In vivo imaging of MmpL transporters reveals distinct subcellular locations for export of mycolic acids and non-essential trehalose polyphleates in the mycobacterial outer membrane.

The mycobacterial cell envelope consists of a typical plasma membrane, surrounded by a complex cell wall and a lipid-rich outer membrane. The biogenesis of this multilayer structure is a tightly regulated process requiring the coordinated synthesis and assembly of all its constituents. Mycobacteria grow by polar extension and recent studies showed that cell envelope incorporation of mycolic acids, the major constituent of the cell wall and outer membrane, is coordinated with peptidoglycan biosynthesis at the cell poles. However, there is no information regarding the dynamics of incorporation of other families of outer membrane lipids during cell elongation and division. Here, we establish that the translocation of non-essential trehalose polyphleates (TPP) occurs at different subcellular locations than that of the essential mycolic acids. Using fluorescence microscopy approaches, we investigated the subcellular localization of MmpL3 and MmpL10, respectively involved in the export of mycolic acids and TPP, in growing cells and their colocalization with Wag31, a protein playing a critical role in regulating peptidoglycan biosynthesis in mycobacteria. We found that MmpL3, like Wag31, displays polar localization and preferential accumulation at the old pole whereas MmpL10 is more homogenously distributed in the plasma membrane and slightly accumulates at the new pole. These results led us to propose a model in which insertion of TPP and mycolic acids into the mycomembrane is spatially uncoupled.

T Cell-Mediated Targeted Delivery of Anti-PD-L1 Nanobody Overcomes Poor Antibody Penetration and Improves PD-L1 Blocking at the Tumor Site.

Monoclonal antibodies (mAbs) blocking immune checkpoints such as programmed death ligand 1 (PD-L1) have yielded strong clinical benefits in many cancer types. Still, the current limitations are the lack of clinical response in a majority of patients and the development of immune-related adverse events in some. As an alternative to PD-L1-specific antibody injection, we have developed an approach based on the engineering of tumor-targeting T cells to deliver intratumorally an anti-PD-L1 nanobody. In the MC38-OVA model, our strategy enhanced tumor control as compared with injection of PD-L1-specific antibody combined with adoptive transfer of tumor-targeting T cells. As a possible explanation for this, we demonstrated that PD-L1-specific antibody massively occupied PD-L1 in the periphery but failed to penetrate to PD-L1-expressing cells at the tumor site. In sharp contrast, locally delivered anti-PD-L1 nanobody improved PD-L1 blocking at the tumor site while avoiding systemic exposure. Our approach appears promising to overcome the limitations of immunotherapy based on PD-L1-specific antibodies.

The final assembly of trehalose polyphleates takes place within the outer layer of the mycobacterial cell envelope.

Trehalose polyphleates (TPP) are high-molecular-weight, surface-exposed glycolipids present in a broad range of nontuberculous mycobacteria. These compounds consist of a trehalose core bearing polyunsaturated fatty acyl substituents (called phleic acids) and a straight-chain fatty acid residue and share a common basic structure with trehalose-based glycolipids produced by Mycobacterium tuberculosis TPP production starts in the cytosol with the formation of a diacyltrehalose intermediate. An acyltransferase, called PE, subsequently catalyzes the transfer of phleic acids onto diacyltrehalose to form TPP, and an MmpL transporter promotes the export of TPP or its precursor across the plasma membrane. PE is predicted to be an anchored membrane protein, but its topological organization is unknown, raising questions about the subcellular localization of the final stage of TPP biosynthesis and the chemical nature of the substrates that are translocated by the MmpL transporter. Here, using genetic, biochemical, and proteomic approaches, we established that PE of Mycobacterium smegmatis is exported to the cell envelope following cleavage of its signal peptide and that this process is required for TPP biosynthesis, indicating that the last step of TPP formation occurs in the outer layers of the mycobacterial cell envelope. These results provide detailed insights into the molecular mechanisms controlling TPP formation and transport to the cell surface, enabling us to propose an updated model of the TPP biosynthetic pathway. Because the molecular mechanisms of glycolipid production are conserved among mycobacteria, these findings obtained with PE from M. smegmatis may offer clues to glycolipid formation in M. tuberculosis.