Genomics-guided identification of a conserved CptBA-like toxin-antitoxin system in Acinetobacter baumannii,Journal of Advanced Research

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Genomics-guided identification of a conserved CptBA-like toxin-antitoxin system in Acinetobacter baumannii
Journal of Advanced Research ( IF 11.4 ) Pub Date : 2020-11-23 , DOI: 10.1016/j.jare.2020.11.007
Shahira A ElBanna ₁ , Nayera A Moneib ₁ , Ramy K Aziz _{1,

2} , Reham Samir _{1,

2}

Affiliation

Introduction

Toxin-antitoxin (TA) systems are widespread among bacteria, archaea and fungi. They are classified into six types (I-VI) and have recently been proposed as novel drug targets.

Objectives

This study aimed to screen the pathogen Acinetobacter baumannii, known for its alarming antimicrobial resistance, for TA systems and identified a CptBA-like type IV TA, one of the least characterized systems.

Methods

In silico methods included secondary structure prediction, comparative genomics, multiple sequence alignment, and phylogenetic analysis, while in vitro strategies included plasmid engineering and expression of the TA system in Escherichia coli BL21, growth measurement, and transcription analysis with quantitative reverse-transcription polymerase chain reaction.

Results

Comparative genomics demonstrated the distribution of CptBA-like systems among Gram-negative bacteria, while phylogenetic analysis delineated two major groups, in each of which Acinetobacter spp. proteins clustered together. Sequence alignment indicated the conservation of cptA and cptB in 4,732 strains of A. baumannii in the same syntenic order. Using A. baumannii recombinant cptA and cptB, cloned under different promoters, confirmed their TA nature, as cptB expression was able to reverse growth inhibition by CptA in a dose-time dependent manner. Furthermore, transcriptional analysis of cptBA in clinical and standard A. baumannii strains demonstrated the downregulation of this system under oxidative and antibiotic stress.

Conclusion

Combining in silico and in vitro studies confirmed the predicted TA nature of a cptBA-like system in A. baumannii . Transcriptional analysis suggests a possible role of cptBA in response to antibiotics and stress factors in A. baumannii, making it a promising drug target.

中文翻译：

鲍曼不动杆菌中保守的 CptBA 样毒素-抗毒素系统的基因组学指导鉴定

介绍

毒素-抗毒素 (TA) 系统广泛存在于细菌、古细菌和真菌中。它们分为六种类型（I-VI），最近被提出作为新的药物靶点。

目标

本研究旨在为 TA 系统筛选病原体鲍曼不动杆菌，以其令人担忧的抗菌素耐药性而闻名，并确定了 CptBA 样 IV 型 TA，这是特征最少的系统之一。

方法

计算机方法包括二级结构预测、比较基因组学、多序列比对和系统发育分析，而体外策略包括质粒工程和 TA 系统在大肠杆菌BL21中的表达、生长测量和使用定量逆转录聚合酶链的转录分析反应。

结果

比较基因组学证明了 CptBA 样系统在革兰氏阴性细菌中的分布，而系统发育分析描绘了两个主要群体，在每个群体中不动杆菌属。蛋白质聚集在一起。序列比对表明cptA和cptB在 4,732 株鲍曼不动杆菌中以相同的同线顺序保守。使用在不同启动子下克隆的鲍曼不动杆菌重组cptA和cptB证实了它们的 TA 性质，因为cptB表达能够以剂量时间依赖性方式逆转 CptA 的生长抑制。此外，cptBA 的转录分析在临床和标准鲍曼不动杆菌菌株中证明了该系统在氧化和抗生素应激下的下调。