Complete Title of Thesis:
"The PhoPQ two-component regulatory system: At the crossroads of nitrosative stress and Salmonella pathogenesis"
Prepared under the direction of Andrés Vázquez-Torres, D.V.M., Ph.D.
Nitric oxide (NO) and the reactive nitrogen species (RNS) derived from this diatomic radical are critical members of the antimicrobial armamentarium of mammalian hosts. Gastric RNS arise from the decomposition of salivary nitrite (NO2-) in acidic milieu of the stomach, while professional phagocytes produce RNS through the enzymatic activity of the inducible nitric oxide synthase (iNOS). Because Salmonella enterica is exposed to RNS in these two anatomical locations, I attempted to identify targets of RNS that mediate host defense against Salmonella enterica serovar Typhimurium. Using the genetically tractable acid tolerance response (ATR) that protects Salmonella from extreme acidity, I identified the PhoPQ two-component regulatory system as a critical target of RNS. RNS were found to antagonize PhoPQ-dependent gene transcription, thereby rendering Salmonella hypersensitive to acid stress. My data provide a mechanism by which RNS synergize with gastric acidity to form a formidable barrier against infection.
Since Salmonella encounters similar RNS in both the gastric lumen and activated macrophages, I investigated whether the PhoPQ signal transduction pathway is also targeted in IFNγ-stimulated macrophages. These studies revealed that RNS produced by activated phagocytes repress intracellular PhoP-activated gene transcription, thereby abrogating the intracellular survival advantage associated with the PhoPQ two-component regulatory system. RNS also repress the transcription of the Salmonella pathogenicity island 2 (SPI2) type III secretion system that is partially controlled by PhoPQ. The majority of intracellular SPI2 transcription occurs, nonetheless, independently of PhoPQ, suggesting that RNS target hitherto unidentified signaling pathways contributing to SPI2 transcription. Overall, the RNS-dependent inhibition of PhoPQ signaling and the SPI2 type III secretion system underlie most of the anti-Salmonella activity of IFNγ-primed macrophages. Despite being a major target of RNS generated by activated macrophages PhoPQ is also the most important factor to be identified for the protection of Salmonella from innate oxidative stress arising from the iNOS hemoprotein expressed in the innate response of professional phagocytes.
Collectively, this thesis underscores the importance of the PhoPQ two-component regulatory system in the defense of Salmonella against innate responses associated with the enzymatic activity of iNOS, while serving as a major target of RNS generated under acidic conditions typical of the gastric lumen or in acidified phagosomes of IFNγ-primed macrophages.