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.     

Fusion between phagosomes, early and late endosomes: a role for actin in fusion between late, but not early endocytic organelles

Actin is implicated in membrane fusion, but the precise mechanisms remain unclear. We showed earlier that membrane organelles catalyze the de novo assembly of F-actin that then facilitates the fusion between latex bead phagosomes and a mixture of early and late endocytic organelles. Here, we correlated the polymerization and organization of F-actin with phagosome and endocytic organelle fusion processes in vitro by using biochemistry and light and electron microscopy. When membrane organelles and cytosol were incubated at 37 degrees C with ATP, cytosolic actin polymerized rapidly and became organized into bundles and networks adjacent to membrane organelles. By 30-min incubation, a gel-like state was formed with little further polymerization of actin thereafter. Also during this time, the bulk of in vitro fusion events occurred between phagosomes/endocytic organelles. The fusion between latex bead phagosomes and late endocytic organelles, or between late endocytic organelles themselves was facilitated by actin, but we failed to detect any effect of perturbing F-actin polymerization on early endosome fusion. Consistent with this, late endosomes, like phagosomes, could nucleate F-actin, whereas early endosomes could not. We propose that actin assembled by phagosomes or late endocytic organelles can provide tracks for fusion-partner organelles to move vectorially toward them, via membrane-bound myosins, to facilitate fusion.     

Ezrin promotes actin assembly at the phagosome membrane and regulates phago-lysosomal fusion

Phagosome maturation is defined as the process by which phagosomes fuse sequentially with endosomes and lysosomes to acquire an acidic pH and hydrolases that degrade ingested particles. While the essential role of actin cytoskeleton remodeling during particle internalization is well established, its role during the later stages of phagosome maturation remains largely unknown. We have previously shown that purified mature phagosomes assemble F-actin at their membrane, and that the ezrin-radixin-moesin (ERM) proteins ezrin and moesin participate in this process. Moreover, we provided evidence that actin assembly on purified phagosomes stimulates their fusion with late endocytic compartments in vitro. In this study, we further investigated the role of ezrin in phagosome maturation. We engineered a structurally open form of ezrin and demonstrated that ezrin binds directly to the actin assembly promoting factor N-WASP (Neural Wiskott-Aldrich Syndrome Protein) by its FERM domain. Using a cell-free system, we found that ezrin stimulates F-actin assembly on purified phagosomes by recruiting the N-WASP-Arp2/3 machinery. Accordingly, we showed that the down-regulation of ezrin activity in macrophages by a dominant-negative approach caused reduced F-actin accumulation on maturing phagosomes. Furthermore, using fluorescence and electron microscopy, we found that ezrin is required for the efficient fusion between phagosomes and lysosomes. Live-cell imaging analysis supported the notion that ezrin is necessary for the fusogenic process itself, promoting the transfer of the lysosome content into the phagosomal lumen.     

Phosphoinositides regulate membrane-dependent actin assembly by latex bead phagosomes

Actin assembly on membrane surfaces is an elusive process in which several phosphoinositides (PIPs) have been implicated. We have reconstituted actin assembly using a defined membrane surface, the latex bead phagosome (LBP), and shown that the PI(4,5)P(2)-binding proteins ezrin and/or moesin were essential for this process (). Here, we provide several lines of evidence that both preexisting and newly synthesized PI(4,5)P(2), and probably PI(4)P, are essential for phagosomal actin assembly; only these PIPs were routinely synthesized from ATP during in vitro actin assembly. Treatment of LBP with phospholipase C or with adenosine, an inhibitor of type II PI 4-kinase, as well as preincubation with anti-PI(4)P or anti-PI(4,5)P(2) antibodies all inhibited this process. Incorporation of extra PI(4)P or PI(4,5)P(2) into the LBP membrane led to a fivefold increase in the number of phagosomes that assemble actin. An ezrin mutant mutated in the PI(4,5)P(2)-binding sites was less efficient in binding to LBPs and in reconstituting actin assembly than wild-type ezrin. Our data show that PI 4- and PI 5-kinase, and under some conditions also PI 3-kinase, activities are present on LBPs and can be activated by ATP, even in the absence of GTP or cytosolic components. However, PI 3-kinase activity is not required for actin assembly, because the process was not affected by PI 3-kinase inhibitors. We suggest that the ezrin-dependent actin assembly on the LBP membrane may require active turnover of D4 and D5 PIPs on the organelle membrane.     

Differential requirements for actin polymerization, calmodulin, and Ca2+ define distinct stages of lysosome/phagosome targeting

Fusion of phagosomes with late endocytic organelles is essential for cellular digestion of microbial pathogens, senescent cells, apoptotic bodies, and retinal outer segment fragments. To further elucidate the biochemistry of the targeting process, we developed a scintillation proximity assay to study the stepwise association of lysosomes and phagosomes in vitro. Incubation of tritium-labeled lysosomes with phagosomes containing scintillant latex beads led to light emission in a reaction requiring cytosol, ATP, and low Ca(2+) concentrations. The nascent complex was sensitive to disruption by alkaline carbonate, indicating that the organelles had "docked" but not fused. Through inhibitor studies and fluorescence microscopy we show that docking is preceded by a tethering step that requires actin polymerization and calmodulin. In the docked state ongoing actin polymerization and calmodulin are no longer necessary. The tethering/docking activity was purified to near homogeneity from rat liver cytosol. Major proteins in the active fractions included actin, calmodulin and IQGAP2. IQGAPs are known to bind calmodulin and cross-link F-actin, suggesting a key coordinating role during lysosome/phagosome attachment. The current results support the conclusion that lysosome/phagosome interactions proceed through distinct stages and provide a useful new approach for further experimental dissection.     

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     

Mechanically induced actin-mediated rocketing of phagosomes

Actin polymerization can be induced in Dictyostelium by compressing the cells to bring phagosomes filled with large particles into contact with the plasma membrane. Asymmetric actin assembly results in rocketing movement of the phagosomes. We show that the compression-induced assembly of actin at the cytoplasmic face of the plasma membrane involves the Arp2/3 complex. We also identify two other proteins associated with the mechanically induced actin assembly. The class I myosin MyoB accumulates at the plasma membrane-phagosome interface early during the initiation of the response, and coronin is recruited as the actin filaments are disassembling. The forces generated by rocketing phagosomes are sufficient to push the entire microtubule apparatus forward and to dislocate the nucleus.     

Microfilaments and microtubules regulate recycling from phagosomes

It is clear that the uptake of large particles is driven by a finely controlled rearrangement of the actin cytoskeleton. Here, we present evidence that myosin motors and microtubules also participate in the Fcgamma-mediated internalization process in macrophages. During phagocytosis, a substantial amount of plasma membrane is internalized without a net reduction in cell surface area, implying an active mechanism for membrane recycling. Despite the importance of this recycling pathway in phagosome maturation and in the retrieval of immunogenic peptides from phagosomes, the cytoskeletal requirements are largely unknown. To study this vesicle-mediated recycling transport, we used a biochemical assay and we developed a method to follow this process by confocal fluorescence microscopy. Interestingly, recycling from the phagosomal compartment was increased when the actin cortex was thinned by inhibitors of F-actin. In contrast, depolymerization of microtubules diminished both phagocytosis and recycling from phagosomes. Our results suggest that actin and microtubules are needed not only for phagosome biogenesis but also at other steps along the phagocytic pathway.     

Anti-inflammatory effects of phosphatidylcholine

We recently showed that mucus from patients with ulcerative colitis, a chronic inflammatory disorder of the colon, is characterized by a low level of phosphatidylcholine (PC) while clinical studies reveal that therapeutic addition of PC using slow release preparations is beneficial. The positive role of PC in this disease is still elusive. Here we tested the hypothesis that exogenous application of PC has anti-inflammatory properties using three model systems. First, human Caco-2 cells were treated with tumor necrosis factor-alpha (TNF-alpha) to induce a pro-inflammatory response via activation of NF-kappaB. Second, latex bead phagosomes were analyzed for their ability to assemble actin in vitro, a process linked to pro-inflammatory signaling and correlating with the growth versus killing of mycobacteria in macrophages. The third system used was the rapid assembly of plasma membrane actin in macrophages in response to sphingosine 1-phosphate. TNF-alpha induced a pro-inflammatory response in Caco-2 cells, including 1) assembly of plasma membrane actin; 2) activation of both MAPKs ERK and p38; 3) transport of NF-kappaB subunits to the nucleus; and 4) subsequent up-regulation of the synthesis of pro-inflammatory gene products. Exogenous addition of most PCs tested significantly inhibited these processes. Other phospholipids like sphingomyelin or phosphatidylethanolamine showed no effects in these assays. PC also inhibited latex bead phagosome actin assembly, the killing of Mycobacterium tuberculosis in macrophages, and the sphingosine 1-phosphate-induced actin assembly in macrophages. TNF-alpha induces the activation of signaling molecules and the reorganization of the actin cytoskeleton in human intestinal cells. Exogenous application of PC blocks pro-inflammatory signaling in Caco-2 cells, in phagosomes in vitro and facilitates intracellular survival of mycobacteria. We provide further evidence that actin assembly by membranes is part of the pro-inflammatory response. Collectively, these results provide a molecular foundation for the clinical studies showing a beneficial effect of PC therapy in ulcerative colitis.     

Phagosomal Proteins of Dictyostelium discoideum

ABSTRACT. In recognizing food particles, Dictyostelium cell-surface molecules initiate cytoskeletal rearrangements that result in phagosome formation. After feeding D. discoideum cells latex beads, early phagosomes were isolated on sucrose step gradietns. Protein analyses of these vesicles showed that they contained glycoproteins and surface-labeled species corresponding to integral plasma membrane proteins. Cytoskeletal proteins also were associated with phagosomes, including myosin II, actin and a 30 kDa-actin bundling protein. As seen by the acridine orange fluorescence of vesicles containing bacteria, phagosomes were acidified rapidly by a vacuolar H⁺-ATPase that was detected by immunoblotting. Except for the loss of cytoskeletal proteins, few other changes over time were noted in the protein profiles of phagosomes, suggesting that phagosome maturation was incomplete. The indigestibility of the beads possibly inhibited further endocytic processing, which has been observed by others. Since nascent phagosomes contained molecules of both the cytoskeleton and plasma membrane, they will be useful in studies aimed at identifying specific protein associations occurring between membrane proteins and the cytoskeleton during phagocytosis.     

Actin assembly induced by polylysine beads or purified phagosomes: quantitation by a new flow cytometry assay

BACKGROUND: Actin assembly on biological membranes is a poorly understood process. We have previously shown that phagosomal membranes could induce actin assembly in the presence of thymosin beta4 (an actin sequestering protein that inhibits nonspecific nucleation), via the barbed ends of actin filaments. METHODS: Here, we have developed an in vitro system based on fluorescein-labeled G (monomeric) actin and flow cytometry analysis, which allowed us to quantify de novo actin assembly on the cytoplasmic side of purified phagosomes. To standardize the system, we also used latex beads covalently coupled with polylysine, which efficiently promote actin nucleation. RESULTS: Flow cytometry analysis showed that the percentage of polylysine beads positive for F-actin filaments increased in a time- and G-actin concentration-dependent manner. Incubation of phagosomes with reagents affecting actin dynamics allowed us to extend our previous data showing that the phagosomal membranes assemble actin filaments de novo. Finally, our results pin-point a potential role for gelsolin as a positive regulator of actin assembly on the phagosomal membrane. CONCLUSIONS: We propose that our system could facilitate the development of other in vitro assays for the analysis of actin assembly and its links to signaling in cells.     

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.     

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.     

Localized exocytosis of primary (lysosomal) granules during phagocytosis: role of Ca2+-dependent tyrosine phosphorylation and microtubules

The uptake and killing of bacteria by human neutrophils are dependent on the fusion of secretory granules with forming phagosomes. The earliest component of exocytosis was found to precede phagosome closure, so that granular membrane constituents were detectable on the plasmalemma. We show that during phagocytosis of IgG-opsonized particles, this early secretory response is highly polarized in the case of primary granules, but less so for specific granules. The vectorial discharge of primary granules was dependent on calcium, but no evidence was found that calcium is involved in determining the polarity of exocytosis. In particular, a redistribution of endomembrane calcium stores toward forming phagosomes could not be detected. Polarized granule exocytosis was accompanied by focal tyrosine phosphorylation and actin polymerization, although the latter was not required for the response. Instead, microtubules seemed to contribute to the vectorial nature of the response. During particle ingestion, the microtubule-organizing center relocated toward forming phagosomes, and colchicine treatment altered the pattern of exocytosis, reducing its directionality. We hypothesize that the focal activation of tyrosine kinases generates localized signals that induce exocytosis in a calcium-dependent manner, and that reorientation of microtubules facilitates preferential delivery of granules toward the forming phagosome.     

Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis

The Rho GTPases play a critical role in initiating actin polymerization during phagocytosis. In contrast, the factors directing the disassembly of F-actin required for fission of the phagocytic vacuole are ill defined. We used fluorescent chimeric proteins to monitor the dynamics of association of actin and active Cdc42 and Rac1 with the forming phagosome. Although actin was found to disappear from the base of the forming phagosome before sealing was complete, Rac1/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly. Furthermore, fully internalized phagosomes engineered to associate constitutively with active Rac1 showed little associated F-actin. The disappearance of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) from the phagosomal membrane closely paralleled the course of actin disassembly. Furthermore, inhibition of PI(4,5)P(2) hydrolysis or increased PI(4,5)P(2) generation by overexpression of phosphatidylinositol phosphate kinase I prevented the actin disassembly necessary for the completion of phagocytosis. These observations suggest that hydrolysis of PI(4,5)P(2) dictates the remodeling of actin necessary for completion of phagocytosis.     

A vesicle surface tyrosine kinase regulates phagosome maturation

Phagocytosis is crucial for host defense against microbial pathogens and for obtaining nutrients in Dictyostelium discoideum. Phagocytosed particles are delivered via a complex route from phagosomes to lysosomes for degradation, but the molecular mechanisms involved in the phagosome maturation process are not well understood. Here, we identify a novel vesicle-associated receptor tyrosine kinase-like protein, VSK3, in D. discoideum. We demonstrate how VSK3 is involved in phagosome maturation. VSK3 resides on the membrane of late endosomes/lysosomes with its C-terminal kinase domain facing the cytoplasm. Inactivation of VSK3 by gene disruption reduces the rate of phagocytosis in cells, which is rescued by re-expression of VSK3. We found that the in vivo function of VSK3 depends on the presence of the kinase domain and vesicle localization. Furthermore, VSK3 is not essential for engulfment, but instead, is required for the fusion of phagosomes with late endosomes/lysosomes. Our findings suggest that localized tyrosine kinase signaling on the surface of endosome/lysosomes represents a control mechanism for phagosome maturation.     

An electrostatic switch displaces phosphatidylinositol phosphate kinases from the membrane during phagocytosis

Plasmalemmal phosphatidylinositol (PI) 4,5-bisphosphate (PI4,5P₂) synthesized by PI 4-phosphate (PI4P) 5-kinase (PIP5K) is key to the polymerization of actin that drives chemotaxis and phagocytosis. We investigated the means whereby PIP5K is targeted to the membrane and its fate during phagosome formation. Homology modeling revealed that all PIP5K isoforms feature a positively charged face. Together with the substrate-binding loop, this polycationic surface is proposed to constitute a coincidence detector that targets PIP5Ks to the plasmalemma. Accordingly, manipulation of the surface charge displaced PIP5Ks from the plasma membrane. During particle engulfment, PIP5Ks detached from forming phagosomes as the surface charge at these sites decreased. Precluding the change in surface charge caused the PIP5Ks to remain associated with the phagosomal cup. Chemically induced retention of PIP5K-γ prevented the disappearance of PI4,5P₂ and aborted phagosome formation. We conclude that a bistable electrostatic switch mechanism regulates the association/dissociation of PIP5Ks from the membrane during phagocytosis and likely other processes.     

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.     

Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

Phagocytes engulf unwanted particles into phagosomes that then fuse with lysosomes to degrade the enclosed particles. Ultimately, phagosomes must be recycled to help recover membrane resources that were consumed during phagocytosis and phagosome maturation, a process referred to as “phagosome resolution.” Little is known about phagosome resolution, which may proceed through exocytosis or membrane fission. Here, we show that bacteria-containing phagolysosomes in macrophages undergo fragmentation through vesicle budding, tubulation, and constriction. Phagosome fragmentation requires cargo degradation, the actin and microtubule cytoskeletons, and clathrin. We provide evidence that lysosome reformation occurs during phagosome resolution since the majority of phagosome-derived vesicles displayed lysosomal properties. Importantly, we show that clathrin-dependent phagosome resolution is important to maintain the degradative capacity of macrophages challenged with two waves of phagocytosis. Overall, our work suggests that phagosome resolution contributes to lysosome recovery and to maintaining the degradative power of macrophages to handle multiple waves of phagocytosis.     

The ClpC ATPase of Listeria monocytogenes is a general stress protein required for virulence and promoting early bacterial escape from the phagosome of macrophages

Under stress conditions, the facultative intracellular pathogen Listeria monocytogenes produces a ClpC ATPase, which is a general stress protein encoded by clpC and belonging to the HSP-100/Clp family. A ClpC-deficient mutant was obtained by gene disruption in strain LO28, which became highly susceptible to stress conditions in vitro . Intracellular growth of this mutant was restricted within macrophages, one of the major target cells of L . monocytogenes , during the infectious process. A quantitative electron microscope study showed that, contrary to wild-type bacteria that rapidly gain access to the cytoplasm of macrophages, mutant bacteria remained confined to membrane-bound phagosomes. Only a few mutant bacteria disrupted the phagosome membrane after 4 h of incubation, then polymerized actin filaments and multiplied within the cytoplasm. The ClpC ATPase, therefore, promotes early bacterial escape from the phagosome of macrophages, thus enhancing intracellular survival. The ClpC ATPase was produced in vivo during experimental infection by wild-type bacteria. The virulence of the ClpC-deficient mutant was severely attenuated in mice, with a three-log decrease in its 50% lethal dose compared with wild-type bacteria. Bacterial growth of mutant bacteria was strongly restricted in organs, presumably because of an impairment of intracellular survival in host tissues. Our results provide evidence that a general stress protein is required for the virulence of L . monocytogenes , which behaves as a virulence factor promoting intracellular survival of this pathogen.