Allelic variations and gene cluster modularity act as nonlinear bottlenecks for cholera emergence.

The underlying factors that lead to specific strains within a species to emerge as human pathogens remain mostly enigmatic. The diarrheal disease cholera is caused by strains from a phylogenetically confined group within the Vibrio cholerae species, the pandemic cholera group (PCG), making it an ideal model system to tackle this puzzling phenomenon. Comprehensive analyses of over 1,840 V. cholerae genomes, including environmental isolates from this study, reveal that the species consists of eleven groups, with the PCG belonging to the largest and located within a lineage shared with environmental strains. This hierarchical classification provided us with a framework to unravel the ecoevolutionary dynamics of the genetic determinants associated with the emergence of toxigenic V. cholerae. Our analyses indicate that this phenomenon is largely dependent on the acquisition of unique modular gene clusters and allelic variations that confer a competitive advantage during intestinal colonization. We determined that certain PCG-associated alleles are essential for successful colonization whereas others provide a nonlinear competitive advantage, acting as a critical bottleneck that clarifies the isolated emergence of PCG. For instance, toxigenic strains encoding non-PCG alleles of a) tcpF or b) a sextuple allelic exchange mutant for genes tcpA, toxT, VC0176, VC1791, rfbT, and ompU, lose their ability to colonize the intestine. Interestingly, these alleles do not play a role in the colonization of newly established model environmental reservoirs. Our study uncovers the evolutionary roots of toxigenic V. cholerae offering a tractable approach for investigating the emergence of pathogenic clones within an environmental population.