Investigating the mycomembrane protein assembly pathway

PROJECT SUMMARY The bacterial cell envelope functions as the cell’s primary defense against environmental threats, including antibiotics. Rising levels of resistance to antimicrobial treatments demand the development of new therapeutics that target this formidable barrier. Protein transport to the outer layers of the cell envelope is a high-priority candidate for further therapeutic development as proteins that reside within the outer layers of the cell envelope are critical in mediating interactions between the intra- and extracellular environment. The transport and assembly of proteins that reside within the outer layers of diderm bacterial species is highly regulated due to their importance for cell viability. Gram-positive microbial species in the Mycobacteriales order contain a protein- rich outer layer called the mycomembrane (MM) that is functionally analogous to the Gram-negative outer membrane. However, despite this diderm organization, the lipid composition and proteins that reside within the MM are structurally distinct from Gram-negative bacteria. Additionally, Gram-negative outer membrane protein transport pathways are not conserved and the mechanism through which mycolate outer membrane proteins (MOMPs) are transported to the MM is an outstanding question in the field. By cross-referencing my preliminary datasets from a FACS-based Tn-seq screen and immunoprecipitation for interaction partner with a surface- exposed MOMP, I hypothesize that a family of redundant enzymes called mycoloyltransferases are MOMP assembly factors. I will validate binding between mycoloyltransferases and MOMPs, characterize the binding interfaces, and utilize a spheroplast-based transport assay to test the hypothesis that MOMPs are transported across the cell envelope by mycoloyltransferases. Additionally, I have demonstrated through mass spectrometry and immunoblotting that, in addition to the previously described lipidation of these proteins, MOMPs undergo an additional post-translational modification that I propose is glycosylation. I will investigate the identity of the sugar modification, determine the residues that are glycosylated, and identify the responsible glycosyltransferase. Finally, I will investigate the dynamics of MOMPs within the MM using a surface exposed tag on a model MOMP in relation to other layers of the cell envelope by fluorescence microscopy. The results of this work will elucidate the essential process of MM biogenesis in the Mycobacteriales suborder, identifying novel targets for antibiotic development against pathogenic members such as Mycobacterium tuberculosis. The Specific Aims of this K99 application are: Aim 1: Investigate mycomembrane protein assembly factors Aim 2: Characterize mycomembrane protein post-translational modifications Aim 3: Analysis of in vivo global dynamics of proteins in the mycomembrane