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.     

Identification of Icm protein complexes that play distinct roles in the biogenesis of an organelle permissive for Legionella pneumophila intracellular growth

Legionella pneumophila is a bacterial pathogen that can enter the human lung and grow inside alveolar macrophages. To grow within phagocytic host cells, the bacteria must create a specialized organelle that restricts fusion with lysosomes. Biogenesis of this replicative organelle is controlled by 24 dot and icm genes, which encode a type IV-related transport apparatus. To understand how this transporter functions, isogenic L. pneumophila dot and icm mutants were characterized, and three distinct phenotypic categories were identified. Our data show that, in addition to genes that encode the core Dot/Icm transport apparatus, subsets of genes are required for pore formation and modulation of phagosome trafficking. To understand activities required for virulence at a molecular level, we investigated protein-protein interactions. Specific interactions between different Icm proteins were detected by yeast two-hybrid and gel overlay analysis. These data support a model in which the IcmQ-IcmR complex regulates the formation of a translocation channel that delivers proteins into host cells, and the IcmS-IcmW complex is required for export of virulence determinants that modulate phagosome trafficking.     

Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila

Legionella pneumophila mutants specifically defective for intracellular replication were isolated using an intracellular thymineless death enrichment strategy. Mutants belonging to two distinct phenotypic classes were unable to grow in macrophage-like cultured cells. One class of mutants was defective for both inhibition of phagosome–lysosome fusion and association of host cell organelles with bacteria-containing phagosomes (‘recruitment’). Another class of mutants was defective only for organelle recruitment, suggesting that recruitment may be necessary for intracellular growth. Recombinant clones were identified that complemented the intracellular growth defects of these mutants. A single genetic locus, designated dot (for defect in organelle trafficking), restored wild-type phenotypes for intracellular growth, organelle recruitment, and inhibition of phagosome–lysosome fusion to mutants belonging to both phenotypic classes.     

Manipulation of host vesicular trafficking and innate immune defence by Legionella Dot/Icm effectors

Legionella pneumophila, the causative agent of Legionnaires' disease, infects and replicates in macrophages and amoebas. Following internalization, L. pneumophila resides in a vacuole structure called Legionella-containing vacuole (LCV). The LCV escapes from the endocytic maturation process and avoids fusion with the lysosome, a hallmark of Legionella pathogenesis. Interference with the secretory vesicle transport and avoiding lysosomal targeting render the LCV permissive for L. pneumophila intracellular replication. Central to L. pneumophila pathogenesis is a defect in the organelle trafficking/intracellular multiplication (Dot/Icm) type IV secretion system that translocates a large number of effector proteins into host cells. Many of the Dot/Icm effectors employ diverse and sophisticated biochemical strategies to manipulate the host vesicular transport system, playing an important role in LCV biogenesis and trafficking. Similar to other bacterial pathogens, L. pneumophila also delivers effector proteins to modulate or counteract host innate immune defence pathways such as the NF-κB and apoptotic signalling. This review summarizes the known functions and mechanisms of Dot/Icm effectors that target host membrane trafficking and innate immune defence pathways.     

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.     

The phagosome containing Legionella pneumophila within the protozoan Hartmannella vermiformis is surrounded by the rough endoplasmic reticulum.

Legionella pneumophila is an intracellular parasite of protozoa and human phagocytes. To examine adaptation of this bacterium to parasitize protozoa, the sequence of events of the intracellular infection of the amoeba Hartmannella vermiformis was examined. The previously described uptake phenomenon of coiling phagocytosis by human monocytes was not detected. A 1 h postinfection with wild-type strain AA100, mitochondria were observed within the vicinity of the phagosome. At 2.5 h postinfection, numerous vesicles surrounded the phagosomes and mitochondria were in close proximity to the phagosome. At 5 h postinfection, the bacterium was surrounded by a ribosome-studded multilayer membrane. Bacterial multiplication was evident by 8 h postinfection, and the phagosome was surrounded by a ribosome-studded multilayer membrane until 15 h postinfection. The recruitment of organelles and formation of the ribosome-studded phagosome was defective in an isogenic attenuated mutant of L. pneumophila (strain AA101A) that failed to replicate within amoebae. At 20 h postinfection with wild-type strain AA100, numerous bacteria were present in the phagosome and ribosome were not detected around the phagosome. These data showed that, at the ultrastructural level, the intracellular infection of protozoa by L. pneumophila is highly similar to that of infection of macrophages. Immunocytochemical studies provided evidence that at 5 h postinfection the phagosome containing L. pneumophila acquired an abundant amount of the endoplasmic reticulum-specific protein (BiP). Similar to phagosomes containing heat-killed wild-type L. pneumophila, the BiP protein was not detectable in phagosomes containing the mutant strain AA101A. In addition to the absence of ribosomes and mitochondria, the BiP protein was not detected in the phagosomes at 20 h postinfection with wild-type L. pneumophila. The data indicated that the ability of L. pneumophila to establish the intracellular infection of amoebae is dependent on its capacity to reside and multiply within a phagosome surrounded by the rough endoplasmic reticulum. This compartment may constitute a rich source of nutrients for the bacteria and is probably recognized as cellular compartment. The remarkable similarity of the intracellular infections of macrophages and protozoa by L. pneumophila strongly supports the hypothesis that adaptation of the bacterium to the intracellular environment of protozoa may be the mechanism for its ability to adapt to the intracellular environment of human alveolar macrophages and causes pneumonia.     

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.     

Early trafficking and intracellular replication of Legionella longbeachaea within an ER-derived late endosome-like phagosome

Legionella pneumophila is the predominant cause of Legionnaires' disease in the USA and Europe in contrast to Legionella longbeachaea, which is the leading cause of the disease in Western Australia. The ability of L. pneumophila to replicate intracellularly is triggered at the post-exponential phase along with expression of other virulence traits, such as motility. We show that while motility of L. longbeachaea is triggered upon growth transition into post-exponential phase, its ability to proliferate intracellularly is totally independent of the bacterial growth phase. Within macrophages, L. pneumophila replicates in a phagosome that excludes early and late endocytic markers and is surrounded by the rough endoplasmic reticulum (RER). In contrast, the L. longbeachaea phagosome colocalizes with the early endosomal marker early endosomal antigen 1 (EEA1) and the late endosomal markers lysosomal associated membrane glycoprotein 2 (LAMP-2) and mannose 6-phosphate receptor (M6PR), and is surrounded by the RER. The L. longbeachaea phagosome does not colocalize with the vacuolar ATPase (vATPase) proton pump, and the lysosomal luminal protease Cathepsin D, or the lysosomal tracer Texas red Ovalbumin (TROV). Intracellular proliferation of L. longbeachaea occurs in LAMP-2-positive phagosomes that are remodelled by the RER. Despite their distinct trafficking, both L. longbeachaea and L. pneumophila can replicate in communal phagosomes whose biogenesis is predominantly modulated by L. longbeachaea into LAMP-2-positive phagosomes. In addition, the L. pneumophila dotA mutant is rescued for intracellular replication if it co-inhabits the phagosome with L. longbeachaea. During late stages of infection, L. longbeachaea escape into the cytoplasm, prior to lysis of the macrophage, similar to L. pneumophila. We conclude that the L. longbeachaea phagosome matures to a non-acidified late endosome-like stage that is remodelled by the RER, indicating an idiosyncratic trafficking of L. longbeachaea compared with other intracellular pathogens, and a divergence in its intracellular lifestyle from L. pneumophila. In addition, re-routing biogenesis of the L. pneumophila phagosome into a late endosome controlled by L. longbeachaea has no effect on intracellular replication.     

Host cell processes that influence the intracellular survival of Legionella pneumophila

Key to the pathogenesis of intracellular pathogens is their ability to manipulate host cell processes, permitting the establishment of an intracellular replicative niche. In turn, the host cell deploys defence mechanisms that limit intracellular infection. The bacterial pathogen Legionella pneumophila, the aetiological agent of Legionnaire's Disease, has evolved virulence mechanisms that allow it to replicate within protozoa, its natural host. Many of these tactics also enable L. pneumophila's survival and replication inside macrophages within a membrane-bound compartment known as the Legionella-containing vacuole. One of the virulence factors indispensable for L. pneumophila's intracellular survival is a type IV secretion system, which translocates a large repertoire of bacterial effectors into the host cell. These effectors modulate multiple host cell processes and in particular, redirect trafficking of the L. pneumophila phagosome and mediate its conversion into an ER-derived organelle competent for intracellular bacterial replication. In this review, we discuss how L. pneumophila manipulates host cells, as well as host cell processes that either facilitate or impede its intracellular survival.     

Regulation of Legionella phagosome maturation and infection through flagellin and host Ipaf

Legionella pneumophila is an intracellular bacterium that causes an acute form of pneumonia called Legionnaires' disease. After infection of human macrophages, the Legionella-containing phagosome (LCP) avoids fusion with the lysosome allowing intracellular replication of the bacterium. In macrophages derived from most mouse strains, the LCP is delivered to the lysosome resulting in Legionella degradation and restricted bacterial growth. Mouse macrophages lacking the NLR protein Ipaf or its downstream effector caspase-1 are permissive to intracellular Legionella replication. However, the mechanism by which Ipaf restricts Legionella replication is not well understood. Here we demonstrate that the presence of flagellin and a competent type IV secretion system are critical for Legionella to activate caspase-1 in macrophages. Activation of caspase-1 in response to Legionella infection also required host Ipaf, but not TLR5. In the absence of Ipaf or caspase-1 activation, the LCP acquired endoplasmic reticulum-derived vesicles, avoided fusion with the lysosome, and allowed Legionella replication. Accordingly a Legionella mutant lacking flagellin did not activate caspase-1, avoided degradation, and replicated in wild-type macrophages. The regulation of phagosome maturation by Ipaf occurred within 2 h after infection and was independent of macrophage cell death. In vivo studies confirmed that flagellin and Ipaf play an important role in the control of Legionella clearance. These results reveal that Ipaf restricts Legionella replication through the regulation of phagosome maturation, providing a novel function for NLR proteins in host defense against an intracellular bacterium.     

The perplexing functions and surprising origins of Legionella pneumophila type IV secretion effectors

Only a limited number of bacterial pathogens evade destruction by phagocytic cells such as macrophages. Legionella pneumophila is a Gram-negative γ-proteobacterial species that can infect and replicate in alveolar macrophages, causing Legionnaires' disease, a severe pneumonia. L. pneumophila uses a complex secretion system to inject host cells with effector proteins capable of disrupting or altering the host cell processes. The L. pneumophila effectors target multiple processes but are essentially aimed at modifying the properties of the L. pneumophila phagosome by altering vesicular trafficking, gradually creating a specialized vacuole in which the bacteria replicate robustly. In nature, L. pneumophila is thought to parasitize free-living protists, which may have selected for traits that promote virulence of L. pneumophila in humans. Indeed, many effector genes encode proteins with eukaryotic domains and are likely to be of protozoan origin. Sustained horizontal gene transfer events within the protozoan niche may have allowed L. pneumophila to become a professional parasite of phagocytes, simultaneously giving rise to its ability to infect macrophages, cells that constitute the first line of cellular defence against bacterial infections.     

Mycobacterium tuberculosis phagosome

The arrest of Mycobacterium tuberculosis phagosome maturation in infected macrophages is a phenomenon of dual significance both for the pathogenesis of tuberculosis and as a model system to study interference of microbes with membrane trafficking and organelle biogenesis in host cells. Among other factors, compartment-specialized regulators of vesicular trafficking and other parts of membrane fusion machinery are likely to play a role in these processes. Here we summarize the emerging view of mycobacterial phagosome maturation arrest in the context of the dynamic processes of intracellular membrane trafficking.     

LegC3, an Effector Protein from Legionella pneumophila,Inhibits Homotypic Yeast Vacuole Fusion In Vivo and In Vitro

During infection, the intracellular pathogenic bacterium Legionella pneumophila causes an extensive remodeling of host membrane trafficking pathways, both in the construction of a replication-competent vacuole comprised of ER-derived vesicles and plasma membrane components, and in the inhibition of normal phagosome:endosome/lysosome fusion pathways. Here, we identify the LegC3 secreted effector protein from L. pneumophila as able to inhibit a SNARE- and Rab GTPase-dependent membrane fusion pathway in vitro, the homotypic fusion of yeast vacuoles (lysosomes). This vacuole fusion inhibition appeared to be specific, as similar secreted coiled-coiled domain containing proteins from L. pneumophila, LegC7/YlfA and LegC2/YlfB, did not inhibit vacuole fusion. The LegC3-mediated fusion inhibition was reversible by a yeast cytosolic extract, as well as by a purified soluble SNARE, Vam7p. LegC3 blocked the formation of trans-SNARE complexes during vacuole fusion, although we did not detect a direct interaction of LegC3 with the vacuolar SNARE protein complexes required for fusion. Additionally, LegC3 was incapable of inhibiting a defined synthetic model of vacuolar SNARE-driven membrane fusion, further suggesting that LegC3 does not directly inhibit the activity of vacuolar SNAREs, HOPS complex, or Sec17p/18p during membrane fusion. LegC3 is likely utilized by Legionella to modulate eukaryotic membrane fusion events during pathogenesis.     

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.     

The HL-60 model for the interaction of human macrophages with the Legionnaires' disease bacterium

The facultative intracellular pathogen, Legionella pneumophila, multiplies within and kills human monocytes and alveolar macrophages. We show that L. pneumophila strain Philadelphia-1 infects, multiplies within and kills the promyelocyte HL-60 cell line after its differentiation into macrophage-like cells. The characteristics of the interaction between L. pneumophila and differentiated HL-60 cells closely resemble those between L. pneumophila and human peripheral blood monocytes. With both cell types, C receptors and serum C mediate attachment of L. pneumophila, which are taken up by coiling phagocytosis. The replicative phagosome is lined with ribosomes; intracellular multiplication is iron-dependent; and replicating bacteria ultimately destroy the host cell. As in human monocytes, an avirulent mutant derivative of L. pneumophila Philadelphia-1, 25D, does not replicate in and is not cytopathic for differentiated HL-60 cells. Differentiated HL-60 cells therefore provide a convenient and faithful model for the study of L. pneumophila-mononuclear phagocyte interaction.     

The Legionella pneumophila Effector Protein,LegC7, Alters Yeast Endosomal Trafficking

The intracellular pathogen, Legionella pneumophila, relies on numerous secreted effector proteins to manipulate host endomembrane trafficking events during pathogenesis, thereby preventing fusion of the bacteria-laden phagosome with host endolysosomal compartments, and thus escaping degradation. Upon expression in the surrogate eukaryotic model Saccharomyces cerevisiae, we find that the L. pneumophila LegC7/YlfA effector protein disrupts the delivery of both biosynthetic and endocytic cargo to the yeast vacuole. We demonstrate that the effects of LegC7 are specific to the endosome:vacuole delivery pathways; LegC7 expression does not disrupt other known vacuole-directed pathways. Deletions of the ESCRT-0 complex member, VPS27, provide resistance to the LegC7 toxicity, providing a possible target for LegC7 function in vivo. Furthermore, a single amino acid substitution in LegC7 abrogates both its toxicity and ability to alter endosomal traffic in vivo, thereby identifying a critical functional domain. LegC7 likely inhibits endosomal trafficking during L. pneumophila pathogenesis to prevent entry of the phagosome into the endosomal maturation pathway and eventual fusion with the lysosome.     

The DotL protein, a member of the TraG-coupling protein family, is essential for Viability of Legionella pneumophila strain Lp02

Legionella pneumophila is able to survive inside phagocytic cells by an internalization route that bypasses fusion of the nascent phagosome with the endocytic pathway to allow formation of a replicative phagosome. The dot/icm genes, a major virulence system of L. pneumophila, encode a type IVB secretion system that is required for intracellular growth. One Dot protein, DotL, has sequence similarity to type IV secretion system coupling proteins (T4CPs). In other systems, coupling proteins are not required for viability of the organism. Here we report the first example of a strain, L. pneumophila Lp02, in which a putative T4CP is essential for viability of the organism on bacteriological media. This result is particularly surprising since the majority of the dot/icm genes in Lp02 are dispensable for growth outside of a host cell, a condition that does not require a functional Dot/Icm secretion complex. We were able to isolate suppressors of the Delta dotL lethality and found that many contained mutations in other components of the Dot/Icm secretion system. A systematic analysis of dot/icm deletion mutants revealed that the majority of them (20 of 26) suppressed the lethality phenotype, indicating a partially assembled secretion system may be the source of Delta dotL toxicity in the wild-type strain. These results are consistent with a model in which the DotL protein plays a role in regulating the activity of the L. pneumophila type IV secretion apparatus.     

Macroautophagy is dispensable for intracellular replication of Legionella pneumophila in Dictyostelium discoideum

The Gram-negative bacterium Legionella pneumophila is a facultative intracellular pathogen of free-living amoebae and mammalian phagocytes. L. pneumophila is engulfed in phagosomes that initially avoid fusion with lysosomes. The phagosome associates with endoplasmic reticulum (ER) and mitochondria and eventually resembles ER. The morphological similarity of the replication vacuole to autophagosomes, and enhanced bacterial replication in response to macroautophagy-inducing starvation, led to the hypothesis that L. pneumophila infection requires macroautophagy. As L. pneumophila replicates in Dictyostelium discoideum, and macroautophagy genes have been identified and mutated in D. discoideum, we have taken a genetic and cell biological approach to evaluate the relationship between host macroautophagy and intracellular replication of L. pneumophila. Mutation of the apg1, apg5, apg6, apg7 and apg8 genes produced typical macroautophagy defects, including reduced bulk protein degradation and cell viability during starvation. We show that L. pneumophila replicates normally in D. discoideum macroautophagy mutants and produces replication vacuoles that are morphologically indistinguishable from those in wild-type D. discoideum. Furthermore, a green fluorescent protein (GFP)-tagged marker of autophagosomes, Apg8, does not systematically co-localize with DsRed-labelled L. pneumophila. We conclude that macroautophagy is dispensable for L. pneumophila intracellular replication in D. discoideum.     

The mechanism of killing and exiting the protozoan host Acanthamoeba polyphaga by Legionella pneumophila

The ability of Legionella pneumophila to cause legionnaires' disease is dependent on its capacity to replicate within cells in the alveolar spaces. The bacteria kill mammalian cells in two phases: induction of apoptosis during the early stages of infection, followed by an independent and rapid necrosis during later stages of the infection, mediated by a pore-forming activity. In the environment, L. pneumophila is a parasite of protozoa. The molecular mechanisms by which L. pneumophila kills the protozoan cells, after their exploitation for intracellular proliferation, are not known. In an effort to decipher these mechanisms, we have examined induction of both apoptosis and necrosis in the protozoan Acanthamoeba polyphaga upon infection by L. pneumophila. Our data show that, although A. polyphaga undergoes apoptosis following treatment with actinomycin D, L. pneumophila does not induce apoptosis in these cells. Instead, intracellular L. pneumophila induces necrotic death in A. polyphaga, which is mediated by the pore-forming activity. Mutants of L. pneumophila defective in expression of the pore-forming activity are indistinguishable from the parental strain in intracellular replication within A. polyphaga. The parental strain bacteria cause necrosis-mediated lysis of all the A. polyphaga cells within 48 h after infection, and all the intracellular bacteria are released into the tissue culture medium. In contrast, all cells infected by the mutants remain intact, and the intracellular bacteria are 'trapped' within A. polyphaga after the termination of intracellular replication. Failure to exit the host cell after termination of intracellular replication results in a gradual decline in the viability of the mutant strain bacteria within A. polyphaga starting 48h after infection. Our data show that the pore-forming activity of L. pneumophila is not required for intracellular bacterial replication within A. polyphaga but is required for killing and exiting the protozoan host upon termination of intracellular replication.     

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.