Global transmission and distribution of phage-encoded cholera toxin genes constrained by toxin-repression genes and anti-phage defense systems.

Cholera is a severe diarrheal disease caused by toxigenic Vibrio cholerae, whose virulence depends on lysogenic infection by CTXφ bacteriophages encoding the cholera toxin genes (ctxA and ctxB) and associated accessory genes (ace and zot). However, the global distribution and transmission dynamics of phage-encoded cholera toxin genes across environments remain poorly understood. To address this, we performed a large-scale bioinformatic analysis of publicly available whole genomes. We show that both phages and bacteria carrying toxin genes are globally distributed across human-associated, freshwater, fish, and mammalian habitats, with Vibrio and Aeromonas being the dominant bacterial taxa and Inoviridae is the most prevalent phage family. Phage-mediated horizontal gene transfer (HGT) of toxin genes occurred in both Vibrio and non-Vibrio species, with the highest transfer between Inoviridae and V. cholerae occuring predominantly among bacteria from the same habitat. Temporal analysis revealed an increase in candidate HGT events after 2000, peaking at 377845 events during 2010-2019. HGT events negatively correlated with the presence of CRISPR-Cas system and toxin-repression genes (hns, hapR, and tsrA) in host bacteria. Experimental validation indicated that H-NS and HapR inhibit phage infection by repressing phage release. Together, our results suggest that CRISPR-Cas phage defense system and toxin-repression mechanisms could constrain the spread of toxin-carrying phages, with potential implications for the occurrence and severity of cholera outbreaks worldwide.