Evaluation of fungal-specific fluorescent labeled echinocandin probes as diagnostic adjuncts

Pratt, Ayiasha; García‐Effrón, Guillermo; Zhao, Yanan; Park, Steven; Mustaev, Arkady; Pillai, Shyamala; Perlin, David S.

doi:10.3109/13693786.2012.685767

Cited by 15 publications

(16 citation statements)

References 14 publications

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“…Because loss of CDC50 is expected to perturb membrane function, we hypothesized that the cdc50Δ mutant may exhibit enhanced sensitivity to caspofungin because it alters drug uptake into cells. To test this hypothesis, we monitored drug uptake by using boron dipyrromethene difluoride (BODIPY)-labeled caspofungin ( 25 ). We observed that while H99 failed to take up significant amounts of fluorescently labeled caspofungin, producing only limited intracellular fluorescence, the cdc50Δ mutant showed a significantly higher level of fluorescent signal, indicative of an increased intracellular drug concentration ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Lipid Flippase Subunit Cdc50 Mediates Drug Resistance and Virulence in Cryptococcus neoformans

Huang

Liao

Baker

et al. 2016

mBio

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100

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Cryptococcus neoformans is a human fungal pathogen and a major cause of fungal meningitis in immunocompromised individuals. Treatment options for cryptococcosis are limited. Of the two major antifungal drug classes, azoles are active against C. neoformans but exert a fungistatic effect, necessitating long treatment regimens and leaving open an avenue for emergence of azole resistance. Drugs of the echinocandin class, which target the glucan synthase and are fungicidal against a number of other fungal pathogens, such as Candida species, are ineffective against C. neoformans. Despite the sensitivity of the target enzyme to the drug, the reasons for the innate resistance of C. neoformans to echinocandins remain unknown. To understand the mechanism of echinocandin resistance in C. neoformans, we screened gene disruption and gene deletion libraries for mutants sensitive to the echinocandin-class drug caspofungin and identified a mutation of CDC50, which encodes the β-subunit of membrane lipid flippase. We found that the Cdc50 protein localized to membranes and that its absence led to plasma membrane defects and enhanced caspofungin penetration into the cell, potentially explaining the increased caspofungin sensitivity. Loss of CDC50 also led to hypersensitivity to the azole-class drug fluconazole. Interestingly, in addition to functioning in drug resistance, CDC50 was also essential for fungal resistance to macrophage killing and for virulence in a murine model of cryptococcosis. Furthermore, the surface of cdc50Δ cells contained increased levels of phosphatidylserine, which has been proposed to act as a macrophage recognition signal. Together, these results reveal a previously unappreciated role of membrane lipid flippase in C. neoformans drug resistance and virulence.

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Section: Resultsmentioning

confidence: 99%

Lipid Flippase Subunit Cdc50 Mediates Drug Resistance and Virulence in Cryptococcus neoformans

Huang

Liao

Baker

et al. 2016

mBio

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100

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“…Mutations were noted in the 5’-upstream region of CYP51 , and in the coding regions of other genes linked with ergosterol biosynthesis (Supplementary Figure S1 ). However, these did not affect the intracellular level of CYP51, as judged by binding between the enzyme and posaconazole conjugated with the fluorophore boron-dipyrromethene 36 , 37 (Supplementary Figure 2 ). To exclude the possibility that TcNTR-1 depletion might perturb susceptibility to posaconazole, we tested its activity against two cloned T. cruzi cell lines (Methods) in which we had deleted one copy of the gene (Fig.…”

Section: Resultsmentioning

confidence: 98%

Genome-wide mutagenesis and multi-drug resistance in American trypanosomes induced by the front-line drug benznidazole

Campos

Phelan

Francisco

et al. 2017

Sci Rep

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Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and affects 5–8 million people in Latin America. Although the nitroheterocyclic compound benznidazole has been the front-line drug for several decades, treatment failures are common. Benznidazole is a pro-drug and is bio-activated within the parasite by the mitochondrial nitroreductase TcNTR-1, leading to the generation of reactive metabolites that have trypanocidal activity. To better assess drug action and resistance, we sequenced the genomes of T. cruzi Y strain (35.5 Mb) and three benznidazole-resistant clones derived from a single drug-selected population. This revealed the genome-wide accumulation of mutations in the resistant parasites, in addition to variations in DNA copy-number. We observed mutations in DNA repair genes, linked with increased susceptibility to DNA alkylating and inter-strand cross-linking agents. Stop-codon-generating mutations in TcNTR-1 were associated with cross-resistance to other nitroheterocyclic drugs. Unexpectedly, the clones were also highly resistant to the ergosterol biosynthesis inhibitor posaconazole, a drug proposed for use against T. cruzi infections, in combination with benznidazole. Our findings therefore identify the highly mutagenic activity of benznidazole metabolites in T. cruzi, demonstrate that this can result in multi-drug resistance, and indicate that vigilance will be required if benznidazole is used in combination therapy.

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“…Conjugation of CSF with a fluorophore was performed as described previously. 9 Briefly, CSF-DDAO conjugate was produced by incubating 20 mg (15 μmol) of CSF-diacetate and 2.8 mg (8 μmol) of DDAO-isothiocyano derivative in 0.2 ml of dimethylformamide and 4 μl of triethylamine at 50°C during 30 min. Thin liquid chromatography (TLC) analysis in acetonitrile-water (7:1) developing system revealed nearly complete conversion of the original DDAO derivative (Rf = 0.95) to the main reaction product (Rf = 0.6), which was purified by preparative TLC in the same system.…”

Section: Methodsmentioning

confidence: 99%

“…We previously reported that CSF can be modified at the ethylenediamine functionalized position with a reporter BODIPY, while still retaining highly potent antifungal targeting properties. 9 When coupled with the fluorophore label 7-Amino-9H-(1,3-Dichloro-9,9-Dimethylacridin-2-One) (DDAO) at the amino group of 3-hydroxyornithine residue, CSF-DDAO can be used as fungal specific probe, whereby drug-bound cells fluoresce and can be visualized by near-infrared (NIR) fluorescence imaging. Furthermore, a liposome-encapsulated CSF-DDAO (L-CSF-DDAO) was developed to facilitate a greater penetration of the probe into the cornea and to minimize its interaction with corneal cell membranes.…”

Section: Introductionmentioning

confidence: 99%

A novel, tomographic imaging probe for rapid diagnosis of fungal keratitis

et al. 2017

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Fungal keratitis is a leading cause of ocular morbidity and blindness in developing countries. Diagnosing fungal keratitis currently relies on a comparative evaluation of corneal biopsy or scraping using a direct microscopy and culture results. These methods not only carry the risk of developing complications due to the invasive tissue sampling but also are largely limited by diagnostic speed and accuracy, making it difficult to initiate timely appropriate antifungal therapy. Therefore, rapid and noninvasive diagnostic tools are a pressing need for improved outcomes for fungal keratitis. Taking advantage of the highly specific fungal cell targeting properties of caspofungin, we have developed a fluorescent chemical probe with high selectivity against fungal pathogens. Utilizing fluorescence imaging technology, we have demonstrated a highly specific and sensitive detection of Aspergillus in a fungal keratitis model in mice as early as 5 min post-topical application of the probe. Our results indicate that a fluorescence-mediated platform can be used as a rapid (<10 min) alternative to conventional methods for detecting Aspergillus, and potentially other fungi, in fungal infections of the cornea.

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Evaluation of fungal-specific fluorescent labeled echinocandin probes as diagnostic adjuncts

Cited by 15 publications

References 14 publications

Lipid Flippase Subunit Cdc50 Mediates Drug Resistance and Virulence in Cryptococcus neoformans

Lipid Flippase Subunit Cdc50 Mediates Drug Resistance and Virulence in Cryptococcus neoformans

Genome-wide mutagenesis and multi-drug resistance in American trypanosomes induced by the front-line drug benznidazole

A novel, tomographic imaging probe for rapid diagnosis of fungal keratitis

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