Project description:Applying a shotgun proteomics approach, we built a unique database of Brachyspira hyodysenteriae and B. pilosicoli peptides and proteins. A total of 148 LC-MS/MS runs were conducted on an Orbitrap instrument, and more than 29000 different peptides in cell supernatants and partially digested cell suspensions of 3 strains of B. hyodysenteriae and 2 strains of B. pilosicoli were identified. Overall, 16970 and 15493 peptide sequences, which pointed to 1625 and 1338 different protein accessions, were identified in B. hyodysenteriae and B. pilosicoli, respectively. The large-scale characterization of peptides and proteins in the extracellular medium as well as of the exposed proteins on the bacterial surface in these species provides a unique collection of interrelated data on the molecules involved in the interaction of these bacteria with their environment.
Project description:The anaerobic spirochete Brachyspira causes intestinal spirochetosis, characterized by the intimate attachment of bacterial cells to the colonic mucosa, potentially leading to symptoms such as diarrhea, abdominal pain, and weight loss. Despite the clinical significance of Brachyspira infections, the mechanism of the interaction between Brachyspira and the colon epithelium is not known. We characterized the molecular mechanism of the B. pilosicoli-epithelium interaction and its impact on the epithelial barrier during infection. Through a proteomics approach, we identified BPP43_05035 as a candidate B. pilosicoli surface protein that mediates bacterial attachment to cultured human colonic epithelial cells. The crystal structure of BPP43_05035 revealed a globular lipoprotein with a six-bladed beta-propeller domain. Blocking the native BPP43_05035 on B. pilosicoli, either with a specific antibody or via competitive inhibition, abrogated its binding to epithelial cells, which required cell surface-exposed N-glycans. Proximity labeling and interaction assays revealed that BPP43_05035 bound to tight junctions, thereby increasing the permeability of the epithelial monolayer. Extending our investigation to humans, we discovered a downregulation of tight junction and brush border genes in B. pilosicoli-infected patients carrying detectable levels of epithelium-bound BPP43_05035. Collectively, our findings identify BPP43_05035 as a B. pilosicoli adhesin that weakens the colonic epithelial barrier during infection.
