Chronic wounds impact ~6.5M people and cost ~$25B/year in the US alone. Despite significant effort, understanding the mechanisms involved in development of chronic wounds in humans has met with limited success, primarily because we cannot experiment in human chronic wounds and because current animal models are inadequate. We have developed a novel mouse model for diabetic chronic wounds that closely mimics those of humans. High levels of oxidative stress (OS) are important for chronic wound development. Human chronic wounds have high levels of OS. Using diabetic mice, we can generate chronic wounds 100% of the time by creating high levels of OS immediately after wounding by treating with inhibitors specific to two antioxidant enzymes. The wounds become fully chronic within 20 days after treatment and remain chronic until the mouse dies, sometimes >100 days. The wounds in the mouse model feature all of the same problems observed in human chronic diabetic wounds: high levels of OS lead to DNA damage, gene deregulation, protein and lipid damage, cell death, impaired keratinocyte migration (potentially inhibiting re-epithelialization), chronic inflammation, lack of proper angiogenesis and matrix deposition. Equally important, the chronic wounds in the mouse model develop a biofilm from the bacteria present on the skin microbiome by elimination of non-biofilm-forming bacteria in favor of the biofilm-forming species. These biofilm-forming bacteria are also present on human skin and appear in human diabetic chronic wounds. All of these characteristics indicate that the PI's mouse model mimics key aspects of human chronic wounds. We hypothesize that high OS levels affect the microenvironment of the wound resulting in expression of genes that combat OS, that are involved in adhesin and expression of quorum sensing molecules and virulent factors that favor biofilm development by P. aeruginosa. To test this hypothesis, we will:
Aim#1 : Isolate a pure culture of PA from the biofilms in our chronic wound mouse model and sequence its genome. We already isolated PA from one such wound.
Aim#2 : Using RNAseq, perform experiments in sterile wounds infected with the isolated P.A alone in the presence or absence of high OS and: A. Determine whether P.A genes known to be involved in response to high levels of OS, adhesion to surfaces, production of quorum sensing molecules and virulence factors in vitro are also expressed in in vivo in the CW bed during the transition of PA from non-biofilm-forming in the skin microbiome to biofilm-forming in the CW. B. Identify new genes that are expressed by PA in the wound bed versus abiotic surfaces, and if time permits or with future funding determine whether they may be important in the transition of PA to biofilm forming. Our proposal is significant and innovative because with the use of our novel db/db-/- chronic wound model, we will determine how P.A becomes biofilm-forming in the high OS environment of a chronic wound. We will also identify P.A molecules that contribute to biofilm development by this bacterium in the wound bed. Most importantly, our work will impact health care because it will potentially identify biomarkers that are critical for initiation of biofilm development by P.A in diabetic wounds. Such biomarkers have the potential, when verified in humans, to objectively guide treatment after debridement to prevent return of biofilm. Currently, wound bed assessment is subjective.
Chronic wounds impact ~6.5M people and cost ~$25B/year in the US alone and, despite significant effort, development of chronic wounds in humans has met with limited success, primarily because we cannot experiment in humans and because current animal models are inadequate to study initiation and development of chronicity. Using our novel and unique model of chronic wounds, we propose to identify Pseudomonas aeruginosa genes that are involved in this bacterium changing from non-biofilm forming in the skin microbiome to biofilm forming in the presence of high oxidative stress in the chronic wound bed. This information will serve as the basis for mechanistic studies to be done with further funding.
