Table_3.DOCX

Acinetobacter baumannii is one of the major causes of hard to treat multidrug-resistant hospital infections. A. baumannii features contributing to its spread and persistence in clinical environment are only beginning to be explored. Bacterial toxin-antitoxin (TA) systems are genetic loci shown to be involved in plasmid maintenance and proposed to function as components of stress response networks. Here we present a thorough characterization of type II system of A. baumannii, which is the most ubiquitous TA module present in A. baumannii plasmids. higBA of A. baumannii is a reverse TA (the toxin gene is the first in the operon) and shows little homology to other TA systems of RelE superfamily. It is represented by two variants, which both are functional albeit exhibit strong difference in sequence conservation. The higBA2 operon is found on ubiquitous 11 Kb pAB120 plasmid, conferring carbapenem resistance to clinical A. baumannii isolates and represents a higBA variant that can be found with multiple sequence variations. We show here that higBA2 is capable to confer maintenance of unstable plasmid in Acinetobacter species. HigB2 toxin functions as a ribonuclease and its activity is neutralized by HigA2 antitoxin through formation of an unusually large heterooligomeric complex. Based on the in vivo expression analysis of gfp reporter gene we propose that HigA2 antitoxin and HigBA2 protein complex bind the higBA2 promoter region to downregulate its transcription. We also demonstrate that higBA2 is a stress responsive locus, whose transcription changes in conditions encountered by A. baumannii in clinical environment and within the host. We show elevated expression of higBA2 during stationary phase, under iron deficiency and downregulated expression after antibiotic (rifampicin) treatment.

鲍曼不动杆菌（Acinetobacter baumannii）是难治性多重耐药医院感染的主要致病原之一。目前，关于鲍曼不动杆菌在临床环境中传播与持续定植的相关特征仅处于初步探索阶段。细菌毒素-抗毒素（toxin-antitoxin, TA）系统是一类被证实参与质粒维持的基因座，且被认为是应激反应网络的组成部分。本研究对鲍曼不动杆菌的II型TA系统开展了全面表征，该系统是鲍曼不动杆菌质粒中分布最为广泛的TA模块。鲍曼不动杆菌的higBA属于反向TA系统（毒素基因位于操纵子首位），与RelE超家族的其他TA系统同源性极低。该系统存在两种变体，二者均具备功能，但在序列保守性上存在显著差异。higBA2操纵子存在于广泛分布的11 kb pAB120质粒上，可赋予临床分离鲍曼不动杆菌菌株碳青霉烯类抗生素抗性，且该higBA变体存在多种序列变异类型。本研究证实，higBA2可帮助不动杆菌属物种维持不稳定质粒。HigB2毒素具有核糖核酸酶活性，HigA2抗毒素可通过形成异常庞大的异源寡聚复合体中和其活性。基于绿色荧光蛋白（green fluorescent protein, GFP）报告基因的体内表达分析，本研究提出：HigA2抗毒素与HigBA2蛋白复合物可结合higBA2启动子区域，从而下调其转录。本研究同时证实，higBA2是一类应激响应基因座，其转录水平会随鲍曼不动杆菌在临床环境及宿主体内遭遇的环境条件发生改变。研究发现，higBA2在稳定生长期、铁匮乏条件下的表达量升高，而经抗生素（利福平，rifampicin）处理后其表达量则出现下调。