# Systematic identification of Salmonella T6SS effectors uncovers diverse new families and lipid-targeting activities

## TL;DR

This study identifies new Salmonella T6SS effectors, including a toxin that targets lipids and helps Salmonella compete in the gut.

## Contribution

The study discovers new toxin families and directly characterizes a lipid-targeting NlpC/P60 toxin domain in Salmonella T6SS effectors.

## Key findings

- Tox-Act1 is a phospholipase that cleaves phosphatidylglycerol and phosphatidylethanolamine.
- Tox-Act1 is secreted via T6SS and provides a competitive advantage during gut colonization in mice.
- Evolutionary analysis links Tox-Act1 to acyltransferases and reveals new toxin domain diversity.

## Abstract

Bacterial warfare is a widespread phenomenon in which bacteria deploy toxins to inhibit or kill competitors. These toxins disrupt essential cellular processes, and their diversification is driven by an evolutionary arms race involving toxin and immunity gene acquisition. Here, we used in-silico approaches to analyze genomes from the 10k Salmonella Project and identify effectors secreted via the Type VI Secretion System (T6SS). We uncovered 128 candidates distributed across diverse Salmonella serovars and other bacterial species. Among them, Tox-Act1 was selected for in-depth characterization. Tox-Act1 contains a permuted NlpC/P60 papain-like catalytic core typical of lipid-targeting enzymes. Evolutionary analysis revealed its relationship with acyltransferases. Biochemical assays and lipidomics of intoxicated cells showed that Tox-Act1 acts as a phospholipase, cleaving phosphatidylglycerol and phosphatidylethanolamine. We further demonstrate that Tox-Act1 is secreted in a T6SS-dependent manner and provides a competitive advantage during mouse gut colonization. This study broadens our understanding of toxin domain diversity and provides the first direct characterization of a lipid-targeting NlpC/P60 toxin domain.

During microbial warfare, bacteria deploy toxins to inhibit or kill competitors. This study reveals lipid-targeting antibacterial toxins and previously unknown toxin domains within Salmonella T6SS effectors, broadening our understanding of enzymatic diversity and the molecular strategies bacteria use to compete in the gut.