Isolation and characterization of the mycobacterial phagosome: segregation from the endosomal/lysosomal pathway

Mycobacteria have the ability to persist within host phagocytes, and their success as intracellular pathogens is thought to be related to the ability to modify their intracellular environment. After entry into phagocytes, mycobacteria-containing phagosomes acquire markers for the endosomal pathway, but do not fuse with lysosomes. The molecular machinery that is involved in the entry and survival of mycobacteria in host cells is poorly characterized. Here we describe the use of organelle electrophoresis to study the uptake of Mycobacterium bovis bacille Calmette Guerin (BCG) into murine macrophages. We demonstrate that live, but not dead, mycobacteria occupy a phagosome that can be physically separated from endosomal/lysosomal compartments. Biochemical analysis of purified mycobacterial phagosomes revealed the absence of endosomal/lysosomal markers LAMP-1 and β-hexosaminidase. Combining subcellular fractionation with two-dimensional gel electrophoresis, we found that a set of host proteins was present in phagosomes that were absent from endosomal/lysosomal compartments. The residence of mycobacteria in compartments outside the endosomal/lysosomal system may explain their persistence inside host cells and their sequestration from immune recognition. Furthermore, the approach described here may contribute to an improved understanding of the molecular mechanisms that determine the intracellular fate of mycobacteria during infection.     

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.     

Jamming the endosomal system: lipid rafts and lysosomal storage diseases

Some lysosomal storage diseases result from the accumulation of lipids in degradative compartments of the endocytic pathway. Particularly striking is the example of the Niemann-Pick (NP) syndrome. NP syndromes types A and B are characterized by the accumulation of sphingomyelin, whereas cholesterol typically accumulates in NP type C. These two different lipids, sphingomyelin and cholesterol, are normal constituents of specific lipid microdomains called rafts. Because accumulation of raft lipids is observed not only in NP diseases but also in many other lipidoses, we forward the hypothesis that lysosomal storage diseases can be caused by the accumulation of lipid rafts in late endosomes/lysosomes.     

Isolation and characterization of lysosomal alpha-mannosidase of placental tissue

The acidic α-mannosidase was purified 4400-fold by affinity chromatography on Concavalin A-Sepharose and heat treatment at 65‡C in the presence of 1 mM zinc ion. The enzyme did not resolve into multiple forms as in the case of enzymes from human liver and human kidney. The pH optimum of the enzyme was 4.2 in citrate-phosphate buffer. The K_m value for p-nitrophenyl-α-D-mannose was 1.9 mM. The molecular weight of the enzyme determined by gel filtration was 300,000. The enzyme contained 10.6% neutral sugars.     

Biosynthesis of phosphatidylcholine from a phosphocholine precursor pool derived from the late endosomal/lysosomal degradation of sphingomyelin

Previous studies suggest that the steps of the CDP- choline pathway of phosphatidylcholine synthesis are tightly linked in a so-called metabolon. Evidence has been presented that only choline that enters cells through the choline transporter, and not phosphocholine administered to cells by membrane permeabilization, is incorporated into phosphatidylcholine. Here, we show that [(14)C]phosphocholine derived from the lysosomal degradation of [(14)C]choline-labeled sphingomyelin is incorporated as such into phosphatidylcholine in human and mouse fibroblasts. Low density lipoprotein receptor-mediated endocytosis was used to specifically direct [(14)C]sphingomyelin to the lysosomal degradation pathway. Free labeled choline was not found either intracellularly or in the medium, not even when the cells were energy-depleted. Deficiency of lysosomal acid phosphatases in mouse or alkaline phosphatase in human fibroblasts did not affect the incorporation of lysosomal [(14)C]sphingomyelin-derived [(14)C]phosphocholine into phosphatidylcholine, supporting our finding that phosphocholine is not degraded to choline prior to its incorporation into phosphatidylcholine. Inhibition studies and analysis of molecular species showed that exogenous [(3)H]choline and sphingomyelin-derived [(14)C]phosphocholine are incorporated into phosphatidylcholine via a common pathway of synthesis. Our findings provide evidence that, in fibroblasts, phosphocholine derived from sphingomyelin is transported out of the lysosome and subsequently incorporated into phosphatidylcholine without prior hydrolysis of phosphocholine to choline. The findings do not support the existence of a phosphatidylcholine synthesis metabolon in fibroblasts.     

Brucella intracellular replication requires trafficking through the late endosomal/lysosomal compartment

Upon entry into mammalian cells, the intracellular pathogen Brucella abortus resides within a membrane-bound compartment, the Brucella -containing vacuole (BCV), the maturation of which is controlled by the bacterium to generate a replicative organelle derived from the endoplasmic reticulum (ER). Prior to reaching the ER, Brucella is believed to ensure its intracellular survival by inhibiting fusion of the intermediate BCV with late endosomes and lysosomes, although such BCVs are acidic and accumulate the lysosomal-associated membrane protein (LAMP-1). Here, we have further examined the nature of intermediate BCVs using confocal microscopy and live cell imaging. We show that BCVs rapidly acquire several late endocytic markers, including the guanosine triphosphatase Rab7 and its effector Rab-interacting lysosomal protein (RILP), and are accessible to fluid-phase markers either delivered to the whole endocytic pathway or preloaded to lysosomes, indicating that BCVs interact with late endosomes and lysosomes. Consistently, intermediate BCVs are acidic and display proteolytic activity up to 12 h post-infection. Expression of dominant-negative Rab7 or overexpression of RILP significantly impaired the ability of bacteria to convert their vacuole into an ER-derived organelle and replicate, indicating that BCV maturation requires interactions with functional late endosomal/lysosomal compartments. In cells expressing dominant-negative Rab7[T22N], BCVs remained acidic, yet displayed decreased fusion with lysosomes. Taken together, these results demonstrate that BCVs traffic along the endocytic pathway and fuse with lysosomes, and such fusion events are required for further maturation of BCVs into an ER-derived replicative organelle.     

Inhibition of endosomal/lysosomal degradation increases the infectivity of human immunodeficiency virus

Productive entry of human immunodeficiency virus type 1 (HIV-1) into a host cell is believed to proceed via fusion of the viral envelope with the host cell's plasma membrane. Interestingly, the majority of HIV-1 particles that bind to the cell surface are taken up by the host cell via endocytosis; however, this mode of internalization generally does not result in infection. Presumably, virus particles remain trapped in the endocytic pathway and are eventually degraded. Here, we demonstrate that treatment of cells with various pharmacological agents known to elevate the pH of endosomes and lysosomes allows HIV-1 to efficiently enter and infect the host cell. Pretreatment of cells with bafilomycin A1 results in up to a 50-fold increase in the infectivity of HIV-1(SF2). Similarly, pretreatment of target cells with amantadine, concanamycin A, concanamycin B, chloroquine, and ammonium chloride resulted in increases in HIV-1 infectivity ranging between 2- and 15-fold. Analysis of receptor and coreceptor expression, HIV-long terminal repeat (LTR) transactivation, and transduction with amphotropic-pseudotyped murine leukemia virus (MLV)-based vectors suggests that the increase in infectivity is not artifactual. The increased infectivity under these conditions appears to be due to the ability of HIV-1 and MLV particles to enter via the endocytic pathway when spared from degradation in the late endosomes and lysosomes. These results could have significant implications for the administration of current and future lysosmotropic agents to patients with HIV disease.     

Trafficking defects in WASH-knockout fibroblasts originate from collapsed endosomal and lysosomal networks

The Arp2/3-activator Wiskott-Aldrich syndrome protein and Scar homologue (WASH) is suggested to regulate actin-dependent membrane scission during endosomal sorting, but its cellular roles have not been fully elucidated. To investigate WASH function, we generated tamoxifen-inducible WASH-knockout mouse embryonic fibroblasts (WASHout MEFs). Of interest, although EEA1(+) endosomes were enlarged, collapsed, and devoid of filamentous-actin and Arp2/3 in WASHout MEFs, we did not observe elongated membrane tubules emanating from these disorganized endomembranes. However, collapsed WASHout endosomes harbored segregated subdomains, containing either retromer cargo recognition complex-associated proteins or EEA1. In addition, we observed global collapse of LAMP1(+) lysosomes, with some lysosomal membrane domains associated with endosomes. Both epidermal growth factor receptor (EGFR) and transferrin receptor (TfnR) exhibited changes in steady-state cellular localization. EGFR was directed to the lysosomal compartment and exhibited reduced basal levels in WASHout MEFs. However, although TfnR was accumulated with collapsed endosomes, it recycled normally. Moreover, EGF stimulation led to efficient EGFR degradation within enlarged lysosomal structures. These results are consistent with the idea that discrete receptors differentially traffic via WASH-dependent and WASH-independent mechanisms and demonstrate that WASH-mediated F-actin is requisite for the integrity of both endosomal and lysosomal networks in mammalian cells.     

The Niemann-Pick C proteins and trafficking of cholesterol through the late endosomal/lysosomal system

To maintain proper cellular function, the amount and distribution of cholesterol residing within cellular membranes must be regulated. The principal disorder affecting transport of cholesterol through the late endosomal/lysosomal system and intracellular cholesterol homeostasis is Niemann-Pick type C (NPC) disease. The genes responsible for NPC disease have been identified, and the encoded Niemann-Pick C1 (NPC1) and Niemann-Pick C2 (HE1/NPC2) proteins are currently the subject of intense investigation. This review provides a detailed examination of NPC1 and HE1/NPC2 in regulating the transport of cholesterol through the late endosomal/lysosomal system to other cellular compartments responsible for maintaining intracellular cholesterol homeostasis, and how defective function of these proteins may be responsible for the pathophysiology associated with NPC disease.     

Effective generation of reactive oxygen species in the mycobacterial phagosome requires K+ efflux from the bacterium

Efficient killing of mycobacteria by host macrophages depends on a number of mechanisms including production of reactive oxygen species (ROS) by the phagosomal NADPH oxidase, NOX2. Survival of pathogenic mycobacteria in the phagosome relies on the ability to control maturation of the phagosome such that it is biologically and chemically altered in comparison to phagosomes containing non‐pathogenic bacteria. In this study we show that the action of NOX2 to produce ROS in the mycobacterial phagosome is paradoxically dependent on a bacterial potassium transporter. We show that a Mycobacterium bovis BCG mutant (BCGΔkef), deficient in a Kef‐type K+ transporter, exhibits an increased intracellular survival phenotype in resting and activated macrophages, yet retains the ability to inhibit phagosome acidification, and does not show increased resistance to acidic conditions or ROS. Addition of a ROS scavenger replicates this phenotype in macrophages infected with wild‐type BCG, and the production of ROS by macrophages infected with BCGΔkef is substantially decreased compared with those infected with wild‐type BCG. Our results suggest that increased intracellular survival of BCGΔkef is mediated by inducing a decreased macrophage oxidative burst, and are consistent with Kef acting to alter the ionic contents of the phagosome and promoting NOX2 production of ROS.     

Isolation, affinity purification and biochemical characterization of a lysosomal cathepsin D from the deuterostome Asterias rubens

Cathepsin D (EC 3.4.23.5) is one of the lysosomal enzymes responsible for proteolytic degradation in cells. By virtue of its mannose 6-phosphate residues, shortly after its synthesis, it is recognized by the receptors in the trans-Golgi network that mediate its transport to the lysosomes. The mammalian enzyme has been extensively characterized and several forms of cathepsin have also been identified. Cathepsins have also been isolated from other vertebrates and invertebrates and recent studies suggest that the lysosomal sorting machinery is evolutionarily conserved from fish to mammals. We recently characterized the putative mannose 6-phosphate receptors from the invertebrate starfish (Asterias rubens). In the present study we affinity purified the cathepsin D from this animal and biochemically characterized the same. Purified enzyme migrated as a single band on SDS-PAGE corresponding to a molecular mass of 45 kDa. The protein bound specifically to Con A-Sepharose gel and is glycosylated. The deglycosylated enzyme showed a molecular mass of ~ 40 kDa. Furthermore, an antibody raised for the purified enzyme in a rabbit recognizes the crude, the purified enzyme as well as the deglycosylated product in a western blot experiment. The enzyme in the extracts of different tissues can also be quantified by ELISA. We have further evaluated the binding of purified starfish cathepsin D with its receptor, MPR 300 (mannose 6-phosphate receptor) by immunoprecipitation. Cross-linking experiments using purified cathepsin D and MPR 300 revealed a cross-linked product that migrated with a higher molecular mass (345 kDa) compared to the enzyme (45 kDa). Furthermore the specificity of this interaction was also tested in a ligand blot experiment.     

HLA-DM - an endosomal and lysosomal chaperone for the immune system.

Molecular chaperones are involved in a variety of cellular processes, including stabilization of newly synthesized polypeptide chains, assembly of oligomers, transport of proteins and organelle biogenesis. They are known to exert their activity in the cytosol, endoplasmic reticulum, mitochondria and chloroplasts. HLA-DM is the first example of a molecular chaperone that operates in lysosomes: it plays a crucial role in endosomal and lysosomal compartments during loading of major histocompatibility complex (MHC) class II molecules, specialized peptide receptors that are expressed by antigen-presenting cells of the immune system.     

Isolation and characterization of a rough microsomal fraction from rat kidney that is enriched in lysosomal enzymes

1. A special population of rough microsomal material (microsomes) rich in lysosomal acid hydrolases was separated by isopycnic centrifugation as a discrete fraction (RM(2)) from the bulk of rough microsomal material in rat kidney because of its greater density. 2. The specific activities of five acid hydrolases in the RM(2) fraction were approximately one-half those of a purified lysosomal (L) fraction and 10- to 30-fold greater than those of an ordinary rough microsomal (RM(1)) fraction. 3. These special rough microsomes have a distinctive ultrastructure and electron-cytochemical properties. Their cisternal content resembles the matrix of lysosomes in that it is electron-dense, osmiophilic and plumbophilic and gives a positive reaction for acid phosphatase activity. 4. Polyacrylamide-gel electrophoresis of soluble proteins from the L fraction resolved nine anionic glycoproteins, most of which exhibit acid hydrolase activities (Goldstone & Koenig, 1970, 1973; Goldstone et al., 1971a). The most anionic glycoprotein is the acidic lipoglycoprotein of the lysosomal matrix (Goldstone et al., 1970). 5. Polyacrylamide-gel electrophoresis of soluble proteins from the RM(2) fraction resolved two cationic glycoproteins with acid hydrolase activities (Goldstone & Koenig, 1973) and an anionic glycoprotein with the same electrophoretic mobility as the lysosomal lipoglycoprotein, but without its lipid constituents or capacity to bind the basic fluorochrome Acridine Orange. These constituents are considered to be the precursors of the lysosomal glycoproteins.     

Isolation and characterization of rabbit H of the alternative complement pathway

Rabbit factor H, a control protein of the alternative complement pathway, was isolated from rabbit serum by polyethylene glycol precipitation, DEAE-Sephacel chromatography, and gel chromatography on Sephadex G200. The protein migrated as a single-chain polypeptide with a molecular weight of 160,000 on sodium dodecyl sulfate-gel electrophoresis with Laemmli's buffer system, but hardly migrated into the gel with Fairbanks' buffer system. Physical and chemical properties of rabbit H were similar to those of human H, except that fragments produced by limited tryptic digestion from rabbit H had different molecular sizes from those produced from human H. Significant species-specificity was observed in the functional activity of factor H; activation of the alternative complement pathway was inhibited more efficiently with homologous H than with heterologous H. In contrast, factor H inhibited the hemolysis of homologous erythrocytes less than that of heterologous erythrocytes.     

Modulation of the endosomal and lysosomal distribution of cathepsins B,L and S in human monocytes/macrophages

Endosomal and lysosomal fractions of human monocytes/macrophages prepared from buffy coats were analyzed for activities of cathepsins B, L and S, and expression of cathepsin proteins along with major histocompatibility complex class I and class II molecules under control and immunomodulatory conditions. While the total activity of cathepsins B, L, and S together remained unchanged in lysates of control cells during culture for 72 h, the subcellular distribution of cathepsin activities underwent a shift from a predominantly endosomal localization in freshly isolated cells to a lysosomal pattern after 72 h of culture. Interferon-gamma treatment for 72 h resulted in an upregulation of both major histocompatibility complex proteins and cathepsins with differential changes in cathepsin B, L and S activities in endosomes versus lysosomes. These changes suggest a remodeling of the endocytic machinery and imply different functions of cathepsins B, L and S during monocyte differentiation.     

Isolation and phenotypic characterization ofChlamydomonas reinhardtii mutants defective in chloroplast DNA segregation

Summary Each wild-typeChlamydomonas reinhardtii cell has one large chloroplast containing several nuclei (nucleoids). We used DNA insertional mutagenesis to isolate Chlamydomonas mutants which contain a single, large chloroplast (cp) nucleus and which we namedmoc (monokaryotic chloroplast). DAPI-fluorescence microscopy and microphotometry observations revealed thatmoc mutant cells only contain one cp-nucleus throughout the cell division cycle, and that unequal segregation of cpDNA occurred during cell division in themoc mutant. One cell with a large amount of cpDNA and another with a small amount of cpDNA were produced after the first cell division. Unequal segregation also occurred in the second cell division, producing one cell with a large amount (about 70 copies) of cpDNA and three other cells with a small amount (only 2–8 copies) of cpDNA. However, most individualmoc cells contained several dozen cpDNA copies 12 h after the completion of cell division, suggesting that cpDNA synthesis was activated immediately after chloroplast division. In contrast to the cpDNA, the mitochondrial (mt) DNA of themoc mutants was observed as tiny granules scattered throughout the entire cell. These segregated to each daughter cell equally during cell division. Electron-microscopic observation of the ultrastructure ofmoc mutants showed that a low-electron-density area, which was identified as the cp-nucleus by immunoelectron microscopy with anti-DNA antibody, existed near the pyrenoid. However, there were no other structural differences between the chloroplasts of wild-type cells andmoc mutants. The thylakoid membranes and pyrenoid were identical. Therefore, we propose that the novelmoc mutants are only defective in the dispersion and segregation of cpDNA. This strain should be useful to elucidate the mechanism for the segregation of cpDNA.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon-counting system     

Protein traffic from the secretory pathway to the endosomal system in pancreatic beta-cells

Constitutive-like secretion involves vesicular trafficking corresponding kinetically and biochemically with a post-trans-Golgi network (TGN) origin. In pancreatic beta-cells, the budding of AP-1/clathrin-coated vesicles, a portion of which is derived from immature secretory granules, has been hypothesized to initiate constitutive-like trafficking. However, approximately 30 min after release of a 20 degrees C intracellular transport block in pancreatic beta-cells (to synchronize protein egress from the TGN), addition of brefeldin A (BFA) (which inhibits AP-1 recruitment) was reported not to block subsequent constitutive-like secretion. To further explore post-TGN trafficking in pancreatic beta-cell lines, we have followed the fate of pulse-labeled procathepsin B (ProB, a lysosomal proenyzme) after postpulse wortmannin treatment or the BFA treatment described above. We find that continuous wortmannin treatment allows ProB to reach immature secretory granules but inhibits its egress from maturing granules. Remarkably, BFA treatment causes augmented unstimulated secretion of newly synthesized ProB that is not paralleled by insulin. This effect requires a delay of 25-35 min after release from the 20 degrees C block. Further, when ProB delivery to endosomes is inhibited, its BFA-augmented secretion is eliminated. We hypothesize that the constitutive-like pathway involves an endosomal intermediate.