Phagocyte meets prey: uptake, internalization, and killing of bacteria by Dictyostelium amoebae

Dictyostelium cells are professional phagocytes that avidly consume bacteria, their natural prey. Fluorescent probes have allowed us to monitor the initial steps in this process in living cells. Using probes that bind to F-actin, we have visualized the assembly and disassembly of actin filaments responsible for extending the phagocytic cup to engulf a bacterium, and, after the phagosome has sealed, the assembly of new actin filaments to propel the phagosome away from the site of uptake. Using bacteria expressing fluorescent proteins that are susceptible to proteolysis, we have monitored the loss of that fluorescent signal and the staining of the bacterial contents with neutral red, indicating permeabilization of the bacterial cell wall and acidification of the cytoplasm. We find that acidification occurs during a period of microtubule-based transport that promotes fusion of the phagosome with microtubule-associated acidic endosomes. Actin-powered phagosome internalization, transport of the phagosome along microtubules, proteolysis and acidification of bacterial contents, all typically occur within the first six or seven minutes after formation of the phagosome. Thus, tracking individual phagosomes has revealed that early steps in phagosome maturation occur much more rapidly than had been inferred from previous population studies.     

Repeated cycles of rapid actin assembly and disassembly on epithelial cell phagosomes

We have found that early in infection of the intracellular pathogen Listeria monocytogenes in Madin-Darby canine kidney epithelial cells expressing actin conjugated to green fluorescent protein, F-actin rapidly assembles (approximately 25 s) and disassembles (approximately 30 s) around the bacteria, a phenomenon we call flashing. L. monocytogenes strains unable to perform actin-based motility or unable to escape the phagosome were capable of flashing, suggesting that the actin assembly occurs on the phagosome membrane. Cycles of actin assembly and disassembly could occur repeatedly on the same phagosome. Indirect immunofluorescence showed that most bacteria were fully internalized when flashing occurred, suggesting that actin flashing does not represent phagocytosis. Escherichia coli expressing invA, a gene product from Yersinia pseudotuberculosis that mediates cellular invasion, also induced flashing. Furthermore, polystyrene beads coated with E-cadherin or transferrin also induced flashing after internalization. This suggests that flashing occurs downstream of several distinct molecular entry mechanisms and may be a general consequence of internalization of large objects by epithelial cells.     

Involvement of ezrin/moesin in de novo actin assembly on phagosomal membranes

The current study focuses on the molecular mechanisms responsible for actin assembly on a defined membrane surface: the phagosome. Mature phagosomes were surrounded by filamentous actin in vivo in two different cell types. Fluorescence microscopy was used to study in vitro actin nucleation/polymerization (assembly) on the surface of phagosomes isolated from J774 mouse macrophages. In order to prevent non-specific actin polymerization during the assay, fluorescent G-actin was mixed with thymosin beta4. The cytoplasmic side of phagosomes induced de novo assembly and barbed end growth of actin filaments. This activity varied cyclically with the maturation state of phagosomes, both in vivo and in vitro. Peripheral membrane proteins are crucial components of this actin assembly machinery, and we demonstrate a role for ezrin and/or moesin in this process. We propose that this actin assembly process facilitates phagosome/endosome aggregation prior to membrane fusion.     

Host cell-dependent secretion and translocation of the LepA and LepB effectors of Legionella pneumophila

Legionella pneumophila is the Gram-negative bacterial agent of Legionnaires' disease, an acute, often fatal pneumonia. L. pneumophila infects alveolar macrophages, evading the antimicrobial defences of the phagocyte by preventing fusion of the phagosome with lysosomes and avoiding phagosome acidification. The bacteria then modulate the composition of the vacuole so that it takes on the characteristics of the endoplasmic reticulum. Similar events occur when the bacteria infect unicellular protozoa. It is thought that replication in fresh water protozoa provides an environmental reservoir for the organism. Several effector proteins are delivered to the host by the Icm/Dot type IV secretion system (TFSS). Some of these have been shown to participate in the trafficking of the Legionella phagosome. Here we describe the ability of the Icm/Dot TFSS to translocate two effectors, LepA and LepB, that play a role in the non-lytic release of Legionella from protozoa. We report that translocation of the Lep proteins is inhibited by agents that depolymerize actin filaments and that effectors may be secreted into the extracellular medium upon cell contact. Depletion of the Lep proteins by deletion of their genes results in increased ability to lyse red blood cells. In contrast, overexpression of Lep-containing hybrid proteins appears to specifically inhibit the activity of the Icm/Dot TFSS and may prevent the delivery of other effectors that are critical for intracellular multiplication.     

Selected lipids activate phagosome actin assembly and maturation resulting in killing of pathogenic mycobacteria

Pathogenic mycobacteria such as Mycobacterium tuberculosis and Mycobacterium avium facilitate disease by surviving intracellularly within a potentially hostile environment: the macrophage phagosome. They inhibit phagosome maturation processes, including fusion with lysosomes, acidification and, as shown here, membrane actin assembly. An in vitro assay developed for latex bead phagosomes (LBPs) provided insights into membrane signalling events that regulate phagosome actin assembly, a process linked to membrane fusion. Different lipids were found to stimulate or inhibit actin assembly by LBPs and mycobacterial phagosomes in vitro. In addition, selected lipids activated actin assembly and phagosome maturation in infected macrophages, resulting in a significant killing of M. tuberculosis and M. avium. In contrast, the polyunsaturated sigma-3 lipids behaved differently and stimulated pathogen growth. Thus, lipids can be involved in both stimulatory and inhibitory signalling networks in the phagosomal membrane.     

Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis

Phagocytosis requires localized and transient remodeling of actin filaments. Phosphoinositide signaling is believed to play an important role in cytoskeletal organization, but it is unclear whether lipids, which can diffuse along the membrane, can mediate the focal actin assembly required for phagocytosis. We used imaging of fluorescent chimeras of pleckstrin homology and C1 domains in live macrophages to monitor the distribution of phosphatidylinositol-4,5-bisphosphate (4,5-PIP(2)) and diacylglycerol, respectively, during phagocytosis. Our results reveal a sequence of exquisitely localized, coordinated steps in phospholipid metabolism: a focal, rapid accumulation of 4,5-PIP(2) accompanied by recruitment of type Ialpha phosphatidylinositol phosphate kinase to the phagosomal cup, followed by disappearance of the phosphoinositide as the phagosome seals. Loss of 4,5-PIP(2) correlated with mobilization of phospholipase Cgamma (PLCgamma) and with the localized formation of diacylglycerol. The presence of 4, 5-PIP(2) and active PLCgamma at the phagosome was shown to be essential for effective particle ingestion. The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP(2) is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling.     

TPL‐2 kinase induces phagosome acidification to promote macrophage killing of bacteria

Tumour progression locus 2 (TPL‐2) kinase mediates Toll‐like receptor (TLR) activation of ERK1/2 and p38α MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase‐independent regulatory function for TPL‐2 in phagosome maturation, an essential process for killing of phagocytosed microbes. TPL‐2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Quantitative proteomics revealed that blocking TPL‐2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V‐ATPase proton pump subunits. Furthermore, TPL‐2 stimulated the phosphorylation of DMXL1, a regulator of V‐ATPases, to induce V‐ATPase assembly and phagosome acidification. Consistent with these results, TPL‐2 catalytic activity was required for phagosome acidification and the efficient killing of Staphylococcus aureus and Citrobacter rodentium following phagocytic uptake by macrophages. TPL‐2 therefore controls innate immune responses of macrophages to bacteria via V‐ATPase induction of 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.     

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.     

Co-loss of profilin I, II and cofilin with actin from maturing phagosomes inDictyostelium discoideum

Summary Although it is known that actin polymerizes rapidly at the plasma membrane during the ingestion phase of phagocytosis, not yet fully understood are the mechanisms by which actin is recruited to form a phagoeytic cup and subsequently is dissociated from the phagosome. The aim of this study was to identify actin-binding proteins that mediated actin filament dynamics during phagosome formation and processing. We report that profilins I and II, which promote filament assembly, and cofilin, which stimulates filament disassembly, were constituents of phagosomes isolated fromDictyostelium discoideum fed latex beads, and associated with actin. Biochemical analyses detected one isoform only of cofilin, which bound actin in unstimulated cells as well as in cells engaged in phagocytosis, subjected to various stress treatments, and through development. At membranes of young phagosomes, profilins I and II colocalized with monomeric actin labeled with fluorescent DNase I, and cofilin colocalized with filamentous actin labeled with rhodamine phalloidin. Both immunocytochemical and quantitative immunoblotting data indicated that the kinetic loss of profilins I, II, and cofilin of maturing phagosomes closely followed the falling levels of actin associated with the vesicles. As evidence of vesicle processing,D. discoideum crystal protein (an esterase) was recruited rapidly to phagosomes and its levels increased while those of actin, profilins I, II, and cofilin jointly decreased. The localization data and concurrent losses of profilins and cofilin with actin from phagosomes are consistent with the roles of these actin-binding proteins in filament dynamics and indicated that they were involved in regulating the assembly and disassembly of the actin coat of phagosomes.Abbreviations DNase deoxyribonuclease - FITC fluorescein isothiocyanate - NEpHGE nonequilibrium pH gradient gel electrophoresis - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Scaffolds and chaperones in myofibril assembly: putting the striations in striated muscle

Sarcomere assembly in striated muscles has long been described as a series of steps leading to assembly of individual proteins into thick filaments, thin filaments and Z-lines. Decades of previous work focused on the order in which various structural proteins adopted the striated organization typical of mature myofibrils. These studies led to the view that actin and α-actinin assemble into premyofibril structures separately from myosin filaments, and that these structures are then assembled into myofibrils with centered myosin filaments and actin filaments anchored at the Z-lines. More recent studies have shown that particular scaffolding proteins and chaperone proteins are required for individual steps in assembly. Here, we review the evidence that N-RAP, a LIM domain and nebulin repeat protein, scaffolds assembly of actin and α-actinin into I-Z-I structures in the first steps of assembly; that the heat shock chaperone proteins Hsp90 & Hsc70 cooperate with UNC-45 to direct the folding of muscle myosin and its assembly into thick filaments; and that the kelch repeat protein Krp1 promotes lateral fusion of premyofibril structures to form mature striated myofibrils. The evidence shows that myofibril assembly is a complex process that requires the action of particular catalysts and scaffolds at individual steps. The scaffolds and chaperones required for assembly are potential regulators of myofibrillogenesis, and abnormal function of these proteins caused by mutation or pathological processes could in principle contribute to diseases of cardiac and skeletal muscles.     

Dynamic life and death interactions between Mycobacterium smegmatis and J774 macrophages

After internalization into macrophages non-pathogenic mycobacteria are killed within phagosomes. Pathogenic mycobacteria can block phagosome maturation and grow inside phagosomes but under some conditions can also be killed by macrophages. Killing mechanisms are poorly understood, although phago-lysosome fusion and nitric oxide (NO) production are implicated. We initiated a systematic analysis addressing how macrophages kill 'non-pathogenic'Mycobacterium smegmatis. This system was dynamic, involving periods of initial killing, then bacterial multiplication, followed by two additional killing stages. NO synthesis represented the earliest killing factor but its synthesis stopped during the first killing period. Phagosome actin assembly and fusion with late endocytic organelles coincided with the first and last killing phase, while recycling of phagosome content and membrane coincided with bacterial growth. Phagosome acidification and acquisition of the vacuolar (V) ATPase followed a different pattern coincident with later killing phases. Moreover, V-ATPase localized to vesicles distinct from classical late endosomes and lysosomes. Map kinase p38 is a crucial regulator of all processes investigated, except NO synthesis, that facilitated the host for some functions while being usurped by live bacteria for others. A mathematical model argues that periodic high and low cellular killing activity is more effective than is a continuous process.     

Rearrangements of actin cytoskeleton during infection with Escherichia coli O157 in macrophages

The lamina propria of the large intestine is rich in macrophages, and they might be one of the first lines of the host defense in enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection. Although macrophages were infected with them, they can survive the EHEC O157 infection. We examined the structural rearrangements of the actin cytoskeleton during the microbial infection process. Macrophage actin filaments were rearranged in the following sequence; 1) disappearance of the actin filament bundles in the cytoplasm, 2) accumulation of actin filaments under the cell surface, and 3) construction of actin networks underlying the endosome membrane. Before infection, actin filaments were distributed under the cell surface and in bundles located in the macrophage cytoplasm. Within 2 min, infection caused a rapid and marked loss of the actin filament bundles that had run parallel to the long axis of the cell. Concomitant with the loss, actin filaments became more markedly distributed under the cell surface. In the formation of the endosome, new networks of actin filaments were constructed below the phagosome membrane. The networks contained a large amount of actin as well as a fodrin-like immunoreactivity. The thickness of the networks reached about 400 nm under the phagosome membrane. The actin networks disappeared again after the bacterial digestion. The results of this study showed that actin filaments undergo three major rearrangements of the actin filaments during the infection in macrophages, and suggested that the third rearrangement is mediated by actin-binding proteins, such as a fodrin-like molecules. These morphological changes in macrophages were not clear after infection with other strains of Escherichia coli.     

Temporal and spatial trigger of post‐exponential virulence‐associated regulatory cascades by Legionella pneumophila after bacterial escape into the host cell cytosol

During late stages of infection and prior to lysis of the infected macrophages or amoeba, the Legionella pneumophila‐containing phagosome becomes disrupted, followed by bacterial escape into the host cell cytosol, where the last few rounds of bacterial proliferation occur prior to lysis of the plasma membrane. This coincides with growth transition into the post‐exponential (PE) phase, which is controlled by regulatory cascades including RpoS and the LetA/S two‐component regulator. Whether the temporal expression of flagella by the regulatory cascades at the PE phase is exhibited within the phagosome or after bacterial escape into the host cell cytosol is not known. We have utilized fluorescence microscopy‐based phagosome integrity assay to differentiate between vacuolar and cytosolic bacteria/or bacteria within disrupted phagosomes. Our data show that during late stages of infection, expression of FlaA is triggered after bacterial escape into the macrophage cytosol and the peak of FlaA expression is delayed for few hours after cytosolic residence of the bacteria. Importantly, bacterial escape into the host cell cytosol is independent of flagella, RpoS and the two‐component regulator LetA/S, which are all triggered by L. pneumophila upon growth transition into the PE phase. Disruption of the phagosome and bacterial escape into the cytosol of macrophages is independent of the bacterial pore‐forming activity, and occurs prior to the induction of apoptosis during late stages of infection. We conclude that the temporal and spatial engagement of virulence‐associated regulatory cascades by L. pneumophila at the PE phase is temporally and spatially triggered after phagosomal escape and bacterial residence in the host cell cytosol.     

Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes

Listeria monocytogenes was used as a model intracellular parasite to study stages in the entry, growth, movement, and spread of bacteria in a macrophage cell line. The first step in infection is phagocytosis of the Listeria, followed by the dissolution of the membrane surrounding the phagosome presumably mediated by hemolysin secreted by Listeria as nonhemolytic mutants remain in intact vacuoles. Within 2 h after infection, each now cytoplasmic Listeria becomes encapsulated by actin filaments, identified as such by decoration of the actin filaments with subfragment 1 of myosin. These filaments are very short. The Listeria grow and divide and the actin filaments rearrange to form a long tail (often 5 microns in length) extending from only one end of the bacterium, a "comet's tail," in which the actin filaments appear randomly oriented. The Listeria "comet" moves to the cell surface with its tail oriented towards the cell center and becomes incorporated into a cell extension with the Listeria at the tip of the process and its tail trailing into the cytoplasm behind it. This extension contacts a neighboring macrophage that phagocytoses the extension of the first macrophage. Thus, within the cytoplasm of the second macrophage is a Listeria with its actin tail surrounded by a membrane that in turn is surrounded by the phagosome membrane of the new host. Both these membranes are then solubilized by the Listeria and the cycle is repeated. Thus, once inside a host cell, the infecting Listeria and their progeny can spread from cell to cell by remaining intracellular and thus bypass the humoral immune system of the organism. To establish if actin filaments are essential for the spread of Listeria from cell to cell, we treated infected macrophages with cytochalasin D. The Listeria not only failed to spread, but most were found deep within the cytoplasm, rather than near the periphery of the cell. Thin sections revealed that the net of actin filaments is not formed nor is a "comet" tail produced.     

The Human Fungal Pathogen Cryptococcus neoformans Escapes Macrophages by a Phagosome Emptying Mechanism That Is Inhibited by Arp2/3 Complex-Mediated Actin Polymerisation

The lysis of infected cells by disease-causing microorganisms is an efficient but risky strategy for disseminated infection, as it exposes the pathogen to the full repertoire of the host''s immune system. Cryptococcus neoformans is a widespread fungal pathogen that causes a fatal meningitis in HIV and other immunocompromised patients. Following intracellular growth, cryptococci are able to escape their host cells by a non-lytic expulsive mechanism that may contribute to the invasion of the central nervous system. Non-lytic escape is also exhibited by some bacterial pathogens and is likely to facilitate long-term avoidance of the host immune system during latency. Here we show that phagosomes containing intracellular cryptococci undergo repeated cycles of actin polymerisation. These actin ‘flashes’ occur in both murine and human macrophages and are dependent on classical WASP-Arp2/3 complex mediated actin filament nucleation. Three dimensional confocal imaging time lapse revealed that such flashes are highly dynamic actin cages that form around the phagosome. Using fluorescent dextran as a phagosome membrane integrity probe, we find that the non-lytic expulsion of Cryptococcus occurs through fusion of the phagosome and plasma membranes and that, prior to expulsion, 95% of phagosomes become permeabilised, an event that is immediately followed by an actin flash. By using pharmacological agents to modulate both actin dynamics and upstream signalling events, we show that flash occurrence is inversely related to cryptococcal expulsion, suggesting that flashes may act to temporarily inhibit expulsion from infected phagocytes. In conclusion, our data reveal the existence of a novel actin-dependent process on phagosomes containing cryptococci that acts as a potential block to expulsion of Cryptococcus and may have significant implications for the dissemination of, and CNS invasion by, this organism.     

Sequential Assembly of Myosin II, an IQGAP-like Protein, and Filamentous Actin to a Ring Structure Involved in Budding Yeast Cytokinesis

We have identified a Saccharomyces cerevisiae protein, Cyk1p, that exhibits sequence similarity to the mammalian IQGAPs. Gene disruption of Cyk1p results in a failure in cytokinesis without affecting other events in the cell cycle. Cyk1p is diffused throughout most of the cell cycle but localizes to a ring structure at the mother–bud junction after the initiation of anaphase. This ring contains filamentous actin and Myo1p, a myosin II homologue. In vivo observation with green fluorescent protein–tagged Myo1p showed that the ring decreases drastically in size during cell division and therefore may be contractile. These results indicate that cytokinesis in budding yeast is likely to involve an actomyosin-based contractile ring. The assembly of this ring occurs in temporally distinct steps: Myo1p localizes to a ring that overlaps the septins at the G1-S transition slightly before bud emergence; Cyk1p and actin then accumulate in this ring after the activation of the Cdc15 pathway late in mitosis. The localization of myosin is abolished by a mutation in Cdc12p, implicating a role for the septin filaments in the assembly of the actomyosin ring. The accumulation of actin in the cytokinetic ring was not observed in cells depleted of Cyk1p, suggesting that Cyk1p plays a role in the recruitment of actin filaments, perhaps through a filament-binding activity similar to that demonstrated for mammalian IQGAPs.     

Hypaphorine, an indole-3-acetic acid antagonist delivered by the ectomycorrhizal fungus Pisolithus tinctorius, induces reorganisation of actin and the microtubule cytoskeleton in Eucalyptus globulus ssp bicostata root hairs

Hypaphorine, an indole alkaloid from the ectomycorrhizal fungus Pisolithus tinctorius Coker & Couch., counteracts indole-3-acetic acid (IAA) activity and controls the rate of root hair elongation in Eucalyptus globulus ssp. bicostata. The present investigation shows that hypaphorine changes cytoskeletal organisation in elongating root hairs of the host. The actin cytoskeleton was investigated by two different fixation and labelling procedures, which gave similar results. In control root hairs, actin organisation was characterised by (i) an actin cap at the very tip region, (ii) a subapical region with reduced labelling and containing fine actin filaments, and (iii) axial bundles of actin filaments running from the subapical part to the base of the root hair. In the hypaphorine-treated root hairs no actin cap was distinguished. The fine actin filaments occurring in the subapical region were replaced by a few thick actin filament bundles that extended from the subapical region toward the root hair tip. In the hypaphorine-treated hairs the total number of actin filament bundles along most of the root hair length was significantly reduced, presumably due to aggregation of pre-existing actin filaments. The first signs of alteration to the cytoskeleton could be detected as soon as 15 min after hypaphorine treatment. In hypaphorine-treated, but not in control root hairs, a patch of aggregated microtubules regularly occurred at a distance of approximately 10 m from the tip, possibly as a consequence of changes induced by hypaphorine in the actin cytoskeleton. The hypaphorine-induced aggregations in the actin and microtubule cytoskeletons could stabilise the structure of cytoskeletal elements, which in turn could hinder the vesicle delivery at the tip necessary for elongation. Such cytoskeletal alterations may be a consequence of the antagonism between IAA and hypaphorine. The latter view was supported by restoration of the actin cytoskeleton in hypaphorine-treated root hairs by IAA application.     

Heparin-binding hemagglutinin HBHA from Mycobacterium tuberculosis affects actin polymerisation

HBHA is a mycobacterial cell surface protein that mediates adhesion to epithelial cells and that has been implicated in the dissemination of Mycobacterium tuberculosis (Mtb) from the site of primary infection. In this work, we demonstrate that HBHA is able to bind G-actin whereas its shorter form, deprived of the lysine-rich C-terminal region (HBHAΔC), does not bind. Consistently, interaction of actin with HBHA is competitive with heparin binding. Notably, we also observe that HBHA, but not HBHAΔC, clearly hampers G-actin polymerisation into F-actin filaments. Since Mtb escapes from the phagosome into the cytosol of host cells, where it can persist and replicate, HBHA is properly localised on the bacterial surface to regulate the dynamic process of cytoskeleton formation driven by actin polymerisation and depolymerisation.