Elucidating factors affecting H-NS-mediated regulation of horizontally acquired genes in Vibrio cholerae

Horizontal gene transfer amongst bacteria plays a critical role in the evolution, emergence, and virulence of both currently recognized and novel pathogens. Vibrio cholerae exemplifies this process, as benign environmental isolates emerge as pandemic pathogens through the acquisition and incorporation of genetic elements encoding virulence factors into their progenitor genomes. In V. cholerae, these genes are localized to distinct areas of the chromosome, known as horizontally acquired islands (HAIs), which are characterized by a lower GC-content than the progenitor genome. In cholera and many other Gram-negative enterics, AT-rich DNA is bound by the H-NS protein immediately upon entrance into the cell, silencing the expression of this potentially toxic DNA. In other enterics, some proteins can remove H-NS from the DNA, allowing the cell to probe these novel genes, while others can interact with H-NS to maintain regulatory control and ensure derepression only happens when appropriate. However, such interactions with H-NS have not been observed in V. cholerae. Here, we discover and characterization of the genetic relationships between H-NS and an H-NS antagonist (ToxR, an essential protein for virulence), as well as an H-NS enhancer (TsrA, a poorly understood protein) in V. cholerae. ToxR is known to be a master activator of virulence, but we demonstrate that ToxR’s critical role is not direct activation, but rather to antagonize H-NS binding at shared binding loci related to host colonization and biofilm formation. Furthermore, TsrA, previously shown to be involved in regulation of type-VI secretion, was detected through immunoprecipitation of H-NS, followed by LC-MS/MS. Subsequent genetic analyses revealed that TsrA enhances H-NS repression of virulence genes on HAIs. Interestingly, TsrA enhances H-NS enrichment at areas of low-GC content, similar to the H-NS interactors in other enterics that V. cholerae was thought to lack. TsrA deletion also affects uptake of a mobile genetic element, establishing the first Vibrio-specific modulator of H-NS function that influences the regulation and acquisition of virulence-defining genetic elements. Knowledge of these interactions sheds light on HNS’ role in defining the virulence potential of V. cholerae and reveals a novel H-NS interactor similar to those present in other Gram-negative bacteria