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Host-Pathogen Interaction in Pseudomonas aeruginosa Infection

Vareechon, Chairut Charles

Abstract Details

, Doctor of Philosophy, Case Western Reserve University, Pathology.
Pseudomonas aeruginosa is a common opportunistic pathogen that is a major cause of acute infections, such as hospital-acquired pneumonia, blood stream infections, and microbial keratitis. P. aeruginosa relies on a type III secretion system (T3SS) to directly inject effector proteins into the cytoplasm of targeted host cells. These effectors paralyze normal cellular functions, thereby enabling successful establishment of infection. To date four effector proteins have been described in P. aeruginosa: ExoS, ExoT, ExoU, and ExoY. We investigated the molecular mechanisms by which ExoS and ExoT impair neutrophil killing. Furthermore, we studied the role of ExoU in P. aeruginosa keratitis infection. Neutrophils are the first responders in bacterial infections and are the primary target of injection by the T3SS in early stages of P. aeruginosa infections. Injection of ExoS and ExoT promotes survival of P. aeruginosa murine keratitis, as well as in neutrophils in vitro. Using peripheral blood human neutrophils from healthy volunteers, we found that the injection of ExoS or ExoT into the cytoplasm of neutrophils result in inhibition of reactive oxygen species (ROS) production. We demonstrate that P. aeruginosa targets the Ras-mediated PI3K signaling cascade that is responsible for the assembly of NADPH oxidase complex which leads to ROS production. Specifically, in human neutrophils, ExoS and ExoT, prevent the phosphorylation of the PI3K associated regulatory kinase Akt and the cytosolic NADPH oxidase component p40phox thereby rendering both inactive and preventing ROS generation. Importantly, in a murine model of corneal infection and in vitro, preventing ROS production by neutrophils lead to increased survival of P. aeruginosa. Our in vitro studies revealed that ExoS targets Ras for ADP-ribosylation in human neutrophils. ExoS had been shown previously to ADP-ribosylate Ras in epithelial cells at either Arg41 or Arg128. Intracellular delivery of a mutated Ras (R41K), which is unable to be ribosylated at Arg41, rescued ROS production in neutrophils infected with P. aeruginosa. This increase in ROS production was accompanied by a decrease in intracellular survival of P. aeruginosa in human neutrophils harboring Ras (R41K). Among the effector proteins, ExoU contributes the most to disease severity, in both the clinic and P. aeruginosa infection models. ExoU is encoded on a genomic island, provoking the notion that additional virulence factors found on the island may also contribute to the virulence of ExoU-producing strains. Using a murine model of keratitis, we found that ExoU expression lowers the minimal dose required to cause corneal disease. In addition, we found that other genes on the exoU-genomic island of strain 19660 contribute to the severity of the infection. In conclusion, our data indicae that P. aeruginosa utilizes its T3SS to inject ExoS into the neutrophil cytoplasm which directly targets Ras. Riboyslation of Ras at Arg41 leads to the inhibition of ROS production and, therefore, increased intracellular survival within the neutrophil. We also demonstrate that exoU and the genes on the ExoU island increases the virulence of strain 19660.
Eric Pearlman, PhD (Advisor)
Arne Rietsch, PhD (Advisor)

Recommended Citations

Citations

  • Vareechon, C. C. (n.d.). Host-Pathogen Interaction in Pseudomonas aeruginosa Infection [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1499427972888735

    APA Style (7th edition)

  • Vareechon, Chairut. Host-Pathogen Interaction in Pseudomonas aeruginosa Infection. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1499427972888735.

    MLA Style (8th edition)

  • Vareechon, Chairut. "Host-Pathogen Interaction in Pseudomonas aeruginosa Infection." Doctoral dissertation, Case Western Reserve University. Accessed MARCH 28, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=case1499427972888735

    Chicago Manual of Style (17th edition)