Session: Session 3

Interactome characterisation of a protein involved in lipid transport across the mycobacterial membrane: an XL-AP-MS approach

Marion BEAUPERE¹, Eve RANDRIANARIDERA², Emmanuelle MOUTON-BARBOSA¹, Alexandre STELLA¹, Odile BURLET-SCHILTZ¹, Christian CHALUT¹, Manuelle DUCOUX-PETIT¹

¹IPBS, Toulouse, France
²Institut de Science des matériaux de Mulhouse, Mulhouse, France

Nowadays, tuberculosis, caused by species belonging to the “Mycobacterium tuberculosis complex” (including M. tuberculosis or M. bovis), remains a significant public health problem, both in human and veterinary medicine (World Health Organization 2022). This is partly due to the resistance to antibiotics targeting the activity of some specific compounds in the membrane. This mycomembrane is one of the specific features of mycobacteria because of the cell wall thickness. It is especially rich in various lipids contributing to the organization and integrity of the bacterial shape (Chiaradia et al. 2017).

Therefore, some new therapeutic research is focusing on the interaction between proteins involved in the transport and organization of lipids across the mycomembrane (Bories et al. 2024). However, these proteins remain poorly characterized. Furthermore, study of dynamics and transitory phenomena, such as protein-protein interactions, still presents a challenge for research at an analytical level.

In this study, we focused on a transmembrane protein of Mycobacterium smegmatis, a non-pathogenic strain, involved in lipid transport across the mycomembrane: MmpL10. After stabilizing protein-protein interaction by crosslinking, cytosolic proteins were removed and the mycomembrane was isolated by ultracentrifugation. Then, the proteins complexed with the bait (MmpL10) were enriched by affinity purification, identified and quantified by mass spectrometry.

This interatomic approach allowed us to identify over 1,500 proteins, 48 of which were significantly enriched with the bait, compared to the control. The data highlighted the presence of proteins that could be involved in lipid transport across the mycobacterial membrane. Precise characterization of the protein complexes interacting with MmpL10 may lead to a better understanding of mycomembrane organization and open up new perspectives for therapeutic research.