An ART-fold Rhs toxin from Pluralibacter gergoviae defines Tne5, a novel family of NAD(P) glycohydrolases effectors

The type VI secretion system (T6SS) is a widespread bacterial nanomachine that mediates interbacterial competition by delivering toxic effectors into neighboring cells. Among these, enzymes targeting nicotinamide adenine dinucleotide (NAD) cofactors and NAD phosphate (NAD(P)) are particularly potent because they rapidly disrupt redox homeostasis and central metabolism. Several families of T6SS-associated NAD(P)-consuming effectors (Tne1-Tne4) have been described. Here, we characterize a T6SS-associated Rearrangement hot spot toxin from Pluralibacter gergoviae. Competition assays show that P. gergoviae kills Escherichia coli in a T6SS-dependent manner. Heterologous production reveals that the Rearrangement hot spot C-terminal extension is toxic in the Escherichiacoli cytoplasm and that co-production with the protein encoded downstream neutralizes this activity. AlphaFold3 modeling predicts that the toxin adopts an ADP-ribosyltransferase-like α/β fold with a putative catalytic pocket accommodating NADH. By contrast to T6SS ART toxins described so far, the toxin does not inhibit transcription or translation but instead depletes NAD(P) in vitro and causes intracellular NAD depletion in intoxicated cells. Nuclear magnetic resonance analyses further show that the toxin hydrolyzes NAD ⁺ into nicotinamide and ADP-ribose. Phylogenetic analyses and structural modeling show that this effector defines a new ART-related family of NAD(P) glycohydrolases broadly distributed across antagonistic systems, which we propose to name Tne5.