Genomics-guided identification of a conserved CptBA-like toxin-antitoxin system in Acinetobacter baumannii

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.