Database Mining Reveals Distinct Clusters of Putative Diphthamide-Specific Mono-ADP-Ribosylating Toxins in Vibrio and Aeromonas

Diphthamide-specific mono-ADP-ribosylating toxins form a structurally conserved but phylogenetically diverse family of AB-type bacterial toxins, with Pseudomonas exotoxin A (PE) serving as a prototypical member. Here, we expand the known diversity of PE-like toxins (PLTs) through comprehensive genome mining and phylogenetic, structural and sequence analyses of PE, Chx, and their Aeromonas and Vibrio cholerae homologues. Aeromonas toxins separate into two distinct groups: a PE-like cluster (AEI) and a cholix-like cluster (AEII), while a fourth cholix subgroup (ChxIV) is identified within V. cholerae, expanding the previously described ChxI–III classification. Although all newly identified toxins retain the core catalytic architecture and hallmark residues essential for ADP-ribosylation of eEF2, they differ in key functional motifs, including receptor-binding residues, furin cleavage loops, and C-terminal ER-retrieval signals, suggesting variation in host-cell uptake routes and intracellular trafficking. Signal peptides were present in all toxins but varied modestly between clusters, consistent with normal species-level Sec-pathway variation rather than divergent secretion mechanisms. Together, our findings provide a significantly expanded overview of PLT diversity and indicate that Aeromonas-derived toxins comprise at least two evolutionarily and mechanistically distinct groups. These insights offer a framework for future functional studies and highlight PLTs as emerging virulence factors in environmentally and clinically relevant bacteria.