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

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.

The cause and effect of Cryptococcus interactions with the host

“…neoformans infection occurs via inhalation of desiccated yeast or spores, which germinate in the host lung (reviewed in [5][6][7]). Two recent studies from the Hull lab have significantly improved overall understanding of this transition [1,2]. Barkal et al developed a high throughput, computational method for in vitro spore germination and analysis [2].…”

“…Two recent studies from the Hull lab have significantly improved overall understanding of this transition [1,2]. Barkal et al developed a high throughput, computational method for in vitro spore germination and analysis [2].…”

“…They show that C. neoformans germination, defined as a switch from ovoid spores to round yeast, is a synchronous process characterized by change in overall shape followed by isotropic growth. Germination requires microtubule reorganization, chromatin remodelling, and protein turn-over, and is slow relative to the typical yeast cell cycle, occurring over 12 hours [2]. Germination is triggered by nutrient availability rather than surface contact or temperature shift, and although human body temperature (37¡C) modestly reduces spore viability, sensitivity appears to be strain dependent [2,8].…”

“…Fungal spores enter this milieu with limited nutritional reserves, germinate, and begin proliferating by budding as yeast. Although relatively little is known about the initial stages of infection, recent work has characterized changes that occur upon germination [1,2]. This program and subsequent yeast-phase proliferation progress in a dynamic environment as host nutrient immunity responds to the infection via toxic accumulation or sequestration of essential micronutrients (reviewed in [3]) and innate immune cells are recruited to the site of infection [4].…”

Aspergillosis and stem cell transplantation: An overview of experimental pathogenesis studies

“…87 A similar system has been used to study the dynamics of germination in the pathogenic yeast Cryptococcus neoformans. 88 The use of this and other similar microsystems may provide a useful tool to improve our understanding of the importance of the microenvironment on the expression and function of virulence factors of A. fumigatus.…”