The Transcriptional Regulator HlyU Positively Regulates Expression of exsA , Leading to Type III Secretion System 1 Activation in Vibrio parahaemolyticus

is a marine bacterium that is globally recognized as the leading cause of seafood-borne gastroenteritis. uses various toxins and two type 3 secretion systems (T3SS-1 and T3SS-2) to subvert host cells during infection. We previously determined that T3SS-1 activity is upregulated by increasing the expression level of the master regulator ExsA under specific growth conditions. In this study, we set out to identify genes responsible for linking environmental and growth signals to gene expression. Using transposon mutagenesis in combination with a sensitive and quantitative luminescence screen, we identify HlyU and H-NS as two antagonistic regulatory proteins controlling the expression of and, hence, T3SS-1 in Disruption of leads to constitutive unregulated gene expression, consistent with its known role in repressing transcription. In contrast, genetic disruption of completely abrogated expression and T3SS-1 activity. A null mutant was significantly deficient for T3SS-1-mediated host cell death during infection. DNA footprinting studies with purified HlyU revealed a 56-bp protected DNA region within the promoter that contains an inverted repeat sequence. Genetic evidence suggests that HlyU acts as a derepressor, likely by displacing H-NS from the promoter, leading to gene expression and appropriately regulated T3SS-1 activity. Overall, the data implicate HlyU as a critical positive regulator of T3SS-1-mediated pathogenesis. Many species are zoonotic pathogens, infecting both animals and humans, resulting in significant morbidity and, in extreme cases, mortality. While many species virulence genes are known, their associated regulation is often modestly understood. We set out to identify genetic factors of that are involved in activating gene expression, a process linked to a type III secretion system involved in host cytotoxicity. We discover that employs a genetic regulatory switch involving H-NS and HlyU to control promoter activity. While HlyU is a well-known positive regulator of species virulence genes, this is the first report linking it to a transcriptional master regulator and type III secretion system paradigm.