Lipoarabinomannan regulates septation in <i>Mycobacterium smegmatis</i>

Abstract The growth and division of mycobacteria, which include several clinically relevant pathogens, deviate significantly from that of canonical bacterial models. Despite their Gram-positive ancestry, mycobacteria synthesize and elongate a diderm envelope asymmetrically from the poles, with the old pole elongating more robustly than the new pole. In addition to being structurally distinct, the molecular components of the mycobacterial envelope are also evolutionarily unique, including the phosphatidylinositol-anchored lipoglycans lipomannan (LM) and lipoarabinomannan (LAM). LM and LAM modulate host immunity during infection, but their role outside of intracellular survival remains poorly understood, despite their widespread conservation among non-pathogenic and opportunistically pathogenic mycobacteria. Previously, Mycobacterium smegmatis and Mycobacterium tuberculosis mutants producing structurally altered LM and LAM were shown to grow slowly under certain conditions and to be more sensitive to antibiotics, suggesting that mycobacterial lipoglycans may support cellular integrity or growth. To test this, we constructed multiple biosynthetic lipoglycan mutants of M. smegmatis and determined the effect of each mutation on cell wall biosynthesis, envelope integrity, and division. We found that mutants deficient in LAM, but not LM, fail to maintain cell wall integrity in a medium-dependent manner, with envelope deformations specifically associated with septa and new poles. Conversely, a mutant producing abnormally large LAM formed multiseptated cells in way distinct from that observed in a septal hydrolase mutant. These results show that LAM plays critical and distinct roles at subcellular locations associated with division in mycobacteria, including maintenance of local cell envelope integrity and septal placement. Significance Mycobacteria cause many diseases including tuberculosis (TB). Lipoarabinomannan (LAM) is a lipoglycan of mycobacteria and related bacteria, playing important roles as a surface-exposed pathogen-associated molecular pattern during host-pathogen interactions. Its importance is highlighted by the facts that anti-LAM antibody appears to be protective against TB disease progression, and urine LAM serves as a diagnostic marker for active TB. Given the clinical and immunological relevance of the molecule, it was a striking gap in knowledge that we did not know the cellular function of this lipoglycan in mycobacteria. In this study, we demonstrated that LAM regulates septation, a principle potentially generalizable to other lipoglycans widely found in a group of Gram-positive bacteria that lack lipoteichoic acids.