16 (BN 16), and virion-associated protein VPR 12. Strain CaTK1 was resistant to killing with these peptides (VPR 12 > LFcn 11 > BN 16), showing the requirement of Trk1p for fungicidal activity. In contrast, human neutrophil defensin 1 (HNP-1), human β-defensin 2 (hBD-2), and hBD-3 effects on viability of and membrane permeability to PI were not different between mutant and wild-type strains, clearly showing that their candidacidal mechanism does not involve Trk1p as a functional effector. To test whether defensins require binding to Candida surface Ssa1/2 proteins for their activity, we measured the killing effectiveness in SSA1/2 mutant strains. Both hBD-2 and hBD-3, but not HNP-1, exhibited reduced killing of ssa1Δ and ssa2Δ strains compared to the wild type, showing that Ssa1 and Ssa2 proteins are required for their fungicidal activity. These results demonstrate that (i) Trk1p mediates candidacidal activities of cysteine-free peptides, but not of defensins, and (ii) hBD-2 and hBD-3, but not HNP-1, require Ssa1/2p for antifungal activity.

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