Biogenesis of Afipia-containing phagosomes in non-professional phagocytes

Afipia felis is a Gram-negative alpha-proteobacterium, a rare cause of human cat scratch disease (CSD), and likely a pathogen of amoeba. Here, we show that various members of the genus Afipia attach to and are taken up by various non-professional phagocytic mammalian cells (epithelial CHO, endothelial EA.hy926, epithelial HeLa, epithelial INT407 cells, endothelial HMEC-1, endothelial HUVEC, and fibroblast L929 cells). However, only A. felis was able to do this efficiently. Invasion depended on a functional actin cytoskeleton and much less on microtubule dynamics. Bacteria were slowly taken up into HMEC-1 (and HUVEC) via pocket-like structures and they resided within membrane-surrounded phagosomes. While A. felis was found in a non-canonical endocytic compartment in macrophage cells, Afipia-containing phagosomes in HMEC-1 were transiently positive for early endosomal EEA1 and then became and remained positive for lysosome-associated membrane protein-1 (LAMP1) and the proton-pumping ATPase, suggesting undisturbed, albeit slowed, phagosome biogenesis in these cells. Similarly, at 24h of infection, most phagosomes in HeLa, INT407, HUVEC and in EA.hy926 cells were positive for LAMP1. In summary, A. felis enters various non-professional phagocytes and its compartmentation differs between macrophages and non-professional phagocytes.     

Reprogramming the phagocytic pathway—intracellular pathogens and their vacuoles (Review)

Phagocytic immune cells (particularly macrophages and neutrophils) take up and digest particles that have invaded our bodies. In doing so, they represent a very early line of defence against a microbial attack. During uptake, the particles are wrapped by a portion of the phagocyte's plasma membrane, and a new endocytic compartment, the phagosome, is formed. The typical fate of a phagosome is its fusion with lysosomes to yield a phagolysosome in which the particle is digested. Recent data show that some ‘intracellular microorganisms’ that can cause severe illnesses (tuberculosis, leprosy, legionaire's disease and others) manage to reprogramme the host phagocytes not to deliver them to the lysosomal compartment. This probably results in increased survival of the pathogens. The analysis of the composition of such ‘novel’ compartments and research on the molecular mechanisms underlying the microbial interference with host cell functions are likely to yield important insights into: (1) which endocytic/phagocytic compartments phagocytes employ to handle ingested material in general; (2) how some pathogenic microorganisms can reprogramme the phagocytic pathway; and possibly (3) how infections caused by these microorganisms can be treated more effectively. Here, some studies are presented analysing which compartments intracellular pathogens inhabit and how microbes might be able to reprogramme their host cells.     

Porins facilitate nitric oxide-mediated killing of mycobacteria

Non-pathogenic mycobacteria such us Mycobacterium smegmatis reside in macrophages within phagosomes that fuse with late endocytic/lysosomal compartments. This sequential fusion process is required for the killing of non-pathogenic mycobacteria by macrophages. Porins are proteins that allow the influx of hydrophilic molecules across the mycobacterial outer membrane. Deletion of the porins MspA, MspC and MspD significantly increased survival of M. smegmatis in J774 macrophages. However, the mechanism underlying this observation is unknown. Internalization of wild-type M. smegmatis (SMR5) and the porin triple mutant (ML16) by macrophages was identical indicating that the viability of the porin mutant in vivo was enhanced. This was not due to effects on phagosome trafficking since fusion of phagosomes containing the mutant with late endocytic compartments was unaffected. Moreover, in ML16-infected macrophages, the generation of nitric oxide (NO) was similar to the wild type-infected cells. However, ML16 was significantly more resistant to the effects of NO in vitro compared to SMR5. Our data provide evidence that porins render mycobacteria vulnerable to killing by reactive nitrogen intermediates within phagosomes probably by facilitating uptake of NO across the mycobacterial outer membrane.     

Membrane trafficking along the phagocytic pathway

Phagosome maturation involves extensive remodelling of the phagosomal membrane as a result of intracellular transport events. Newly formed phagosomes exchange membrane-associated and soluble proteins with early endosomes by fusion. Budding of vesicles from the phagosome and fusion with Golgi-derived vesicles may also contribute to the remodelling of the phagosomal compartment. As a consequence of changes in membrane composition, phagosomes acquire the ability to fuse with late endocytic compartments. In vitro reconstitution and other studies suggest that the trafficking events underlying phagosome maturation require several GTP-binding proteins, including Rab5 and Galphas', NSF-SNAP-SNARE complexes and coatomers.     

Endocytic membrane traffic with respect to phagosomes in macrophages infected with non-pathogenic bacteria: phagosomal membrane acquires the same composition as lysosomal membrane

A morphometric analysis was made to study membrane traffic in bone marrow-derived macrophages, containing phagosomes with partially degraded Bacillus subtilis. Cell surface glycoproteins, labeled with radioactive galactose by terminal glycosylation, provided a covalent autoradiographic membrane marker. Membrane compartments were characterized in terms of cytochemical staining for horseradish peroxidase taken up by receptor-mediated endocytosis. The area, composition, and exchange rates of endocytic membrane compartments were measured as in a previous analysis for non-infected macrophages, devoid of phagosomes. In direct comparison with this earlier study, the present data allowed an assessment of the involvement of phagosomes in the interactions between endocytic membrane compartments. The presence of phagosomes led to a 30% reduction of lysosomal membrane area. The rate at which cell surface-derived label flowed into the lysosomal membrane pool was reduced by the same fractional amount. This suggested a linear relationship between flow rate and membrane area. The initial flow rate of label into phagosomes was higher than expected, based on their membrane area being only about 60% that of lysosomes. This rate could only be measured during the early phase of the experiments when phagosomes were younger, therefore displaying a fast exchange rate, reminiscent of the endosome compartment. However, steady-state conditions, at late times, strongly suggested that phagosomes with degraded contents finally acquire membrane of lysosomal origin. First, the composition of phagosome membrane became the same as that of lysosomes, remaining unchanged as compared to non-infected cells. Second, the membrane area of phagosomes amounted to the loss of lysosomal membrane area in infected cells.     

Shaping cups into phagosomes and macropinosomes

The ingestion of particles or cells by phagocytosis and of fluids by macropinocytosis requires the formation of large endocytic vacuolar compartments inside cells by the organized movements of membranes and the actin cytoskeleton. Fc-receptor-mediated phagocytosis is guided by the zipper-like progression of local, receptor-initiated responses that conform to particle geometry. By contrast, macropinosomes and some phagosomes form with little or no guidance from receptors. The common organizing structure is a cup-shaped invagination of the plasma membrane that becomes the phagosome or macropinosome. Recent studies, focusing on the physical properties of forming cups, indicate that a feedback mechanism regulates the signal transduction of phagocytosis and macropinocytosis.     

TI-VAMP/VAMP7 is required for optimal phagocytosis of opsonised particles in macrophages

Phagocytosis relies on extension of plasmalemmal pseudopods generated by focal actin polymerisation and delivery of membranes from intracellular pools. Here we show that compartments of the late endocytic pathway, bearing the tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP/VAMP7), are recruited upon particle binding and undergo exocytosis before phagosome sealing in macrophages during Fc receptor (FcR)-mediated phagocytosis. Expression of the dominant-negative amino-terminal domain of TI-VAMP or depletion of TI-VAMP with small interfering RNAs inhibited phagocytosis mediated by Fc or complement receptors. In addition, inhibition of TI-VAMP activity led to a reduced exocytosis of late endocytic vesicles and this resulted in an early blockade of pseudopod extension, as observed by scanning electron microscopy. Therefore, TI-VAMP defines a new pathway of membrane delivery required for optimal FcR-mediated phagocytosis.     

Maturation of Rhodococcus equi-containing vacuoles is arrested after completion of the early endosome stage

Rhodococcus equi is a facultative intracellular bacterium that can cause bronchopneumonia in foals and AIDS patients. Here, we have analyzed R. equi-containing vacuoles (RCVs) in murine macrophages by confocal laser scanning microscopy, by transmission electron microscopy and by immunochemistry upon purification. We show that RCVs progress normally through the early stages of phagosome maturation acquiring PI3P, early endosome antigen-1, and Rab5, and loosing all or much of them within minutes. Although mature RCVs possess the normally late endocytic markers, lysosome-associated membrane proteins, lysobisphosphatidic acid and Rab7, they lack other hallmark features of late endocytic organelles such as possession of cathepsin D, acid beta-glucuronidase, proton-pumping ATPase and the ability to fuse with prelabeled lysosomes. Bacterial strains possessing a virulence-associated plasmid maintain a nonacidified compartment for 48 h, whereas isogenic strains lacking such plasmids acidify progressively. In summary, RCVs represent a novel phagosome maturation stage positioned after completion of the early endosome stage and before reaching a fully mature late endosome compartment. In addition, vacuole biogenesis can be influenced by bacterial plasmids.     

Early intracellular events during internalization of Listeria monocytogenes by J774 cells.

The gram-positive bacillus Listeria monocytogenes gains entry into host cells through a phagosome membrane that forms around entering bacteria. During the early stages of internalization the invading bacteria appear to modify the protein composition of the forming phagosome membrane in J774 cells. MHC class II molecules on the cell surface and exposed surface molecules available for biotinylation are excluded from the bacteria-host cell membrane interface and from the forming phagosome. This exclusion of MHC class II molecules from the early phagosome may partially help to explain previous reports suggesting that L. monocytogenes is able to interfere with antigen presentation. Inside the host cell, MHC class II molecules are delivered to the phagosome membrane. This is followed by delivery of LAMP 1, a marker of late endocytic compartments, and fusion with low-pH compartments. The bacteria then escape into the cell cytoplasm, possibly assisted by rapid delivery of this low-pH environment.     

Osmotic swelling of endocytic compartments induced by internalized sucrose is restricted to mature lysosomes in cultured mammalian cells.

To determine which endocytic compartments are sensitive to sucrose-induced osmotic swelling, CHO and Vero cells were cultured for 1-3 days in media containing 0.03 to 0.05 M sucrose. (Sucrose is internalized but not digested by these cells.) To immunolocalize late endocytic compartments, cells were fixed with formaldehyde and labeled with antibodies against the 215-kDa mannose 6-phosphate receptor (prelysosomal compartment) and LAMP-1 and -2 (mature lysosomes). Early endosomes were labeled by a 2-min uptake of lucifer yellow, mature lysosomes by greater than or equal to 16-h uptake of lucifer yellow followed by a 2-h chase. The data showed that sucrose induced swelling of mature lysosomes only (mannose 6-phosphate receptor negative, LAMP-1 and LAMP-2 positive); early endosomes and the prelysosomal compartment were not affected by the presence of sucrose, i.e., osmotically swollen. Accumulation of lucifer yellow in the swollen compartment was insensitive to cycloheximide. These results suggest, by inference, that the complement of membrane transport proteins that regulate the osmotic properties of endocytic organelles must be discontinuously distributed along the endocytic pathway.     

Immunocytochemical characterization of the endocytic and phagolysosomal compartments in peritoneal macrophages

We have used endocytic and phagocytic tracers in an EM immunocytochemical study to define the compartments of the phagocytic and endocytic pathways in mouse peritoneal macrophages. Endocytosed BSA-gold appeared successively in early endosomes, spherical endosomal vesicles, a late endosomal tubuloreticular compartment (TC), and terminal lysosomes. The TC appeared as an elaborate structure enriched for the lysosomal membrane glycoproteins Lamp 1 and Lamp 2, and expressing significant levels of rab7, a late endosome-specific GTP-binding protein. The cation-independent mannose-6-phosphate receptor was restricted to specialized regions of the TC that were predominantly adjacent to the Golgi complex. Both the early endosome and the TC had coated bud structures whose composition and function are presently unknown. Phagolysosomes containing latex beads expressed the same membrane antigens and received endocytic tracers simultaneously with the TC. Since the membrane surrounding both organelles was also in direct continuity, we assume that both structures form one functional compartment. Macrosialin, an antigen confined to macrophages and dendritic cells, was heavily expressed in TC and phagolysosomal membranes with low levels being detected in other endosomal compartments and on the cell surface. Treatment of cells with wheat germ agglutinin drastically altered the morphology of the TC, giving rise to sheets of tightly adherent membrane and greatly expanded vesicles, in which cell-associated wheat germ agglutinin was concentrated. The spherical endosomal carrier vesicles loaded with internalized gold tracers clustered nearby, often making contact without fusing. Since the delivery of endocytic tracer to the TC was significantly delayed these experiments suggest that the lectin is somehow preventing the endosome vesicles from fusing with the TC. Collectively, our data argue first that the PLC is equivalent to the "tubular lysosomes" commonly described in macrophages, and second that the meeting of the phagocytic and endocytic pathway occurs in this compartment.     

Dynamin A, Myosin IB and Abp1 couple phagosome maturation to F-actin binding

The role of actin, class I myosins and dynamin in endocytic uptake processes is well characterized, but their role during endo-phagosomal membrane trafficking and maturation is less clear. In Dictyostelium, knockout of myosin IB (myoB) leads to a defect in membrane protein recycling from endosomes back to the plasma membrane. Here, we show that actin plays a central role in the morphology and function of the endocytic pathway. Indeed, latrunculin B (LatB) induces endosome tubulation, a phenotype also observed in dynamin A (dymA)-null cells. Knockout of dymA impairs phagosome acidification, whereas knockout of myoB delays reneutralization, a phenotype mimicked by a low dose of LatB. As a read out for actin-dependent processes during maturation, we monitored the capacity of purified phagosomes to bind F-actin in vitro, and correlated this with the presence of actin-binding and membrane-trafficking proteins. Phagosomes isolated from myoB-null cells showed an increased binding to F-actin, especially late phagosomes. In contrast, early phagosomes from dymA-null cells showed reduced binding to F-actin while late phagosomes were unaffected. We provide evidence that Abp1 is the main F-actin-binding protein in this assay and is central for the interplay between DymA and MyoB during phagosome maturation.     

Linking exocytosis and endocytosis during phagocytosis

Phagocytosis is used by macrophages, dendritic cells and neutrophils to capture and destroy pathogens and particulate antigens. Although localized assembly of actin filaments is the driving force for particle internalization, exocytosis of intracellular compartments, and in particular endocytic compartments, has been shown recently to be required for the early steps of phagosome formation. Here we report on the different compartments undergoing exocytosis during phagocytosis, with a special focus on late endosomes. We then compare this process with secretion from lysosomes or lysosome-related organelles in specialized cells. Finally, we discuss how some of the molecular mechanisms responsible for lysosome-related organelle secretion could also be implicated in phagosome formation.     

Membrane sorting in the endocytic and phagocytic pathway of Dictyostelium discoideum

To study sorting in the endocytic pathway of a phagocytic and macropinocytic cell, monoclonal antibodies to membrane proteins of Dictyostelium discoideum were generated. Whereas the p25 protein was localized to the cell surface, p80 was mostly present in intracellular endocytic compartments as observed by immunofluorescence as well as immunoelectron microscopy analysis. The p80 gene was identified and encodes a membrane protein presumably involved in copper transport. Expression of chimeric proteins revealed that the cytoplasmic domain of p80 was sufficient to cause constitutive endocytosis and localization of the protein to endocytic compartments. Dileucine- and tyrosine-based endocytic signals described previously in mammalian systems were also capable of targeting chimera to endocytic compartments. In phagocytosing cells no membrane sorting was observed during formation of the phagosome. Both p25 and p80 were incorporated non-selectively in nascent phagosomes, and then retrieved shortly after phagosome closure. Our results emphasize the fact that very active membrane traffic takes place in phagocytic and macropinocytic cells. This is coupled with precise membrane sorting to maintain the specific composition of endocytic compartments.     

Evidence for pore-forming ability by Legionella pneumophila

Legionella pneumophila is the cause of Legionnaires' pneumonia. After internalization by macrophages, it bypasses the normal endocytic pathway and occupies a replicative phagosome bound by endoplasmic reticulum. Here, we show that lysis of macrophages and red blood cells by L . pneumophila was dependent on dotA and other loci known to be required for proper targeting of the phagosome and replication within the host cell. Cytotoxicity occurred rapidly during a high-multiplicity infection, required close association of the bacteria with the eukaryotic cell and was a form of necrotic cell death accompanied by osmotic lysis. The differential cytoprotective ability of high-molecular-weight polyethylene glycols suggested that osmotic lysis resulted from insertion of a pore less than 3 nm in diameter into the plasma membrane. Results concerning the uptake of membrane-impermeant fluorescent compounds of various sizes are consistent with the osmoprotection analysis. Therefore, kinetic and genetic evidence suggested that the apparent ability of L . pneumophila to insert a pore into eukaryotic membranes on initial contact may play a role in altering endocytic trafficking events within the host cell and in the establishment of a replicative vacuole.     

Morphology and dynamics of the endocytic pathway in Dictyostelium discoideum

Dictyostelium discoideum is a genetically and biochemically tractable social amoeba belonging to the crown group of eukaryotes. It performs some of the tasks characteristic of a leukocyte such as chemotactic motility, macropinocytosis, and phagocytosis that are not performed by other model organisms or are difficult to study. D. discoideum is becoming a popular system to study molecular mechanisms of endocytosis, but the morphological characterization of the organelles along this pathway and the comparison with equivalent and/or different organelles in animal cells and yeasts were lagging. Herein, we used a combination of evanescent wave microscopy and electron microscopy of rapidly frozen samples to visualize primary endocytic vesicles, vesicular-tubular structures of the early and late endo-lysosomal system, such as multivesicular bodies, and the specialized secretory lysosomes. In addition, we present biochemical and morphological evidence for the existence of a micropinocytic pathway, which contributes to the uptake of membrane along side macropinocytosis, which is the major fluid phase uptake process. This complex endosomal compartment underwent continuous cycles of tubulation/vesiculation as well as homo- and heterotypic fusions, in a way reminiscent of mechanisms and structures documented in leukocytes. Finally, egestion of fluid phase from the secretory lysosomes was directly observed.     

GFP-golvesin constructs to study Golgi tubulation and post-Golgi vesicle dynamics in phagocytosis

Dictyostelium cells are professional phagocytes that are optimally suited for the imaging of phagosome processing from particle uptake to exocytosis. In order to design fluorescent probes for monitoring membrane trafficking in the endocytic pathway, we have dissected a membrane protein, golvesin, and have linked fragments of its sequence to GFP. Endogenous golvesin is partitioned between the ER, the Golgi apparatus, endosomes, and the contractile vacuole complex. We have localized signals that are required for exit from the Golgi to post-Golgi compartments to the C-terminal region of the golvesin sequence. One GFP-tagged fragment turned out to be a highly specific Golgi marker and was used to demonstrate the interaction of Golgi tubules with phagosomes. Signals essential for the retrieval of golvesin at the end of phagosome processing were localized to the N-terminal region. A truncated golvesin construct escaping retrieval was employed in recording the delivery of a phagosomal protein to the plasma membrane. Applying this construct to a phagosome filled with multiple particles, we observed that the phagosome is segmented during exocytosis, meaning that sequential release of particles alternates with membrane fusion.     

Mycobacterium's arrest of phagosome maturation in macrophages requires Rab5 activity and accessibility to iron

Many mycobacteria are intramacrophage pathogens that reside within nonacidified phagosomes that fuse with early endosomes but do not mature to phagolysosomes. The mechanism by which mycobacteria block this maturation process remains elusive. To gain insight into whether fusion with early endosomes is required for mycobacteria-mediated inhibition of phagosome maturation, we investigated how perturbing the GTPase cycles of Rab5 and Rab7, GTPases that regulate early and late endosome fusion, respectively, would affect phagosome maturation. Retroviral transduction of the constitutively activated forms of both GTPases into primary murine macrophages had no effect on Mycobacterium avium retention in an early endosomal compartment. Interestingly, expression of dominant negative Rab5, Rab5(S34N), but not dominant negative Rab7, resulted in a significant increase in colocalization of M. avium with markers of late endosomes/lysosomes and increased mycobacterial killing. This colocalization was specific to mycobacteria since Rab5(S34N) expressing cells showed diminished trafficking of endocytic tracers to lysosomes. We further demonstrated that maturation of M. avium phagosomes was halted in Rab5(S34N) expressing macrophages supplemented with exogenous iron. These findings suggest that fusion with early endosomes is required for mycobacterial retention in early phagosomal compartments and that an inadequate supply of iron is one factor in mycobacteria's inability to prevent the normal maturation process in Rab5(S34N)-expressing macrophages.     

Internalization of Bordetella pertussis adenylate cyclase–haemolysin into endocytic vesicles contributes to macrophage cytotoxicity

Bordetella pertussis adenylate cyclase–haemolysin is a critical virulence factor in the murine model of intranasal infection, where it is required for several pathological effects, including macrophage apoptosis. Based on biochemical and immunological properties, it was proposed that the toxin was delivered directly to the cytoplasm of eukaryotic cells without trafficking through the endocytic pathway. In the present study, we analysed the cellular distribution of the adenylate cyclase–haemolysin during intoxication of macrophages. We showed that, shortly after its initial binding to the plasma membrane of macrophages, the toxin gains access to intracellular compartments that become progressively positive for the endosomal marker transferrin, but not for the lysosomal membrane protein CD107a/Lamp1. Importantly, the vesicular trafficking of the adenylate cyclase–haemolysin appears to be required for its ability to induce macrophage death. Inhibitors of actin polymerization and of macropinocytosis, as well as depletion of plasma membrane cholesterol and disruption of the Golgi network, reduce the toxin's ability to kill macrophages. Altogether, these results suggest that internalization of the adenylate cyclase–haemolysin into endocytic vesicles, at least partly through macropinocytosis, contributes to cytotoxicity.     

Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake

Numerous intracellular bacterial pathogens modulate the nature of the membrane-bound compartment in which they reside, although little is known about the molecular basis for this control. Legionella pneumophila is a bacterial pathogen able to grow within human alveolar macrophages and residing in a phagosome that does not fuse with lysosomes. This study demonstrates that the dotA product is required to regulate trafficking of the L. pneumophila phagosome. Phagosomes containing L. pneumophila dotA ⁺ bacteria exhibited differential trafficking profiles when compared with isogenic dotA mutants. Phagosomes containing dotA mutants showed rapid accumulation of the lysosomal glycoprotein LAMP-1 as early as 5 min after uptake, whereas the majority of wild-type L. pneumophila phagosomes did not acquire LAMP-1. The association of LAMP-1 with phagosomes containing dotA mutant bacteria was concomitant with the appearance of the small GTP-binding protein Rab7 on the vacuolar membrane. These data demonstrate that phagosomes containing replication-competent L. pneumophila evade early endocytic fusion events. In contrast, the kinetics of LAMP-1 and Rab7 association indicate that the dotA mutants are routed along a well-characterized endocytic pathway leading to fusion with lysosomes. Genetic studies show that L. pneumophila requires DotA expression before macrophage uptake in order to establish an intracellular site for replication. However, the bacteria do not appear to require continuous expression of the DotA protein to maintain a replicative phagosome. These data indicate that DotA is one factor that plays a fundamental role in regulating initial phagosome trafficking decisions either upon or immediately after macrophage uptake.