Identification of novel toxin domains and characterization of a broadly distributed family of lipid-targeting NlpC/P60.

Abstract

Bacteria live in complex communities and use antagonistic strategies to compete for resources. Gram-negatives encode a type VI secretion system (T6SS) that delivers toxins into eukaryotic and prokaryotic cells. Previous studies highlighted the importance ofSalmonellaT6SSs in bacterial antagonism and host cell infection, but few antibacterial effectors have been described. Here, we used bioinformatic strategies to identify the T6SS effectors in 10,000Salmonellagenomes. As a result, we identified and manually curated 128 candidates. SpecificSalmonellaserovars harbor unique effectors that are target-specific (antibacterial or anti-eukaryotic), depending on the T6SS subtype (e.g. i1 or i3). In addition, 45 previously uncharacterized toxin domains were identified and designated STox (Salmonellatoxins). STox15 was among the most frequent candidates and was selected for in-depth characterization. STox15 is an antibacterial effector belonging to the NlpC/P60 papain-like fold superfamily with a permuted catalytic core typical of lipid-targeting versions rather than peptidases or amidases. Biochemical analysis with recombinant protein and lipidomics of intoxicatedEscherichia colirevealed that STox15 displays phospholipase activity cleaving off acyl groups from phosphatidylglycerol and phosphatidylethanolamine. This work improves our understanding of theSalmonellaT6SS effector repertoire and provides the first direct characterization of a lipid-targeting NlpC/P60 domain in biological conflicts.