Structural basis of the VapBC5 complex from Mycobacterium abscessus and its role in regulating persister cell...

Toxin-antitoxin (TA) systems contribute to bacterial stress adaptation and persistence, yet the structural and regulatory features of the VapBC5 module from Mycobacterium abscessus remain incompletely defined. Here, we characterize the VapBC5 toxin-antitoxin system using an integrated structural and functional approach. The VapBC5 complex was coexpressed, purified, crystallized, and its structure was determined at 2.24 Å resolution, revealing a heterotetrameric 2:2 assembly with a locally asymmetric interface. The C-terminal region of the antitoxin VapB5 engages the VapC5 toxin through an extensive network of hydrogen bonds and salt bridges, consistent with suppression of toxin activity. Structure-guided mutagenesis demonstrates that VapBC5 interface stability relies on a cooperative and distributed interaction network, and that disruption of multiple antitoxin-toxin contacts is required to unmask VapC5 activity in vivo. Functional assays in a heterologous Escherichia coli model show that VapC5 promotes antibiotic-tolerant survival under fluoroquinolone stress, whereas VapB5 counteracts this phenotype. Antitoxin-derived peptides were first screened in E. coli, and a selected peptide was subsequently evaluated in a laboratory M. abscessus strain, where it attenuated VapC5-associated persistence-related phenotypes. Together, these findings define the structural basis of VapBC5 regulation and support the use of antitoxin-derived peptides as molecular probes to modulate toxin-antitoxin-associated antibiotic tolerance in a mycobacterial context.