鲍曼不动杆菌无痕基因敲除及hcp基因相关功能

Marker-less gene deletion and the function of hcp gene in Acinetobacter baumannii

摘要:

【背景】鲍曼不动杆菌耐药严重，基因敲除是研究细菌毒力与耐药的重要方式。但现有的大部分细菌基因敲除方法基于抗生素抗性筛选，导致不适用于多重耐药菌株的基因敲除。【目的】旨在建立一种非依赖于抗生素抗性筛选的方法，用于敲除多重耐药鲍曼不动杆菌基因。【方法】运用同源重组和自杀载体pMo130-TelR对亚碲酸钾的抗性，使用两步筛选法，构建鲍曼不动杆菌VI型分泌系统溶血素共调节蛋白(Hemolysin-Coregulated protein，Hcp)基因敲除突变体，并对缺失株的生长能力、细菌竞争能力以及血清抵抗能力进行测试。【结果】通过构建含同源片段的重组pMo130-TelR载体，成功敲除了鲍曼不动杆菌标准株ATCC 17978中的hcp基因，获得了ATCC 17978 hcp基因缺失突变体。突变体生长能力没有显著改变，但细菌竞争能力显著下降，血清抵抗能力显著升高。【结论】pMo130-TelR可成功用于鲍曼不动杆菌无痕基因敲除，对于研究鲍曼不动杆菌的耐药机制等相关问题具有深远意义。

Abstract:

[Background] Acinetobacter baumannii is highly resistant to antibiotics. Gene knockout is an important way to study the virulence and antibiotic resistance of A. baumannii. Currently, most gene knockout methods rely on the use of an antibiotic selection marker and are not suited for multidrug resistant strains. [Objective] This study aims to establish a marker-less gene deletion method to knock out the genes of multidrug resistant A. baumannii for subsequent experiments. [Methods] We used a two-step screening method using homologous recombination and the resistance of pMo130-TelR to potassium tellurite to construct the type VI secretion system hcp gene deletion mutant of A. baumannii. Then we tested the growth ability, bacterial competition ability and serum resistance of the mutant strain. [Results] By constructing a tellurite-resistant suicide vector inserted with recombinant fragment, pMo130-TelR-(hcp up-down), we successfully knocked out the hcp gene in A. baumannii. There were no significant changes in the growth ability of mutants, in contrast, the bacterial competitive ability decreased and the serum resistance increased significantly. [Conclusion] The marker-less gene deletion method using pMo130-TelR is applicable for creating gene deletion mutants in A. baumannii. It will be very useful in multidrug resistant A. baumannii gene deletion and will have profound significance for A. baumannii research such as the antibiotic-resistance mechanism.