Leveraging phenazine cycling to understand maintenance metabolism in Pseudomonas aeruginosa

About This Grant

PROJECT SUMMARY Antibiotic tolerance hinders the treatment of chronic infections of diverse types, contributing to poor patient outcomes and burdening the healthcare system. Underpinning the failure of many conventional drugs to eradicate opportunistic pathogens in these contexts is that they target machinery needed to support rapid growth, yet cells in chronic infections typically exist as slow- or non-growing multicellular aggregates (biofilms). Cells in the biofilm interior experience anoxia yet maintain a low level of metabolic activity. How these cells conserve energy and how they spend energy economically is poorly understood, in part because we have lacked a good experimental system through which to quantify and study maintenance physiology. But thanks to recent technological advances, we now have one. Pseudomonas aeruginosa is an opportunistic pathogen found in chronic infections of the foot (diabetic ulcers), ear and lung (cystic fibrosis) and is a model biofilm-forming bacterium that is notoriously difficult to eradicate with standard antibiotics. A defining aspect of P. aeruginosa is its ability to make phenazines, redox-active pigments that promote biofilm development and energy conservation under anoxia, shifting it into a physiological state that is highly antibiotic tolerant. Phenazines are extracellular electron shuttles that transfer electrons from the cell interior (thereby promoting intracellular redox-balance and permitting ATP to be generated) to the exterior (where they pass electrons to an extracellular oxidant). When the extracellular oxidant is an electrode, the current measured at the electrode reflects the cellular metabolic rate. Using a novel 96-well electrode system, we have demonstrated that we can study phenazine maintenance metabolism under anoxic conditions in high-throughput. The mass-specific power output of this non-growth metabolism is amongst the lowest ever recorded for any organism. We now seek to leverage the unique properties of this phenazine-cycling platform to gain a mechanistic understanding of how maintenance physiology is achieved. What limits the metabolic rate generated by cycling different phenazines over a physiologically relevant concentration range? How do cells economize their anabolic expenditures in a way that harmonizes with their low-power output? Aim 1 will explore what sets the energetic threshold for cellular viability, and how the physicochemical properties of phenazines impact how they are processed by the cell. Aim 2 will test the hypothesis that cells engaged in maintenance metabolism rely on anabolic recycling to economically spend their low energy resources; we predict that many of the key anabolic strategies needed for phenazine maintenance metabolism overlap those needed to survive other growth- arrested, phenazine-independent states, whereas others are specific for phenazine maintenance metabolism. The proposed work will provide us with new insights into the physiology of maintenance, an underexplored yet pervasive biological state, pointing us in the direction of new targets for drug development to treat devastating chronic infections.