The Gram-negative bacteria, which include Pseudomonas aeruginosa, cause substantial morbidity and mortality: 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 opportunistic bacterial 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 infection transitions from an acute infection to a chronic infection. This transition is hallmarked by the progressive loss, or down-regulation, of the bacterial flagellar swimming motility which can result in the formation of microcolonies or biofilms. Importantly, loss of bacterial motility enables P. aeruginosa to evade phagocytic clearance both in vitro and 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 as chronic infections presents an obvious opportunity and need. Loss of bacterial motility confers resistance to phagocytosis due primarily to decreased bacterial association with the phagocytic cells, e.g. the non-motile bacteria avoid cell-surface interactions with the phagocytes that lead to ingestion. The proposed studies are based on the unprecedented observation that cell surface polyphosphoinositide lipids can promote a 30-fold clearance of non-motile P. aeruginosa. Moreover, this enables non-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 novel mechanism 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 of polyphosphoinositide lipids enable phagocytosis of non-motile P. aeruginosa.
In Specific Aim 2 we provide preliminary data that support the hypothesis that P. aeruginosa interactions with phagocytic cells, in the absence of polyphosphoinositide treatment, are dependent upon endogenous cell-surface polyanions. This further reinforces the proposed mechanism by which polyphosphoinositides induce binding and, importantly, the proposed experiments will identify the cell-surface molecules that mediate bacterial interactions with host cells that result in phagocytosis. Achievement of these Aims will reveal from the host cells the mechanism by which phagocytes preferentially interact with motile bacteria and initiate their clearance while, concomitantly, non-motile P. aeruginosa evade phagocytosis. Additionally, these findings will enable and inform future studies directed at utilization of efficacious polyanions as a novel therapeutic opportunity to eradicate antibiotic-tolerant chronic infections.
Attachment Bacterial infections result in pneumonia, septicemia and chronic respiratory diseases that are major causes of death and mortality in the USA; the rates of infection are even higher in less developed countries. Specific to this grant proposal, Pseudomonas aeruginosa is a bacterial species that continues to cause substantial morbidity and mortality. Thus it is evident and imperative that we develop a better understanding of the host interactions with bacterial pathogens, how bacteria avoid or manipulate the host's response, and to develop new strategies to prevent disease and improve patient care. Therefore this proposal focuses on the identification of novel host/pathogen interactions, and therapeutic interventions into these interactions, that can be clinically utilized to improve public health.
