Chemosensitization of Fluconazole Resistance in
            <i>Saccharomyces cerevisiae</i>
            and Pathogenic Fungi by a
            <scp>d</scp>
            -Octapeptide Derivative

Niimi, Kyoko; Harding, David; Parshot, Rekha; King, Amanda M.; Lun, David J.; Decottignies, Anabelle; Niimi, Masakazu; Lin, Susan; Cannon, Richard D.; Goffeau, André; Monk, Brian C.

doi:10.1128/aac.48.4.1256-1271.2004

Cited by 110 publications

(148 citation statements)

References 58 publications

(53 reference statements)

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“…The germlings were further incubated with PCZ (R. oryzae, 0.125 to 0.5 g/ml; C. bertholletiae, 1 to 4 g/ml; M. circinelloides, 1 to 4 g/ml) or ICZ (R. oryzae, 0.06 to 0.25 g/ml; C. bertholletiae, 0.25 to 1 g/ml; M. circinelloides, 1 to 4 g/ml) in the presence of TCR (0.015 g/ml) for 3 h at 37°C. The quantities of cyt c in the supernatant and mitochondria were determined by the method of Niimi et al (29). The absorbance at 550 nm was measured with a POLARstar Omega spectrophotometer.…”

Section: Methodsmentioning

confidence: 99%

“…Translocation of mitochondrial cytochrome c (cyt c) to the cytosol is considered a critical event in apoptosis (26). For estimation of cyt c, isolation of mitochondria from R. oryzae, C. bertholletiae, and M. circinelloides germlings was performed according to the method described by Niimi et al (29). We used R. oryzae, C. bertholletiae, and M. circinelloides germlings grown in RPMI 1640 broth at 37°C for 5 h and then resuspended in fresh RPMI 1640 broth.…”

Section: Methodsmentioning

confidence: 99%

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The Calcineurin Pathway Inhibitor Tacrolimus Enhances the In Vitro Activity of Azoles against Mucorales via Apoptosis

Shirazi

Kontoyiannis

2013

Eukaryot Cell

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The calcineurin pathway regulates antifungal drug resistance and the virulence of several major human-pathogenic fungi, including the recalcitrant Mucorales. We hypothesized that the fungistatic triazoles posaconazole (PCZ) and itraconazole (ICZ) become fungicidal in the setting of the calcineurin inhibitor tacrolimus (TCR) and that such an effect is mediated through apoptosis. Fungicidal activity and apoptosis were studied using standard microbiological techniques and hyphal metabolic and vital dye reduction assays at 37°C in RPMI 1640. Apoptosis was characterized by detecting intracellular Ca 2؉ , phosphatidylserine (PS) externalization, DNA fragmentation, plasma membrane integrity, chromatin condensation, reactive oxygen species (ROS) generation, caspase-like activity, ATP, and cytochrome c release. MICs for PCZ and ICZ alone were significantly higher (8 to 128 g/ml) than those of PCZ or ICZ plus TCR (0.25 to 4 g/ml) for Rhizopus oryzae, Cunninghamella bertholletiae, and Mucor circinelloides. Both PCZ and ICZ in combination with TCR became fungicidal, and their activity was mediated through increased apoptotic cell death of R. oryzae (10 to 50%), C. bertholletiae (5 to 50%), and M. circinelloides (5 to 55%) germlings, with morphological apoptotic changes characterized by externalization of PS, nuclear condensation, and DNA fragmentation. Moreover, activation of the caspase-like activity was correlated with cell death induced by TCR plus PCZ or ICZ. These changes correlated with elevated intracellular Ca 2؉ and ROS levels and disturbance of mitochondrial potential. We found that PCZ or ICZ in combination with TCR renders Mucorales sensitive to triazoles via apoptotic death. These observations could serve as a new paradigm for the development of new therapeutic strategies.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The Calcineurin Pathway Inhibitor Tacrolimus Enhances the In Vitro Activity of Azoles against Mucorales via Apoptosis

Shirazi

Kontoyiannis

2013

Eukaryot Cell

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“…Plasma membranes were isolated as described elsewhere (Niimi et al, 2004). Briefly, overnight cultures in YPD at 30 uC were harvested and pellets were concentrated 20 times and washed with cold H 2 O.…”

Section: Methodsmentioning

confidence: 99%

The GPI-modified proteins Pga59 and Pga62 of Candida albicans are required for cell wall integrity

et al. 2009

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The fungal cell wall is essential in maintaining cellular integrity and plays key roles in the interplay between fungal pathogens and their hosts. The PGA59 and PGA62 genes encode two short and related glycosylphosphatidylinositol-anchored cell wall proteins and their expression has been previously shown to be strongly upregulated when the human pathogen Candida albicans grows as biofilms. Using GFP fusion proteins, we have shown that Pga59 and Pga62 are cell-walllocated, N-and O-glycosylated proteins. The characterization of C. albicans pga59D/pga59D, pga62D/pga62D and pga59D/pga59D pga62D/pga62D mutants suggested a minor role of these two proteins in hyphal morphogenesis and that they are not critical to biofilm formation. Importantly, the sensitivity to different cell-wall-perturbing agents was altered in these mutants. In particular, simultaneous inactivation of PGA59 and PGA62 resulted in high sensitivity to Calcofluor white, Congo red and nikkomicin Z and in resistance to caspofungin. Furthermore, cell wall composition and observation by transmission electron microscopy indicated an altered cell wall structure in the mutant strains. Collectively, these data suggest that the cell wall proteins Pga59 and Pga62 contribute to cell wall stability and structure. INTRODUCTIONThe cell wall is an essential component of fungal cells, preserving cellular integrity and playing a central role in the interaction of fungi with their environment. This is particularly the case for pathogenic fungi such as the opportunistic yeast pathogen Candida albicans, where the cell wall has been shown to play central roles in adhesion, virulence, biofilm formation, infection and immunomodulation (Albrecht et al., 2006;Douglas, 2003;Netea et al., 2006;Richard et al., 2002b;Sundstrom, 2002). Because of the essential role of the cell wall in cellular integrity and fungal specificity of some enzymes involved in its biogenesis, it is a recognized target for the development of novel antifungals (e.g. echinocandins that target the b-1,3-glucan synthase) (Latge, 2007).The organization of the fungal cell wall has been mainly characterized in the yeasts Saccharomyces cerevisiae and C. albicans and in the filamentous fungus Aspergillus fumigatus (Klis et al., 2006;Latge, 2007;Lesage & Bussey, 2006;Ruiz-Herrera et al., 2006). The yeast cell wall has a bilayered structure. The inner part is composed of a network of b-1,3-glucan molecules linked by hydrogen bonds. These chains can be bound covalently to b-1,6-glucan molecules and to chitin chains. The outer part of the cell wall is composed mainly of mannoproteins (Klis et al., 2006Lesage & Bussey, 2006). Most proteins in the cell wall of ascomycetous yeasts are glycosylphosphatidylinositolanchored proteins (GPI-modified proteins) that become covalently linked to b-1,6-glucan through a remnant of their GPI anchor. As the b-1,6-glucan moiety can be linked to b-1,3-glucan or chitin, the cell wall GPI-modified proteins are strongly linked to the cell wall Klis et al., 2001;Richard & Plaine, 2007).GPI-...

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“…Analysis, visualization, and purification of PM proteins. Plasma membrane (PM) fractions of S. cerevisiae cells were prepared as described previously (40). Protein samples (30 g) were separated on 8% acrylamide gels and stained with Coomassie blue R250.…”

Section: Cloning Of Ckabc1 For Expression In Yeastmentioning

confidence: 99%

Characterization of Three Classes of Membrane Proteins Involved in Fungal Azole Resistance by Functional Hyperexpression inSaccharomyces cerevisiae

et al. 2007

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The study of eukaryotic membrane proteins has been hampered by a paucity of systems that achieve consistent high-level functional protein expression. We report the use of a modified membrane protein hyperexpression system to characterize three classes of fungal membrane proteins (ABC transporters Pdr5p, CaCdr1p, CaCdr2p, CgCdr1p, CgPdh1p, CkAbc1p, and CneMdr1p, the major facilitator superfamily transporter CaMdr1p, and the cytochrome P450 enzyme CaErg11p) that contribute to the drug resistance phenotypes of five pathogenic fungi and to express human P glycoprotein (HsAbcb1p). The hyperexpression system consists of a set of plasmids that direct the stable integration of a single copy of the expression cassette at the chromosomal PDR5 locus of a modified host Saccharomyces cerevisiae strain, AD⌬. Overexpression of heterologous proteins at levels of up to 29% of plasma membrane protein was achieved. Membrane proteins were expressed with or without green fluorescent protein (GFP), monomeric red fluorescent protein, His, FLAG/His, Cys, or His/Cys tags. Most GFP-tagged proteins tested were correctly trafficked within the cell, and His-tagged proteins could be affinity purified. Kinetic analysis of ABC transporters indicated that the apparent K m value and the V max value of ATPase activities were not significantly affected by the addition of His tags. The efflux properties of seven fungal drug pumps were characterized by their substrate specificities and their unique patterns of inhibition by eight xenobiotics that chemosensitized S. cerevisiae strains overexpressing ABC drug pumps to fluconazole. The modified hyperexpression system has wide application for the study of eukaryotic membrane proteins and could also be used in the pharmaceutical industry for drug screening.The resolution and exploitation of protein structure and function are among the greatest biological challenges in the postgenomic era. These challenges, and their potential dividends, are greatest for membrane proteins, which are notoriously difficult to functionally express and purify in the quantities and forms needed for drug discovery or for high-resolution X-ray crystallography (1, 16). About a quarter of the cellular proteome consists of membrane proteins (5), which often play vital physiological roles: from environmental sensing to energy transduction, from nutrient uptake to drug efflux, and from cellular proliferation to programmed cell death. Membrane proteins are involved in many prominent diseases, including cystic fibrosis (48), type 2 diabetes (49), heart disease (52), and the drug resistance of numerous cancers (57). Hence, they are the targets for many therapies and constitute up to 70% of the drug targets used in medicine today. Membrane proteins also play key roles in drug modification, detoxification, and resistance in a wide variety of prokaryotic and eukaryotic systems (7). A fundamental understanding of cell biology, cell physiology, and cell-drug interactions therefore requires a detailed analysis of membrane protein function. Fu...

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Chemosensitization of Fluconazole Resistance in Saccharomyces cerevisiae and Pathogenic Fungi by a d -Octapeptide Derivative

Abstract: Hyperexpression of the

Cited by 110 publications

References 58 publications

The Calcineurin Pathway Inhibitor Tacrolimus Enhances the In Vitro Activity of Azoles against Mucorales via Apoptosis

The Calcineurin Pathway Inhibitor Tacrolimus Enhances the In Vitro Activity of Azoles against Mucorales via Apoptosis

The GPI-modified proteins Pga59 and Pga62 of Candida albicans are required for cell wall integrity

Characterization of Three Classes of Membrane Proteins Involved in Fungal Azole Resistance by Functional Hyperexpression inSaccharomyces cerevisiae

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