PROJECT SUMMARY
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.
