The role of cytochrome c in leukocyte induced Aspergillus fumigatus cell death

Author:

M James1*, KW Liu1, E Vesley1, T Hohl2, RA Cramer1

Author address:

1Microbiology and Immunology , Geisel School of Medicine at Dartmouth College, Hanover, United States

2Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, United States

Full conference title:

10th Advances Against Aspergillosis and Mucormycosis

Date: 2 February 2022

Abstract:

Purpose:

Aspergillus fumigatus is a ubiquitous environmental mold that can cause a life-threatening infection known as invasive aspergillosis (IA). IA is caused by defects in innate immune system function that result in failed clearance of inhaled conidia from the lung. While it is known that innate immune function, particularly NADPH oxidase activity, is responsible for clearance of these conidia from the lung, the mechanism by which these conidia are killed by leukocytes remains unknown. While processes of cell death in have been identified in animals, these processes remain largely unknown in outside of metazoa. Contributing to this question regarding non-metazoan cell death, many canonical components of regulated cell death are not conserved outside of metazoa. Here we investigate the role of A. fumigatus cytochrome c (cycA) in both hydrogen peroxide-induced and leukocyte-induced fungal cell death. Cytochrome c is a canonical cell death effector in higher order metazoa that functions in programmed and regulated forms of cell death and is conserved across eukaryotes.

 

Methods:

To examine the effects of hydrogen peroxide (H2O2) on cell death, we generated a cycA null strain in a background strain that contains a histone H2A:mRFP reporter to monitor histone fragmentation, a canonical marker of cell death. Swollen conidia were exposed to 10mM H2O2 for 8hrs in LGMM media. Every two hours, conidia were gathered and stained with caspACE and sytox blue to quantify caspase-like activity and cell viability, respectively, and were analyzed by flow cytometry. Long term viability was examined by treating swollen conidia with 10mM H2O2 for 2.5hrs, followed by monitoring long term growth by absorbance at 405nm in a germination assay. For in vivo experiments, a FLuorescent Aspergillus REporter (FLARE) experiment was conducted in C57BL/6J mice by staining H2A:mRFP conidia with Alexa Flour 633 and inoculating at 3e7 conidia per mouse. 36hrs post inoculation, cells were gathered from murine lungs and stained for corresponding leukocyte subsets. Analysis was conducted by flow cytometry.

 

Results:

We observed that the cycA null strain displays altered cell death phenotypes including reduced histone fragmentation, reduced caspase-like activity, and reduced sytox blue staining after 6hr and 8hr exposure to 10mM H2O2. However, using a germination assay to monitor growth 30hrs after acute 2.5hr exposure to 10mM H2O2, we observed that loss of cycA results in no observable growth after treatment, suggesting a loss of viability as compared to the WT and complement strains. Examining in vivo leukocyte killing by FLARE technology, we observe that loss of cycA results in lower in viability in leukocyte subsets including neutrophils and total macrophages as compared to WT and complement strains. However, the cycA null strain displayed higher viability in specifically the alveolar macrophage subset as compared to other leukocyte subsets.

 

Conclusion:

Taken together, these data suggest cytochrome c presence in A. fumigatus contributes to cell survival under death inducing conditions and future studies will seek to define the underlying mechanisms.

Abstract Number: 88

Conference Year: 2022

Link to conference website: https://aaam2022.org/

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