Promoting the Phagocytic Clearance of Non-Motile P. Aeruginosa

Project Leader

Brent L. Berwin, Ph.D.
Associate Professor of Microbiology and Immunology
Geisel School of Medicine at Dartmouth

Co-Investigator

Scott Gerber, Ph.D.
Associate Professor of Genetics and Biochemistry
Geisel School of Medicine at Dartmouth

A well-characterized, but poorly understood, observation is that serial Pseudomonas aeruginosa (Pa) isolates taken from chronically infected Cystic Fibrosis (CF) patients progressively lose, or down-regulate, their flagellar swimming motility and become sessile during the course of infection. The observed transition of Pa from a motile lifestyle to a sessile lifestyle is thought to be necessary for the establishment of chronic infection and is a critical initial step toward subsequent antibiotic-tolerant biofilm formation. Importantly, there is currently no clinical treatment that effectively eradicates chronic Pa infections from CF patients, and this is likely linked to the resistance of sessile Pa to phagocytic and antibiotic-mediated clearance.

Our fundamental, published observation that forms the basis for these studies is that loss of flagellar motility by Pa, independent of flagellar expression, confers resistance to phagocytosis. We have identified a phagocytic pathway, dependent upon PI3K/Akt activity, which is specifically activated in response and proportionally to Pa flagellar motility. Importantly, our data support that PI3K/Akt activity is a critical bottleneck for phagocytic uptake of Pa: activation of Akt with exogenous PIP3 promotes phagocytic clearance of non-motile Pa. The rationale for the proposed studies is that a mechanistic understanding of the phagocytic pathways engaged through flagellar motility and evaded by non-motile bacteria, will contribute to the rational design of innovative and effective interventions for chronic Pa infections.

We will:

(1) Test the hypothesis that activation of phagocytic pathways will enable clearance of non-motile P. aeruginosa. We have identified that the PI3K/Akt pathway and the c-Abl pathway in phagocytes (macrophages and PMN) are activated in response to Pa flagellar motility and are required for phagocytosis of Pa. Therefore we hypothesize that activation of the Akt and c-Abl pathways will increase phagocytic uptake of non-motile Pa. We will test this hypothesis utilizing non-motile clinical isolates of Pa from CF patients, and will use small-molecule activators of Akt and c-Abl to provide the first proof-of-principle test of how druggable activation of these pathways promotes phagocytic clearance of non-motile Pa.

(2) Test the hypothesis that CIN85 regulates the Pa motility-induced activation of PI3K. In collaboration between the Berwin and Gerber labs, we employed phospho-proteomic analyses to identify that CIN85, a molecular scaffold of signaling complexes, is required for hagocytic uptake of Pa. We will employ mass spectrometry to simultaneously: A) test the specific hypothesis that phagocyte engagement with motile Pa induces CIN85 to sequester the regulatory subunit of PI3K thereby de-repressing PI3K activity and promoting phagocytosis; and B) provide an unbiased approach to determine how motility-dependent signaling alters the composition of the CIN85 molecular complex, thereby providing both new insights into the phagocytic mechanism and providing novel molecular targets by which to induce the phagocytic clearance of Pa.

Publications
Brent Berwin: Pubmed
Scott Gerber: Pubmed