Heptose 1,7-bisphosphate sensing in gram-negative bacterial infections – HBPsensing

Bacterial infections constitute a major problem for human health. With the increase of antibiotic resistance, there is an urgent need for academic research to better characterize the virulence determinants associated to pathogens and envision new therapeutic strategies that are based on a deep understanding of the host-pathogen interactions involved in infection.
Since innate immunity constitutes the first line of defense against bacteria, the molecular interactions underlying its activation are of particular importance. During infection, bacteria are detected through the recognition of pathogen-associated molecular patterns (PAMPs) by pathogen recognition receptors. Pathogen sensing leads to the secretion of inflammatory cytokines allowing the recruitment of immune cells to sites of infection.
By applying a genome wide RNAi screening strategy, C. Arrieumerlou (coordinator, Institut Cochin) et al. recently identified a new cellular pathway regulating innate immunity during infection by several gram-negative bacteria known as major pathogens for humans. They showed that the proteins ALPK1, TIFA and TRAF6 act sequentially to induce the activation of NF-KB and the secretion of inflammatory cytokines in response to the detection of D-glycero-D-manno-heptose-1,7-bisphosphate (HBP), a newly described PAMP from gram-negative bacteria. HBP is a metabolic intermediate in the lipopolysaccharide (LPS) biosynthesis pathway.
In this context, the HBPsensing proposal aims at characterizing the mechanism of HBP sensing that regulates innate immunity during infection by gram-negative bacteria. As with other PAMPs, we hypothesize that HBP sensing occurs through binding of HBP to (a) receptor(s) and that this recognition triggers a signaling cascade regulating inflammation, and thereby the onset of adaptive immunity. This new challenge of innate immunity will be tackled by a consortium of four teams, three of which at Institut Pasteur, with complementary expertise in chemistry, biophysics, biochemistry, infection biology and imaging.
Our first aim consists in using state-of-the-art multistep chemical synthesis to obtain HBP and several analogues thereof. HBP is not commercially available and its chemical synthesis has not been described so far. Chemically defined HBP and the generation of different analogues will be essential throughout the project to dissect the HBP sensing pathway in absence of bacterial contaminants.
The second aim is the identification of the cellular receptor(s) for HBP. This will first be addressed by RNAi screens and proteomics on ALPK1 binding proteins. If these approaches are not successful, we will capture HBP binding proteins from cell lysates by making use of HBP-based probes harboring a UV-reactive cross-linking group and biotin for purification. The HBP/receptor(s) interaction will then be addressed by biophysical methods.
Our third aim is the molecular characterization of the HBP sensing pathway in epithelial cells and macrophages. In particular, we will investigate the role of ALPK1, an atypical kinase that is poorly described. We will explore the ALPK1/TIFA functional interaction and the role of ALPK1 in the regulation of IL-1b expression.
Finally, our fourth aim is to obtain a spatiotemporal model for the mechanism of HBP sensing during S. flexneri infection of epithelial cells. Here, we will explore how HBP is delivered into host cells and monitor the localization of the main players of the HBP sensing pathway during infection by different imaging modalities, including super-resolution microscopy and correlative light electron microscopy.
The HBPsensing project will reveal a new pathway regulating innate immunity during gram-negative bacterial infections and will provide a novel set of chemical tools to facilitate research in this emerging field. Moreover, the gained knowledge should help identifying new targets for treatments aiming at modulating inflammation in bacterial infections and sepsis.