Insights into copper sensing and tolerance in Pneumocystis species

Abstract

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.

Keywords: Pneumocystis; copper sensing; copper tolerance; cuproproteins; host-pathogen interaction; metalloproteome.

Figure 1

SEC-ICP-MS chromatograms were generated for P. murina samples (in red), lung homogenates from infected mice (in black), and lung homogenates from uninfected mice (in blue) to investigate the Cu metalloproteome. Each analysis was conducted in two technical replicates, and the chromatograms have been offset for clarity. The peaks observed at 23–24 min correspond to free copper. Molecular weight markers are indicated based on the Bio-Rad Gel filtration standard mixture containing thyroglobulin (bovine), g-globulin (bovine), ovalbumin (chicken) and myoglobin (horse).

Figure 2

A PCA plot of the RNA-seq data from P. murina organisms exposed to CuSO₄ at concentrations of 0 (controls), 1, 10, and 100 μM over time durations of 0.5, 2, and 5 h. Results are based on three biological replicates, i.e., each derived from organisms originating from three distinct animals. The PCA plot highlights the grouping of samples by the duration of exposure to CuSO₄, marked in orange.

Figure 3

RT-qPCR of select genes found over-expressed in P. murina treated with 100 μM CuSO4 at 5 h. Reference gene used is thymidylate synthase. Measurements were made in triplicates. Relative quantity is shown as 2ΔΔCT. * – statistical significance at p-value <0.05.

Figure 4

Efficacy of Cu(II) sulfate on P. murina viability. Viability assessed via ATP luciferase assay, indicated by relative light units (RLU) which correlate with the quantity of metabolically active organisms. Data derived from four biological replicates in each group. Adjustments made by deducting mean RLU of blank medium wells from all other values. Error bars represent standard deviations. Control comprises untreated organisms.