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

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 , 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 strategies included plasmid engineering and expression of the TA system in 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 spp. proteins clustered together. Sequence alignment indicated the conservation of and in 4,732 strains of in the same syntenic order. Using recombinant and , cloned under different promoters, confirmed their TA nature, as expression was able to reverse growth inhibition by CptA in a dose-time dependent manner. Furthermore, transcriptional analysis of in clinical and standard strains demonstrated the downregulation of this system under oxidative and antibiotic stress.

CONCLUSION

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