The Dynamics and Components of Intracellular Lipid Inclusion Formation in Mycobacteria

Mycobacterium tuberculosis is an airborne bacterial pathogen responsible for the infectious disease tuberculosis. Tuberculosis is the second leading infectious killer after COVID-19 and was responsible for over 1.6 million deaths in 2021. Astoundingly, a quarter of the world is thought to have been infected by M. tuberculosis. If left untreated, TB has a high mortality rate of up to 50%. While a strict regime of antibiotics can treat the disease, the rise of multidrug resistant clinical M. tuberculosis strains threatens the world. Typically, a majority of the population can quell the disease into a latent TB infection (LTBI), although in 5-10% of cases, an infected individual is unable to prevent disease progression and the active bacteria can start their infection anew. These harrowing statistics demand the need to understand the biology of M. tuberculosis and the development of model systems to study the bacterium and its pathogenesis. Intracellular lipid inclusions (ILI), receptacles of concentrated lipids, have long been perceived as hallmarks of non-replicating persistent bacteria found in LTBI. These organelles are thought to provide fuel for basal metabolic processes while the dormant bacteria withstand the immunological and chemical artillery launched by host immune cells. Understanding how and why ILI are formed would promote identification of tactical therapeutic targets against dormant bacilli and allow for sustained prevention and control of TB. Here, we provide surprising evidence that refutes this longstanding belief and suggests that ILI can be formed in actively replicating Mycobacterium marinum. We find this discovery is corroborated in several clinical M. tuberculosis strains and through next generation RNA sequencing we uncover some key differences in the transcriptional landscape of ILI producing and ILI barren bacteria, allowing us to decouple non-replicating persistence from ILI formation. Separately, we utilize an arrayed transposon mutagenesis library in M. marinum to identify four genes involved in the ILI formation pathway. M. marinum mutant strains with disruptions in these genes were used to study the role of ILI, allowing us to add mounting evidence to the importance of ILI in mycobacterial infection. Together, these findings suggest an alternative function of ILI in replicating M. marinum and M. tuberculosis, as well as provide novel models to study the purpose of ILI in M. tuberculosis pathogenesis