The higBA Toxin-Antitoxin Module From the Opportunistic Pathogen Acinetobacter baumannii – Regulation, Activity, and Evolution

Abstract

<i>Acinetobacter baumannii</i> is one of the major causes of hard to treat multidrug-resistant hospital infections. <i>A. baumannii</i> 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 <i>A. baumannii</i>, which is the most ubiquitous TA module present in <i>A. baumannii</i> plasmids. <i>higBA</i> of <i>A. baumannii</i> 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 <i>higBA2</i> operon is found on ubiquitous 11 Kb pAB120 plasmid, conferring carbapenem resistance to clinical <i>A. baumannii</i> isolates and represents a <i>higBA</i> variant that can be found with multiple sequence variations. We show here that <i>higBA2</i> is capable to confer maintenance of unstable plasmid in <i>Acinetobacter</i> 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 <i>in vivo</i> expression analysis of <i>gfp</i> reporter gene we propose that HigA2 antitoxin and HigBA2 protein complex bind the <i>higBA2</i> promoter region to downregulate its transcription. We also demonstrate that <i>higBA2</i> is a stress responsive locus, whose transcription changes in conditions encountered by <i>A. baumannii</i> in clinical environment and within the host. We show elevated expression of <i>higBA2</i> during stationary phase, under iron deficiency and downregulated expression after antibiotic (rifampicin) treatment.