Molecular dissection of Pseudomonas aeruginosa Exotoxin T virulence functions

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 DESCRIPTION (provided by applicant): Pseudomonas aeruginosa (PA) is the most common cause of hospital-acquired pneumonia and respiratory failure, a leading cause of bacteremia and sepsis in patients receiving cancer drugs, and a killer of immunocompromised patients. PA possesses a number of cell-associated and secreted virulence factors. Chief amongst them is a Type III Secretion System (T3SS) apparatus which functions as a conduit, allowing PA to directly translocate a set of effector exotoxins, namely ExoS, ExoT, ExoU, and ExoY, into the target host cytoplasm where they modify host cellular processes and advance PA infections. ExoT is the only T3SS effector toxin that is present in all P. aeruginosa isolates examined thus far, suggesting a more fundamental role for this virulence factor in PA pathogenesis. Recently, we demonstrated that PA inhibits wound healing in a manner that is primarily dependent on its T3SS effector toxins. We found that ExoT playes a pivotal role in the T3SS-dependent inhibition of wound repair. We and others have further demonstrated that ExoT disrupts the actin cytoskeleton, prevents cell migration, interferes with cell-substratum contacts, and blocks cytokinesis at multiple steps. More recently, we have demonstrated that ExoT blocks the necrotic toxicity associated with T3SS apparatus insertion into target host membrane and reroutes the cell into apoptotic cell death. We propose that although T3SS is essential for PA infection, its activity triggers host inflammatory responses which are harmful to PA pathogenesis. To circumvent these harmful events, we hypothesize that PA employs ExoT as an anti-inflammatory agent to deal with this undesirable side effect of T3SS during infection. In this proposal, we will determine the mechanism(s) by which ExoT inhibits T3SS-induced pro-inflammatory associated cytotoxicity (Aim 1) and determine the role of ExoT as an anti-inflammatory agent in Pseudomonas aeruginosa in the context of wound infection in vivo (Aim 2). Findings from these studies will be essential to define a unique role of ExoT in PA pathogenesis, one that can be exploited for therapeutic intervention to treat PA in the setting of immune compromised patients and/or exploit the powerful ability of ExoT to induce none inflammatory apoptosis for cancer therapy.