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.