Flotillin-1-enriched lipid raft domains accumulate on maturing phagosomes

Flotillin-1 was recently shown to be enriched on detergent-resistant domains of the plasma membrane called lipid rafts. These rafts, enriched in sphingolipids and cholesterol, sequester certain proteins while excluding others. Lipid rafts have been implicated in numerous cellular processes including signal transduction, membrane trafficking, and molecular sorting. In this study, we demonstrate both morphologically and biochemically that lipid rafts are present on phagosomes. These structures are enriched in flotillin-1 and devoid of the main phagosomes membrane protein lysosomal-associated membrane protein (LAMP1). The flotillin-1 present on phagosomes does not originate from the plasma membrane during phagocytosis but accumulates gradually on maturing phagosomes. Treatment with bafilomycin A1, a compound that inhibits the proton pump ATPase and prevents the fusion of phagosomes with late endocytic organelles, prevents the acquisition of flotillin-1 by phagosomes, indicating that this protein might be recruited on phagosomes from endosomal organelles. A proteomic characterization of the lipid rafts of phagosomes indicates that actin, the alpha- and beta-subunits of heterotrimeric G proteins, as well as subunits of the proton pump V-ATPase are among the constituents of these domains. Remarkably, the intracellular parasite Leishmania donovani can actively inhibit the acquisition of flotillin-1-enriched lipid rafts by phagosomes and the maturation of these organelles. These results indicate that specialized functions required for phagolysosome biogenesis may occur at focal points on the phagosome membrane, and therefore represent a potential target of intracellular pathogens.     

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.     

A phagosome of one's own: a microbial guide to life in the macrophage

Macrophages protect their host by engulfing foreign bodies within phagosomes that rapidly develop into microbicidal organelles. Numerous pathogens, such as species of Toxoplasma, Leishmania, Mycobacterium, Salmonella and Legionella, thrive in human macrophages, sometimes with disastrous effects. Defining the survival tactics of intracellular parasites is one approach to understanding macrophage function. Here, we briefly review phagosome maturation, then discuss how particular microbes may target particular host factors to short-circuit membrane traffic in macrophages. Recent studies support a new paradigm in which pathogens evade lysosomal degradation by entering macrophages within specialized lipid microdomains of the plasma membrane.     

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

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.     

Rab27a regulates phagosomal pH and NADPH oxidase recruitment to dendritic cell phagosomes

To prevent excessive degradation of internalized antigens, which could destroy the peptides recognized by T lymphocytes, dendritic cells have developed several strategies that limit proteolytic activity in phagosomes. The recruitment of the NADPH oxidase NOX2 prevents acidification of phagosomes, limiting antigen degradation. Here, we show that dendritic cells derived from Rab27a-deficient ashen mice show increased phagosome acidification and antigen degradation, causing a defect in antigen cross-presentation. Enhanced acidification results from a delay in the recruitment to phagosomes of a subset of lysosome-related organelles containing the membrane subunits of NOX2. The Rab27a-dependent recruitment of these "inhibitory lysosome-related organelles" to phagosomes continuously limits acidification and degradation of ingested particles in dendritic cells, thus promoting antigen cross-presentation.     

Lipid Body–Phagosome Interaction in Macrophages during Infectious Diseases: Host Defense or Pathogen Survival Strategy?

Phagocytosis of invading microorganisms by specialized cells such as macrophages and neutrophils is a key component of the innate immune response. These cells capture and engulf pathogens and subsequently destroy them in intracellular vacuoles—the phagosomes. Pathogen phagocytosis and progression and maturation of pathogen-containing phagosomes, a crucial event to acquire microbicidal features, occurs in parallel with accentuated formation of lipid-rich organelles, termed lipid bodies (LBs), or lipid droplets. Experimental and clinical infections with different pathogens such as bacteria, parasites, and viruses induce LB accumulation in cells from the immune system. Within these cells, LBs synthesize and store inflammatory mediators and are considered structural markers of inflammation. In addition to LB accumulation, interaction of these organelles with pathogen-containing phagosomes has increasingly been recognized in response to infections and may have implications in the outcome or survival of the microorganism within host cells. In this review, we summarize our current knowledge on the LB-phagosome interaction within cells from the immune system, with emphasis on macrophages, and discuss the functional meaning of this event during infectious diseases.     

Single-phagosome imaging reveals that homotypic fusion impairs phagosome degradative function

Immune cells degrade internalized pathogens in vesicle compartments called phagosomes. Many intracellular bacteria induce homotypic phagosome fusion to survive in host cells, but the fusion interaction between phagosomes and its consequence for phagosome function have scarcely been studied. Here, we characterize homotypic fusion between phagosomes in macrophages and identify how such interactions impact the degradative capacity of phagosomes. By developing a series of particle sensors for measuring biochemical changes of single phagosomes, we show that phagosomes undergo stable fusion, transient “kiss-and-run” fusion, or both in succession. Super-resolution three-dimensional fluorescence microscopy revealed that stably fused phagosomes are connected by membrane “necks” with submicron–sized fusion pores. Furthermore, we demonstrate that, after stable fusion, phagosomes have leaky membranes and thereby impaired degradative functions. Our findings, based on phagosomes that contain synthetic particles, illustrate that homotypic fusion is not exclusive to phagosomes that encapsulate pathogens, as previously believed. The physical process of homotypic fusion is alone sufficient to perturb the degradative functions of phagosomes.     

Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus

We have examined the modifications occurring during the transformation of phagosomes into phagolysosomes in J-774 macrophages. The use of low density latex beads as markers of phagosomes (latex bead compartments, LBC) allowed the isolation of these organelles by flotation on a simple sucrose gradient. Two-dimensional gel electrophoresis, immunocytochemistry, and biochemical assays have been used to characterize the composition of LBC at different time points after their formation, as well as their interactions with the organelles of the endocytic pathway. Our results show that LBC acquire and lose various markers during their transformation into phagolysosomes. Among these are members of the rab family of small GTPases as well as proteins of the lamp family. The transfer of the LBC of lamp 2, a membrane protein associated with late endocytic structures, was shown to be microtubule dependent. Video-microscopy showed that newly formed phagosomes were involved in rapid multiple contacts with late components of the endocytic pathway. Collectively, these observations suggest that phagolysosome formation is a highly dynamic process that involves the gradual and regulated acquisition of markers from endocytic organelles.     

Biogenesis of phagolysosomes: the 'kiss and run' hypothesis

Particles such as microorganisms that are taken up by the cell into phagosomes are usually ultimately degraded in phagolysosomes. However, despite its importance, phagolysosome biogenesis is poorly understood. This article presents a model for phagosome maturation into phagolysosomes that involves multiple transient fusion-fission interactions of phagosomes with endocytic organelles via a fusion-pore-like structure. This dynamic process may be modulated by the sequential appearance and disappearance of key phagosome proteins.     

Quantitative and dynamic assessment of the contribution of the ER to phagosome formation

Phagosomes were traditionally thought to originate from an invagination and scission of the plasma membrane to form a distinct intracellular vacuole. An alternative model implicating the endoplasmic reticulum (ER) as a major component of nascent and maturing phagosomes was recently proposed (Gagnon et al., 2002). To reconcile these seemingly disparate hypotheses, we used a combination of biochemical, fluorescence imaging, and electron microscopy techniques to quantitatively and dynamically assess the contribution of the plasmalemma and of the ER to phagosome formation and maturation. We could not verify even a transient physical continuity between the ER and the plasma membrane, nor were we able to detect a significant contribution of the ER to forming or maturing phagosomes in either macrophages or dendritic cells. Instead, our data indicate that the plasma membrane is the main constituent of nascent and newly formed phagosomes, which are progressively remodeled by fusion with endosomal and eventually lysosomal compartments as phagosomes mature into acidic, degradative organelles.     

Regulation of vacuolar pH and its modulation by some microbial species.

To survive within the host, pathogens such as Mycobacterium tuberculosis and Helicobacter pylori need to evade the immune response and find a protected niche where they are not exposed to microbicidal effectors. The pH of the microenvironment surrounding the pathogen plays a critical role in dictating the organism's fate. Specifically, the acidic pH of the endocytic organelles and phagosomes not only can affect bacterial growth directly but also promotes a variety of host microbicidal responses. The development of mechanisms to avoid or resist the acidic environment generated by host cells is therefore crucial to the survival of many pathogens. Here we review the processes that underlie the generation of organellar acidification and discuss strategies employed by pathogens to circumvent it, using M. tuberculosis and H. pylori as examples.     

Contributions of proteomics to understanding phagosome maturation

In metazoans macrophage cells use phagocytosis, the process of engulfing large particles, to control the spread of pathogens in the body, to clear dead or dying cells, and to aid in tissue remodelling, while the same process is also used by unicellular eukaryotes to ingest food. Phagocytosing cells essentially swallow the particles, trapping them in vacuoles called phagosomes that go through a series of maturation steps, culminating in the destruction of the internalized cargo. Because of their central role in innate immunity and their relatively simple structure (one membrane bilayer surrounding a single particle), phagosomes have been a popular subject for organelle proteomics studies. Qualitative proteomic technologies are now very sensitive so hundreds of different proteins have been identified in phagosomes from several species, revealing new properties of these intriguing compartments. More recently, quantitative proteomic approaches have also been applied, shedding new light on the dynamics and composition of maturing phagosomes. In this review we summarize the studies that have applied proteomic technologies to phagosomes and how they have changed our understanding of phagosome biology.     

PIKfyve Inhibition Interferes with Phagosome and Endosome Maturation in Macrophages

Macrophages eliminate pathogens and cell debris through phagocytosis, a process by which particulate matter is engulfed and sequestered into a phagosome. Nascent phagosomes are innocuous organelles resembling the plasma membrane. However, through a maturation process, phagosomes are quickly remodeled by fusion with endosomes and lysosomes to form the phagolysosome. Phagolysosomes are highly acidic and degradative leading to particle decomposition. Phagosome maturation is intimately dependent on the endosomal pathway, during which diverse cargoes are sorted for recycling to the plasma membrane or for degradation in lysosomes. Not surprisingly, various regulators of the endosomal pathway are also required for phagosome maturation, including phosphatidylinositol‐3‐phosphate, an early endosomal regulator. However, phosphatidylinositol‐3‐phosphate can be modified by the lipid kinase PIKfyve into phosphatidylinositol‐3,5‐bisphosphate, which controls late endosome/lysosome functions. The role of phosphatidylinositol‐3,5‐bisphosphate in macrophages and phagosome maturation remains basically unexplored. Using Fcγ receptor‐mediated phagocytosis as a model, we describe our research showing that inhibition of PIKfyve hindered certain steps of phagosome maturation. In particular, PIKfyve antagonists delayed removal of phosphatidylinositol‐3‐phosphate and reduced acquisition of LAMP1 and cathepsin D, both common lysosomal proteins. Consistent with this, the degradative capacity of phagosomes was reduced but phagosomes appeared to still acidify. We also showed that trafficking to lysosomes and their degradative capacity was reduced by PIKfyve inhibition. Overall, we provide evidence that PIKfyve, likely through phosphatidylinositol‐3,5‐bisphosphate synthesis, plays a significant role in endolysosomal and phagosome maturation in macrophages.      

The different autophagic roads by which phagosomes travel to lysosomes

Autophagy delivers cytoplasmic constituents, like damaged mitochondria, to lysosomes. Recently, it was noted that the same molecular machinery also regulates phagosome delivery for degradation. In this issue of The EMBO Journal, Brooks and colleagues demonstrate that KIM‐1/TIM‐1 receptor‐mediated phagocytosis in epithelial cells does not seem to cause modification of the phagosomal membrane itself by the autophagic machinery, but engulfment of phagosomes by autophagosomes for delivery to lysosomes and MHC restricted antigen presentation. This study suggests that the autophagic machinery can regulate phagocytosis via two pathways, modification of phagosomes during LC3‐associated phagocytosis (LAP) and macroautophagy of phagosomes.     

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.     

Intracellular Transformation of Phagosomes

The transformation of nascent phagosomes into forms capable of interacting with antimicrobial organelles of phagocytes, peroxisomes, depends on certain interactions between phagosomes and other vacuolar organelles. Phagosomes repeatedly interact with early and late endosomes through temporary contacts, which allows them to gain and lose complex sets of proteins. In addition, certain polypeptides are eliminated from phagosomes through recycling. New proteins enter phagosomes from the organelles of the biosynthetic pathway or are recruited from the cytoplasm. In addition, phagosomes receive proteins in the process of interaction with endosomes. The overall result of such transformation is acquiring new properties that make possible their interaction with peroxisomes.     

Phagosome‐endoplasmic reticulum contacts: Kissing and not running

The role of the endoplasmic reticulum (ER) in phagocytosis has been the subject of debate for over a decade. Proteomic determinations and dynamic microscopy of live cells led to conflicting conclusions. Recent insights into the existence of a variety of membrane contact sites (MCS) may help reconcile the seemingly disparate views. Specifically, earlier results can be rationalized considering that the ER forms specialized MCS with nascent and maturing phagosomes, without undergoing fusion. The composition and function of documented ER‐to‐phagosome contact sites is described. In addition, we speculate about the possible existence of additional phagosomal contact sites, based on available knowledge of interactions between the ER and other endocytic compartments. The interaction between phagosomes and the ER has been the subject of debate. Earlier observations that led to the suggestion that the ER fuses with the phagosomal membrane can now be explained in the light of recent evidence that intimate contacts form between the two organelles.     

Alteration of epithelial cell lysosomal integrity induced by bacterial cholesterol‐dependent cytolysins

Bacterial pathogens can interfere during infection with host cell organelles, such as mitochondria, the endoplasmic reticulum‐Golgi system or nuclei. As important cellular functions are often compartmentalized in these organelles, their targeting allows pathogens to manipulate key host functions during infection. Here, we identify lysosomes as a new class of organelles targeted by the pathogenic bacterium Listeria monocytogenes. We demonstrate that extracellular Listeria, via secretion of the pore‐forming toxin listeriolysin O, alters lysosomal integrity in epithelial cells but not in macrophages. Listeriolysin O induces lysosomal membrane permeabilization and release of lysosomal content, such as cathepsins proteases, which remain transiently active in the host cytosol. We furthermore show that other bacterial pore‐forming toxins, such as perfringolysin O and pneumolysin, also induce lysosomes alteration. Together, our data unveil a novel activity of bacterial cholesterol‐dependent cytolysins.     

Bacterial Endocytic Systems in Plants and Animals: Ca2+ as a Common Theme?

Intracellular interactions between bacteria and host cells are widespread in nature. In this review, the similarity between the infection processes of bacteria in plant and animal cells will be addressed. As paradigms, we selected the symbiosis between rhizobia and leguminous plants, and the survival of intracellular pathogenic bacteria in animal cells. The rhizobial symbiosis with leguminous plants is a model system for the study of plant-bacterium interactions. Through this interaction, the bacteria are released in a vacuole-like structure, called the symbiosome. The molecular processes, which lead to a functional symbiosome, are far from known. However, membrane fusion processes, and therefore also Ca²⁺, are crucial to establish this highly specialized organelle-like structure. A homologous system is the infection by certain bacterial pathogens of animal cells. These bacteria enter their host via phagocytosis and avoid the fusion with lysosomes, resulting in a membrane-bound vacuole in which the pathogens survive. The origin and maturation of this phagosome depends on Ca²⁺-signaling processes in the host cell and on proteins that regulate membrane fusion processes, such as SNAREs, Rab proteins, synaptotagmins and calmodulin. The aim of this review is to compare the endosymbiosis in leguminous plants with the surviving pathogens in animal host cells with a focus on Ca²⁺-signaling and membrane fusion-related processes. For both systems, the interaction starts with a bacterial entry of the host cell. It will be demonstrated that in both cases Ca²⁺ is a crucial second messenger. However, more emphasis will be put on the comparison of the later stages of infection, i.e., the formation of specialized bacteria-containing vacuoles. From structural, functional, and proteomic data, it is clear that phagosomes and symbiosomes are more related to each other than originally assumed. Proteins such as V-ATPases, calreticulin, phosphatidylinositol-3-kinase, Rab proteins, and SNAREs are present in both the phagosome and the symbiosome membrane, indicating that common cellular processes are used for building these intracellular organelles.