通过GST下拉鉴定肺炎支原体P1相互作用蛋白，并分析其在A549细胞中的转录组调控

摘要(中文译文)

肺炎支原体（Mycoplasma pneumoniae，M. pneumoniae）是引起儿童社区获得性肺炎的主要病原体。其致病过程依赖于对宿主呼吸道上皮细胞的黏附与定植。P1蛋白是肺炎支原体的主要黏附素，直接介导其与宿主细胞的结合。为探究P1重组蛋白与宿主细胞的相互作用机制，我们进行了蛋白表达与纯化、谷胱甘肽S-转移酶（GST）pull-down实验以及转录组测序。在确认的诱导条件下（16°C，0.1 mM IPTG），rP1-GST融合蛋白成功表达。GST pull-down实验在rP1-GST组中鉴定出22个差异表达的膜蛋白，其中膜联蛋白A2（ANXA2）和C-C趋化因子受体5型（CCR5）变化显著，并与P1黏附素发生相互作用。ANXA2和CCR5均具有多种功能，包括蛋白结合、受体活性及信号感受器活性。转录组分析表明，rP1-A549细胞相互作用产生的差异表达基因在多个基因本体论（GO）条目和KEGG通路中显著富集。这些结果提示，ANXA2和CCR5可能是P1黏附素的潜在结合伴侣。P1黏附素可能调控参与线粒体和能量代谢的宿主基因。这些发现为理解肺炎支原体的黏附及致病机制提供了线索。

Abstract

Mycoplasma pneumoniae (M. pneumoniae) is a major pathogen causing community-acquired pneumonia in children. Its pathogenic process relies on the adherence to and colonization of host respiratory epithelial cells. P1 protein is the primary adhesin of M. pneumoniae, directly mediating its binding to host cells. To explore the interaction mechanism between P1 recombinant protein and host cells, we conducted protein expression and purification, glutathione S-transferase (GST) pull-down assay, and transcriptome sequencing. The rP1-GST fusion protein was expressed under confirmed induction conditions (16°C, 0.1 mM IPTG). GST pull-down assay identified 22 differentially expressed membrane proteins in the rP1-GST group, among which annexin A2 (ANXA2) and C-C chemokine receptor type 5 (CCR5) were significantly altered and interact with P1 adhesin. Both ANXA2 and CCR5 possessed multiple functions including protein binding, receptor activity and signal sensor activity. Transcriptome analysis indicated that differentially expressed genes from rP1-A549 cell interaction were significantly enriched in multiple Gene Ontology (GO) terms and KEGG pathways. These results suggest that ANXA2 and CCR5 may serve as potential binding partners of P1 adhesin. P1 adhesin may regulate host genes involved in mitochondria and energy metabolism. These findings provide clues for understanding the adhesion and pathogenesis of M. pneumoniae.

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