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.