Vibrio vulnificus is an opportunistic marine pathogen that causes severe wound-associated and systemic infections. Following entry into the host, the bacterium must rapidly adapt to host-associated stresses that differ substantially from those encountered in aquatic environments. However, the physiological functions supporting bacterial fitness during infection remain incompletely understood. Previously, we applied signature-tagged mutagenesis (STM) to identify genes required for V. vulnificus survival during host infection, revealing numerous loci that did not correspond to classical toxin-encoding genes. In the present study, we extended this genome-wide screen by linking STM-identified mutations to observable fitness-related phenotypes. Functional annotation revealed enrichment of genes associated with chemotaxis, flagellar motility, regulation, metabolism, and poorly characterized functions. Phenotypic analyses showed that many STM-derived mutants exhibited defects in swimming motility and altered colony surface properties. Bioluminescence imaging further revealed distinct patterns of impaired persistence and dissemination within host tissues, while several mutants displayed increased susceptibility to phagocytic stress in an HL-60-derived neutrophil model. Notably, some regulatory mutants affecting global signaling pathways exhibited impaired tissue dissemination despite retaining resistance to phagocytic stress, indicating the presence of fitness determinants that operate independently of classical surface-associated or cytotoxic traits. Together, these findings demonstrate that V. vulnificus fitness during infection depends on diverse physiological pathways beyond classical virulence factors, highlighting the value of phenotype-centered analyses for understanding bacterial adaptation in host-associated environments.IMPORTANCEVibrio vulnificus causes rapidly progressive wound infections and septicemia, yet the bacterial functions that support fitness within host environments remain incompletely defined. While substantial effort has focused on canonical virulence factors and regulation, increasing evidence suggests that successful infection also depends on broader physiological adaptation. In this study, we link signature-tagged mutagenesis with systematic phenotypic analyses to define physiological determinants of V. vulnificus fitness during host-associated infection. Our results demonstrate that genes involved in motility, regulatory signaling, metabolism, and stress tolerance collectively shape bacterial persistence and dissemination in host tissues. Notably, we identify regulatory and metabolic determinants that influence fitness independently of classical surface-associated or cytotoxic traits, highlighting noncanonical pathways that contribute to pathogenic success. By integrating genome-wide screening with phenotype-centered analyses, this work advances understanding of how physiological adaptation underpins V. vulnificus infection and provides a framework for studying bacterial fitness alongside established concepts of bacterial virulence.
Yamazaki et al. (Mon,) studied this question.