Jennifer Chua

Mycobacterium tuberculosis glycosylated phosphatidylinositol causes phagosome maturation arrest

The tubercle bacillus parasitizes macrophages by inhibiting phagosome maturation into the phagolysosome. This phenomenon underlies the tuberculosis pandemic involving 2 billion people. We report here how Mycobacterium tuberculosis causes phagosome maturation arrest. A glycosylated M. tuberculosis phosphatidylinositol [mannose-capped lipoarabinomannan (ManLAM)] interfered with the phagosomal acquisition of the lysosomal cargo and syntaxin 6 from the trans-Golgi network. ManLAM specifically inhibited the pathway dependent on phosphatidylinositol 3-kinase activity and phosphatidylinositol 3-phosphate-binding effectors. These findings identify ManLAM as the M. tuberculosis product responsible for the inhibition of phagosomal maturation.

Tuberculosis Toxin Blocking Phagosome Maturation Inhibits a Novel Ca2+/Calmodulin-PI3K hVPS34 Cascade

The capacity of Mycobacterium tuberculosis to infect latently over one billion people and cause two million fatalities annually rests with its ability to block phagosomal maturation into the phagolysosome in infected macrophages. Here we describe how M. tuberculosis toxin lipoarabinomannan (LAM) causes phagosome maturation arrest, interfering with a new pathway connecting intracellular signaling and membrane trafficking. LAM from virulent M. tuberculosis, but not from avirulent mycobacteria, blocked cytosolic Ca2+ increase. Ca2+ and calmodulin were required for a newly uncovered Ca2+/calmodulin phosphatidylinositol (PI)3 kinase hVPS34 cascade, essential for production of PI 3 phosphate (PI3P) on liposomes in vitro and on phagosomes in vivo. The interference of the trafficking toxin LAM with the calmodulin-dependent production of PI3P described here ensures long-term M. tuberculosis residence in vacuoles sequestered away from the bactericidal and antigen-processing organelles in infected macrophages.

Rab14 is critical for maintenance of Mycobacterium tuberculosis phagosome maturation arrest

Mycobacterium tuberculosis arrests phagosomal maturation in infected macrophage, and, apart from health significance, provides a superb model system to dissect the phagolysosomal biogenesis pathway. Here, we demonstrate a critical role for the small GTPase Rab14 in maintaining mycobacterial phagosome maturation block. Four‐dimensional microscopy showed that phagosomes containing live mycobacteria accumulated Rab14 following phagocytosis. The recruitment of Rab14 had strong functional consequence, as a knockdown of endogenous Rab14 by siRNA or overexpression of Rab14 dominant‐negative mutants (Rab14S25N and Rab14N125I) released the maturation block and allowed phagosomes harboring live mycobacteria to progress into phagolysosomes. Conversely, overexpression of the wild‐type Rab14 and the constitutively active mutant Rab14Q70L prevented phagosomes with dead mycobacteria from undergoing default maturation into phagolysosomal organelles. Mechanistic studies demonstrated a role for Rab14 in stimulating organellar fusion between phagosomes and early endosomes but not with late endosomes. Rab14 enables mycobacterial phagosomes to maintain early endosomal characteristics and avoid late endosomal/lysosomal degradative components.

Mechanism of Inducible Nitric Oxide Synthase Exclusion from Mycobacterial Phagosomes

Mycobacterium tuberculosis is sensitive to nitric oxide generated by inducible nitric oxide synthase (iNOS). Consequently, to ensure its survival in macrophages, M. tuberculosis inhibits iNOS recruitment to its phagosome by an unknown mechanism. Here we report the mechanism underlying this process, whereby mycobacteria affect the scaffolding protein EBP50, which normally binds to iNOS and links it to the actin cytoskeleton. Phagosomes harboring live mycobacteria showed reduced capacity to retain EBP50, consistent with lower iNOS recruitment. EBP50 was found on purified phagosomes, and its expression increased upon macrophage activation, paralleling expression changes seen with iNOS. Overexpression of EBP50 increased while EBP50 knockdown decreased iNOS recruitment to phagosomes. Knockdown of EBP50 enhanced mycobacterial survival in activated macrophages. We tested another actin organizer, coronin-1, implicated in mycobacterium-macrophage interaction for contribution to iNOS exclusion. A knockdown of coronin-1 resulted in increased iNOS recruitment to model latex bead phagosomes but did not increase iNOS recruitment to phagosomes with live mycobacteria and did not affect mycobacterial survival. Our findings are consistent with a model for the block in iNOS association with mycobacterial phagosomes as a mechanism dependent primarly on reduced EBP50 recruitment.

Higher order Rab programming in phagolysosome biogenesis

Phagosomes offer kinetically and morphologically tractable organelles to dissect the control of phagolysosome biogenesis by Rab GTPases. Model phagosomes harboring latex beads undergo a coordinated Rab5–Rab7 exchange, which is akin to the process of endosomal Rab conversion, the control mechanisms of which are unknown. In the process of blocking phagosomal maturation, the intracellular pathogen Mycobacterium tuberculosis prevents Rab7 acquisition, thus, providing a naturally occurring tool to study Rab conversion. We show that M. tuberculosis inhibition of Rab7 acquisition and arrest of phagosomal maturation depends on Rab22a. Four-dimensional microscopy revealed that phagosomes harboring live mycobacteria recruited and retained increasing amounts of Rab22a. Rab22a knockdown in macrophages via siRNA enhanced the maturation of phagosomes with live mycobacteria. Conversely, overexpression of the GTP-locked mutant Rab22aQ64L prevented maturation of phagosomes containing heat-killed mycobacteria, which normally progress into phagolysosomes. Moreover, Rab22a knockdown led to Rab7 acquisition by phagosomes harboring live mycobacteria. Our findings show that Rab22a defines the critical checkpoint for Rab7 conversion on phagosomes, allowing or disallowing organellar transition into a late endosomal compartment. M. tuberculosis parasitizes this process by actively recruiting and maintaining Rab22a on its phagosome, thus, preventing Rab7 acquisition and blocking phagolysosomal biogenesis.

Mycobacterium tuberculosis phagosome maturation arrest: selective targeting of PI3P-dependent membrane trafficking.

The ability of Mycobacterium tuberculosis to enter host macrophages, and reside in a phagosome, which does not mature into a phagolysosome, is central to the spread of tuberculosis and the associated pandemic involving billions of people worldwide. Tuberculosis can be viewed as a disease with a significant intracellular trafficking and organellar biogenesis component. Current understanding of the block in M. tuberculosis phagosome maturation also sheds light on fundamental aspects of phagolysosome biogenesis. The maturation block involves interference with the recruitment and function of rabs, rab effectors (phosphatidylinositol 3‐kinases and tethering molecules such as EEA1), SNAREs (Syntaxin 6 and cellubrevin) and Ca2+/calmodulin signaling. M. tuberculosis analogs of mammalian phosphatidylinositols interfere with these systems and associated processes.

Cellubrevin Alterations and Mycobacterium tuberculosis Phagosome Maturation Arrest

The intracellular trafficking processes controlling phagosomal maturation remain to be fully delineated.Mycobacterium tuberculosis var. bovis BCG, an organism that causes phagosomal maturation arrest, has emerged as a tool for dissection of critical phagosome biogenesis events. In this work, we report that cellubrevin, a v-SNARE functioning in endosomal recycling and implicated in endosomal interactions with post-Golgi compartments, plays a role in phagosomal maturation and that it is altered on mycobacterial phagosomes. Both mycobacterial phagosomes, which undergo maturation arrest, and model phagosomes containing latex beads, which follow the normal pathway of maturation into phagolysosomes, acquired cellubrevin. However, the mycobacterial and model phagosomes differed, as a discrete proteolytic degradation of this SNARE was detected on mycobacterial phagosomes. The observed cellubrevin alteration on mycobacterial phagosomes was not a passive event secondary to a maturation arrest at another checkpoint of the phagosome maturation pathway, since pharmacological inhibitors of phagosomal/endosomal pathways blocking phagosomal maturation did not cause cellubrevin degradation on model phagosomes. Cellubrevin status on phagosomes had consequences on phagosomal membrane and lumenal content trafficking, involving plasma membrane marker recycling and delivery of lysosomal enzymes. These results suggest that cellubrevin plays a role in phagosomal maturation and that it is a target for modification by mycobacteria or by infection-induced processes in the host cell.

Endosomal membrane traffic: convergence point targeted by Mycobacterium tuberculosis and HIV

Inhibition of phagolysosome biogenesis in infected macrophages is a classical pathogenesis determinant of Mycobacterium tuberculosis. In this review we primarily cover the cellular mechanisms of M. tuberculosis phagosome maturation arrest. A detailed picture is beginning to emerge, involving regulators of membrane trafficking in mammalian cells and phagosomal interactions with endosomal organelles and the trans‐Golgi network. We also present a hypothesis that overlaps may exist between the mycobacterial interference with the host cell membrane trafficking processes and the targeting of the late endosomal sorting machinery by HIV during viral budding in macrophages. We propose that interference with the endosomal sorting machinery contributes to the synergism between the two significant human diseases – AIDS and tuberculosis.

Regulators of membrane trafficking and Mycobacterium tuberculosis phagosome maturation block.

The biogenesis and maturation of phagosomes is an area of study which has been employing aspects of proteomic analyses and variations on that theme by identifying components on isolated organelles and following their dynamic changes and interactions with the endocytic pathway. In the case of Mycobacterium tuberculosis phagosome, the arrest of its maturation in infected macrophages, referred to in classical texts as the inhibition of phagosome‐lysosome fusion, represents a phenomenon that is used to functionally dissect the phagosomal maturation pathway. In this review, we summarize the recent studies on regulators of membrane trafficking and other organelle components in the context of phagosomal biogenesis and mycobacterial phagosome maturation arrest.

Dynamin 2 orchestrates the global actomyosin cytoskeleton for epithelial maintenance and apical constriction

The mechanisms controlling cell shape changes within epithelial monolayers for tissue formation and reorganization remain unclear. Here, we investigate the role of dynamin, a large GTPase, in epithelial morphogenesis. Depletion of dynamin 2 (Dyn2), the only dynamin in epithelial cells, prevents establishment and maintenance of epithelial polarity, with no junctional formation and abnormal actin organization. Expression of Dyn2 mutants shifted to a non-GTP state, by contrast, causes dramatic apical constriction without disrupting polarity. This is due to Dyn2s interactions with deacetylated cortactin and downstream effectors, which cause enhanced actomyosin contraction. Neither inhibitors of endocytosis nor GTP-shifted Dyn2 mutants induce apical constriction. This suggests that GTPase-dependent changes in Dyn2 lead to interactions with different effectors that may differentially modulate endocytosis and/or actomyosin dynamics in polarized cells. We propose this enables Dyn2 to coordinate, in a GTPase-dependent manner, membrane recycling and actomyosin contractility during epithelial morphogenesis.