NrtR Regulates the Type III Secretion System Through cAMP/Vfr Pathway in Pseudomonas aeruginosa

Abstract

The type III secretion system (T3SS) plays an important role in the pathogenesis of <i>Pseudomonas aeruginosa</i>. Expression of the T3SS is controlled under a complicate regulatory network. In this study, we demonstrate that NrtR (PA4916) is involved in the T3SS expression and pathogenesis of <i>P. aeruginosa</i> in a mouse acute pneumonia model. Overexpression of the T3SS central activator ExsA or exogenous supplementation of cAMP restored the expression of T3SS in the Δ<i>nrtR</i> mutant, suggesting that NrtR might regulate T3SS through the cAMP-Vfr signaling pathway. Further experiments demonstrated that the decrease of cAMP content is not due to the expression change of adenylate cyclases or phosphodiesterase in the Δ<i>nrtR</i> mutant. As it has been shown that <i>nadD2</i> is upregulated in the Δ<i>nrtR</i> mutant, we overexpressed <i>nadD2</i> in wild type PAK, which reduced the intracellular cAMP level and the expression of the T3SS genes. Meanwhile, deletion of <i>nadD2</i> in the Δ<i>nrtR</i> mutant restored the expression and secretion of the T3SS. Co-immunoprecipitation assay revealed an interaction between NadD2 and the catalytic domain of the adenylate cyclase CyaB. Further <i>in vitro</i> assay indicated that NadD2 repressed the enzymatic activity of CyaB. Therefore, we have identified a novel regulatory mechanism of T3SS in <i>P. aeruginosa</i>.