Schimmich Cécile, Gondard Mathilde, Caignard Gregory, Valle-Casuso José-Carlos, Vitour Damien, Piumi François
Unité physiopathologie et épidémiologie des maladies équines (PhEED), Laboratoire de Santé Animale, ANSES, Goustranville, France.
UMR Virologie, INRAE, École nationale vétérinaire d'Alfort, Anses, Laboratoire de Santé animale, Université Paris-Est, Maisons-Alfort, France.
PLoS One. 2025 May 30;20(5):e0324917. doi: 10.1371/journal.pone.0324917. eCollection 2025.
Protein-protein interactions (PPI) play a key role in host-pathogens interaction studies, as proteins are essential to many cellular mechanisms. The yeast two-hybrid (Y2H) approach is a well-established method for high-throughput PPI screening and mapping of protein interaction networks. The success of this approach partially depends on the quality and representativeness of the host cDNA library, which can be constructed from the transcriptomic content of a selected host cellular type. However, evaluating the relevance of the cDNA library content remains challenging, and one of the key limitations of this interactomic approach is the occurrence of false-negative results (i.e., the absence of detectable interactions). Here, we report a direct, long read, high-throughput sequencing method using Oxford Nanopore Technologies, to assess the completeness of the host cDNA library used in host-pathogen interactions Y2H screening. This approach enables easy identification of possible downstream screened genes in PPI assays, minimizing sequencing biases and bioinformatics handling of the data. This study was performed on a cDNA library, generated from A549 human lung carcinoma cells. We were able to identify 12,123 protein coding genes from the sequencing of whole plasmids containing the cDNA inserts, that were further analyzed via functional pathways enrichment for deeper characterization. This diversity and relative abundance evaluation method could be a first step when generating new cDNA libraries of interest for PPI studies, ensuring the validity and suitability of the host library before proceeding with all Y2H screening steps.
蛋白质-蛋白质相互作用（PPI）在宿主-病原体相互作用研究中起着关键作用，因为蛋白质对许多细胞机制至关重要。酵母双杂交（Y2H）方法是一种成熟的用于高通量PPI筛选和蛋白质相互作用网络绘制的方法。这种方法的成功部分取决于宿主cDNA文库的质量和代表性，该文库可从选定宿主细胞类型的转录组内容构建。然而，评估cDNA文库内容的相关性仍然具有挑战性，这种相互作用组学方法的关键局限性之一是假阴性结果的出现（即未检测到相互作用）。在这里，我们报告了一种使用牛津纳米孔技术的直接、长读长、高通量测序方法，以评估宿主-病原体相互作用Y2H筛选中使用的宿主cDNA文库的完整性。这种方法能够在PPI测定中轻松识别可能的下游筛选基因，最大限度地减少测序偏差和数据的生物信息学处理。本研究是在从A549人肺癌细胞生成的cDNA文库上进行的。我们能够从包含cDNA插入片段的完整质粒测序中鉴定出12123个蛋白质编码基因，并通过功能途径富集对其进行进一步分析，以进行更深入的表征。这种多样性和相对丰度评估方法可能是为PPI研究生成新的感兴趣的cDNA文库时的第一步，在进行所有Y2H筛选步骤之前确保宿主文库的有效性和适用性。
