Targeted gene disruption in Candida albicans wild-type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates

ABSTRACT Fractalkine (CX3CL1) is the first described chemokine that can exist either as a soluble protein or as a membrane-bound molecule. Both forms of fractalkine can mediate adhesion of cells expressing its receptor, CX3CR1. This activity, together with its expression on endothelial cells, suggests that fractalkine might mediate adhesion of leukocytes to the endothelium during inflammation. Fractalkine is also highly expressed in neurons, and its receptor, CX3CR1, is expressed on glial cells. To determine the biologic role of fractalkine, we used targeted gene disruption to generate fractalkine-deficient mice. These mice did not exhibit overt behavioral abnormalities, and histologic analysis of their brains did not reveal any gross changes compared to wild-type mice. In addition, these mice had normal hematologic profiles except for a decrease in the number of blood leukocytes expressing the cell surface marker F4/80. The cellular composition of their lymph nodes did not differ significantly from that of wild-type mice. Similarly, the responses offractalkine −/− mice to a variety of inflammatory stimuli were indistinguishable from those of wild-type mice.

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Role of melanotransferrin in iron metabolism: studies using targeted gene disruption in vivo

Blood ◽

10.1182/blood-2005-10-4174 ◽

2006 ◽

Vol 107 (7) ◽

pp. 2599-2601 ◽

Cited By ~ 29

Author(s):

Eric O. Sekyere ◽

Louise L. Dunn ◽

Yohan Suryo Rahmanto ◽

Des R. Richardson

Keyword(s):

Metabolic Pathways ◽

Gene Disruption ◽

Vital Role ◽

Plasminogen Activation ◽

Serum Chemistry ◽

Wild Type ◽

Targeted Disruption ◽

Targeted Gene Disruption

AbstractMelanotransferrin (MTf) or tumor antigen p97 is a transferrin homolog that binds one iron (Fe) atom and has been suggested to play roles in a variety of processes, including Fe metabolism, eosinophil differentiation, and plasminogen activation. Considering the vital role of Fe in many metabolic pathways, such as DNA and heme synthesis, it is important to understand the function of MTf. To define this, a MTf knockout (MTf–/–) mouse was generated through targeted disruption of the MTf gene. The MTf–/– mice were viable and fertile and developed normally, with no morphologic or histologic abnormalities. Assessment of Fe indices, tissue Fe levels, hematology, and serum chemistry parameters demonstrated no differences between MTf–/– and wild-type (MTf+/+) mice, suggesting MTf was not essential for Fe metabolism.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.1.324-331.2006 ◽

2006 ◽

Vol 50 (1) ◽

pp. 324-331 ◽

Cited By ~ 62

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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Targeted Gene Disruption of the 14-α Sterol Demethylase (cyp51A) in Aspergillus fumigatus and Its Role in Azole Drug Susceptibility

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.6.2536-2538.2005 ◽

2005 ◽

Vol 49 (6) ◽

pp. 2536-2538 ◽

Cited By ~ 85

Author(s):

E. Mellado ◽

G. Garcia-Effron ◽

M. J. Buitrago ◽

L. Alcazar-Fuoli ◽

M. Cuenca-Estrella ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Type Strain ◽

Gene Disruption ◽

Wild Type Strain ◽

Drug Susceptibility ◽

Wild Type ◽

Targeted Disruption ◽

Targeted Gene Disruption ◽

Resistant Strains

ABSTRACT The role of Aspergillus fumigatus 14α-sterol demethylase (Cyp51A) in azole drug susceptibility was assessed. Targeted disruption of cyp51A in azole-susceptible and -resistant strains decreased MICs from 2- to 40-fold. The cyp51A mutants were morphologically indistinguishable from the wild-type strain, retaining the ability to cause pulmonary disease in neutropenic mice.

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Characterization of the Role of the FluG Protein in Asexual Development of Aspergillus nidulans

Genetics ◽

10.1093/genetics/158.3.1027 ◽

2001 ◽

Vol 158 (3) ◽

pp. 1027-1036 ◽

Cited By ~ 2

Author(s):

Cletus A D'Souza ◽

Bee Na Lee ◽

Thomas H Adams

Keyword(s):

Aspergillus Nidulans ◽

Negative Influence ◽

Asexual Development ◽

Wild Type ◽

Significant Similarity ◽

Developmental Defects ◽

Asexual Sporulation ◽

Type Strains

Abstract We showed previously that a ΔfluG mutation results in a block in Aspergillus nidulans asexual sporulation and that overexpression of fluG activates sporulation in liquid-submerged culture, a condition that does not normally support sporulation of wild-type strains. Here we demonstrate that the entire N-terminal region of FluG (∼400 amino acids) can be deleted without affecting sporulation, indicating that FluG activity resides in the C-terminal half of the protein, which bears significant similarity with GSI-type glutamine synthetases. While FluG has no apparent role in glutamine biosynthesis, we propose that it has an enzymatic role in sporulation factor production. We also describe the isolation of dominant suppressors of ΔfluG(dsg) that should identify components acting downstream of FluG and thereby define the function of FluG in sporulation. The dsgA1 mutation also suppresses the developmental defects resulting from ΔflbA and dominant activating fadA mutations, which both cause constitutive induction of the mycelial proliferation pathway. However, dsgA1 does not suppress the negative influence of these mutations on production of the aflatoxin precursor, sterigmatocystin, indicating that dsgA1 is specific for asexual development. Taken together, our studies define dsgA as a novel component of the asexual sporulation pathway.

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Aspergillus fumigatus Intrinsic Fluconazole Resistance Is Due to the Naturally Occurring T301I Substitution in Cyp51Ap

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00905-16 ◽

2016 ◽

Vol 60 (9) ◽

pp. 5420-5426 ◽

Cited By ~ 26

Author(s):

Florencia Leonardelli ◽

Daiana Macedo ◽

Catiana Dudiuk ◽

Matias S. Cabeza ◽

Soledad Gamarra ◽

...

Keyword(s):

Candida Albicans ◽

Aspergillus Fumigatus ◽

Parental Strain ◽

Amino Acid Residues ◽

Intrinsic Resistance ◽

Single Nucleotide ◽

Fluconazole Resistance ◽

Content Type ◽

Natural Polymorphism

ABSTRACTAspergillus fumigatusintrinsic fluconazole resistance has been demonstrated to be linked to theCYP51Agene, although the precise molecular mechanism has not been elucidated yet. Comparisons betweenA. fumigatusCyp51Ap andCandida albicansErg11p sequences showed differences in amino acid residues already associated with fluconazole resistance inC. albicans. The aim of this study was to analyze the role of the natural polymorphism I301 inAspergillus fumigatusCyp51Ap in the intrinsic fluconazole resistance phenotype of this pathogen. The I301 residue inA. fumigatusCyp51Ap was replaced with a threonine (analogue to T315 atCandida albicansfluconazole-susceptible Erg11p) by changing one single nucleotide in theCYP51Agene. Also, aCYP51Aknockout strain was obtained using the same parental strain. Both mutants' antifungal susceptibilities were tested. The I301T mutant exhibited a lower level of resistance to fluconazole (MIC, 20 μg/ml) than the parental strain (MIC, 640 μg/ml), while no changes in MIC were observed for other azole- and non-azole-based drugs. These data strongly implicate theA. fumigatusCyp51Ap I301 residue in the intrinsic resistance to fluconazole.

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Barrier Activity in Candida albicans Mediates Pheromone Degradation and Promotes Mating

Eukaryotic Cell ◽

10.1128/ec.00090-07 ◽

2007 ◽

Vol 6 (6) ◽

pp. 907-918 ◽

Cited By ~ 28

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.

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Role of Calcineurin in Stress Resistance, Morphogenesis, and Virulence of a Candida albicans Wild-Type Strain

Infection and Immunity ◽

10.1128/iai.00142-06 ◽

2006 ◽

Vol 74 (7) ◽

pp. 4366-4369 ◽

Cited By ~ 58

Author(s):

Teresa Bader ◽

Klaus Schröppel ◽

Stefan Bentink ◽

Nina Agabian ◽

Gerwald Köhler ◽

...

Keyword(s):

Candida Albicans ◽

Type Strain ◽

Pulmonary Infection ◽

Wild Type Strain ◽

Selection Marker ◽

Wild Type ◽

Environmental Signals ◽

Successful Infection ◽

Strain Sc5314

ABSTRACT By generating a calcineurin mutant of the Candida albicans wild-type strain SC5314 with the help of a new recyclable dominant selection marker, we confirmed that calcineurin mediates tolerance to a variety of stress conditions but is not required for the ability of C. albicans to switch to filamentous growth in response to hypha-inducing environmental signals. While calcineurin was essential for virulence of C. albicans in a mouse model of disseminated candidiasis, deletion of CMP1 did not significantly affect virulence during vaginal or pulmonary infection, demonstrating that the requirement for calcineurin for a successful infection depends on the host niche.

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