Perturbing H-NS function reveals roles in restricting virulence heterogeneity and pathogen adaptation.

Xenogeneic silencers, such as histone-like nucleoid structuring protein (H-NS), are critical for maintaining horizontally acquired genes in bacterial genomes and minimizing fitness costs associated with inappropriate expression. For bacterial pathogens, this has enabled the acquisition of costly virulence regulons, with H-NS balancing the need for tight silencing with rapid expression in host environments. For Salmonella enterica serovar Typhimurium (STm), survival in these environments relies on phenotypic heterogeneity in virulence gene expression and evolutionary adaptation. Although complete loss of hns is highly deleterious in STm, how subtle impairments to this global silencer disrupt heterogeneity in virulence gene expression and alter adaptation to host environments remains poorly understood. Here, we identify an STm hns hypomorph strain and find that its reduced H-NS DNA-binding affinity increases the proportion of virulence-expressing cells, resulting in enhanced epithelial cell infection in vitro. Furthermore, through experimental evolution in intracellular-like conditions in vitro, we demonstrate that both wild-type and mutant populations converge on disrupting the SPI-2 virulence regulon to improve fitness; however, the mutant population also acquires distinct adaptive mutations to resolve the underlying dysregulation in gene expression. These results suggest that H-NS sets single-cell virulence activation thresholds and that even minor disruptions to its silencing function impact pathogen adaptation, highlighting its role as a critical evolutionary buffer.