Biomedical subjects

Jennifer Chua

Publications and source records attributed to Jennifer Chua.

14 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

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.

Cells, Cultured↗

PubMed2005

Mechanism of phagolysosome biogenesis block by viable Mycobacterium tuberculosis.

Live Mycobacterium tuberculosis persists in macrophage phagosomes by interfering with phagolysosome biogenesis. Here, using four-dimensional microscopy and in vitro assays, we report the principal difference between phagosomes containing live and dead mycobacteria. Phosphatidylinositol 3-phosphate (PI3P), a membrane trafficking regulatory lipid essential for phagosomal acquisition of lysosomal constituents, is retained on phagosomes harboring dead mycobacteria but is continuously eliminated from phagosomes with live bacilli. We show that the exclusion of PI3P from live mycobacterial phagosomes can be only transiently reversed by Ca2+ fluxes, and that live M. tuberculosis secretes a lipid phosphatase, SapM, that hydrolyzes PI3P, inhibits phagosome-late endosome fusion in vitro, and contributes to inhibition of phagosomal maturation.

Animals↗

PubMed2004

Mycobacterium tuberculosis reprograms waves of phosphatidylinositol 3-phosphate on phagosomal organelles.

The potent human pathogen Mycobacterium tuberculosis persists in macrophages within a specialized, immature phagosome by interfering with the pathway of phagolysosome biogenesis. The molecular mechanisms underlying this process remain to be fully elucidated. Here, using four-dimensional microscopy, we detected on model phagosomes, which normally mature into phagolysosomes, the existence of cyclical waves of phosphatidylinositol 3-phosphate (PI3P), a membrane trafficking regulatory lipid essential for phagosomal acquisition of lysosomal characteristics. We show that mycobacteria interfere with the dynamics of PI3P on phagosomal organelles by altering the timing and characteristics of the PI3P waves on phagosomes. The default program of cyclical PI3P waves on model phagosomes is composed of an initial stage (phase I), represented by a strong PI3P burst occurring only upon the completion of phagosome formation, and a subsequent stage (phase II) of recurring PI3P waves on maturing phagosomes with the average periodicity of 20 min. Mycobacteria alter this program in two ways: (i) by inducing, in a cholesterol-dependent fashion, a neophase I* of premature PI3P production, coinciding with the process of mycobacterial entry into the macrophage, and (ii) by inhibiting the calmodulin-dependent phase II responsible for the acquisition of lysosomal characteristics. We conclude that the default pathway of phagosomal maturation into the phagolysosome includes temporally organized cyclical waves of PI3P on phagosomal membranes and that this process is targeted for reprogramming by mycobacteria as they prevent phagolysosome formation.

Animals↗

PubMed2004

A tale of two lipids: Mycobacterium tuberculosis phagosome maturation arrest.

Mycobacterium tuberculosis persistence in human populations relies on its ability to inhibit phagosomal maturation. M. tuberculosis resides in a pathogen-friendly phagosome escaping lysosomal bactericidal mechanisms and efficient antigen presentation in the host phagocytic cell. M. tuberculosis phagosome maturation arrest includes the action of mycobacterial lipid products, which mimic mammalian phosphatidylinositols, targeting host cell membrane trafficking processes. These products interfere with membrane trafficking and organelle biogenesis processes initiated by Ca(2+) fluxes, and ending with host cell Rab GTP-binding proteins and their effectors. The block includes phosphatidylinositol 3-kinase and membrane tethering molecules that prepare phagosomes for fusion with other organelles. Understanding these processes could provide new targets for pharmacological intervention in tuberculosis.

Humans↗

PubMed2004

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.

Cell Membrane↗

PubMed2004

Cell biology of mycobacterium tuberculosis phagosome.

Phagocytosis and phagolysosome biogenesis represent fundamental biological processes essential for proper tissue homeostasis, development, elimination of invading microorganisms, and antigen processing and presentation. Phagosome formation triggers a preprogrammed pathway of maturation into the phagolysosome, a process controlled by Ca2+ and the regulators of organellar trafficking centered around the small GTP-binding proteins Rabs and their downstream effectors, including lipid kinases, organellar tethering molecules, and membrane fusion apparatus. Mycobacterium tuberculosis is a potent human pathogen parasitizing macrophages. It interferes with the Rab-controlled membrane trafficking and arrests the maturing phagosome at a stage where no harm can be done to the pathogen while the delivery of nutrients and membrane to the vacuole harboring the microorganism continues. This process, referred to as the M. tuberculosis phagosome maturation arrest or inhibition of phagosome-lysosome fusion, is critical for M. tuberculosis persistence in human populations. It also provides a general model system for dissecting the phagolysosome biogenesis pathways. Here we review the fundamental trafficking processes targeted by M. tuberculosis and the mycobacterial products that interfere with phagosomal maturation.

Biological Transport↗

PubMed2003

Mycobacterium tuberculosis phagosome maturation arrest: mycobacterial phosphatidylinositol analog phosphatidylinositol mannoside stimulates early endosomal fusion.

Mycobacterium tuberculosis is a facultative intracellular pathogen that parasitizes macrophages by modulating properties of the Mycobacterium-containing phagosome. Mycobacterial phagosomes do not fuse with late endosomal/lysosomal organelles but retain access to early endosomal contents by an unknown mechanism. We have previously reported that mycobacterial phosphatidylinositol analog lipoarabinomannan (LAM) blocks a trans-Golgi network-to-phagosome phosphatidylinositol 3-kinase-dependent pathway. In this work, we extend our investigations of the effects of mycobacterial phosphoinositides on host membrane trafficking. We present data demonstrating that phosphatidylinositol mannoside (PIM) specifically stimulated homotypic fusion of early endosomes in an ATP-, cytosol-, and N-ethylmaleimide sensitive factor-dependent manner. The fusion showed absolute requirement for small Rab GTPases, and the stimulatory effect of PIM increased upon partial depletion of membrane Rabs with RabGDI. We found that stimulation of early endosomal fusion by PIM was higher when phosphatidylinositol 3-kinase was inhibited by wortmannin. PIM also stimulated in vitro fusion between model phagosomes and early endosomes. Finally, PIM displayed in vivo effects in macrophages by increasing accumulation of plasma membrane-endosomal syntaxin 4 and transferrin receptor on PIM-coated latex bead phagosomes. In addition, inhibition of phagosomal acidification was detected with PIM-coated beads. The effects of PIM, along with the previously reported action of LAM, suggest that M. tuberculosis has evolved a two-prong strategy to modify its intracellular niche: its products block acquisition of late endosomal/lysosomal constituents, while facilitating fusion with early endosomal compartments.

Adenosine Triphosphate↗

PubMed2003

Induction of p38 mitogen-activated protein kinase reduces early endosome autoantigen 1 (EEA1) recruitment to phagosomal membranes.

Mycobacterium tuberculosis survives in the infected host by parasitizing macrophages in which the bacillus resides in a specialized phagosome sequestered from the phagolysosomal degradative pathway. Here we report a role of the stress-induced p38 mitogen-activated protein kinase (p38 MAPK) in the component of M. tuberculosis phagosome maturation arrest that has been linked previously to the reduced recruitment of the endosomal and phagosomal membrane-tethering molecule called early endosome autoantigen 1 (EEA1; Fratti, R. A., Backer, J. M., Gruenberg, J., Corvera, S., and Deretic, V. (2001) J. Cell Biol. 154, 631-644). A pharmacological inhibition of M. tuberculosis var. bovis Bacillus Calmette-Guérin-induced p38 MAPK activity caused a marked increase in EEA1 colocalization with mycobacterial phagosomes. Consistent with the increase in EEA1 association and its role in phagosomal maturation, the pharmacological block of p38 activity caused phagosomal acidification and enrichment of the late endocytic markers lysobisphosphatidic acid and CD63 (lysosomal integral membrane protein 1) on mycobacterial phagosomes. A negative regulatory role of p38 MAPK activation in phagosome maturation was further demonstrated by converse experiments with latex bead phagosomes. Artificial activation of p38 MAPK caused a decrease in EEA1 colocalization with model latex bead phagosomes, which normally acquire EEA1 and subsequently mature into the phagolysosome. These findings show that p38 MAPK activity contributes to the arrest of M. tuberculosis phagosome maturation and demonstrate a negative regulatory role of p38 in phagolysosome biogenesis.

Animals↗

PubMed2003

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.

Antigens, Bacterial↗

PubMed2003

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.

Glycosylation↗

PubMed2003

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.

Animals↗

PubMed2003

Nicotinic induction of preproenkephalin and tyrosine hydroxylase gene expression in butyrate-differentiated rat PC12 cells: a model for adaptation to gut-derived environmental signals.

Accelerated maturation of peripheral sympathoadrenal transmitter levels and function occurs at 7-10 postnatal days in the rat. This event is temporally disconnected from the timing of major changes in physiologic stimuli evident after the birthing process (i.e. temperature, oxygen, sound, light, etc.). Colonization of the gut, fermentation of carbohydrates, and production of short-chain fatty acids (e.g. butyrate) mirrors this postnatal time course. In this report, we examined the interaction between butyrate differentiation of rat pheochromocytoma cells and cholinergic-nicotinic induction of the neuropeptide (enkephalin) and catecholamine-related biosynthetic enzymes (tyrosine hydroxylase, dopamine beta-hydroxylase, phenylethanolamine N-methyltransferase). Our results show that butyrate induces both preproenkephalin and tyrosine hydroxylase mRNA through a proximal promoter region and that this regulatory step is time and dose dependent. Moreover, there is an additional interaction with cholinergic-nicotinic inducible mechanisms consistent with classically described transsynaptic cholinergic regulation of these genes. Dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase promoters were not affected by butyrate treatment. We speculate that colonization of the human gut (along with the attendant fermentation of enteral carbohydrates to short-chain fatty acids) may represent a mechanism through which environmental signals affect postnatal maturation of sympathoadrenal transmitter systems.

Animals↗

PubMed2002

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.

Adenosine Triphosphatases↗