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.     

A coat protein on phagosomes involved in the intracellular survival of mycobacteria.

Mycobacteria are intracellular pathogens that can survive within macrophage phagosomes, thereby evading host defense strategies by largely unknown mechanisms. We have identified a WD repeat host protein that was recruited to and actively retained on phagosomes by living, but not dead, mycobacteria. This protein, termed TACO, represents a component of the phagosome coat that is normally released prior to phagosome fusion with or maturation into lysosomes. In macrophages lacking TACO, mycobacteria were readily transported to lysosomes followed by their degradation. Expression of TACO in nonmacrophages prevented lysosomal delivery of mycobacteria and prolonged their intracellular survival. Active retention of TACO on phagosomes by living mycobacteria thus represents a mechanism preventing cargo delivery to lysosomes, allowing mycobacteria to survive within macrophages.     

Construction of chimeric phagosomes that shelter Mycobacterium avium and Coxiella burnetii (phase II) in doubly infected mouse macrophages: an ultrastructural study.

Dual infection of cells may divert pathogens to intracellular compartments different from those occupied in mono-infected cells. In the present studies, mouse bone marrow in vitro-derived macrophages were first infected with virulent Mycobacterium avium, which are normally singly lodged within tight phagosomes. These phagosomes do not mature; they undergo homotypic fusion with early endosomes and do not fuse with lysosomes. Seven days later, the cultures were superinfected with phase II (non-virulent) Coxiella burnetii, organisms sheltered in lysosome- (or prelysosome)-like, multi-occupancy phagosomes. The latter can attain large size and engage in efficient homo- and heterotypic fusion with other phagosomes. Cultures were fixed for transmission electron microscopy 6, 12, 24, and 48 h later. Other M. avium-infected cultures were superinfected with amastigotes of the trypanosomatid flagellate Leishmania amazonensis, which are also sheltered in lysosome- (or prelysosome)-like multi-occupancy vacuoles, and fixed at the same time periods. Chimeric phagosomes containing both M. avium and C. burnetii, were found already at 6 h and the proportion of M. avium that colocalized with C. burnetii in the same phagosomes reached over 90% after 48 h. In such phagosomes, both organisms were ultrastructurally well preserved. In contrast, colocalization of M. avium and L. amazonensis was rarely found. Speculative scenarios that could underlie the formation of chimeric phagosomes could involve delayed maturation of C. burnetii-containing phagosomes in presence of M. avium, which would allow for fusion of C. burnetii- and M. avium-containing phagosomes; the production, by C. burnetii, of molecules that upregulate the fusion of M. avium-containing phagosomes with those that contain C. burnetii; and the secretion of factors that could favour the survival of M. avium within chimeric vacuoles.     

Isolation and characterization of the mycobacterial phagosome: segregation from the endosomal/lysosomal pathway

Mycobacteria have the ability to persist within host phagocytes, and their success as intracellular pathogens is thought to be related to the ability to modify their intracellular environment. After entry into phagocytes, mycobacteria-containing phagosomes acquire markers for the endosomal pathway, but do not fuse with lysosomes. The molecular machinery that is involved in the entry and survival of mycobacteria in host cells is poorly characterized. Here we describe the use of organelle electrophoresis to study the uptake of Mycobacterium bovis bacille Calmette Guerin (BCG) into murine macrophages. We demonstrate that live, but not dead, mycobacteria occupy a phagosome that can be physically separated from endosomal/lysosomal compartments. Biochemical analysis of purified mycobacterial phagosomes revealed the absence of endosomal/lysosomal markers LAMP-1 and β-hexosaminidase. Combining subcellular fractionation with two-dimensional gel electrophoresis, we found that a set of host proteins was present in phagosomes that were absent from endosomal/lysosomal compartments. The residence of mycobacteria in compartments outside the endosomal/lysosomal system may explain their persistence inside host cells and their sequestration from immune recognition. Furthermore, the approach described here may contribute to an improved understanding of the molecular mechanisms that determine the intracellular fate of mycobacteria during infection.     

Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system

Mycobacterium avium and Mycobacterium tuberculosis are human pathogens that infect and replicate within macrophages. Both organisms live in phagosomes that fail to fuse with lysosomes and have adapted their lifestyle to accommodate the changing environment within the endosomal system. Among the many environmental factors that could influence expression of bacterial genes are the concentrations of single elements within the phagosomes. We used a novel hard x-ray microprobe with suboptical spatial resolution to analyze characteristic x-ray fluorescence of 10 single elements inside phagosomes of macrophages infected with M. tuberculosis and M. avium or with avirulent M. smegmatis. The iron concentration decreased over time in phagosomes of macrophages infected with Mycobacterium smegmatis but increased in those infected with pathogenic mycobacteria. Autoradiography of infected macrophages incubated with (59)Fe-loaded transferrin demonstrated that the bacteria could acquire iron delivered via the endocytic route, confirming the results obtained in the x-ray microscopy. In addition, the concentrations of chlorine, calcium, potassium, manganese, copper, and zinc were shown to differ between the vacuole of pathogenic mycobacteria and M. smegmatis. Differences in the concentration of several elements between M. avium and M. tuberculosis vacuoles were also observed. Activation of macrophages with recombinant IFN-gamma or TNF-alpha before infection altered the concentrations of elements in the phagosome, which was not observed in cells activated following infection. Siderophore knockout M. tuberculosis vacuoles exhibited retarded acquisition of iron compared with phagosomes with wild-type M. tuberculosis. This is a unique approach to define the environmental conditions within the pathogen-containing compartment.     

Cholesterol depletion in Mycobacterium avium-infected macrophages overcomes the block in phagosome maturation and leads to the reversible sequestration of viable mycobacteria in phagolysosome-derived autophagic vacuoles

Phagocytic entry of mycobacteria into macrophages requires the presence of cholesterol in the plasma membrane. This suggests that pathogenic mycobacteria may require cholesterol for their subsequent intra-cellular survival in non-maturing phagosomes. Here we report on the effect of cholesterol depletion on pre-existing phagosomes in mouse bone marrow-derived macrophages infected with Mycobacterium avium. Cholesterol depletion with methyl-beta-cyclodextrin resulted in a loosening of the close apposition between the phagosome membrane and the mycobacterial surface, followed by fusion with lysosomes. The resulting phagolysosomes then autonomously executed autophagy, which did not involve the endoplasmic reticulum. After 5 h of depletion, intact mycobacteria had accumulated in large auto-phagolysosomes. Autophagy was specific for phagolysosomes that contained mycobacteria, as it did not involve latex bead-containing phagosomes in infected cells. Upon replenishment of cholesterol, mycobacteria became increasingly aligned to the lysosomal membrane, from where they were individually sequestered in phagosomes with an all-around closely apposed phagosome membrane and which no longer fused with lysosomes. These observations indicate that, cholesterol depletion (i) resulted in phagosome maturation and fusion with lysosomes and (ii) caused mycobacterium-containing phagolysosomes to autonomously undergo autophagy. Furthermore, (iii) mycobacteria were not killed in auto-phagolysosomes, and (iv) cholesterol replenishment enabled mycobacterium to rescue itself from autophagic phagolysosomes to again reside individually in phagosomes which no longer fused with lysosomes.     

Acquisition of Hrs, an essential component of phagosomal maturation, is impaired by mycobacteria

Pathogenic mycobacteria survive within macrophages by precluding the fusion of phagosomes with late endosomes or lysosomes. Because the molecular determinants of normal phagolysosome formation are poorly understood, the sites targeted by mycobacteria remain unidentified. We found that Hrs, an adaptor molecule involved in protein sorting, associates with phagosomes prior to their fusion with late endosomes or lysosomes. Recruitment of Hrs required the interaction of its FYVE domain with phagosomal phosphatidylinositol 3-phosphate, but two other attachment sites were additionally involved. Depletion of Hrs by use of small interfering RNA impaired phagosomal maturation, preventing the acquisition of lysobisphosphatidic acid and reducing luminal acidification. As a result, the maturation of phagosomes formed in Hrs-depleted cells was arrested at an early stage, characterized by the acquisition and retention of sorting endosomal markers. This phenotype is strikingly similar to that reported to occur in phagosomes of cells infected by mycobacteria. We therefore tested whether Hrs is recruited to phagosomes containing mycobacteria. Hrs associated readily with phagosomes containing inert particles but poorly with mycobacterial phagosomes. Moreover, Hrs was found more frequently in phagosomes containing avirulent Mycobacterium smegmatis than in phagosomes with the more virulent Mycobacterium marinum. These findings suggest that the inability to recruit Hrs contributes to the arrest of phagosomal maturation induced by pathogenic mycobacteria.     

The Trojan horse: survival tactics of pathogenic mycobacteria in macrophages

Mycobacterium tuberculosis, the causative agent of tuberculosis, has infected billions of people worldwide. A key to the success of M. tuberculosis and related pathogenic mycobacteria lies in their ability to persist within the hostile environment of the host macrophage. After internalization by macrophages, most microbes are rapidly transported to lysosomes in which they are destroyed. By contrast, pathogenic mycobacteria prevent fusion of phagosomes with lysosomes, thereby surviving intracellularly. Recent progress in understanding the molecular biology of host-mycobacteria interactions is providing insights into these survival tactics.     

TB or not TB: calcium regulation in mycobacterial survival

Mycobacterium tuberculosis (Mtb)-the bacterium that causes tuberculosis-resides in phagosomes inside macrophages. This bacterium evades destruction by preventing phagosome maturation, which involves the fusion of phagosomes with lysosomes. In this issue of Cell, Jayachandran et al. (2007) suggest that mycobacteria co-opt the actin-binding protein coronin 1 to activate the phosphatase calcineurin, thereby preventing phagosomal maturation.     

Mycobacterium's arrest of phagosome maturation in macrophages requires Rab5 activity and accessibility to iron

Many mycobacteria are intramacrophage pathogens that reside within nonacidified phagosomes that fuse with early endosomes but do not mature to phagolysosomes. The mechanism by which mycobacteria block this maturation process remains elusive. To gain insight into whether fusion with early endosomes is required for mycobacteria-mediated inhibition of phagosome maturation, we investigated how perturbing the GTPase cycles of Rab5 and Rab7, GTPases that regulate early and late endosome fusion, respectively, would affect phagosome maturation. Retroviral transduction of the constitutively activated forms of both GTPases into primary murine macrophages had no effect on Mycobacterium avium retention in an early endosomal compartment. Interestingly, expression of dominant negative Rab5, Rab5(S34N), but not dominant negative Rab7, resulted in a significant increase in colocalization of M. avium with markers of late endosomes/lysosomes and increased mycobacterial killing. This colocalization was specific to mycobacteria since Rab5(S34N) expressing cells showed diminished trafficking of endocytic tracers to lysosomes. We further demonstrated that maturation of M. avium phagosomes was halted in Rab5(S34N) expressing macrophages supplemented with exogenous iron. These findings suggest that fusion with early endosomes is required for mycobacterial retention in early phagosomal compartments and that an inadequate supply of iron is one factor in mycobacteria's inability to prevent the normal maturation process in Rab5(S34N)-expressing macrophages.     

Elemental analysis of the Mycobacterium avium phagosome in Balb/c mouse macrophages

Using a hard X-ray microprobe, we showed recently that in unstimulated peritoneal macrophages from C57BL/6 mice, the phagosome of pathogenic mycobacteria (Mycobacterium tuberculosis and Mycobacterium avium) can accumulate iron. We expanded our studies to the M. avium infection of peritoneal macrophages of Balb/c mice that show a similar degree of M. tuberculosis and M. avium-related chronic disease, but a higher susceptibility towards other intracellular pathogens such as Listeria monocytogenes, Leishmania major, or Brucella abortus as compared to C57BL/6 mice. Similar to C57BL/6 macrophages, the iron concentration in Balb/c macrophages increased significantly after 24 h of infection. A significant increase of the chlorine and potassium concentrations was observed in the Balb/c phagosomes between 1 and 24 h, in contrast with macrophages from C57BL/6 mice. The absolute elemental concentrations of calcium and zinc were higher in the mycobacterial phagosomes of Balb/c mice. We hypothesize that a potassium channel is abundant in the phagosome in macrophages that may be related to microbiocidal killing, similar to the requirement of potassium channels for microbiocidal function in neutrophils.     

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.     

Influence of phagosomal contents on the apparent inhibition of phagosome-lysosome fusion mediated by polyanionic substances in mouse peritoneal macrophages.

The study of fusion of phagosomes with secondary lysosomes in macrophages is facilitated by assessing transfer of fluorescent or electron-opaque markers (or both) from the lysosomes to the phagosomes. When certain virulent viable pathogens are phagocytosed by mouse peritoneal macrophages, phagosome-lysosome fusion (P-LF) is inhibited. Nonviable counterparts ordinarily cannot impose this block. A similar, but spurious, block to P-LF seems to be mediated from the lysosomal domain following sequestration of certain polyanionic substances. This block has been judged to be relieved by, for example, heat-killed yeasts and various viable bacteria designated as fusion-inducing microorganisms, acting from the phagosome. In this study we tested this concept and believed it to be unfounded. Macrophages labeled with Thorotrast and incubated with dextran sulfate were offered a variety of viable and heat-killed microorganisms for phagocytosis: Saccharomyces cerevisiae, Mycobacterium lepraemurium, Streptococcus faecalis, and Escherichia coli. By electron microscopy, a transfer of Thorotrast to phagosomes up to 18 h was seen to be highly suppressed as compared with controls, but was not notably different for any of the targets, whether viable or not. Instead, inert 0.45-micron carboxylated polystyrene beads (the smallest target) showed the most delivery of marker. If polyanionic agents truly inhibited fusion, then "fusiogenic" microorganisms should free the marker for delivery. If polyanions do not inhibit P-LF and only trap the marker, the behavior of the various targets would correspond to what we found.     

Mycobacterial Nucleoside Diphosphate Kinase Blocks Phagosome Maturation in Murine Raw 264.7 Macrophages

Background

Microorganisms capable of surviving within macrophages are rare, but represent very successful pathogens. One of them is Mycobacterium tuberculosis (Mtb) whose resistance to early mechanisms of macrophage killing and failure of its phagosomes to fuse with lysosomes causes tuberculosis (TB) disease in humans. Thus, defining the mechanisms of phagosome maturation arrest and identifying mycobacterial factors responsible for it are key to rational design of novel drugs for the treatment of TB. Previous studies have shown that Mtb and the related vaccine strain, M. bovis bacille Calmette-Guérin (BCG), disrupt the normal function of host Rab5 and Rab7, two small GTPases that are instrumental in the control of phagosome fusion with early endosomes and late endosomes/lysosomes respectively.

Methodology/Principal Findings

Here we show that recombinant Mtb nucleoside diphosphate kinase (Ndk) exhibits GTPase activating protein (GAP) activity towards Rab5 and Rab7. Then, using a model of latex bead phagosomes, we demonstrated that Ndk inhibits phagosome maturation and fusion with lysosomes in murine RAW 264.7 macrophages. Maturation arrest of phagosomes containing Ndk-beads was associated with the inactivation of both Rab5 and Rab7 as evidenced by the lack of recruitment of their respective effectors EEA1 (early endosome antigen 1) and RILP (Rab7-interacting lysosomal protein). Consistent with these findings, macrophage infection with an Ndk knocked-down BCG strain resulted in increased fusion of its phagosome with lysosomes along with decreased survival of the mutant.

Conclusion

Our findings provide evidence in support of the hypothesis that mycobacterial Ndk is a putative virulence factor that inhibits phagosome maturation and promotes survival of mycobacteria within the macrophage.     

On phagosome individuality and membrane signalling networks

Cells such as macrophages take up pathogens into specialized membrane organelles (phagosomes) that fuse with other organelles, including lysosomes, in a process termed maturation. The fully matured phagolysosome is a low-pH, hydrolase-rich killing device that some pathogens can bypass. One might expect that phagosomes containing a given type of particle that entered cells simultaneously via the same receptor would behave the same, at least in a single cell. Surprisingly, however, recent data show that phagosomes formed via the same receptors can find themselves in different chemical states even within the same macrophage. Here, I argue that each phagosome is an individual entity whose behaviour depends on a finite number of stable equilibrium states in its membrane signalling networks.     

Leishmania promastigotes require lipophosphoglycan to actively modulate the fusion properties of phagosomes at an early step of phagocytosis

The lipophosphoglycan (LPG) of Leishmania promastigotes plays key roles in parasite survival in both insect and mammalian hosts. Evidence suggests that LPG decreases phagosome fusion properties at the onset of infection in macrophages. The mechanisms of action of this molecule are, however, poorly understood. In the present study, we used a panoply of Leishmania mutants displaying modified LPG structures to determine more precisely how LPG modulates phagosome-endosome fusion. Using an in vivo fusion assay measuring, at the electron microscope, the transfer of solute materials from endosomes to phagosomes, we provided further evidence that the repeating Gal(beta1,4)Man(alpha1-PO4) units of LPG are responsible for the alteration in phagosome fusion. The inhibitory effect of LPG on phagosome fusion was shown to be more potent towards late endocytic organelles and lysosomes than early endosomes, explaining how Leishmania promastigotes can avoid degradation in hydrolase-enriched compartments. The involvement of other repeating unit-containing molecules, including the secreted acid phosphatase, in the inhibition process was ruled out, as an LPG-defective mutant (Ipg1-) which secretes repeating unit-containing glycoconjugates was present in highly fusogenic phagosomes. In L. major, oligosaccharide side-chains of LPG did not contribute to the inhibition process, as Spock, an L. major mutant lacking LPG side-chains, blocked fusion to the same extent as wild-type parasites. Finally, dead parasites internalized from the culture medium were not as efficient as live parasites in altering phagosome-endosome fusion, despite the presence of LPG. However, the killing of parasites with vital dyes after their sequestration in phagosomes had no effect on the fusion properties of this organelle. Collectively, these results suggest that living promastigotes displaying full-length cell surface LPG can actively influence macrophages at an early stage of phagocytosis to generate phagosomes with poor fusogenic properties.     

The metabolic activity of Mycobacterium tuberculosis, assessed by use of a novel inducible GFP expression system, correlates with its capacity to inhibit phagosomal maturation and acidification in human macrophages

Mycobacterium tuberculosis generally reside in phagosomes within human macrophages that resist maturation and acidification, but exhibit significant heterogeneity. In this study we have constructed an IPTG-inducible GFP expression system in M. tuberculosis to assess the relationship between the metabolic status of M. tuberculosis and the degree of phagosomal maturation. Using these recombinant bacteria, we have found that, in human macrophages, M. tuberculosis that respond to IPTG with expression of GFP fluorescence, and hence are metabolically active, reside in non-acidified phagosomes that have not fused with Texas red dextran pre-labelled lysosomes. In contrast, M. tuberculosis that fail to express GFP in response to IPTG, and hence are metabolically inactive, reside within acidified phagosomes that have fused with Texas red dextran labelled lysosomes. These studies demonstrate that metabolic activity of M. tuberculosis correlates strongly with phagosomal maturation and that the inducible GFP expression system is useful for assessing metabolic activity of intracellular M. tuberculosis.     

Iron acquisition within host cells and the pathogenicity of Leishmania

Iron is an essential cofactor for several enzymes and metabolic pathways, in both microbes and in their eukaryotic hosts. To avoid toxicity, iron acquisition is tightly regulated. This represents a particular challenge for pathogens that reside within the endocytic pathway of mammalian cells, because endosomes and lysosomes are gradually depleted in iron by host transporters. An important player in this process is Nramp1 (Slc11a1), a proton efflux pump that translocates Fe^{2 +} and Mn²⁺ ions from macrophage lysosomes/phagolysosomes into the cytosol. Mutations in Nramp1 cause susceptibility to infection with the bacteria Salmonella and Mycobacteria and the protozoan Leishmania , indicating that an available pool of intraphagosomal iron is critical for the intracellular survival and replication of these pathogens . Salmonella and Mycobacteria are known to express iron transporter systems that effectively compete with host transporters for iron. Until recently, however, very little was known about the molecular strategy used by Leishmania for survival in the iron-poor environment of macrophage phagolysosomes. It is now clear that intracellular residence induces Leishmania amazonensis to express LIT1, a ZIP family membrane Fe²⁺ transporter that is required for intracellular growth and virulence.