Table_1_Insights into copper sensing and tolerance in Pneumocystis species.XLSX

IntroductionPneumocystis 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 resultsThe 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.
DiscussionThe 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.

引言
肺孢子菌属（Pneumocystis）是一类致病真菌，已知可引发免疫受损哺乳动物罹患肺炎。它们为专性寄生菌，于肺泡胞外增殖，并在哺乳动物肺部的富铜微环境中存活繁衍。本研究以感染小鼠的模式生物鼠肺孢子菌（Pneumocystis murina）为研究对象，探究其铜感知与铜耐受相关的蛋白质组（proteome）。

材料与方法及结果
首先，在FungiDB数据库中检索铜相关基因注释并经人工手动整理后，仅在鼠肺孢子菌中鉴定出21个基因，该数量显著少于其他临床相关真菌病原体或系统发育相近的自由生活真菌。随后，我们采用尺寸排阻色谱-电感耦合等离子体质谱法（Size-Exclusion Chromatography Inductively Coupled Plasma Mass Spectrometry，SEC-ICP-MS）、固定化金属亲和色谱法（Immobilized Metal Affinity Chromatography，IMAC）以及液相色谱-串联质谱法（Liquid Chromatography–Tandem Mass Spectrometry，LC–MS/MS）等仪器分析手段，从新鲜提取的菌体中分离并鉴定铜结合蛋白（cuproprotein），最终得到29种不同的铜结合蛋白。
对暴露于不同浓度硫酸铜（CuSO4）、并分别在0.5、2、5小时三个时间节点取样的鼠肺孢子菌进行转录组测序（RNA sequencing，RNA-seq）分析后发现，仅在所使用的最高硫酸铜浓度（100μM）与最长暴露时长（5小时）条件下，才出现显著的基因表达变化。与未处理对照组相比，该刺激条件下共有43个基因上调表达，27个基因下调表达。实时定量聚合酶链反应（Quantitative PCR，qPCR）验证了8个筛选出的上调基因中的4个的表达情况，其中包括3个假定转录因子（PNEG_01236、PNEG_01675与PNEG_01730）以及1个推定铜转运蛋白（PNEG_02609）。值得注意的是，本研究采用的三种方法——基于同源性的基因注释、SEC-ICP-MS/IMAC/LC–MS/MS仪器分析以及RNA-seq——所得结果重合度较低，仅4个基因（PNEG_02587、PNEG_03319、PNEG_02584与PNEG_02989）同时被两种仪器分析方法鉴定到。

讨论
本研究所得结果丰富了肺孢子菌铜稳态（copper homeostasis）相关的认知，并为胞外病原体的宿主-病原体互作研究提供了新视角。我们建议，未来针对肺孢子菌致病性与铜胁迫存活的研究，应纳入本研究鉴定到的全部基因。