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Insights into copper sensing and tolerance in Pneumocystis species
Aleksey Porollo1,2,3, Steven G Sayson4,5, Alan Ashbaugh4,5
1Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States.
Introduction:
Pneumocystis species are pathogenic fungi known to cause pneumonia in immunocompromised mammals. They are obligate to their host, replicate extracellularly in lung alveoli and thrive in the copper-enriched environment of mammalian lungs. In this study, we investigated the proteome of Pneumocystis murina, a model organism that infects mice, in the context of its copper sensing and tolerance.
Methods And Results:
The query for copper-associated annotations in FungiDB followed by a manual curation identified only 21 genes in P. murina, significantly fewer compared to other clinically relevant fungal pathogens or phylogenetically similar free-living fungi. We then employed instrumental analyses, including Size-Exclusion Chromatography Inductively Coupled Plasma Mass Spectrometry (SEC-ICP-MS), Immobilized Metal Affinity Chromatography (IMAC), and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), to isolate and identify copper-binding proteins from freshly extracted organisms, revealing 29 distinct cuproproteins. The RNA sequencing (RNA-seq) analysis of P. murina exposed to various CuSO4 concentrations at three temporal intervals (0.5, 2, and 5 h) indicated that significant gene expression changes occurred only under the highest CuSO4 concentration probed (100 μM) and the longest exposure duration (5 h). This stimulus led to the upregulation of 43 genes and downregulation of 27 genes compared to untreated controls. Quantitative PCR (qPCR) confirmed the expression of four out of eight selected upregulated genes, including three assumed transcription factors (PNEG_01236, PNEG_01675, and PNEG_01730) and a putative copper transporter (PNEG_02609). Notably, the three applied methodologies - homology-based annotation, SEC-ICP-MS/IMAC/LC-MS/MS, and RNA-seq - yielded largely distinct findings, with only four genes (PNEG_02587, PNEG_03319, PNEG_02584, and PNEG_02989) identified by both instrumental methods.
Discussion:
The insights contribute to the broader knowledge of Pneumocystis copper homeostasis and provide novel facets of host-pathogen interactions for extracellular pathogens. We suggest that future studies of Pneumocystis pathogenicity and copper stress survival should consider the entire spectrum of identified genes.
Insights
This study reveals how Pneumocystis murina, a fungus causing pneumonia, handles copper. Researchers identified new copper-binding proteins and genes involved in copper tolerance, offering insights into host-pathogen interactions.
Area of Science:
- Mycology
- Proteomics
- Genomics
Background:
- Pneumocystis species are pathogenic fungi causing pneumonia in immunocompromised mammals.
- These fungi are obligate parasites thriving in copper-rich lung environments.
Purpose of the Study:
- To investigate the proteome of Pneumocystis murina concerning its copper sensing and tolerance mechanisms.
- To identify copper-binding proteins and genes involved in copper homeostasis in P. murina.
Main Methods:
- Proteomic analysis using Size-Exclusion Chromatography Inductively Coupled Plasma Mass Spectrometry (SEC-ICP-MS) and Immobilized Metal Affinity Chromatography (IMAC).
- Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) for protein identification.
- RNA sequencing (RNA-seq) to analyze gene expression changes under copper sulfate (CuSO4) exposure.
- Quantitative PCR (qPCR) to validate gene expression.
Main Results:
- Identified 29 distinct cuproproteins in P. murina, exceeding initial homology-based predictions.
- RNA-seq revealed significant gene expression changes at high CuSO4 concentrations (100 μM) and prolonged exposure (5 h), with 43 genes upregulated and 27 downregulated.
- qPCR confirmed the upregulation of key genes, including transcription factors and a putative copper transporter.
Conclusions:
- The study expands knowledge of Pneumocystis copper homeostasis and host-pathogen interactions.
- Multiple methodologies yielded distinct findings, highlighting the complexity of copper management in P. murina.
- Future research on Pneumocystis pathogenicity and copper stress survival should consider the full spectrum of identified genes.
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