Showing papers on "Phagosome published in 1987"

In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2.

Abstract: Penetration and replication of Listeria monocytogenes within intestinal epithelial cells were studied by infecting the human enterocyte-like cell line Caco-2. Entry was due to directed phagocytosis, as suggested by the inhibiting effect of cytochalasin D on bacterial entry and by electron microscopy showing bacteria inside membrane-limiting vacuoles at the early stage of infection. Only bacteria from pathogenic species (L. monocytogenes and Listeria ivanovii) were able to induce their own phagocytosis by Caco-2 cells, as opposed to Listeria seeligeri, Listeria welshimeri, and Listeria innocua. L. monocytogenes multiplied readily within Caco-2 cells, with an apparent generation time of about 90 min. Listeriolysin O was found to be a major factor promoting intracellular growth of L. monocytogenes. After being internalized at the same rate as that of its hemolytic revertant strain, a nonhemolytic mutant from L. monocytogenes failed to replicate significantly within Caco-2 cells. Electron microscopic study demonstrated that bacteria from the nonhemolytic mutant remained inside phagosomes during cellular infection, whereas hemolytic bacteria from L. monocytogenes were released free within the cytoplasm. This indicates that disruption of vacuole membranes by listeriolysin O-producing strains of L. monocytogenes might be a key mechanism allowing bacteria to escape from phagosomes and to multiply unrestricted within cell cytoplasm.

Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes.

Abstract: Legionella pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative bacterium and a facultative intracellular parasite that multiplies in human monocytes and alveolar macrophages. In this paper, mutants of L. pneumophila avirulent for human monocytes were obtained and extensively characterized. The mutants were obtained by serial passage of wild-type L. pneumophila on suboptimal artificial medium. None of 44 such mutant clones were capable of multiplying in monocytes or exerting a cytopathic effect on monocyte monolayers. Under the same conditions, wild-type L. pneumophila multiplied 2.5-4.5 logs, and destroyed the monocyte monolayers. The basis for the avirulent phenotype was an inability of the mutants to multiply intracellularly. Both mutant and wild-type bacteria bound to and were ingested by monocytes, and both entered by coiling phagocytosis. Thereafter, their intracellular destinies diverged. The wild-type formed a distinctive ribosome-lined replicative phagosome, inhibited phagosome-lysosome fusion, and multiplied intracellularly. The mutant did not form the distinctive phagosome nor inhibit phagosome-lysosome fusion. The mutant survived intracellularly but did not replicate in the phagolysosome. In all other respects studied, the mutant and wild-type bacteria were similar. They had similar ultrastructure and colony morphology; both formed colonies of compact and diffuse type. They had similar structural and secretory protein profiles and LPS profile by PAGE. Both the mutant and wild-type bacteria were completely resistant to human complement in the presence or absence of high titer anti-L. pneumophila antibody. The mutant L. pneumophila have tremendous potential for enhancing our understanding of the intracellular biology of L. pneumophila and other parasites that follow a similar pathway through the mononuclear phagocyte. Such mutants also show promise for enhancing our understanding of immunity to L. pneumophila, and they may serve as prototypes in the development of safe and effective vaccines against intracellular pathogens.

Inhibition of phagosome-lysosome fusion in macrophages by certain mycobacteria can be explained by inhibition of lysosomal movements observed after phagocytosis.

Abstract: We have investigated the mechanism of the inhibition of phagosome-lysosome (P-L) fusion in macrophages known to occur after infection by Mycobacterium tuberculosis and by the mouse pathogen Mycobacterium microti. We have used an M. microti infection and have studied, first, the saltatory movements of periphagosomal secondary lysosomes by means of visual phase-contrast microscopy (a similar use of the method having been previously supported by computer analyses). The movements became slow or static after ingestion of live but not of heat-killed M. microti. They were unaffected by a fusiogenic mycobacterium M. lepraemurium. Second, we studied the behavior of a normally fusiogenic unrelated organism, Saccharomyces cerevisiae, after its phagocytosis by cells already containing live M. microti ingested 18 h previously. We observed, using a fluorescent assay of fusion, that many of these yeast phagosomes now also failed to fuse with the lysosomes; in contrast, when the host M. microti had been heat killed the yeast phagosomes fused normally. These observations were extended by ultrastructural quantitative analyses of P-L fusion, which confirmed the nonfusion of phagosomes of live M. microti and, more particularly, the change to nonfusion from the normal fusion behavior of the separate phagosomes of accompanying yeasts. Third, we have assembled evidence against the likelihood that these M. microti-induced phenomena are nonspecific, i.e., secondary to a general depression of activity of heavily infected host cells. The evidence includes the feasibility of adjusting the degree of infection so as to facilitate visual assessment of organelle movements without the presence of detectable damage to the cells studied; the absence of lysosomal stasis after comparable infection with another mycobacterium of comparable virulence (M. lepraemurium); and the reversibility of the stasis. We conclude that inhibition of lysosome saltatory movements (and consequently its secondary effect on the associated yeasts) is a significant, specifically induced phenomenon. From these observations and considerations, therefore, in conjunction with the analogous inhibition of lysosomal movements in normal macrophages by some chemical inhibitors of P-L fusion, and our suggestion that this association is causally related, we now suggest that M. microti-induced focal lysosomal stasis is also the main means by which the inhibition of P-L fusion is brought about by this organism. This concept is strengthened by the observations on S. cerevisiae, which provide strong evidence that stasis can cause suppression of fusion.

Differential and sequential delivery of fluorescent lysosomal probes into phagosomes in mouse peritoneal macrophages.

Abstract: It has previously been inferred that the fusion of a macrophage secondary lysosome with a phagosome delivers the entire lysosomal contents uniformly to the phagosome. We found, however, that different fluorescent lysosomal probes can enter phagosomes at remarkably different rates, even though they are initially sequestered together in the same organelles. Thus, sulforhodamine is almost exclusively delivered to yeast-containing phagosomes within 2 h of phagocytosis. But fluoresceinated, high molecular weight dextran accumulates in the same phagosomes only over a period of approximately 24 h. We postulate that the delivery of lysosomal contents may involve an intermittent and incremental process in which individual components can be selectively and sequentially transferred.

Mycobacterium leprae surface components intervene in the early phagosome-lysosome fusion inhibition event.

Abstract: Bone marrow-derived cultured macrophages were infected with Mycobacterium leprae. The bacteria were either used as freshly isolated organisms or incubated with M. leprae antiserum (1:5) for 30 min prior to phagocytosis. Immediately after inoculation (1 to 4 h) and at 1 to 8 days later, macrophages were stained for acid phosphatase activity to assess fusions between phagosomes and lysosomes. Inhibition of fusions was essentially apparent as an early event, which was partially reversed by antiserum treatment of the bacteria, suggesting a role for M. leprae immunogenic surface components in this early phenomenon. Later incubation times (1 to 8 days) did not show any considerable difference between antiserum-treated and nontreated bacteria. The formation of an electron-transparent zone around phagocytized bacteria and its role in phagosome-lysosome fusion was investigated, and a direct relationship could not be established.

In vivo killing and degradation of Mycobacterium aurum within mouse peritoneal macrophages.

Abstract: We studied the in vivo killing and degradation of Mycobacterium aurum, a nonpathogenic, acid-fast bacillus, within macrophages after inoculation into the peritoneal cavity of CD-1 mice. The degradative process could be divided in five successive steps that were characterized on ultrastructural and cytochemical grounds and the relative contributions of which were determined by quantitative electron microscopy of samples taken at different times. The main ultrastructural alterations observed during the degradative process were ribosome disaggregation, coagulation of the cytoplasmic matrix, and change in the membrane profile from asymmetric to symmetric, with loss of the polysaccharide components from the outer layer, followed by membrane solubilization and intracellular clearing, followed by digestion of the innermost (peptidoglycan) layer of the cell wall, and at the end of the process, disorganization and collapse of the remaining layers of the cell wall. The correlation between viability and morphology indicated that the first ultrastructural signs of viability loss are cytoplasmic coagulation, change in the membrane geometry, and disappearance of ribosomes. The labeling of lysosomes of peritoneal macrophages with ferritin or by the cytochemical demonstration of inorganic trimetaphosphatase showed that fusion of lysosomes with phagosomes containing mycobacteria occurs in the phagocytes in the mouse peritoneal cavity and is already extensive as soon as 1 h after the inoculation of the bacilli.

Polyanionic agents as inhibitors of phagosome-lysosome fusion in cultured macrophages: evolution of an alternative interpretation.

Abstract: Various natural and synthetic substances classified as polyanionics have been implicated in antagonizing phagosome-lysosome fusion in cultured macrophages. The phenomenon has been judged by comparing the transfer of selected markers from secondary lysosomes to phagosomes in control and in "polyanion" cells. Our earlier studies showed that use of one of the markers, the membrane-permeating acridine orange, was plagued with artifacts that were especially misleading in the presence of polyanionic agents. We now question the validity of data obtained by the alternative technique, electron microscopy. Our present evidence shows that nonionic hydrocolloids of sufficiently high molecular weight prevent the transfer of various colloidal electron-opaque markers from lysosomes to phagosomes in the same manner as does the powerful polyanionic "fusion inhibitor" dextran sulfate. Both kinds of hydrocolloids, however, allow delivery of lysosomal, low-molecular-weight highly charged non-permeant fluorescent markers to phagosomes, probably by a fusion process. We propose that neither type of hydrocolloid inhibits fusion; instead, when sufficiently concentrated, they trap particulate electron-opaque markers in a gelatinous matrix, which may move only slowly out of lysosomes. The polyanionics trap the electron-opaque markers physically and acridine orange ionically. Hence, the semblance of "fusion inhibition."

Polyanionic agents do not inhibit phagosome-lysosome fusion in cultured macrophages.

Abstract: The survival of some intracellular pathogens within macrophages may be aided by an ability of the organism to antagonize, from within the entrapping phagosome, its fusion with lysosomes. On the other hand, certain polyanionic agents have been implicated in imposing a similar block to fusion from the lysosomal domain--because the transfer of various foreign markers from lysosomes to newly formed phagosomes is remarkably inhibited in these polyanion-containing cells. Based on an analysis of various observations and our own recent data, we propose that the polyanionics do not, in fact, prevent phagosome-lysosome fusion but, instead, physically entrap the usual markers in a gelatinous matrix within the lysosomes. This view accounts for many paradoxical consequences of polyanionic accumulation and for the curiously normal behavior of macrophages that are presumed to be suffering from such a crucial intracellular dysfunction.

Trypanosoma cruzi: phagolysosomal fusion after invasion into non professional phagocytic cells.

Abstract: Parasite-containing endocytic vacuoles are formed during the process of in vitro interiorization of the trypomastigote forms of Trypanosoma cruzi by primary culture of mouse fibroblasts, heart and skeletal muscle cells. Fusion of these vacuoles with host cell lysosomes takes place. The process of T. cruzi-muscle cell interaction was analysed by ultrastructural cytochemistry. Two lysosomal enzymes, acid phosphatase and aryl sulphatase and the fusion of peroxidase-labeled secondary lysosomes with the parasitophorus vacuoles were studied. These finding indicate that the basic mechanism of interaction of T. cruzi with the so called non phagocytic cells is similar to that which occurs with phagocytic cells.

Discrepancy in assessment of phagosome-lysosome fusion with two lysosomal markers in murine macrophages infected with Candida albicans.

Abstract: Phagosome-lysosome fusion (P-LF) was studied in cultured mouse resident peritoneal macrophages after phagocytosis of Candida albicans. The macrophages were labeled with acridine orange (AO), the electronopaque colloidal Thorotrast, or both markers. After phagocytosis of heat-killed C. albicans, both markers were delivered to more than 95% of phagosomes. After ingestion of viable C. albicans by labeled cells, delivery of AO to phagosomes was highly suppressed (90%), and yet Thorotrast delivery was almost universal. After phagocytosis and 60 min of incubation, about 10 to 20% of the yeasts were killed, and a similar fraction of phagosomes was stained by the fluorescent marker. The evidence from Thorotrast transfer and assessment of yeast viability indicates that C. albicans largely resists intracellular killing by resident macrophages in the face of entirely uninhibited P-LF. We infer that AO must transfer to nearly all of the phagosomes but that it is evidently recognizable only in those in which the yeasts have been killed or possibly severely injured. This conclusion constitutes yet another limitation in the usefulness of AO for studying P-LF.

Gamma interferon reverses inhibition of leukocyte bactericidal activity by a 25-kilodalton fraction from Mycobacterium tuberculosis.

Abstract: In this study we examined the effects of Mycobacterium tuberculosis cell extracts on the phagocytic activity of polymorphonuclear leukocytes and cultured peripheral blood monocytes. M. tuberculosis cell extracts were fractionated on Sephacryl S-200 columns, and a 25-kilodalton glycolipoprotein was shown to inhibit the intracellular killing ability of these leukocytes but had no effect on their phagocytic potential. This same fraction inhibited fusion of phagosomes with lysosomes, as assessed by noting the transfer of acridine orange from lysosomes to phagosomes. This fraction was shown to have a maximal inhibitory effect when it was in the form of an intact carbohydrate-lipid-protein complex. Gamma interferon (IFN-gamma), but not IFN-alpha, reversed the inhibitory effect of the mycobacterial component on bactericidal activity and on fusion of phagosomes and lysosomes. Thus, this 25-kilodalton fraction of M. tuberculosis cell extract may be important in protecting organisms against phagocytic degradation, an effect which can be reversed by IFN-gamma.

The Roles of Ca2+Dependent Membrane-Binding Proteins in the Regulation and Mechanism of Exocytosis

Abstract: Exocytosis is the process of release of A. stored secretory product from A. cell by fusion of the membrane of the storage organelle (secretory vesicle) with the surface membrane of the cell. This process appears to have been almost universally adopted as the method for secretion of products that are stored and then suddenly released upon stimulation of the cell. For example, it is the basis for the release of neurotransmitters, such as norepinephrine and acetylcholine (ACH); hormones, such as insulin or other polypeptide hormones; and digestive enzymes, such as amylase and trypsin. Therefore, this is one of the most important membrane fusion events that occurs in mature, differentiated cells. Furthermore, some aspects of this process may provide A. model for other intracellular membrane fusion events that occur during regeneration of the plasma membrane, movement of secretory material between the stacks of the Golgi apparatus, fusion of phagosomes with lysosomes, or, in general, any intracellular transport process that involves membrane fusion.

Unusual mechanism of pathogenicity of Pseudomonas aeruginosa isolates from patients with cystic fibrosis.

Abstract: A Pseudomonas aeruginosa strain was isolated from a patient with cystic fibrosis and was found capable of growing within polymorphonuclear leucocytes. As illustrated by electron microscopy, several mechanisms accounted for the intracellular growth: (a) Cell division occurred within the lysosomes; (b) The fusion of the phagosome with the primary lysosome was inhibited; (c) The phagosomal membrane disintegrated, and the bacteria grew within the cytosol; (d) Cytosolic material was separated from the surroundings by bacterial membranes, and subsequently the bacteria coated with host material were released from the leucocyte. The patient suffered from several severe acute pulmonary exacerbations as long as the intracellularly growing variant was detectable in his sputum. After the strain had been eliminated by antibiotic therapy the patient improved (from stage two to one).

Electron microscopic analysis of in vitro interaction of Rickettsia prowazekii with guinea pig macrophages. I. Macrophages from nonimmune animals

Abstract: Alike to macrophages from intact animals, reproduction, destruction and formation of spheroplast-like forms were observed in macrophages from immune guinea pigs 2 months post-infection (p.i.) with the virulent Breinl strain of Rickettsia prowazekii. Unlike to the former, immune macrophages revealed phagolysosomes which were larger in size and contained more rickettsiae showing morphologic signs of destruction. Spheroplast-like forms occurred more often and were more numerous than in intact animals. Structures morphologically similar to L-forms of gram-negative bacteria and that of chlamydiae were also detected. After adding immune serum, more intact rickettsiae and spheroplasts were found in phagosomes as well as more phagolysosomes contained rickettsiae and spheroplasts with morphologic signs of destruction. It is suggested that clearance of immune macrophages from rickettsiae is mediated by at least two processes: on one hand by destruction of rod-shaped rickettsiae within phagolysosomes and, on the other hand, by formation and subsequent destruction of spheroplast-like forms within vacuoles, which probably also function as phagolysosomes.