Identification and characterization of VC1123, a novel gene required for colonization in Vibrio cholerae

A novel lineage of serogroup O1 El Tor Vibrio cholerae, genetically distinct from the seventh-pandemic strain, has recently emerged in China and has been associated with diarrheal outbreaks. However, the molecular basis underlying its virulence potential remains incompletely understood. This study aimed to identify virulence-associated determinants in the new lineage through comparative transcriptomic and functional analyses.

We compared the transcriptomes of two new-lineage isolates-VC6050 (ctxAB⁺ ) and VC6055 (ctxAB^- )-with the representative seventh-pandemic strain N16961 (ctxAB⁺ ) following growth in virulence-inducing (AKI) and non-inducing (LB) media. Differential expression patterns were analyzed, with particular focus on the toxigenic new-lineage strain VC6050 and N16961 under AKI conditions. A highly expressed gene shared across all three strains, VC1123, was selected for functional characterization. A targeted deletion mutant (ΔVC1123) was constructed in N16961, and its intestinal colonization capacity was evaluated in the neonatal mouse model. Complementation analysis was performed to confirm phenotype specificity. To investigate the underlying regulatory mechanisms, comparative RNA-seq was conducted between wild-type and ΔVC1123 strains grown in LB medium.

Under AKI conditions, VC6050 exhibited more pronounced repression of chemotaxis- and biofilm-associated genes compared with N16961. Among genes highly expressed across all strains in AKI, VC1123 was selected for further study. Deletion of VC1123 significantly enhanced intestinal colonization in neonatal mice, and this phenotype was fully restored upon genetic complementation. Transcriptomic analysis revealed that VC1123 deletion led to marked upregulation of the outer membrane porin gene ompU and concomitant downregulation of ompT, without affecting expression of the master regulators toxR or toxS. Given that ompU enhances bile resistance, adhesion, and intestinal survival, whereas ompT impairs gut colonization, these transcriptional alterations provide a mechanistic explanation for the enhanced colonization phenotype.

Our findings demonstrate that VC1123 functions as a negative regulator of intestinal colonization in V. cholerae. The data suggest that VC1123 modulates ompU and ompT expression through a pathway independent of the canonical ToxR/ToxS regulatory system. This study uncovers a previously underexplored layer of virulence regulation mediated by a conserved gene of unknown function and provides new insight into adaptive strategies employed by emerging V. cholerae lineages.