The BAMBI project aims to characterize a new moonlighting protein in Mycobacterium abscessus (Mab), a highly drug-resistant bacterium causing severe pulmonary diseases. We have recently uncovered a novel mechanism facilitating Mab's entry into host cells, relying on the macrophage tetraspanin CD81. Furthermore, we have identified an interaction between CD81 and Mab's SufA protein, an unexpected characteristic for a member of the iron-sulfur (Fe-S) cluster biogenesis protein family.

First, we will investigate at molecular-level the interaction between SufA and CD81, using advanced structural mass spectrometry to explore binding interfaces and conformational changes. Invasion assays will be conducted to understand the role of SufA and the contribution of the residues interacting with CD81 in the uptake of Mab by macrophages.

Second, we propose to thoroughly characterize SufA's role in Fe-S cluster biogenesis, assessing the presence, properties, and transfer of Fe-S clusters within SufA and target proteins.

Third, we will employ the APEX2 proximity labeling technique to determine the SufA’s interaction network under various conditions, shedding light into its dual function in both internalization by host cells and in the Fe-S cluster transport.

Overall, BAMBI will highlight the central role of the SufA/CD81 complex in early host-pathogen interactions, revealing the unexpected function of a Fe-S protein as a bacterial adhesin. Additionally, we delve into SufA's contribution to the biogenesis of Fe-S clusters and their transfer to metalloproteins, providing a comprehensive understanding of its dual functionality. This research will offer insights into SufA's role in pathogenesis and essential cellular processes, bridging proteomics, biochemistry, spectroscopy, microbiology, host-pathogen interactions, and protein-protein interactions, ultimately advancing our understanding of Mab-caused diseases and potential therapeutic strategies.