Association of Mycobacterium Proteins with Lipid Droplets.

Mycobacterium tuberculosis is a global pathogen of significant medical importance. A key aspect of its life cycle is the ability to enter into an altered physiological state of nonreplicating persistence during latency and resist elimination by the host immune system. One mechanism by which M. tuberculosis facilitates its survival during latency is by producing and metabolizing intracytoplasmic lipid droplets (LDs). LDs are quasi-organelles consisting of a neutral lipid core such as triacylglycerol surrounded by a phospholipid monolayer and proteins. We previously reported that PspA (phage shock protein A) associates with LDs produced in Mycobacterium In particular, the loss or overproduction of PspA alters LD homeostasis in Mycobacterium smegmatis and attenuates the survival of M. tuberculosis during nonreplicating persistence. Here, M. tuberculosis PspA (PspAMtb) and a ΔpspA M. smegmatis mutant were used as model systems to investigate the mechanism by which PspA associates with LDs and determine if other Mycobacterium proteins associate with LDs using a mechanism similar to that for PspA. Through this work, we established that the amphipathic helix present in the first α-helical domain (H1) of PspA is both necessary and sufficient for the targeting of this protein to LDs. Furthermore, we identified other Mycobacterium proteins that also possess amphipathic helices similar to PspA H1, including a subset that localize to LDs. Altogether, our results indicate that amphipathic helices may be an important mechanism by which proteins target LDs in prokaryotes.IMPORTANCEMycobacterium spp. are one of the few prokaryotes known to produce lipid droplets (LDs), and their production has been linked to aspects of persistent infection by M. tuberculosis Unfortunately, little is known about LD production in these organisms, including how LDs are formed, their function, or the identity of proteins that associate with them. In this study, an established M. tuberculosis LD protein and a surrogate Mycobacterium host were used as model systems to study the interactions between proteins and LDs in bacteria. Through these studies, we identified a commonly occurring protein motif that is able to facilitate the association of proteins to LDs in prokaryotes.

Rv2744c Is a PspA Ortholog That Regulates Lipid Droplet Homeostasis and Nonreplicating Persistence in Mycobacterium tuberculosis.

UNLABELLED: Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a significant cause of morbidity and mortality worldwide, despite the availability of a live attenuated vaccine and anti-TB antibiotics. The vast majority of individuals infected with M. tuberculosis develop an asymptomatic latent infection in which the bacterium survives within host-generated granulomatous lesions in a physiologically altered metabolic state of nonreplicating persistence. The granuloma represents an adverse environment, as M. tuberculosis is exposed to various stressors capable of disrupting the essential constituents of the bacterium. In Gram-negative and Gram-positive bacteria, resistance to cell envelope stressors that perturb the plasma membrane is mediated in part by proteins comprising the phage shock protein (Psp) system. PspA is an important component of the Psp system; in the presence of envelope stress, PspA localizes to the inner face of the plasma membrane, homo-oligomerizes to form a large scaffold-like complex, and helps maintain plasma membrane integrity to prevent a loss of proton motive force. M. tuberculosis and other members of the Mycobacterium genus are thought to encode a minimal functional unit of the Psp system, including an ortholog of PspA. Here, we show that Rv2744c possesses structural and physical characteristics that are consistent with its designation as a PspA family member. However, although Rv2744c is upregulated under conditions of cell envelope stress, loss of Rv2744c does not alter resistance to cell envelope stressors. Furthermore, Rv2744c localizes to the surface of lipid droplets in Mycobacterium spp. and regulates lipid droplet number, size, and M. tuberculosis persistence during anaerobically induced dormancy. Collectively, our results indicate that Rv2744c is a bona fide ortholog of PspA that may function in a novel role to regulate lipid droplet homeostasis and nonreplicating persistence (NRP) in M. tuberculosis IMPORTANCE: Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease associated with significant morbidity and mortality worldwide. M. tuberculosis is capable of establishing lifelong asymptomatic infections in susceptible individuals and reactivating during periods of immune suppression to cause active disease. The determinants that are important for persistent infection of M. tuberculosis or for reactivation of this organism from latency are poorly understood. In this study, we describe our initial characterizations of Rv2744c, an ortholog of phage shock protein A (PspA) that regulates the homeostasis of lipid bodies and nonreplicating persistence in M. tuberculosis This function of PspA in M. tuberculosis is novel and suggests that PspA may represent a unique bacterial target upon which to base therapeutic interventions against this organism.