Development of an Imaging Flow Cytometry Method for Fungal Cytological Profiling and Its Potential Application in Antifungal Drug Development

Courtney L. McMahon; Marisol Esqueda; Jieh-Juen Yu; Gina Wall; Jesus A. Romo; Taissa Vila; Ashok Chaturvedi; Jose L. Lopez-Ribot; Floyd Wormley; Chiung-Yu Hung

doi:10.3390/jof9070722

journal article Open Access Jun 30, 2023

Development of an Imaging Flow Cytometry Method for Fungal Cytological Profiling and Its Potential Application in Antifungal Drug Development

Courtney L. McMahon

Marisol Esqueda Jieh-Juen Yu

Gina Wall Jesus A. Romo

Taissa Vila Ashok Chaturvedi Jose L. Lopez-Ribot

Floyd Wormley Chiung-Yu Hung

Journal of Fungi Vol. 9 No. 7 pp. 722 · MDPI AG

View at Publisher Save 10.3390/jof9070722

Abstract

Automated imaging techniques have been in increasing demand for the more advanced analysis and efficient characterization of cellular phenotypes. The success of the image-based profiling method hinges on assays that can rapidly and simultaneously capture a wide range of phenotypic features. We have developed an automated image acquisition method for fungal cytological profiling (FCP) using an imaging flow cytometer that can objectively measure over 250 features of a single fungal cell. Fungal cells were labeled with calcofluor white and FM4-64FX, which bind to the cell wall and lipophilic membrane, respectively. Images of single cells were analyzed using IDEAS® software. We first acquired FCPs of fungal cells treated with fluconazole, amphotericin B, and caspofungin, each with a distinct mode of action, to establish FCP databases of profiles associated with specific antifungal treatment. Once fully established, we investigated the potential application of this technique as a screening methodology to identify compounds with novel antifungal activity against Candida albicans and Cryptococcus neoformans. Altogether, we have developed a rapid, powerful, and novel image-profiling method for the phenotypic characterization of fungal cells, also with potential applications in antifungal drug development.

Topics

No keywords indexed for this article. Browse by subject →

References

54

[1]

Richardson "Changing patterns and trends in systemic fungal infections" J. Antimicrob. Chemother. (2005) 10.1093/jac/dki218

[2]

Gintjee, T.J., Donnelley, M.A., and Thompson, G.R. (2020). Aspiring Antifungals: Review of Current Antifungal Pipeline Developments. J. Fungi, 6. 10.3390/jof6010028

[3]

Rauseo "Hope on the Horizon: Novel Fungal Treatments in Development" Open Forum Infect. Dis. (2020) 10.1093/ofid/ofaa016

[4]

Morris "Echinocandins in the management of invasive fungal infections, Part 2" Am. J. Health Syst. Pharm. (2006) 10.2146/ajhp050464.p2

[5]

Morris "Echinocandins in the management of invasive fungal infections, Part 1" Am. J. Health Syst. Pharm. (2006) 10.2146/ajhp050464.p1

[6]

Hamill "Amphotericin B formulations: A comparative review of efficacy and toxicity" Drugs (2013) 10.1007/s40265-013-0069-4

[7]

Alexander "Increasing echinocandin resistance in Candida glabrata: Clinical failure correlates with presence of FKS mutations and elevated minimum inhibitory concentrations" Clin. Infect. Dis. (2013) 10.1093/cid/cit136

[8]

Sanglard "Emerging Threats in Antifungal-Resistant Fungal Pathogens" Front. Med. (2016) 10.3389/fmed.2016.00011

[9]

Mitchison "Small-molecule screening and profiling by using automated microscopy" Chembiochem (2005) 10.1002/cbic.200400272

[10]

Perlman "Multidimensional drug profiling by automated microscopy" Science (2004) 10.1126/science.1100709

[11]

Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes

Mark-Anthony Bray, Shantanu Singh, Han Han et al.

Nature Protocols 2016 10.1038/nprot.2016.105

[12]

Willis "Phenotypic Profiling of Reference Chemicals across Biologically Diverse Cell Types Using the Cell Painting Assay" SLAS Discov. Adv. Sci. Drug Discov. (2020) 10.1177/2472555220928004

[13]

Gustafsdottir, S.M., Ljosa, V., Sokolnicki, K.L., Anthony Wilson, J., Walpita, D., Kemp, M.M., Petri Seiler, K., Carrel, H.A., Golub, T.R., and Schreiber, S.L. (2013). Multiplex cytological profiling assay to measure diverse cellular states. PLoS ONE, 8. 10.1371/journal.pone.0080999

[14]

Lutgring "FDA-CDC Antimicrobial Resistance Isolate Bank: A Publicly Available Resource To Support Research, Development, and Regulatory Requirements" J. Clin. Microbiol. (2018) 10.1128/jcm.01415-17

[15]

Wayne, P. (2008). Approved Standard: CLSI Document M38–A2, CLSI.

[16]

Harrington "Calcofluor White: A Review of its Uses and Applications in Clinical Mycology and Parasitology" Lab. Med. (2003) 10.1309/eph2tdt8335gh0r3

[17]

Zheng "Isolation of yeast mutants defective for localization of vacuolar vital dyes" Proc. Natl. Acad. Sci. USA (1998) 10.1073/pnas.95.20.11721

[18]

Wall "Screening a Repurposing Library for Inhibitors of Multidrug-Resistant Candida auris Identifies Ebselen as a Repositionable Candidate for Antifungal Drug Development" Antimicrob. Agents Chemother. (2018) 10.1128/aac.01084-18

[19]

Butts "A repurposing approach identifies off-patent drugs with fungicidal cryptococcal activity, a common structural chemotype, and pharmacological properties relevant to the treatment of cryptococcosis" Eukaryot. Cell (2013) 10.1128/ec.00314-12

[20]

Jabes "Thioridazine inhibits gene expression control of the cell wall signaling pathway (CWI) in the human pathogenic fungus Paracoccidioides brasiliensis" Mol. Genet. Genom. MGG (2016) 10.1007/s00438-016-1184-1

[21]

Vitale "Activity and post antifungal effect of chlorpromazine and trifluopherazine against Aspergillus, Scedosporium and zygomycetes" Mycoses (2007) 10.1111/j.1439-0507.2007.01371.x

[22]

Galgoczy "In vitro antifungal activity of phenothiazines and their combination with amphotericin B against different Candida species" Mycoses (2011) 10.1111/j.1439-0507.2010.02010.x

[23]

Eilam "Activity of phenothiazines against medically important yeasts" Antimicrob. Agents Chemother. (1987) 10.1128/aac.31.5.834

[24]

Wang "Susceptibility of melanized and nonmelanized Cryptococcus neoformans to the melanin-binding compounds trifluoperazine and chloroquine" Antimicrob. Agents Chemother. (1996) 10.1128/aac.40.3.541

[25]

Fuchs "Inhibition of bacterial and fungal pathogens by the orphaned drug auranofin" Future Med. Chem. (2016) 10.4155/fmc.15.182

[26]

Thangamani "Ebselen exerts antifungal activity by regulating glutathione (GSH) and reactive oxygen species (ROS) production in fungal cells" Biochim. Biophys. Acta (2017) 10.1016/j.bbagen.2016.09.029

[27]

Wiederhold "Repurposing auranofin as an antifungal: In vitro activity against a variety of medically important fungi" Virulence (2017) 10.1080/21505594.2016.1196301

[28]

Orie "In vitro assessment of the growth and plasma membrane H+-ATPase inhibitory activity of ebselen and structurally related selenium- and sulfur-containing compounds in Candida albicans" J. Biochem. Mol. Toxicol. (2017) 10.1002/jbt.21892

[29]

Wall, G., Herrera, N., and Lopez-Ribot, J.L. (2019). Repositionable Compounds with Antifungal Activity against Multidrug Resistant Candida auris Identified in the Medicines for Malaria Venture’s Pathogen Box. J. Fungi, 5. 10.3390/jof5040092

[30]

Mendonca "Nimesulide inhibits pathogenic fungi: PGE2-dependent mechanisms" Folia Microbiol. (2017) 10.1007/s12223-016-0483-6

[31]

Vu "Astemizole and an analogue promote fungicidal activity of fluconazole against Cryptococcus neoformans var. grubii and Cryptococcus gattii" Med. Mycol. (2010) 10.3109/13693780903081968

[32]

Dennis "Synergistic combinations of azoles and antihistamines against Candida species in vitro" Med. Mycol. (2018) 10.1093/mmy/myy088

[33]

Garcia "A phenotypic small-molecule screen identifies halogenated salicylanilides as inhibitors of fungal morphogenesis, biofilm formation and host cell invasion" Sci. Rep. (2018) 10.1038/s41598-018-29973-8

[34]

Beggs "Anti-Candida activity of the anti-cancer drug tamoxifen" Res. Commun. Chem. Pathol. Pharmacol. (1993)

[35]

Dolan "Antifungal activity of tamoxifen: In vitro and in vivo activities and mechanistic characterization" Antimicrob. Agents Chemother. (2009) 10.1128/aac.01564-08

[36]

Maguire "Quantifying nuclear p65 as a parameter for NF-κB activation: Correlation between ImageStream cytometry, microscopy, and Western blot" Cytom. Part A (2011) 10.1002/cyto.a.21068

[37]

George "Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow" J. Immunol. Methods (2006) 10.1016/j.jim.2006.01.018

[38]

Cerveira "An imaging flow cytometry-based approach to measuring the spatiotemporal calcium mobilisation in activated T cells" J. Immunol. Methods (2015) 10.1016/j.jim.2015.04.030

[39]

Filby "An imaging flow cytometric method for measuring cell division history and molecular symmetry during mitosis" Cytom. Part A (2011) 10.1002/cyto.a.21091

[40]

Patterson "An Imaging Flow Cytometry-based approach to analyse the fission yeast cell cycle in fixed cells" Methods (2015) 10.1016/j.ymeth.2015.04.026

[41]

Chia "Imaging flow cytometry for the screening of compounds that disrupt the Plasmodium falciparum digestive vacuole" Methods (2017) 10.1016/j.ymeth.2016.07.002

[42]

Barteneva "Imaging flow cytometry: Coping with heterogeneity in biological systems" J. Histochem. Cytochem. (2012) 10.1369/0022155412453052

[43]

McFarlin "Flow cytometry what you see matters: Enhanced clinical detection using image-based flow cytometry" Methods (2017) 10.1016/j.ymeth.2016.09.001

[44]

Georgopapadakou "Antifungals: Mechanism of action and resistance, established and novel drugs" Curr. Opin. Microbiol. (1998) 10.1016/s1369-5274(98)80087-8

[45]

Demers "Evolution of drug resistance in an antifungal-naive chronic Candida lusitaniae Infection" Proc. Natl. Acad. Sci. USA (2018) 10.1073/pnas.1807698115

[46]

Dromer "Antifungal drug resistance in pathogenic fungi" Med. Mycol. (1998)

[47]

Lackner "In vitro antifungal susceptibility of Candida glabrata to caspofungin and the presence of FKS mutations correlate with treatment response in an immunocompromised murine model of invasive infection" Antimicrob. Agents Chemother. (2014) 10.1128/aac.02666-13

[48]

Chowdhary, A., Sharma, C., and Meis, J.F. (2017). Candida auris: A rapidly emerging cause of hospital-acquired multidrug-resistant fungal infections globally. PLoS Pathog., 13. 10.1371/journal.ppat.1006290

[49]

Klepser "Antifungal pharmacodynamic characteristics of fluconazole and amphotericin B against Cryptococcus neoformans" J. Antimicrob. Chemother. (1998) 10.1093/jac/41.3.397

[50]

Kordalewska "Understanding Echinocandin Resistance in the Emerging Pathogen Candida auris" Antimicrob. Agents Chemother. (2018) 10.1128/aac.00238-18

Showing 50 of 54 references

Metrics

10

Citations

54

References

Details

Published: Jun 30, 2023
Vol/Issue: 9(7)
Pages: 722
License: View

Authors

C

Courtney L. McMahon