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PubMed1991

Ammonium chloride, an inhibitor of phagosome-lysosome fusion in macrophages, concurrently induces phagosome-endosome fusion, and opens a novel pathway: studies of a pathogenic mycobacterium and a nonpathogenic yeast.

The weak base ammonium chloride has been previously reported to inhibit lysosomal movements and phagosome-lysosome (Ph-L) fusion in cultured mouse macrophages (M phi), thus reducing delivery, to an intraphagosomal infection, of endocytosed solutes that have concentrated in secondary lysosomes. We have now addressed the question, whether NH4Cl might affect any direct interaction (if it exists) between such infection phagosomes and earlier, nonlysosomal compartments of the endocytic pathway, i.e., solute-containing endosomes. The phagosomes studied were formed after ingestion of the mouse pathogen Mycobacterium microti and the nonpathogenic yeast Saccharomyces cerevisiae; and the endosomes were formed after nonreceptor-mediated endocytosis of electronopaque and fluorescent soluble markers. By electron microscopy, survey of the cell profiles of M phi that had been treated with 10 mM NH4Cl so that Ph-L fusion was prevented, and that displayed many ferritin-labeled endosomes, revealed numerous examples of the fusion of electronlucent endosomes, revealed numerous examples of the fusion of electronlucent vesicles with phagosomes, whether containing M. microti bacilli or S. cerevisiae yeasts. Fusion was recognized by transfer of label and by morphological evidence of fusion in progress. The fusing vesicles were classed as endosomes, not NH4Cl-lysosomes, by their appearance and provenance, and because lysosome participation was excluded by the concurrent, NH4Cl-caused block of Ph-L fusion and associated lysosomal stasis. No evidence of such phagosome-endosome (Ph-E) fusion was observed in profiles from M phi treated with chloroquine, nor in those from normal, untreated M phi. NH4Cl-treated living M phi that had ingested yeasts at 37 degrees C, followed by endocytosis of lucifer yellow at 17 degrees C (to accumulate labeled endosomes and postpone label passing to lysosomes), were then restored to 37 degrees C. Fluorescence microscopy showed that as many as half of the yeast phagosomes (previously unlabeled) rapidly became colored. We inferred that this transfer was from endosomes (by Ph-E fusion) because Ph-L passage was blocked (by the NH4Cl). We conclude that NH4Cl induces Ph-E fusion at the same time as it suppressed Ph-L fusion. We discuss the mechanisms of these concurrent effects and suggest that they are independent; and we consider the implications of NH4Cl opening a direct route for endocytosed molecules to reach an intraphagosomal infection without involving lysosomes.

Ammonium Chloride↗
PubMed1990

Influence of phagosomal contents on the apparent inhibition of phagosome-lysosome fusion mediated by polyanionic substances in mouse peritoneal macrophages.

The study of fusion of phagosomes with secondary lysosomes in macrophages is facilitated by assessing transfer of fluorescent or electron-opaque markers (or both) from the lysosomes to the phagosomes. When certain virulent viable pathogens are phagocytosed by mouse peritoneal macrophages, phagosome-lysosome fusion (P-LF) is inhibited. Nonviable counterparts ordinarily cannot impose this block. A similar, but spurious, block to P-LF seems to be mediated from the lysosomal domain following sequestration of certain polyanionic substances. This block has been judged to be relieved by, for example, heat-killed yeasts and various viable bacteria designated as fusion-inducing microorganisms, acting from the phagosome. In this study we tested this concept and believed it to be unfounded. Macrophages labeled with Thorotrast and incubated with dextran sulfate were offered a variety of viable and heat-killed microorganisms for phagocytosis: Saccharomyces cerevisiae, Mycobacterium lepraemurium, Streptococcus faecalis, and Escherichia coli. By electron microscopy, a transfer of Thorotrast to phagosomes up to 18 h was seen to be highly suppressed as compared with controls, but was not notably different for any of the targets, whether viable or not. Instead, inert 0.45-micron carboxylated polystyrene beads (the smallest target) showed the most delivery of marker. If polyanionic agents truly inhibited fusion, then "fusiogenic" microorganisms should free the marker for delivery. If polyanions do not inhibit P-LF and only trap the marker, the behavior of the various targets would correspond to what we found.

Animals↗
PubMed2000

Rab5 regulates the kiss and run fusion between phagosomes and endosomes and the acquisition of phagosome leishmanicidal properties in RAW 264.7 macrophages.

Phagolysosome biogenesis is essential for the killing and degradation of intracellular pathogens. It involves the fusion of phagosomes with various endocytic organelles, a process known to be regulated in part by Rab proteins. We generated RAW 264.7 macrophages expressing an active mutant of Rab5 (Rab5(Q79L)) to determine the role of Rab5 in phagocytosis and phagolysosome biogenesis. Our results indicate that Rab5 stimulates phagocytosis of latex beads but not Fc or C3 receptor-mediated phagocytosis. Rab5 also acts to restrict the complete fusion of phagosomes with endosomes, a phenomenon allowing exchange of solutes from the two compartments without complete intermixing of their membrane (kiss and run). In Rab5(Q79L)-expressing macrophages, uncontrolled fusion events occurred, leading to the appearance of giant phagosomes. These phagosomes could initiate their maturation and acquire LAMP1, but failed to generate the microbicidal conditions needed to kill intracellular parasites. These results identify Rab5 as a key molecule regulating phagosome-endosome fusion and as an essential component in the innate ability of macrophages to restrict the growth of intracellular parasites.

Animals↗
PubMed1996

Translocation of annexin I to plasma membranes and phagosomes in human neutrophils upon stimulation with opsonized zymosan: possible role in phagosome function.

Annexin I in the cytosol of resting neutrophils was translocated to the plasma membranes upon addition of opsonized zymosan (OZ). Maximum translocation could be detected 1 min after stimulation with OZ, and decreased thereafter. Subcellular fractionation studies demonstrated that annexin I could not be detected in the granule fractions in either resting or activated cells, but was found in association with the phagosome fraction. The marked translocation of annexin I was unique to OZ, since formyl-Met-Leu-Phe induced only slight translocation of annexin I to the plasma membranes, and phorbol 12-myristate 13-acetate had no effect at all. The mechanism regulating the translocation of annexin I is not clear. Annexin I is not phosphorylated in resting or stimulated cells. The correlation between the elevation in the intracellular calcium ion concentration ([Ca2+]i) and the degree of translocation of annexin I to the plasma membranes induced by the different stimuli, together with the inhibition of these processes by the addition of EGTA, indicate that the translocation of annexin I can probably be attributed to the rise in [Ca2+]i. However, this cannot be the sole mechanism since ionomycin, which caused an increase in [CA2+]i similar to that induced by OZ, was less efficient than OZ in inducing translocation of annexin I. The induction of annexin I translocation to the plasma membrane by OZ, which was the only agent that induced phagosome formation, and the detection of annexin I in the phagosome fraction, suggest that annexin I participates in phagosome function.

Annexin A1↗
PubMed1996

Mycobacterium-containing phagosomes are accessible to early endosomes and reflect a transitional state in normal phagosome biogenesis.

The success of Mycobacterium as a pathogen hinges on its ability to modulate its intracellular environment. Mycobacterium avium reside in vacuoles with limited proteolytic activity, maintain cathepsin D in an immature form and remain accessible to internalized transferrin. Artificial acidification of isolated phagosomes facilitated processing of cathepsin D, demonstrating that pH alone limits proteolysis in these vacuoles. Moreover, analysis of IgG-bead phagosomes at early time points during their formation indicates that these phagosomes also acquire LAMP 1 and cathepsin D prior to the accumulation of proton-ATPases, and are transiently accessible to sorting endosomes. This suggests that the anomolous distribution of endosomal proteins in M. avium-containing vacuoles results from their arrested differentiation in an early transitional stage through which all phagosomes pass.

Animals↗
PubMed2004

Rab coupling protein associates with phagosomes and regulates recycling from the phagosomal compartment.

The Rab coupling protein (RCP) is a recently identified novel protein that belongs to the Rab11-FIP family. RCP interacts specifically with Rab4 and Rab11, small guanosine-5'-triphosphatases that function as regulators along the endosomal recycling pathway. We used fluorescence confocal microscopy and biochemical approaches to evaluate the participation of RCP during particle uptake and phagosome maturation. In macrophages, RCP is predominantly membrane-bound and displays a punctuate vesicular pattern throughout the cytoplasm. RCP is mainly associated with transferrin-containing structures and Rab11-labeled endosomes. Overexpression of H13, the carboxyl-terminal region of RCP that contains the Rab binding domain, results in an abnormal endosomal compartment. Interestingly, we found that RCP is associated as discrete patches or protein domains to early phagosomal membranes. In macrophages, overexpression of full-length RCP stimulates recycling from the phagosomal compartment, whereas overexpression of H13 diminishes this vesicular transport step. It is likely that acting as an intermediate between Rab4 and Rab11, RCP regulates membrane flux along the phagocytic pathway via recycling events.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Role of the Brucella suis lipopolysaccharide O antigen in phagosomal genesis and in inhibition of phagosome-lysosome fusion in murine macrophages.

Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both cell types. However, the molecular mechanisms and the microbial factors involved are poorly understood. Smooth lipopolysaccharide (LPS) of Brucella has been reported to be an important virulence factor, although its precise role in pathogenesis is not yet clear. In this study, we show that the LPS O side chain is involved in inhibition of the early fusion between Brucella suis-containing phagosomes and lysosomes in murine macrophages. In contrast, the phagosomes containing rough mutants, which fail to express the O antigen, rapidly fuse with lysosomes. In addition, we show that rough mutants do not enter host cells by using lipid rafts, contrary to smooth strains. Thus, we propose that the LPS O chain might be a major factor that governs the early behavior of bacteria inside macrophages.

Animals↗
PubMed2006

Role for RpoS but not RelA of Legionella pneumophila in modulation of phagosome biogenesis and adaptation to the phagosomal microenvironment.

The induction of virulence traits by Legionella pneumophila at the post-exponential phase has been proposed to be triggered by the stringent response mediated by RelA, which triggers RpoS. We show that L. pneumophila rpoS but not relA is required for early intracellular survival and replication within human monocyte-derived macrophages and Acanthamoeba polyphaga. In addition, L. pneumophila rpoS but not relA is required for expression of the pore-forming activity. We provide evidence that RpoS plays a role in the modulation of phagosome biogenesis and in adaptation to the phagosomal microenvironment. Thus, there is no functional link between the stringent response and RpoS in the pathogenesis of L. pneumophila.

Acanthamoeba↗
PubMed1983

Simultaneous measurement of phagocytosis and phagosomal pH by flow cytometry: role of polymorphonuclear neutrophilic leukocyte granules in phagosome acidification.

Human polymorphonuclear neutrophilic leukocytes (PMNLs) phagocytosed fluorescein-isothiocyanate (FITC)-labelled Staphylococcus aureus. Free bacteria, phagocytes, and nonphagocytes were discriminated and quantified by flow cytometry (FCM). The relative fluorescence of phagocyte-associated and free bacteria (Nf:N) was calculated by dividing the mean phagocyte fluorescence by that of the free bacteria and the number of phagocytosed bacteria. Bactericidal capacity and chemiluminescence were measured by standard methods. The red-to-green fluorescence ratio of acridine orange-stained PMNLs (R/G) was measured by FCM. Degradation of bacteria was monitored by the reduction in FITC and ethidiumbromide fluorescence of bacteria liberated from the phagocytes. Bacterial FITC fluorescence was pH dependent. Nf:N was 0.5 to 0.7. Using a standard curve for the interrelationship between bacterial fluorescence and pH, phagosomal pH was 5.0-5.5. Phagocytes, kept at 4 degrees C for 24 h had Nf:N approximately 1, did not degrade bacteria, but killed them and emitted chemiluminescence. NH4Cl increased phagocyte fluorescence by 27% and decreased R/G by 50%. Cyanide and azide did not affect Nf:N. Nf:N of phagocytes from a patient with chronic granulomatous disease was 32% below, and R/G was 32% higher than the controls. Acidification of the phagosomes seems to be related to discharge of PMNL granule contents and independent of the respiratory burst.

Acridine Orange↗
PubMed1996

Phagocytosed live Listeria monocytogenes influences Rab5-regulated in vitro phagosome-endosome fusion.

Survival or destruction of a pathogen following phagocytosis depends, in part, on fusion events between the phagosome and the endosomal or lysosomal compartments. Here we use an in vitro assay to show that phagosome-endosome fusion is regulated by the small GTPase rab5 and that fusion events are influenced by an internalized live organism, Listeria monocytogenes (LM). We compare the in vitro fusion of phagosomes containing heat-killed organisms (dead LM) with that of phagosomes containing a live nonhemolytic mutant (live LMhly-). Unlike the wild-type organism, LMhly- remains trapped inside the phagosome. Phagosome-endosome fusion was reconstituted using biotinylated organisms and endosomes containing horseradish peroxidase conjugated with avidin. With both live LMhly- and dead LM preparations, in vitro phagosome-endosome fusion was time-, temperature-, and cytosol-dependent. Live LMhly- phagosomes exhibited a faster rate of fusion. Fusion in both preparations was regulated by rab5 and possibly by other GTPases. Anti-rab5 antibodies and immunodepletion of cytosolic rab5 inhibited fusion. Addition of glutatione S-transferase-rab5 in the GTP form stimulated phagosome-endosome fusion, whereas addition of a dominant negative mutant of rab5 blocked fusion. Purified live LMhly- phagosomal membranes were enriched in rab5 as revealed by Western blotting, compared with dead LM phagosomes. Fusion of endosomes with dead LM containing phagosomes required ATP and was inhibited by ATP depletion and by N-ethylmaleimide (NEM) and anti-NEM-sensitive factor (NSF) antibodies. Unexpectedly, phagosome-endosome fusion with live LMhly--containing phagosomes was not inhibited by ATP depletion nor by NEM or anti-NSF antibodies. Western blot analysis revealed that live LMhly--containing phagosomes were enriched for membrane-bound NSF, while dead LM containing phagosomes contained low or undetectable quantities. Washing live LMhly--containing phagosomes with 0.5 M KCl removed NSF associated with the membranes and rendered them NEM, ATP, anti-NSF antibody sensitive for fusion. We conclude that rab5 regulates phagosome-endosome fusion and that live microorganisms can up-regulate this process by recruiting rab5 to the membrane.

Animals↗
PubMed1998

Flow analysis of MHC molecules and other membrane proteins in isolated phagosomes.

A method was developed to apply flow cytometry analysis to the characterization of individual phagosomes. Macrophages were incubated with latex beads and homogenized to release the phagosomes. Intact cells and nuclei were removed by low speed centrifugation, and a crude phagosome preparation was fixed with paraformaldehyde. Distinct optical properties of latex bead phagosomes allowed their analytic isolation from other organelles and cell fragments by flow analysis using a narrow gate based on scatter parameters. Furthermore, separate gates were established for phagosomes containing one, two and even three beads, which were sorted and examined by electron microscopy (EM). EM showed that the phagosomal membrane was closely apposed to the latex bead in most phagosomes, but some more spacious phagosomes were also observed. Phagosomes were immunolabeled and subjected to flow analysis for MHC-I and MHC-II molecules and lysosomal membrane markers (LAMPs). The proportion of LAMP-positive phagosomes increased with incubation time, reflecting maturation of phagolysosomes. Significant staining for MHC-I and MHC-II was demonstrated and remained relatively constant with time. Flow analysis of phagosomes allows the characterization and comparison of individual phagosomes, and the identification of subpopulations of phagosomes with differing membrane compositions. It also provides the advantage of analytically isolating phagosomes from other components of the cell without the need for extensive prior physical purification. Thus, it can be used to rapidly assess changes in phagosomal membrane composition as a function of phagosome maturation.

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↗
PubMed1959

Rapid cytochemical identification of phagosomes in various tissues of the rat and their differentiation from mitochondria by the peroxidase method.

1. Granules characterized by their ability to segregate foreign proteins (phagosomes) were identified in the cells of many rat organs after intravenous administration of horseradish peroxidase, by using the conventional test with benzidine for the histochemical detection of peroxidase. The largest numbers of phagosomes were identified in kidney and liver. Considerable numbers were observed cytochemically in pancreas, prostate, epididymis, thymus, spleen, bone marrow, small intestine, heart, pituitary, and mouse mammary carcinoma. 2. The variation in size of the phagosomes ranging from the limit of microscopic visibility up to 5 micro diameter, previously described for kidney, was also observed to occur in many of the other organs. The average size of the phagosomes in different organs was also different, the phagosomes of the liver, for example being on the average smaller than those of the kidney, pancreas, and prostate. 3. In squash preparations of kidney and liver, the phagosomes appeared often in curved rows following the course of the cell membranes of epithelial cells. In several other organs, they appeared aggregated in cells located in the vicinity of blood or lymphatic vessels or capillaries. 4. After injection of peroxidase directly into the brain of a rabbit, a striking concentration of peroxidase was observed in phagosomes of endothelial cells of capillaries and vessels, surrounding the site of injection. It was suggested that this localization may offer an explanation for the so called blood-brain barrier. 5. The cytochemical peroxidase method was applied to smears of isolated fractions of kidney and liver. Only the isolated phagosomes, but not the isolated nuclei, mitochondria, and microsomes, reacted with benzidine after administration of peroxidase. The contamination of conventionally prepared nuclear, mitochondrial, and microsomal fractions of kidney and liver with phagosomes of different sizes was observed. By correlating the cytochemical peroxidase test of smears of isolated fractions with the colorimetric determination of peroxidase, acid phosphatase, and cytochrome oxidase in the same fractions, the differentiation of the phagosomes from mitochondria and other cell granules was facilitated. 6. The marked difference in the osmotic properties of phagosomes and mitochondria, detectable after treatment with 70 per cent alcohol, and the difference in their affinities towards basic fuchsin, made it possible to differentiate the phagosomes from the mitochondria. It was found by this simple procedure that kidney cells of normal rats contain a large number of phagosomes ranging in size from 0.5 to 3 micro, whereas liver cells of normal rats contain relatively few phagosomes of this size but many smaller ones (0.2 to 0.5 micro diameter). These increased in size after treatment of the rats with horseradish peroxidase.

Acid Phosphatase↗
PubMed2005

Directed motility of phagosomes in Tetrahymena thermophila requires actin and Myo1p, a novel unconventional myosin.

The phagosome cycle was investigated in Tetrahymena thermophila, which had internalized fluorescent latex beads. Confocal microscopy of cells from a GFP-actin strain revealed actin filaments that extended 3-5 mum from the periphery of fluorescent phagosomes. In GFP-actin cells and in wild-type cells, motility of fluorescent phagosomes was directed from the oral cavity to the posterior end of the cell. Although 60% of fluorescent phagosomes in the MYO1-knockout strain were motile, movement of phagosomes was not directed toward the posterior end of the cell and was random. Forty percent of fluorescent phagosomes in knockout cells were non-motile in contrast to only 20% non-motile phagosomes in wild-type cells. The increased incidence of non-motile phagosomes in the knockout strain could reflect absence of Myo1p as a motor. Another myosin or other molecular motors could power random movement of phagosomes in the MYO1-knockout strain. In latrunculin-treated GFP-actin cells, movement of fluorescent phagosomes was random. Average velocity of random movement of fluorescent phagosomes in the knockout strain and in latrunculin-treated cells was statistically the same as the average velocity (2.0 +/- 1.9 microm/min) of phagosomes in GFP-actin cells. These findings are an indication that dynamic actin and Myo1p are required for directed motility of phagosomes.

Actins↗