Pseudomonas aeruginosa is an opportunistic pathogen found in acute infections (burns, wounds, ventilator associated pneumonia, eye infections) and chronic infections of the foot (diabetic ulcers) and lung (cystic fibrosis) (1). This bacterium commonly survives in these contexts as a biofilm, the formation and high-level antibiotic tolerance of which interferes with effective patient treatment. The hypoxic/anoxic, slowly-growing biofilm core defines the subpopulation that is most tolerant of conventional drugs (2, 3). Yet few therapies currently exist that target this recalcitrant group. Chronic nonhealing wounds typically contain biofilms comprising facultative anaerobes such as P. aeruginosa, and affect millions of people annually worldwide (4). Diabetic foot ulcers alone contribute to 80% of nontraumatic lower-extremity amputations and are associated with 5-year mortality rates of 43-55%, higher than Hodgkin's disease, breast cancer or prostate cancer (5). Nar-mediated nitrate respiration is a widespread anaerobic metabolism used by bacterial pathogens (6); mammals lack a Nar homolog (7). Our recent in vitro work shows that chlorate specifically kills hypoxic/anoxic biofilm subpopulations by co-opting Nar activity to produce a toxic product (8). The overall objective of this proposal is to better understand the mechanism of chlorate toxicity and assess its utility as a pro-drug in a chronic wound diabetic mouse model. Our hypothesis is that chlorate kills anoxic biofilm subpopulations because it triggers protein aggregation, that biofilm development in vivo depends on Nar expression, and, therefore, that topical application of chlorate will facilitate wound healing. Our hypothesis has been formulated based on preliminary data that showed chlorate exposure upregulates genes involved in protein folding and that P. aeruginosa develops into biofilm aggregates in the mouse model with dimensions that would be expected to comprise hypoxic/anoxic subpopulations expressing Nar.
In Aim 1, we will use targeted biochemical approaches to test the hypothesis that chlorate toxicity is linked to protein aggregation, and an un- biased genetic Tn-seq screen to identify determinants of chlorate resistance under nitrate-replete conditions.
In Aim 2, we will assess whether P. aeruginosa expresses Nar in vivo, and whether its expression is required for biofilm development and can be hijacked by chlorate to kill antibiotic tolerant biofilm subpopulations, thus accelerating wound healing. The proposed research is significant because it will provide new basic understanding of the mechanisms underpinning chlorate toxicity, and allow us to better evaluate chlorate's potential to be an effective pro-drug to target P. aeruginosa biofilms in the context of infection. The long-term outcomes generated by this research are likely to provide insights that may be relevant to developing chlorate as a novel treatment for polymicrobial biofilms in disparate infections.
This proposal seeks to understand how chlorate harms Pseudomonas aeruginosa and whether chlorate can be used to help clear P. aeruginosa biofilm infections. Specifically, our studies will give us a basic understanding of the molecular mechanisms whereby chlorate treatment induces cellular damage, how P. aeruginosa may resist it, and if P aeruginosa-infected chronic wounds are sensitive to chlorate. The work proposed here will provide the foundation of knowledge required to critically assess the potential of chlorate to serve as a novel pro-drug to treat P. aeruginosa infections.