Allelic variations and gene cluster modularity act as non-linear bottlenecks for cholera emergence

Cholera, an acute secretory diarrhea, is caused by strains from a phylogenetically confined group within the Vibrio cholerae species, the pandemic cholera group (PCG). To date, the molecular and evolutionary factors that enable the isolated emergence of toxigenic V. cholerae from environmental populations remain mostly enigmatic. Comprehensive analyses of over 1,100 V. cholerae genomes, including novel environmental isolates from this study, reveal that the species consists of four major clades and several minor ones. PCG belongs to a large clade located within a lineage shared with environmental strains, the pandemic cholera lineage. This hierarchical classification provided us with a framework to unravel the eco-evolutionary 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 non-linear competitive advantage, acting as a critical bottleneck that elucidates 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 model environmental reservoirs. Our study uncovers the evolutionary roots of toxigenic V. cholerae and offers a tractable approach for investigating the emergence of pathogenic clones within an environmental population. SIGNIFICANCE The underlying factors that lead to specific strains within a species to emerge as human pathogens remain mostly enigmatic. Toxigenic clones of the cholera agent, Vibrio cholerae , are encompassed within one phylogenomic clade, the pandemic cholera group (PCG). Here, we investigate the molecular and evolutionary factors that explain the confined nature of this group. Our analyses determined that the emergence of PCG is largely dependent on the acquisition of unique modular gene clusters and allelic variations that confer a competitive advantage during intestinal colonization. These allelic variations act as a critical bottleneck that elucidates the isolated emergence of PCG and provides a tractable blueprint for the study of the emergence of pathogenic clones within an environmental population. ### Competing Interest Statement The authors have declared no competing interest.