A Novel Periplasmic Protein involved in the Mannan Chain Elongation Step of Lipomannan and Lipoarabinomannan Biosynthesis in Mycobacterium smegmatis

Mycobacteria are atypical bacteria possessing unusual cell envelopes comprised of an outer membrane, covalently linked to an arabinogalacatan-peptidoglycan structure via waxy mycolic acids, in addition to the conventional inner membrane. This thick and highly impermeable cell envelope is a major deterrent to antibiotic treatment of clinically relevant mycobacterial pathogens, including <em>Mycobacterium tuberculosis </em>(<em>Mtb</em>), which infects a third of the world’s population and kills millions each year. Thus, the regulation of mycobacterial cell envelope biosynthesis is of great interest for the development of more effective therapeutics for treating <em>Mtb</em> infections. Using the model organism <em>Mycobacterium smegmatis</em> (<em>M. smegmatis</em>), we identified a novel protein, Spe2, with an unknown role in the biosynthesis of the cell envelope glycolipids lipomannan (LM) and lipoarabinomannan (LAM). Based on the observation that Δ<em>spe2</em> mutants produce truncated LM/LAM, I speculated Spe2 might enhance the elongation of these products. Here, I use biochemical assays to show Spe2 is localized to the periplasm where it can directly interact with the LM/LAM biosynthetic pathway. I further utilize a genetic approach to demonstrate that Spe2 acts at the stage in which the mannosyltransferase MptA mediates periplasmic LM elongation. Moreover, native polyacrylamide gel electrophoresis (PAGE) and co-immunoprecipitation techniques failed to reveal Spe2 protein binding partners. Together, these data suggest Spe2 is a periplasmic protein involved in regulating LM/LAM biosynthesis, perhaps through direct interactions with LM intermediates.