# The VapBC-4 Characterization Indicates It Is a Bona Fide Toxin-Antitoxin Module of Leptospira interrogans: Initial Evidence for a Role in Bacterial Adaptation

## TL;DR

This study confirms that VapBC-4 is a functional toxin-antitoxin system in Leptospira interrogans, which may help the bacteria adapt to harsh conditions.

## Contribution

The study experimentally characterizes VapBC-4 as a functional toxin-antitoxin module in Leptospira interrogans.

## Key findings

- Overexpression of VapC-4 toxin inhibits E. coli growth, which is reversed by VapB-4 antitoxin.
- VapC-4 is a PIN domain endoribonuclease capable of degrading viral RNA.
- Transcriptional evidence suggests VapC-4 may contribute to bacterial virulence and environmental adaptation.

## Abstract

Toxin-antitoxin (TA) systems are one of the bacterial adaptation mechanisms to adverse conditions. Leptospira interrogans serovar Copenhageni contains nine putative TA systems. To date, only VapBC-3 and VapBC-1 have been experimentally characterized and considered functional modules. This study shows that the VapBC-4 module is a novel bona fide TA system constituted by VapB-4 antitoxin and VapC-4 toxin. Overexpression of the recombinant toxin in Escherichia coli resulted in growth inhibition, which was rescued by co-expression of the VapB-4 antitoxin. The toxin-antitoxin binding capability, essential to TA functionality, was demonstrated by dot blot assay in vitro, while the pull-down assay indicates that the toxin and antitoxin interact in vivo. In addition, we confirmed that VapC-4 is a PIN domain endoribonuclease capable of degrading viral MS2 substrate. The transcriptional studies suggest that vapC-4 may be involved in the virulence and adaptability of L. interrogans serovar Copenhageni for adverse environmental conditions. Taken together, these results show that the VapBC-4 module is functional and can be considered a bona fide module.