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The heat-ramp method to study regulated cell death in a pathogenic yeast Cryptococcus neoformans
Madhura Kulkarni1, Yining Liu1, Quanxi Cheng1
1W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States.
Abstract:
Human fungal pathogens cause a significant public health burden. While no reliable surveilence data are available, estimations suggest that 1 billion infections and over 2 million deaths are attributable to fungal infections annually worldwide. This drove the World Health Organization to generate a priority list of fungal pathogens for reearch, which includes the yeast Cryptococcus neoformans as a top critical priority. With the rise of drug-resistance and emerging fungal pathogens, new conceptual strategies for antifungal therapies are needed in addition to existing antibiotic development pipelines to meet clinical needs. Intrinsic cell death pathways encoded by pathogenic fungi are largely unstudied but could be leveraged for antifungal therapy analogous to anti-cancer therapeutics that activate apoptosis or other cell death mechanisms. Thus far, molecularly defined fungal cell death mechanisms are best characterized for only a few, predominantly model filamentous species. To extend these studies to pathogenic yeast, here we describe and demonstrate a tunable heat-ramp stimulus that when applied to small volumes of yeast cell suspensions reveals a protracted cell death process in the pathogenic yeast Cryptococcus neoformans. This low cost protocol induces robust and reproducible phenotypes to study gene-dependent mechanisms in laboratory strains and clinical isolates.
Insights
This study introduces a new, low-cost method to study cell death in Cryptococcus neoformans, a critical fungal pathogen. This technique helps researchers understand fungal infections and develop new antifungal therapies.
Area of Science:
- Medical Mycology
- Molecular Biology
- Cell Death Research
Background:
- Fungal infections pose a significant global health threat, causing millions of deaths annually.
- Cryptococcus neoformans is a critical fungal pathogen prioritized by the World Health Organization due to rising drug resistance.
- Novel antifungal strategies are urgently needed, and understanding fungal cell death pathways offers a promising avenue.
Purpose of the Study:
- To develop a novel, accessible method for studying cell death mechanisms in pathogenic yeasts.
- To investigate the protracted cell death process in Cryptococcus neoformans.
- To enable the study of gene-dependent cell death mechanisms in both laboratory and clinical fungal isolates.
Main Methods:
- Development of a tunable heat-ramp stimulus protocol.
- Application of the stimulus to small volumes of Cryptococcus neoformans yeast cell suspensions.
- Observation and characterization of induced cell death phenotypes.
Main Results:
- A protracted, reproducible cell death process was observed in Cryptococcus neoformans.
- The heat-ramp stimulus protocol is low-cost and effective.
- The method generates robust phenotypes suitable for genetic studies.
Conclusions:
- The described heat-ramp method provides a valuable tool for studying fungal cell death in Cryptococcus neoformans.
- This approach can facilitate the discovery of new antifungal targets and therapies.
- Understanding intrinsic fungal cell death pathways is crucial for combating pathogenic fungi.
