Brucella suis-impaired specific recognition of phagosomes by lysosomes due to phagosomal membrane modifications

Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells. We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell. By in vitro reconstitution experiments, we observed a specific association between killed B. suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments.     

Role of the Brucella suis lipopolysaccharide O antigen in phagosomal genesis and in inhibition of phagosome-lysosome fusion in murine macrophages

Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both cell types. However, the molecular mechanisms and the microbial factors involved are poorly understood. Smooth lipopolysaccharide (LPS) of Brucella has been reported to be an important virulence factor, although its precise role in pathogenesis is not yet clear. In this study, we show that the LPS O side chain is involved in inhibition of the early fusion between Brucella suis-containing phagosomes and lysosomes in murine macrophages. In contrast, the phagosomes containing rough mutants, which fail to express the O antigen, rapidly fuse with lysosomes. In addition, we show that rough mutants do not enter host cells by using lipid rafts, contrary to smooth strains. Thus, we propose that the LPS O chain might be a major factor that governs the early behavior of bacteria inside macrophages.     

Cyclic beta-1,2-glucan is a Brucella virulence factor required for intracellular survival

Pathogenic brucella bacteria have developed strategies to persist for prolonged periods of time in host cells, avoiding innate immune responses. Here we show that the cyclic beta-1,2-glucans (CbetaG) synthesized by brucella is important for circumventing host cell defenses. CbetaG acted in lipid rafts found on host cell membranes. CbetaG-deficient mutants failed to prevent phagosome-lysosome fusion and could not replicate. However, when treated with purified CbetaG or synthetic methyl-beta-cyclodextrin, the mutants were able to control vacuole maturation by avoiding lysosome fusion, and this allowed intracellular brucella to survive and reach the endoplasmic reticulum. Fusion between the endoplasmic reticulum and the brucella-containing vacuole depended on the brucella virulence type IV secretion system but not on CbetaG. Brucella CbetaG is thus a virulence factor that interacts with lipid rafts and contributes to pathogen survival.     

Localization of CORO1A in the macrophages containing Mycobacterium leprae

Mycobacteria have acquired an intracellular lifestyle within the macrophage, which is best exemplified by the enlarged infected histiocytes seen in lepromatous leprosy. To survive within the cell, mycobacteria must escape intracellular bactericidal mechanisms. In a study of Mycobacterium bovis Bacille Calmette-Guérin (M. bovis BCG) infection, it was shown that the host protein, CORO1A, also known as tryptophan aspartate-containing coat protein (TACO), accumulates on the phagosomal membrane, resulting in inhibition of phagosome-lysosome fusion, and thus augmenting intracellular survival. In this study, we show that CORO1A strongly localizes on the membrane of phagosomes that contain Mycobacterium leprae (M. leprae), where Toll-like receptor 2 was also visualized by immunostaining. When cultured macrophages were infected with M. leprae, CORO1A recruitment from the plasma membrane to the phagosomal membrane was observed. Moderate to strong CORO1A retention was observed in late lesions that contained foamy histiocytes, in which M. leprae were difficult to detect by acid-fast staining. These results suggest that components accumulating within the phagosome rather than viable bacilli are responsible for the retention of CORO1A, and that there is also a bactericidal mechanism in the macrophage that might counter the effects of CORO1A.     

Intracellular fate of Mycobacterium avium: use of dual-label spectrofluorometry to investigate the influence of bacterial viability and opsonization on phagosomal pH and phagosome-lysosome interaction.

Mycobacterium avium is a facultative intracellular pathogen that can survive and replicate within macrophages. We tested the hypotheses that survival mechanisms may include alteration of phagosomal pH or inhibition of phagosome-lysosome fusion. M. avium was surface labeled with N-hydroxysuccinimidyl esters of carboxyfluorescein (CF) and rhodamine (Rho) to enable measurement of the pH of individual M. avium-containing phagosomes and the interactions of bacterium-containing phagosomes with labeled secondary lysosomes. CF fluorescence is pH sensitive, whereas Rho is pH insensitive; pH can be calculated from their fluorescence ratios. Surface labeling of M. avium did not affect viability in broth cultures or within J774, a murine macrophage-like cell line. By fluorescence spectroscopy, live M. avium was exposed to an environmental pH of approximately 5.7 at 6 h after phagocytosis, whereas similarly labeled Salmonella typhimurium, zymosan A, or heat-killed M. avium encountered an environmental pH of < 5.0. Video fluorescence and laser scanning confocal microscopy gave consistent pH results and demonstrated the heterogeneity of intracellular fate early in infection. pH became more homogeneous 6 h after infection. M. avium cells were coated with immunoglobulin G (IgG) or opsonized to investigate whether phagocytosis by the corresponding receptors would alter intracellular fate. Opsonized, unopsonized, and IgG-coated M. avium cells entered compartments of similar pH. Finally, the spatial distribution of intracellular bacteria and secondary lysosomes was compared. Only 18% of live fluorescent M. avium cells colocalized with fluorescent lysosomes, while 98% of heat-killed bacteria colocalized. Thus, both inhibition of phagosome-lysosome fusion and alteration of phagosomal pH may contribute to the intracellular survival of M. avium.     

Intracellular survival of Brucella spp. in human monocytes involves conventional uptake but special phagosomes

Brucella spp. are facultative intracellular parasites of various mammals, including humans, typically infecting lymphoid as well as reproductive organs. We have investigated how B. suis and B. melitensis enter human monocytes and in which compartment they survive. Peripheral blood monocytes readily internalized nonopsonized brucellae and killed most of them within 12 to 18 h. The presence of Brucella-specific antibodies (but not complement) increased the uptake of bacteria without increasing their intracellular survival, whereas adherence of the monocytes or incubation in Ca(2+)- and Mg(2+)-free medium reduced the uptake. Engulfment of all Brucella organisms (regardless of bacterial viability or virulence) initially resulted in phagosomes with tightly apposed walls (TP). Most TP were fully fusiogenic and matured to spacious phagolysosomes containing degraded bacteria, whereas some TP (more in monocyte-derived macrophages, HeLa cells, and CHO cells than in monocytes) remained tightly apposed to intact bacteria. Immediate treatment of infected host cells with the lysosomotropic base ammonium chloride caused a swelling of all phagosomes and a rise in the intraphagosomal pH, abolishing the intracellular survival of Brucella. These results indicate that (i) human monocytes readily internalize Brucella in a conventional way using various phagocytosis-promoting receptors, (ii) the maturation of some Brucella phagosomes is passively arrested between the steps of acidification and phagosome-lysosome fusion, (iii) brucellae are killed in maturing but not in arrested phagosomes, and (iv) survival of internalized Brucella depends on an acidic intraphagosomal pH and/or close contact with the phagosomal wall.     

Survival of Mycobacterium avium and Mycobacterium tuberculosis in Acidified Vacuoles of Murine Macrophages

Despite the antimicrobial mechanisms of vertebrate phagocytes, mycobacteria can survive within the phagosomes of these cells. These organisms use various strategies to evade destruction, including inhibition of acidification of the phagosome and inhibition of phagosome-lysosome fusion. In contrast to mycobacteria, Coxiella burnetii, the etiologic agent of Q fever, inhabits a spacious acidified intracellular vacuole which is prone to fusion with other vacuoles of the host cell, including phagosomes containing mycobacteria. The Coxiella-infected cell thus provides a unique model for investigating the survival of mycobacteria in an acidified phagosome-like compartment. In the present study, murine bone marrow-derived macrophages were infected with either Mycobacterium avium or Mycobacterium tuberculosis and then coinfected with C. burnetii. We observed that the majority of phagocytosed mycobacteria colocalized to the C. burnetii-containing vacuole, which maintained its acidic properties. In coinfected macrophages, the growth of M. avium was not impaired following fusion with the acidified vacuole. In contrast, the growth rate of M. tuberculosis was reduced in acidified vacuoles. These results suggest that although both species of mycobacteria inhibit phagosome-lysosome fusion, they may be differentially susceptible to the toxic effects of the acidic environment in the mature phagolysosome.     

The regulation of phagosome maturation in Dictyostelium

Macropinocytosis (fluid uptake) and phagocytosis (particle uptake) are processes that result in the formation of intracellular membrane enclosed vacuoles termed macropinosomes and phagosomes, respectively. Macropinosomes and phagosomes are modified by fission and fusion reactions with the endo-lysosomal pathway that eventually transform these vacuoles into a lysosomal environment. Many human bacterial pathogens, including species of Mycobacteria, Legionella, and Chlamydia, are thought to survive by disrupting the normal membrane trafficking events that usually result in the formation of phago-lysosomes and death of the microorganism. In addition, a number of important pathogens facilitate homotypic phagosome fusion in order to generate an intracellular environment conducive for survival. A greater understanding of the regulation of phagosomal maturation and fusion will be critical in designing new therapies to treat infections caused by intracellular pathogens. The genetically tractable phagocyte, D. discoideum, has proven extremely useful in dissecting the signaling pathways regulating macropinocytosis, phagocytosis, phagosomal maturation and phagosome–phagosome fusion. A body of knowledge has accumulated and demonstrates important roles for Rab GTPases, the cytoskeleton, phosphoinositide metabolism and pH regulation in regulating phagosome maturation. This review will summarize the current state of knowledge.     

The early phagosomal stage of Francisella tularensis determines optimal phagosomal escape and Francisella pathogenicity island protein expression

Francisella tularensis is an intracellular pathogen that can survive and replicate within macrophages. Following phagocytosis and transient interactions with the endocytic pathway, F. tularensis rapidly escapes from its original phagosome into the macrophage cytoplasm, where it eventually replicates. To examine the importance of the nascent phagosome for the Francisella intracellular cycle, we have characterized early trafficking events of the F. tularensis subsp. tularensis strain Schu S4 in a murine bone marrow-derived macrophage model. Here we show that early phagosomes containing Schu S4 transiently interact with early and late endosomes and become acidified before the onset of phagosomal disruption. Inhibition of endosomal acidification with the vacuolar ATPase inhibitor bafilomycin A1 or concanamycin A prior to infection significantly delayed but did not block phagosomal escape and cytosolic replication, indicating that maturation of the early Francisella-containing phagosome (FCP) is important for optimal phagosomal escape and subsequent intracellular growth. Further, Francisella pathogenicity island (FPI) protein expression was induced during early intracellular trafficking events. Although inhibition of endosomal acidification mimicked the early phagosomal escape defects caused by mutation of the FPI-encoded IglCD proteins, it did not inhibit the intracellular induction of FPI proteins, demonstrating that this response is independent of phagosomal pH. Altogether, these results demonstrate that early phagosomal maturation is required for optimal phagosomal escape and that the early FCP provides cues other than intravacuolar pH that determine intracellular induction of FPI proteins.     

Phagocytic receptors dictate phagosomal escape and intracellular proliferation of Francisella tularensis

Francisella tularensis, the causative agent of tularemia, survives and proliferates within macrophages of the infected host as part of its pathogenic strategy, through an intracellular life cycle that includes phagosomal escape and extensive proliferation within the macrophage cytosol. Various in vitro models of Francisella-macrophage interactions have been developed, using either opsonic or nonopsonic phagocytosis, and have generated discrepant results on the timing and extent of Francisella phagosomal escape. Here we have investigated whether either complement or antibody opsonization of the virulent prototypical type A strain Francisella tularensis subsp. tularensis Schu S4 affects its intracellular cycle within primary murine bone marrow-derived macrophages. Opsonization of Schu S4 with either human serum or purified IgG enhanced phagocytosis but restricted phagosomal escape and intracellular proliferation. Opsonization of Schu S4 with either fresh serum or purified antibodies redirected bacteria from the mannose receptor (MR) to the complement receptor CR3, the scavenger receptor A (SRA), and the Fcγ receptor (FcγR), respectively. CR3-mediated uptake delayed maturation of the early Francisella-containing phagosome (FCP) and restricted phagosomal escape, while FcγR-dependent phagocytosis was associated with superoxide production in the early FCP and restricted phagosomal escape and intracellular growth in an NADPH oxidase-dependent manner. Taken together, these results demonstrate that opsonophagocytic receptors alter the intracellular fate of Francisella by delivering bacteria through phagocytic pathways that restrict phagosomal escape and intracellular proliferation.     

Differential partitioning and trafficking of GM gangliosides and cholesterol-rich lipid rafts in thymic and splenic CD4 T cells

The GM gangliosides and cholesterol components of plasma membrane lipid rafts play an important role in the recruitment and signaling of protein receptors in eukaryotic cells. Herein, we have analyzed at the single-cell level the partitioning and intracellular trafficking of GM gangliosides and cholesterol in quiescent (CD4+CD69-) and CD3-activated (CD4+CD69+) thymic and splenic T cells. First, regardless the gender and the quiescent or activated status of T cells, the GM and cholesterol content in cytosol and plasma membrane as well as the expression levels of GM synthase, Sphingomyelin phosphodiestarase 2 and HMG Co-A reductase genes involved in GM and cholesterol synthesis were constantly lower in CD4 thymocytes than in CD4 splenocytes. Second, we detected variations in the balance between GM and cholesterol in plasma membrane depending on aging, and found that deprivation of cellular cholesterol does not necessarily affect the GM content in both quiescent CD4 thymocytes and splenocytes. Third, CD3 stimulation up-regulated the GM and little if any the cholesterol content in both thymic and splenic CD4 T cells, suggesting a cross talk between the CD3 signaling and GM but not cholesterol biosynthesis pathway. Fourth, partitioning and trafficking of GM to the plasma membrane depended on the transport of ceramide precursors from endoplasmic reticulum to Golgi network, as well as on the synthesis, glycosylation and vesicular assembly in trans-Golgi, and less on the cytoskeleton architecture in both quiescent and activated CD4 thymic and splenic T cells. Together, these findings suggest that the differential partitioning and intracellular trafficking of GM and cholesterol in thymic and splenic CD4 T cells may account for the stage of functional maturation.     

Fate of Listeria monocytogenes in murine macrophages: evidence for simultaneous killing and survival of intracellular bacteria.

The intracellular survival of the ubiquitous pathogen Listeria monocytogenes was studied in primary cultures of bone marrow-derived mouse macrophages. Bacteria were able to grow rapidly in these cells, with an apparent multiplication rate of about 40 min. Electron microscopy demonstrated that intracellular bacterial replication was the consequence of simultaneous intracellular killing and replication of bacteria in the same cells. Within the first hour following phagocytosis, most bacteria were destroyed in the phagosomal compartment to which they were confined. This was due to early transfer of hydrolytic enzymes to phagosomes, undoubtedly via phagosome-lysosome (P-L) fusion, as demonstrated by a quantitative analysis after staining for a lysosomal marker, acid phosphatase. One hour after infection, about 14% of the bacteria were free in the cytoplasm, in which they multiplied and induced actin polymerization and spreading to adjacent macrophages, as in epithelial cells. By using the 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine staining procedure, direct evidence is presented that all phagosomes were acidified immediately after phagocytosis, thus indicating that intraphagosomal bacteria were exposed to an acidic environment that might favor vacuolar lysis by listeriolysin O. Intracellular growth in macrophages, therefore, appears to be the result of a competition between the expression of the hydrolytic activity of these cells following P-L fusion and the capacity of L. monocytogenes to escape from the acidified phagosomal compartment before P-L fusion has occurred. The finding that concomitant intracellular killing and survival of L. monocytogenes occurs in the same macrophages might explain the high immunogenicity observed in vivo with live bacteria, as opposed to killed bacteria.     

Leishmania donovani requires functional Cdc42 and Rac1 to prevent phagosomal maturation

Leishmania donovani promastigotes survive inside macrophage phagosomes by inhibiting phagosomal maturation. The main surface glycoconjugate on promastigotes, lipophosphoglycan (LPG), is crucial for survival and mediates the formation of a protective shell of F-actin around the phagosome. Previous studies have demonstrated that this effect involves inhibition of protein kinase C alpha. The present study shows that functional Cdc42 and Rac1 are required for the formation of F-actin around L. donovani phagosomes. Moreover, we present data showing that phagosomes containing LPG-defective L. donovani, which is unable to induce F-actin accumulation, display both elevated levels of periphagosomal F-actin and impaired phagosomal maturation in macrophages with permanently active forms of Cdc42 and Rac1. We conclude that L. donovani engages Cdc42 and Rac1 to build up a protective coat of F-actin around its phagosome to prevent phagosomal maturation.     

Membrane sorting during swimming internalization of Brucella is required for phagosome trafficking decisions

Brucella infects macrophages by swimming internalization, after which it is enclosed in macropinosomes. We investigated the role of the uptake pathway in phagosome trafficking, which remains unclear. This study found membrane sorting during swimming internalization and is essential in intracellular replication of Brucella. The B. abortus virB mutant replicated intracellularly when it was in the macropinosome established by wild-type B. abortus that retained its ability to alter phagosome trafficking. Lipid rafts-associated molecules, such as GM1 ganglioside, were selectively included into macropinosomes, but Rab5 effector early endosome autoantigen (EEA1) and lysosomal glycoprotein LAMP-1 were excluded from macropinosomes containing B. abortus induced by swimming internalization. In contrast, when the swimming internalization was bypassed by phorbol myristate acetate (PMA)-induced macropinocytosis, lipid raft-associated molecules were excluded, and EEA1 and LAMP-1 were included into macropinosomes containing bacteria. The phosphatidylinositol 3-kinase inhibitor wortmannin that inhibits PMA-induced macropinocytosis blocked internalization of virB mutant, but not of wild-type of B. abortus and wortmannin treatment did not affect intracellular replication. Our results suggest that membrane sorting requires swimming internalization of B. abortus and decides the intracellular fate of the bacterium, and that Brucella -induced macropinosome formation is a different mechanism from PMA-induced macropinocytosis.     

Polyanionic agents do not inhibit phagosome-lysosome fusion in cultured macrophages

The survival of some intracellular pathogens within macrophages may be aided by an ability of the organism to antagonize, from within the entrapping phagosome, its fusion with lysosomes. On the other hand, certain polyanionic agents have been implicated in imposing a similar block to fusion from the lysosomal domain--because the transfer of various foreign markers from lysosomes to newly formed phagosomes is remarkably inhibited in these polyanion-containing cells. Based on an analysis of various observations and our own recent data, we propose that the polyanionics do not, in fact, prevent phagosome-lysosome fusion but, instead, physically entrap the usual markers in a gelatinous matrix within the lysosomes. This view accounts for many paradoxical consequences of polyanionic accumulation and for the curiously normal behavior of macrophages that are presumed to be suffering from such a crucial intracellular dysfunction.     

The Mycobacterium tuberculosis SecA2 system subverts phagosome maturation to promote growth in macrophages

The ability of Mycobacterium tuberculosis to grow in macrophages is critical to the virulence of this important pathogen. One way M. tuberculosis is thought to maintain a hospitable niche in macrophages is by arresting the normal process of phagosomes maturing into acidified phagolysosomes. The process of phagosome maturation arrest by M. tuberculosis is not fully understood, and there has remained a need to firmly establish a requirement for phagosome maturation arrest for M. tuberculosis growth in macrophages. Other intracellular pathogens that control the phagosomal environment use specialized protein export systems to deliver effectors of phagosome trafficking to the host cell. In M. tuberculosis, the accessory SecA2 system is a specialized protein export system that is required for intracellular growth in macrophages. In studying the importance of the SecA2 system in macrophages, we discovered that SecA2 is required for phagosome maturation arrest. Shortly after infection, phagosomes containing a ΔsecA2 mutant of M. tuberculosis were more acidified and showed greater association with markers of late endosomes than phagosomes containing wild-type M. tuberculosis. We further showed that inhibitors of phagosome acidification rescued the intracellular growth defect of the ΔsecA2 mutant, which demonstrated that the phagosome maturation arrest defect of the ΔsecA2 mutant is responsible for the intracellular growth defect. This study demonstrates the importance of phagosome maturation arrest for M. tuberculosis growth in macrophages, and it suggests there are effectors of phagosome maturation that are exported into the host environment by the accessory SecA2 system.     

The functional roles of lipid rafts in T cell activation, immune diseases and HIV infection and prevention

The first appearance of lipid rafts, or lipid rafts-like structure, was occasionally observed by cryo-electronic microscopy in 1980s as cavity, such as caveolae. However, the fully understanding of lipid raft was attributed by the studies of T cell activation, virus entry/budding, and other membrane events. During the interaction of T cell and antigen presenting cell, a highly organized structure is formed at the interface of the two cells, where cholesterol and sphingolipids are enriched, and form a liquid ordered phase that facilitates the signaling proteins on and off. Lipid rafts are also involved in virus entry and assembly. In this review, we will discuss cholesterolsphingolipid floating microdomain, the lipid raft as a unique compartment of the plasma membrane, with biological functions that ensure correct intracellular traffic of proteins and lipids, such as protein-protein interactions by concentrating certain proteins in these microdomains, while excluding others. We also discuss the disruption of lipid rafts is related to different diseases and aging, and we especially exploit the lipid rafts as pharmaceutical targets for anti-virus and anti-inflammation, particularly a new approach to control HIV infection for AIDS prevention and protection by inhibition or disruption of lipid rafts. Cellular ＆ Molecular Immunology.     

Mutant of Salmonella typhimurium lacking the inhibitory function for phagosome-lysosome fusion in murine macrophages.

It has recently been described that Salmonella typhimurium is capable of inhibiting phagosome-lysosome fusion in murine macrophages after ingestion. We selected a mutant of S. typhimurium lacking the phagosome-lysosome fusion inhibitory function from a collection of Tn5-insertion mutants and examined its relevance to the pathogenesis in mice. The Tn5 insertion mutant which has a defect in fusion inhibitory function was found to be significantly sensitive to the intracellular killing by murine macrophages in vitro. However, the loss of the fusion inhibitory function did not reduce the level of virulence for mice in vivo. These results demonstrated that fusion inhibition did not play a critical role in the pathogenesis of S. typhimurium although it might contribute to at least a part of the resistance against macrophage killing mechanisms.     

Inhibition of macrophage phagosome-lysosome fusion by Salmonella typhimurium.

Salmonella typhimurium-infected macrophages were examined by electron microscopy to determine whether intracellular survival of S. typhimurium is associated with failure of bacteria containing phagosomes to fuse with secondary lysosomes. S. typhimurium 14028 actively inhibited phagosome-lysosome fusion and appeared to preferentially divide within unfused phagocytic vesicles. In comparison with Escherichia coli, S. typhimurium inhibited phagosome-lysosome fusion in peritoneal macrophages, J774 macrophages, and bone marrow-derived macrophages from both BALB/c (itys) and SWR/J (ityr) mice. The mechanism responsible for Salmonella inhibition of phagosome-lysosome fusion is unknown but requires viable salmonellae, is not blocked by opsonization with fresh normal mouse serum, and is not due to lipopolysaccharide. Inhibition of phagosome-lysosome fusion may play a critical role in survival of salmonellae within macrophages and in virulence.     

Membrane vesicles shed by Legionella pneumophila inhibit fusion of phagosomes with lysosomes

When cultured in broth to the transmissive phase, Legionella pneumophila infects macrophages by inhibiting phagosome maturation, whereas replicative-phase cells are transported to the lysosomes. Here we report that the ability of L. pneumophila to inhibit phagosome-lysosome fusion correlated with developmentally regulated modifications of the pathogen's surface, as judged by its lipopolysaccharide profile and by its binding to a sialic acid-specific lectin and to the hydrocarbon hexadecane. Likewise, the composition of membrane vesicles shed by L. pneumophila was developmentally regulated, based on binding to the lectin and to the lipopolysaccharide-specific monoclonal antibody 3/1. Membrane vesicles were sufficient to inhibit phagosome-lysosome fusion by a mechanism independent of type IV secretion, since only approximately 25% of beads suspended with or coated by vesicles from transmissive phase wild type or dotA secretion mutants colocalized with lysosomal probes, whereas approximately 75% of beads were lysosomal when untreated or presented with vesicles from the L. pneumophila letA regulatory mutant or E. coli. As observed previously for L. pneumophila infection of mouse macrophages, vesicles inhibited phagosome-lysosome fusion only temporarily; by 10 h after treatment with vesicles, macrophages delivered approximately 72% of ingested beads to lysosomes. Accordingly, in the context of the epidemiology of the pneumonia Legionnaires' disease and virulence mechanisms of Leishmania and Mycobacteria, we discuss a model here in which L. pneumophila developmentally regulates its surface composition and releases vesicles into phagosomes that inhibit their fusion with lysosomes.