The goal of our research proposal is to understand the interactions of Pseudomonas aeruginosa (P. aeruginosa) and its virulent type 3 secretion system (T3SS) with autophagy in airway epithelial cells (AECs). AECs provide initial protection against airborne pathogensby acting as a cellular barrier (1). P. aeruginosa is a gram-negative bacterium which can cause pneumonia in immunocompromised patients, and it is known to target AECs (2). T3SS of P. aeruginosa is a virulence mechanism associated with a high rate of mortality in humans, in which the bacterium inject toxins (effector proteins) into the host cells usually leading to cell death (3). Currently, it is not known whether P. aeruginosa is able to escape autophagy or if its T3SS interferes with autophagy. However, it has been demonstrated that P. aeruginosa T3SS can negatively alter other antimicrobial pathways, including phagocytosis (3). Our hypothesis is that P. aeruginosa T3SS inhibits autophagy thereby protecting P. aeruginosa from elimination in AECs. Recently, our lab has obtained a T3SS mutant P. aeruginosa, which cannot inject effector proteins into host cells as well as the WT strain (4). In addition, we have received plasmids encoding some of the P. aeruginosa T3SS effector proteins (5, 6), which are responsible for the T3SS toxicity (5). We will also obtain P. aeruginosa mutants for individual effectors proteins (3, 5). In the long term, we plan to study the potential mechanism of how P. aeruginosa T3SS is able to modulate autophagy and how this regulation affects P. aeruginosa survival in AECs. In order to reach our goal, the following research strategy has been designed: (a) Determine the interactions between P. aeruginosa and autophagy in AECs by analyzing WT and T3SS mutant P. aeruginosa survival during autophagy induction by colony forming unit assay, as well as by evaluating their colocalization with autophagy membranes using both fluorescence and electron microscopy. (b) Investigate whether P. aeruginosa T3SS effector proteins inhibit autophagy and the mechanism by which this occurs.
This aim will be completed by infecting cells with P. aeruginosa mutants for individual T3SS effector proteins or by over-expressing plasmids containing the sequence of T3SS effector proteins and by evaluating autophagy induction or blockage via Western blotting, fluorescence microscopy and co-Immunoprecipitation. (c) Determine the role of autophagy in ciliated AECs during WT and T3SS mutant P. aeruginosa infection in vivo by infecting normal and autophagy deficient in ciliated AECs mice and evaluating bacteria clearance, inflammatory responses and mouse survival. Understanding whether P. aeruginosa T3SS negatively affect autophagy could provide insight into P. aeruginosa pathogenesis and new therapeutic targets to promote the clearance of these bacteria.
The Type 3 secretion system (T3SS) is a Pseudomonas aeruginosa virulence factor which can negatively affect different processes in Airway epithelial cells. We propose that it may also alter the autophagy pathway, and plan to investigate whether Pseudomonas aeruginosa T3SS has a negative effect on autophagy in airway epithelial cells and can protect Pseudomonas aeruginosa from elimination. This will contribute to the understanding of new mechanisms for Pseudomonas aeruginosa survival in the respiratory tract, and could provide insight into new strategies to promote its elimination.