NDLI: Leveraging a high resolution microfluidic assay reveals insights into pathogenic fungal spore germination

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Leveraging a high resolution microfluidic assay reveals insights into pathogenic fungal spore germination

Content Provider	Royal Society of Chemistry (RSC)
Author	Hull, Christina M. Walsh, Naomi M. Botts, Michael R. Beebe, David J. Barkal, Layla J.
Copyright Year	2016
Abstract	Germination of spores into actively growing cells is a process essential for survival and pathogenesis of many microbes. Molecular mechanisms governing germination, however, are poorly understood in part because few tools exist for evaluating and interrogating the process. Here, we introduce an assay that leverages developments in microfluidic technology and image processing to quantitatively measure germination with unprecedented resolution, assessing both individual cells and the population as a whole. Using spores from Cryptococcus neoformans, a leading cause of fatal fungal disease in humans, we developed a platform to evaluate spores as they undergo morphological changes during differentiation into vegetatively growing yeast. The assay uses pipet-accessible microdevices that can be arrayed for efficient testing of diverse microenvironmental variables, including temperature and nutrients. We discovered that temperature influences germination rate, a carbon source alone is sufficient to induce germination, and the addition of a nitrogen source sustains it. Using this information, we optimized the assay for use with fungal growth inhibitors to pinpoint stages of germination inhibition. Unexpectedly, the clinical antifungal drugs amphotericin B and fluconazole did not significantly alter the process or timing of the transition from spore to yeast, indicating that vegetative growth and germination are distinct processes in C. neoformans. Finally, we used the high temporal resolution of the assay to determine the precise defect in a slow-germination mutant. Combining advances in microfluidics with a robust fungal molecular genetic system allowed us to identify and alter key temporal, morphological, and molecular events that occur during fungal germination.
Starting Page	603
Ending Page	615
Page Count	13
File Format	HTM / HTML PDF
ISSN	17579694
Volume Number	8
Issue Number	5
Journal	Integrative Biology
DOI	10.1039/c6ib00012f
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	Germination Digital image processing Cryptococcus neoformans Carbon Antifungal Amphotericin B Fluconazole Conidium Mutation Microfluidics
Content Type	Text
Resource Type	Article
Subject	Medicine Biochemistry Biophysics

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Triclosan Demonstrates Synergic Effect with Amphotericin B and Fluconazole and Induces Apoptosis-Like Cell Death in Cryptococcus neoformans

Effects of environmental factors on sensitivity of Cryptococcus neoformans to fluconazole and amphotericin B

Combined effect of furanone fluconazole and amphotericin B against biofilms formed from Cryptococcus neoformans.

Different culture media applied to the study of Cryptococcus neoformans susceptibility to amphotericin B and fluconazole

Rates of antifungal resistance among Spanish clinical isolates of Cryptococcus neoformans var. neoformans.

Antifungal pharmacodynamic characteristics of fluconazole and amphotericin B against Cryptococcus neoformans.

Interactions between triazoles and amphotericin B against Cryptococcus neoformans.

Effect of amphotericin B on capsule and cell size in Cryptococcus neoformans during murine infection.

Terbinafine inhibits Cryptococcus neoformans growth and modulates fungal morphology.

Leveraging a high resolution microfluidic assay reveals insights into pathogenic fungal spore germination

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Triclosan Demonstrates Synergic Effect with Amphotericin B and Fluconazole and Induces Apoptosis-Like Cell Death in Cryptococcus neoformans

Effects of environmental factors on sensitivity of Cryptococcus neoformans to fluconazole and amphotericin B

Combined effect of furanone fluconazole and amphotericin B against biofilms formed from Cryptococcus neoformans.

Different culture media applied to the study of Cryptococcus neoformans susceptibility to amphotericin B and fluconazole

Rates of antifungal resistance among Spanish clinical isolates of Cryptococcus neoformans var. neoformans.

Antifungal pharmacodynamic characteristics of fluconazole and amphotericin B against Cryptococcus neoformans.

Interactions between triazoles and amphotericin B against Cryptococcus neoformans.

Effect of amphotericin B on capsule and cell size in Cryptococcus neoformans during murine infection.

Terbinafine inhibits Cryptococcus neoformans growth and modulates fungal morphology.

Leveraging a high resolution microfluidic assay reveals insights into pathogenic fungal spore germination