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.
