A MARTX Toxin rtxA Gene Is Controlled by Host Environmental Signals through a CRP-Coordinated Regulatory Network in Vibrio vulnificus

Abstract

A multifunctional autoprocessing repeats-in-toxin (MARTX) toxin plays an essential role in the virulence of many pathogens, including a fulminating human pathogen Vibrio vulnificus. H-NS and HIyU repress and derepress expression of the MARTX toxin gene rtxA in V. vulnificus, respectively. However, little is known about other regulatory proteins and environmental signals involved in rtxA regulation. In this study, we found that a leucine-responsive regulatory protein (Lrp) activates rtxA by binding directly and specifically to the rtxA promoter, P-rtxA. Phased hypersensitivity resulting from DNase I cleavage of the P(rtxA )regulatory region suggests that Lrp probably induces DNA bending in P-rtxA. Lrp activates independently of H-NS and HlyU, and leucine inhibits Lrp binding to PavA and reduces the Lrp-mediated activation. Furthermore, a cyclic AMP receptor protein (CRP) represses P-rtxA, and exogenous glucose relieves the CRP-mediated repression. Biochemical and mutational analyses demonstrated that CRP binds directly and specifically to the upstream region of P(rtxA )which presumably alters the DNA conformation in P-rtxA and thus represses rixA. Moreover, CRP represses expression of Irp and hlyU by binding directly to their upstream regions, forming coherent feed-forward loops with Lrp and HIyU. In conclusion, expression of rtxA is controlled by a regulatory network comprising CRP, Lrp, H-NS, and HlyU in response to changes in host environmental signals such as leucine and glucose. This collaborative regulation enables the elaborate expression of rtxA, thereby enhancing the fitness and pathogenesis of V. vulnificus during the course of infection. IMPORTANCE A MARTX toxin, RtxA, is an essential virulence factor of many pathogens, including Vibrio species. H-NS and HIyU repress and derepress, respectively, rtxA expression of a life-threatening pathogen, Vibrio vulnificus. We found that Lrp directly activates rtxA independently of H-NS and HIyU, and leucine inhibits the Lrpmediated activation of rtxA. Furthermore, we demonstrated that CRP represses rtxA but derepresses in the presence of exogenous glucose. CRP represses rtxA not only directly by binding to upstream of rtxA but also indirectly by repressing lrp and hlyU. This is the first report of a regulatory network comprising CRP, Lrp, H-NS, and HlyU, which coordinates the rtxA expression in response to environmental signals such as leucine and glucose during infection. This elaborate regulatory network will enhance the fitness of V. vulnificus and contribute to its successful infection within the host.