Developing an in situ assay for determining spatial and temporal autoinducer concentrations in wound biofilms
EMSL Project ID
27692
Abstract
Most living organisms possess sophisticated cell-signaling networks in which lipid-based signals modulate hundreds of biological effects such as cell differentiation, reproduction and immune responses. Mammals use lipid-based hormones for cell signaling, and many pathogenic eukaryotic microbes make similarly structured compounds called eicosanoids. Autoinducers are fatty acid-based signaling molecules synthesized by several Gram-negative bacteria that are used to coordinate gene expression in a process termed quorum sensing (QS). QS signaling pathways have been delineated in many species of bacteria including Pseudomonas aeruginosa. P. aeruginosa uses autoinducers to control the expression of many genes involved in pathogenesis. Recent evidence shows that autoinducers not only control gene expression in bacterial cells, but also alter gene expression in mammalian cells. These alterations include up-regulation of pro-inflammatory cytokines and induction of apoptosis, which may have deleterious effects on the host's immune response, leading to increased pathogenesis of the bacterium. Considering eons of prokaryotic/eukaryotic cohabitation we postulate that pathways have evolved that allow interkingdom signaling between bacteria and eukaryotes and that autoinducers, which exhibit structural resemblance to mammalian hormones, are excellent candidates for mediating this interkingdom communication. We have recently shown that P. aeruginosa autoinducers elicit similar effects on mammalian cells as do some mammalian hormones, including up-regulation of cyclooxygenase and other immune modulators, mobilization of intracellular calcium and eventual cell death. Additionally, utilizing chimeric transcriptional activators based on P. aeruginosa autoinducer receptors we showed that P. aeruginosa autoinducers can enter and function in mammalian cells. However, one major question that remains is what concentration due autoinducers reach in vivo. This information is crucial considering that in cell culture-based assays autoinducers have a wide range of effects on different mammalian cells at different concentrations. Our proposed project will attempt to elucidate the overall concentrations of P. aeruginosa autoinducers within infected mouse tissue. In order to determine in vivo autoinducer concentrations, we will utilize a mouse acute infection model. We have used this model in many studies for almost a decade and published many papers on our findings. Recently we have used this mouse model to demonstrate that P. aeruginosa forms biofilms within the wounded skin 1. In this proposed study wounded and normal skin sections will be removed from mice, placed in humid chambers and sent by overnight carrier to PNNL. Upon receipt, the samples will immediately be transferred to the sample chamber (with or without perfusion) for measurements. Hydrogen-containing metabolite concentrations will be measured for the wounded and control sections, both average and depth resolved. These data will be used to reconstruct difference NMR spectra (wounded minus control). The resulting difference spectra will be assigned to determine the metabolic changes. This will include the determination of the detectability of P. aeruginosa autoinducers for which published NMR spectra exist.
Project Details
Project type
Exploratory Research
Start Date
2008-03-06
End Date
2009-03-08
Status
Closed
Released Data Link
Team
Principal Investigator