ABC转运透性酶DppC2的缺失有利于鼠疫耶尔森氏菌在巨噬细胞内的生存

Deficiency of the ABC transporter permease-like protein DppC2 facilitates the survival of Yersinia pestis in macrophages

摘要:

目的 研究dppC2基因在鼠疫耶尔森氏菌(鼠疫菌)巨噬细胞内生存中的作用。方法 采用自杀质粒同源重组法构建鼠疫菌201株的dppC2基因无痕敲除株(201-ΔdppC2)；通过巨噬细胞内生存实验、酸生存实验、过氧化氢生存实验、酶活测定、活性氧检测、细胞毒力测定和小鼠攻毒实验等，比较201-ΔdppC2与野生株(201-WT)的表型差异；利用转录组学分析和实时定量逆转录PCR (real-time quantitative reverse transcription PCR, RT-qPCR)分析201-ΔdppC2与201-WT的基因表达差异。结果 与201-WT相比，201-ΔdppC2在小鼠单核巨噬细胞RAW264.7和人单核细胞白血病细胞THP-1中的胞内生存率显著升高，酸生存率及过氧化氢生存率升高，耐酸基因hdeD以及过氧化物酶相关基因katA和katG的转录水平上调，过氧化氢酶和过氧化物酶的活力升高，细菌及其感染的巨噬细胞RAW264.7胞内活性氧水平降低；对HeLa细胞的毒性降低，但对小鼠的毒力无差异。结论 dppC2基因缺失后，鼠疫菌对酸性和过氧化氢环境的适应性提高，有利于鼠疫菌在巨噬细胞内的生存和复制。

Abstract:

Objective To investigate the role of dppC2 in the survival of Yersinia pestis in macrophages.Methods The strain (201-ΔdppC2) with traceless knockout of dppC2 was constructed with a suicide plasmid via homologous recombination based on Y. pestis biovar Microtus strain 201. Phenotypes were compared between 201-ΔdppC2 and the wild type (201-WT) by the acid survival assay, hydrogen peroxide survival assay, macrophage intracellular survival assay, reactive oxygen species (ROS) detection, and cytotoxicity and mouse challenge assays. The gene expression was compared between 201-ΔdppC2 and 201-WT by transcriptomics analysis and RT-qPCR.Results Compared with 201-WT, 201-ΔdppC2 exhibited multiple phenotypic alterations, including significantly increases in intracellular survival rates in RAW264.7 and THP-1 cells and under acidic and hydrogen peroxide conditions, upregulation of the acid resistance gene hdeD and the catalase-related genes katA and katG, enhancement of catalase and peroxidase activities, and declines in intracellular ROS levels in 201-ΔdppC2 and RAW264.7 cells infected with the mutant. Furthermore, 201-ΔdppC2 showed reduced cytotoxicity to HeLa cells but no change in the virulence in mice.Conclusion The deletion of dppC2 has been demonstrated to enhance the fitness of Y. pestis to acidic and hydrogen peroxide environments, which promote the survival and replication of Y. pestis in macrophages.