Thursday, May 9, 2024
5/9/2024
PROJECT SUMMARY Metabolism is a well-established regulator of innate immune responses. Previous literature examined metabolic changes upon activation of dendritic cells (DCs) by pathogen associated molecular patterns (PAMPs) from planktonic (free-living) bacteria, like the LPS, while little is still known about the metabolic reprogramming induced by bacterial biofilms. Most bacterial pathogens thrive in biofilms, multicellular communities of bacteria. Biofilms are potent virulence factors associated to recurrent and chronic infections and are resistant to DNAses, proteases and even to antibiotics, providing a formidable challenge for clinical treatment. We have recently reported that a bacterial amyloid termed curli, a dominant component in E. coli and Salmonella biofilms, forms a natural complex with bacterial DNA and is a potent PAMP, activating DCs and macrophages. The objective of this exploratory project is to determine whether DCs acquire a specific metabolic profile upon recognition of biofilm-specific PAMPs vs. PAMPs expressed also by planktonic (free-living) bacteria, with the long-term goals of 1) understanding whether DCs mount distinct responses during biofilm-driven infections vs. planktonic infections, 2) discovering new therapeutics to improve host defense. We hypothesize that curli/DNA induce in inflammatory DCs a specific reprogramming that promotes innate and adaptive immune responses against biofilms. This project will provide the foundation to study murine and human innate responses to biofilms. In Aim 1, we will perform high resolution mass spectrometry to comprehensively profile the metabolic changes that occur in DCs upon recognition of bacterial amyloid curli/DNA. We will compare these results with better-known PAMPs LPS, CpG and Pam3CSK4, the latter two triggering the same TLR2/TLR9 that recognize curli/DNA. We will test murine GM-CSF-monocyte-like DCs and human monocyte-derived DCs, as models of inflammatory DCs. To confirm the metabolomics results, changes in the main metabolic pathways will be followed using stable isotope tracing, the Seahorse Flux Analyzer, and biochemistry assays. In Aim 2, we will analyze the transcriptome and cytokine secretome to identify the molecular pathways associated with the metabolic reprogramming stimulated in DCs by bacterial amyloids and characterize the induced innate response. We will compare these results with those from DCs exposed in vitro to bacterial biofilms. To start dissecting in depth the underlying molecular mechanisms, we will study the purine salvage pathway, a metabolic pathway novel in DC biology that was suggested by our Preliminary studies. This project will explore the innate immune response against bacterial biofilms through the recognition of the most stimulatory component, the bacterial amyloids. It will indicate which metabolic pathways are candidates as therapeutic targets to modulate immune responses to biofilms, starting with the purine salvage pathway, an understudied pathway in DC biology, to strengthen innate responses against biofilm-driven infections.
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