Mechanisms of Phagocyte Interactions with Pseudomonas Mediated by Polyanions

Abstract/Project Summary The Gram-negative bacteria, which include Pseudomonas aeruginosa, cause substantial morbidity andmortality: bacterial pneumonia, septicemia and chronic disease account for ~15% of the total deaths in the USA,and neutropenic patients including those undergoing cancer therapies are especially susceptible to opportunisticbacterial infection. Of particular concern is the ongoing inability to eradicate chronic infections. A well-characterized example of this is P. aeruginosa for which there is no effective clinical therapy once the infectiontransitions from an acute infection to a chronic infection. This transition is hallmarked by the progressive loss, ordown-regulation, of the bacterial flagellar swimming motility which can result in the formation of microcolonies orbiofilms. Importantly, loss of bacterial motility enables P. aeruginosa to evade phagocytic clearance both in vitroand in vivo and to achieve antibiotic-tolerance that exceeds standard-of-care clinical treatments. Therefore,enabling the elimination of the non-motile P. aeruginosa that, despite our best current treatments, persist aschronic infections presents an obvious opportunity and need. Loss of bacterial motility confers resistance to phagocytosis due primarily to decreased bacterial associationwith the phagocytic cells, e.g. the non-motile bacteria avoid cell-surface interactions with the phagocytes thatlead to ingestion. The proposed studies are based on the unprecedented observation that cell surfacepolyphosphoinositide lipids can promote a 30-fold clearance of non-motile P. aeruginosa. Moreover, this enablesnon-motile P. aeruginosa to be phagocytosed by a mechanism previously uniquely accessed by motile P.aeruginosa. This finding guides the central hypothesis of this proposal: that we have identified a novelmechanism by which to induce the phagocytic clearance of non-motile P. aeruginosa by human neutrophils.Thus, the proposed studies in Specific Aim 1 focus on identification of the mechanism by which addition ofpolyphosphoinositide lipids enable phagocytosis of non-motile P. aeruginosa. In Specific Aim 2 we providepreliminary data that support the hypothesis that P. aeruginosa interactions with phagocytic cells, in the absenceof polyphosphoinositide treatment, are dependent upon endogenous cell-surface polyanions. This furtherreinforces the proposed mechanism by which polyphosphoinositides induce binding and, importantly, theproposed experiments will identify the cell-surface molecules that mediate bacterial interactions with host cellsthat result in phagocytosis. Achievement of these Aims will reveal from the host cells the mechanism by whichphagocytes preferentially interact with motile bacteria and initiate their clearance while, concomitantly, non-motileP. aeruginosa evade phagocytosis. Additionally, these findings will enable and inform future studies directed atutilization of efficacious polyanions as a novel therapeutic opportunity to eradicate antibiotic-tolerant chronicinfections.