Stay out of harm’s way: Mycobacteria avoidprogression into phagolysosomes

Nearly 30 years ago, Armstrong and D’Arcy Hart showedthat live intracellular mycobacteria avoid phagosome–lysosome fusion.1 After labelling macrophage lysosomes withferritin, they observed that the electron-dense colloid was nottransferred to phagosomes containing live Mycobacteriumtuberculosis, but it was readily transferred to phagosomescontaining killed mycobacteria. The impaired fusion ofmycobacteria-containing phagosomes with lysosomes hassince been confirmed by others using pinocytosed fluid-phase markers2–4 and soluble endocytic markers.3 The limitedinteraction with lysosomes correlates with mildly acidic pHand paucity of late endocytic and lysosomal markers inmycobacteria-containing phagosomes.3–5 The pH of 6.3–6.5correlated with limited access of the proton pump, v-ATPase,to phagosomes containing Mycobacterium avium.6

Although mycobacteria-containing phagosomes fail tofuse with lysosomes, they interact with early endocytic com-partments.7 Furthermore, plasmalemma-derived tracers, suchas cholera toxin bound to surface GM1 gangliosides orexogenously added transferrin, rapidly enter phagosomes thatharbour M. tuberculosis or M. avium.8–10 The accessibility ofphagosomes to transferrin has suggested a possible role forthe recycling pathway in delivery of iron, a growth-limitingfactor, to the enclosed mycobacteria. Taken together, thesedata indicate that mycobacteria-containing phagosomes arenot secluded from the entire vesicular network of the hostcell, but rather that mycobacteria-containing phagosomesinteract with the early endocytic/recycling system and avoidfusion with late endosomes and lysosomes.

Nascent phagosomes undergo a stepwise maturationprocess via sequential fusion with early endosomes, lateendosomes and finally lysosomes.11 The concept has emergedthat mycobacteria avoid these normal consequences ofphagocytosis. This notion has been corroborated by the trafficking of the acidic protease cathepsin D to phagosomescontaining M. avium or IgG-coated beads. Cathepsin D issynthesized as an inactive 51 kDa proform, which isprocessed to a 46 kDa intermediate form and finally to a30 kDa mature form, in acidic endocytic and lysosomal com-partments, respectively.12 Immunoglobulin G-coated bead-containing phagosomes have been shown to acquire theproform immediately after phagocytosis and this resulted inrapid processing of the proform to the 46 kDa intermediateform (Fig. 1a). Procathepsin D processing was accompaniedby an increase in the phagosomal content of the v-ATPase,suggesting gradual lumenal acidification. Conversely,M. avium-harbouring phagosomes contained the 51 kDaproform and the 46 kDa intermediate form at early (4 h) andlate (6 day) times postinfection (pi; Fig. 1b). At 6 days pi,small amounts of the 30 kDa mature form were present, indi-cating maturation of a fraction of M. avium-containingphagosomes to phagolysosomes. Maintenance of the proformin phagosomes was dependent on bacterial viability, becausephagosomes harboring dead bacilli lost the proform earlyafter infection (Fig. 1b). These data indicate that M. aviumenters via a phagocytic mechanism shared by inert particles,but freezes vacuole maturation at an early developmentallevel. The demonstration that mycobacteria arrest phagosomematuration has changed the ideas on how mycobacteria avoidfusion with lysosomes. Rather than blocking phagosome-lysosome fusion per se, mycobacteria could arrest phago-some maturation at a developmental level, one that does notpermit fusion with lysosomal compartments.

Activation of macrophages overcomes the block to phago-some maturation. Interferon-γ and LPS treatment of infectedmacrophages renders M. avium-containing phagosomes inaccessible to exogenously added transferrin, indicating that

Immunology and Cell Biology (2000) 78, 301–310

Special Feature

How to establish a lasting relationship with your host: Lessonslearned from Mycobacterium spp.

ER RHOADES and HJ ULLRICH

Department of Microbiology, Washington University, St Louis, Missouri, USA

Summary Mycobacterium spp. enjoy an intracellular lifestyle that is fatal to most microorganisms. Bacilli persistand multiply within mononuclear phagocytes in the face of defences ranging from toxic oxygen and nitrogen radicals, acidic proteases and bactericidal peptides. Uptake of Mycobacterium by phagocytes results in the de novoformation of a phagosome, which is manipulated by the pathogen to accommodate its needs for intracellular survival and replication. The present review describes the intracellular compartment occupied by Mycobacteriumspp. and presents current ideas on how mycobacteria may establish this niche, placing special emphasis on theinvolvement of mycobacterial cell wall lipids.

Key words: endocytic trafficking, glycolipid, macrophage, Mycobacterium, phagosome.

Correspondence: Elizabeth Rhoades, Department of Microbiol-ogy and Immunology, Cornell University, C5171 Veterinary MedicalCenter, Ithaca, NY 14853, USA.

Received 26 April 2000; accepted 26 April 2000.

these compartments are transferred deeper into the phago-cytic pathway. The transition to acidic phagolysosomes isindicated by the accumulation of the v-ATPase and the30 kDa lysosomal form of cathepsin D.13,14

How important is the ability of mycobacteria to avoidphagosome–lysosome fusion? Traditionally, it has beenthought that fusion with the lysosome spells the destructionof intracellular bacilli. Accordingly, one study has revealedthat transfer of bacilli to phagolysosomes in humanmacrophages coincides with a drop in mycobacterial viabil-ity.15 However, fusion of lysosomes with M. tuberculosis-containing phagosomes in mouse macrophages does notcause a drop in bacterial viability, indicating that the sourceof macrophages can have a profound effect on mycobacterialsurvival.16 Activated macrophages also vary in their capacityto kill mycobacteria (reviewed by Fenton and Vermeulen17),in support of this idea. Furthermore, the various species ofmycobacteria differ in their ability to withstand the acidicconditions in phagolysosomes. Growth of M. avium in communal vacuoles with Coxiella burnetti is not impaired bythe acidic pH of these spacious compartments. Conversely,growth of M. tuberculosis in compartments shared with Cox-iella is strongly inhibited.18 These differences indicate thatphagosome–lysosome fusion per se does not dictate myco-bacterial killing; rather, it is likely that delivery of bacilli tothis late compartment potentiates antimicrobial defences.

Arresting phagosome maturation could also affect theefficiency of mycobacterial antigen presentation. Class IIMHC heterodimers and HLA-DM, a chaperon of antigenloading, traffic through specialized late endosomal-like compartments (MIIC) on their way to the plasma membrane.

In resting macrophages, M. avium-containing phagosomescontain surface-derived MHC class II heterodimers, but failto acquire MHC class II from intracellular stores. At lateinfection time points, this causes a paucity of phagosomalMHC class II molecules (Ullrich et al., unpubl. data, 1999).It is intriguing to speculate that the arrest in phagosome mat-uration could prevent the fusion of M. avium-containingphagosomes with late endosomal-like MIIC containing MHCclass II molecules. Indeed, phagosomes formed around inertparticles acquire MHC class II molecules and HLA-DM at alate stage during phagosome biogenesis (Ullrich et al.,unpubl. data, 1999). The seclusion of M. avium-harbouringphagosomes from intracellular pools of MHC class II andHLA-DM is no longer maintained after activation of macro-phages (Ullrich et al., unpubl. data, 1999). The sequestrationof mycobacteria-containing phagosomes from the intra-cellular trafficking pathway of MHC class II molecules couldhelp to explain some of the apparent immunosuppressiveactivities of mycobacteria.19

How do mycobacteria arrest phagosomal maturation?

The molecular basis for the block in phagosome maturationremains enigmatic. The identification of virulence factors,which retard phagosome maturation, has been hindered bythe genetic inaccessibility of mycobacteria. Recently, thesegenetic limitations have been overcome and it is likely that aclearer picture of how mycobacteria affect phagosome bio-genesis will soon emerge. Herein, we present current ideas onhow mycobacteria may establish their unique phagosomalniche.

ER Rhoades and HJ Ullrich302

Figure 1 Mycobacteria arrest normal phagosome biogenesis. (a) Immunoglobulin G-coated bead-containing phagosomes isolated at different time points postinternalization were used to analyse cathepsin D acquisition during normal phagosome maturation. Immuno-globulin G-coated bead-containing phagosomes acquired procathepsin D (ProD) early after phagocytosis and processed this form withinless than 2 h to the 46 kDa intermediate form (CathD). (b) Phagosomes containing live or killed Mycobacterium avium from a 4 h infec-tion and live M. avium-containing phagosomes from a 6 day infection were analysed for the different forms of cathepsin D. ProcathepsinD was present in live mycobacteria-containing phagosomes at all times, indicating the arrest in phagosome maturation. Reproduced withpermission from Ullrich et al.14

The intraphagosomal pH

Blocking vesicle acidification by weak bases or BafilomycinA impairs transport of material from early endosomes to lateendosomes.20 This impaired transport is analogous to whatoccurs to phagosomes containing mycobacteria. Hence, it isconceivable that control of phagosomal pH by mycobacteriais essential for arresting phagosome biogenesis. Mycobac-teria express a glutamine synthetase21 and a urease,22 both ofwhich could elevate the intraphagosomal pH by producingammonia. The relevance of the urease for intracellular survival is debatable, because a urease-deficient mutant of Mycobacterium bovis Bacillus Calmette–Guérin (BCG) multiplies normally in macrophages.23 Inhibitors of the glutamine synthetase block mycobacterial growth in macro-phages, but it has not been determined whether intra-phagosomal pH is altered.21 The neutralization capacity ofmycobacteria has been further questioned by co-infections ofmacrophages with mycobacteria and Coxiella. Coxiella-containing phagosomes are acidic and mycobacteria enteringthese spacious compartments in doubly infected macro-phages are unable to raise the intraphagosomal pH, thus questioning the importance of mycobacterial enzymes in preventing phagosome acidification.18

The ‘tight’ phagosome

The mycobacterial cell wall is rich in lipids and thereforevery hydrophobic. Hydrophobic surfaces cause the phagoso-mal membrane to surround a particle tightly24 and indeedmycobacteria reside within phagosomes where the membraneis in close apposition to the bacterium.7 Using bead particleswith different surface properties, de Chastellier and Thilohave shown that ‘tight’ phagosomes intermix with early endo-somes, but fail to fuse with lysosomes.24 A model has beenproposed, in which tight membrane apposition prevents therecycling of early endosomal fusion factors, leading to anaccumulation of these factors on the phagosomal membrane.This could lead to impaired phagosome maturation, due tocontinual fusion of phagosomes with early endosomes. Whilethis model is attractive to explain the maintenance of theMycobacterium-containing phagosome within the earlyendosomal/recycling network, it fails to explain why killedmycobacteria traffic to phagolysosomes.1,4,5,14 Even mycobac-teria that are killed intracellularly by adding an antibiotic orare killed by radiation prior to infection (methods that ensureminimal perturbations of the mycobacterial cell wall) fail toblock phagosome maturation.1,25

Manipulation of the host’s fusion and cytoskeletal system

Phagosome maturation requires the assembly of properfusion machinery on the phagosomal membrane to allowspecific intermixing with early endosomes, then with lateendosomes and finally with lysosomes. Regulation of vesiclefusion within the host vesicular system is determined, in part,by Rab proteins, a family of small GTPases.26 Maturation ofearly endosomes to late endosomes is controlled by the smallGTPase Rab7, which resides on late endocytic compart-ments.27 This is the same step that is blocked in the matura-tion of mycobacteria-containing phagosomes. Indeed, Rab7 is

absent from phagosomes containing M. bovis BCG, butpresent on phagosomes containing inert particles.28 Thesedata suggest that either mycobacteria interfere with Rab7acquisition and that this prevents phagosome maturation or,alternatively, that the bacteria interfere with other endosomaldeterminants that mediate recruitment of Rab7. Furtherinvestigation has revealed that M. bovis BCG-harbouringphagosomes contain Rab 5, another member of the Rabfusion protein family.28 In non-phagocytic cells, Rab5 isexclusively found on early endosomes and it has been proposed to mediate retention of mycobacteria-containingphagosomes within the early endosomal network. Inmacrophages, however, Rab5 has been detected not only onearly endosomes but also on late endosomes and lysosomes.Furthermore, it stimulates fusion of phagosomes harbouringinert particles with early and late endosomes.29 Therefore, itis likely that Rab5 on mycobacteria-containing phagosomesdoes not prevent fusion with late endosomes or lysosomes.Besides affecting the recruitment of fusion factors, mycobac-teria also modify the interaction of phagosomes with thehost’s cytoskeletal system. The cell cortex protein coronin1(in the original paper named TACO for tryptophane aspar-tate-containing coat protein) strongly accumulates aroundlive M. bovis BCG-containing phagosomes.25 The effect isspecific for live mycobacteria, suggesting that coronin1 isactively retained on the phagosomal membrane. The functionof coronin is unknown, but it has been shown to promotephagocytosis and is transiently recruited to nascent bead-containing-phagosomes.30 The fact that there are actin-binding sites on coronin is consistent with the possibility thatcoronin1 may connect phagosomes to the cortical actinnetwork.31 It has been proposed that the accumulation ofcoronin1 around live mycobacteria-containing phagosomesmay prevent the acquisition or hinder the function of phago-some maturation-promoting factors.25 However, the putative‘protective coat’ function of coronin1 does not impair fusionin general, because mycobacteria-containing phagosomesacquire material transported within the early endocytic/recycling network.7,8,10

What are the mycobacterial mediators?

Current models of microbial influence on phagosome manip-ulation focus on secretion systems that transfer proteinaceousvirulence factors into the phagosomal membrane or thecytosol, influencing the trafficking of the enclosedpathogen.32,33 Mycobacteria lack a secretion system that isanalogous to type III or type IV secretion systems, and secre-tion of proteins by mycobacteria is still a largely undefinedprocess.34 Although no mycobacterial proteins or lipids havebeen shown to modify phagosome maturation, lipids are idealcandidates. A mycobacterial sulfolipid, when coated ontoyeast particles, impairs phagosome fusion with lysosomes.35

Furthermore, other mycobacterial glycolipids, includinglipoarabinomannans (LAM) and glycopeptidolipids (GPL),exert pleiotropic effects on macrophages.36 Considering theendotoxin-like activity of these lipids along with theextremely high lipid content of the mycobacterial cell wall, itis possible that these constituents mediate some of thedescribed effects on phagosome maturation.

Phagosomal niche of Mycobacterium spp. 303

Lipids of the mycobacterial cell wall

Although mycobacteria possess a typical cytoplasmic mem-brane and a thin peptidoglyan layer, that is where the simi-larity to the cell walls of Gram-positive or Gram-negativebacteria ends. The popular model of the mycobacterial cellwall positions complex mycobacterial lipids on the outermostlipid leaflet of the envelope.37 A mycolyl arabinogalactan-peptidoglycan (mAGP) complex covers a typical bacterialcytoplasmic membrane, with mycolyl esters (α-alkyl-, β-hydroxy-, C

70-C

90mycolic acids) extending outward, forming

a thick inner lipid leaflet. An outer lipid leaflet, which is com-posed of a mixture of complex waxes, triacylglygerols andbioactive glycolipids, is hydrophobically associated with theinner mycolyl leaflet. Mycobacterial lipoglycans, LAM andrelated lipomannan (LM) and phosphomannosides (PIM) arealso associated with the cell wall. A lipid moiety anchorsthese lipoglycans to the cytoplasmic membrane or the outerlipid leaflet. The nature of the outermost surface of the cellwall remains unresolved. Most agree that the glycosylatedgroups of the complex lipids are exposed on the outermostsurface of the cell wall,37,38 while others assert that a densecapsule-like matrix of free carbohydrates (glucans, mannansand arabinomannans) and secreted proteins conceals theselipids.39 The molecular composition varies among myco-bacterial species and culture conditions,40 which may explainthe controversy.

Mycobacterial lipids exert immunosuppressive andinflammatory/granulomagenic effects. These properties, atfirst glance, appear to be contrary to one another; however,both types of responses contribute to the pathogenesis ofmycobacterial infections. Inflammatory cytokines, includingTNF-α, IL-1β and multiple chemokines, are induced byLAM41,42 and GPL.43 Trehalose dimycolate (TDM) inducesthe formation of granulomas in vivo.44 Immunosuppressiveeffects include suppression of IFN-γ-mediated activation45,46

and suppression of lymphoproliferative responses.47,48 Mostof these reported activities, while directly affectingmacrophage functions, are beyond the scope of the presentreview; excellent reviews by Strohmeier and Fenton,36 Chat-terjee and Khoo49 and Barrow43 describe the plethora ofeffects. We will consider lipid-mediated effects that may alterphagosomal events, first describing the structures of the relevant lipids.

Lipoarabinomannan structure

All LAM molecules are composed of four parts: (i) a uniqueglycerophosphatidyl myo-inositol (GPI) anchor, which is pri-marily esterified to tuberculostearic and palmitic acids; (ii) amannopyranosyl (manp) backbone, which is substituted at C-2 with single manp residues to various degrees; (iii) an arabinofuranosyl (araf) extension (70–80 residues), punctu-ated by linear tetra-araf or biantennary hexa-araf moieties;and (iv) various carbohydrate capping motifs.49 Lipoarabino-mannan molecules from different mycobacterial species arefairly heterogeneous, varying in the extent of carbohydratebranching and capping, the degree of phosphorylation of theoligosaccharide50 and the extent of acylation of the lipoman-nan core (up to four additional acyl groups).51 To date, allcharacterized strains of M. bovis, M. tuberculosis and

Mycobacterium leprae possess mannose-capped forms ofLAM (manLAM), in which mannooligosaccharides areattached to 40–70% of the terminal araf residues. A differentform of LAM, called araLAM, which lacks the terminalmannose capping, is found in all fast growers examined, suchas Mycobacterium smegmatis. A minor portion of the termi-nal tetra-araf groups of araLAM are capped by inositol phos-phate (PI-LAM).52

The various forms of LAM elicit markedly differentresponses and both acyl and carbohydrate termini areinvolved. The acyl moieties of LAM confer the ability to effi-ciently interact with membranes and modulate hostresponses.41,45 Natural deacylated versions of LAM (PI-GAM) and chemically deacylated LAM share the inability toexert effects on the host. Less glycosylated phospholipids,which share a common glycosylphosphatidylinositol (GPI)anchor, are also present on mycobacteria. Lipomannan lacksthe highly branched arabinan extension and PIM are evenshorter versions, consisting of the GPI anchor linked to1–5 manp residues (PIM

1to PIM

5). Both LM and PIM are

also exposed on the surface of mycobacteria, where they maycontribute to binding host macrophages53 or induce inflam-matory mediators;41 however, highly glycosylated forms ofLAM are more potent agonists of cytokine responses,araLAM being much more potent than manLAM.42,54

Trehalose ester structures

Trehalose dimycolate, also known as cord factor, is a bio-active lipid with well-characterized toxic and granuloma-genic properties.44,55 Trehalose dimycolate consists of asingle trehalose residue, a disaccharide of glucose, to whichare esterified mycolic acids at the 6 and 6′ sugar positions.56

The mycolic acid substituents vary among the mycobacterialspecies, with the longest (and most toxic) fatty acyl chainsmade by M. tuberculosis and M. leprae (typically C

70–90).

These fatty acyls consist of α-alkyl, β-hydroxy branchedfatty acids, which may contain double bonds or cyclopropanerings and methoxy or keto groups additional to the β-hydroxygroup and methyl branches in the main carbon backbone.

Sulfated acyl trehaloses, commonly referred to as sulfo-lipids, are quite similar in structure to TDM; acyl chains areesterified to trehalose.56 In the place of mycolyl groups, thereare 2–4 highly branched fatty acids, and a sulfate group isattached to the 2′ position of the disaccharide. Although notas potent as TDM, similar toxic properties have been attrib-uted to this molecule.57

Mycoside structures

Mycosides are type-specific glycolipids of mycobacteria,which contain oligosaccharide moieties that are linked tounusual lipid residues, reviewed by Brennan.58 The expres-sion of particular mycosides is restricted among themycobacterial species. Phenolic glycolipids (PGL), classifiedas mycosides A and B, consist of an oligosaccharide linked,via a phenol group, to a lipid backbone that bears two poly-methyl-branched fatty acids (mycocerosic acids). Theoligosaccharide may be a complex sugar, as in the case of M. kansasii and M. leprae, or a single sugar, such as rham-nose, in the case of M. bovis. The mycoside C group consists

ER Rhoades and HJ Ullrich304

of GPL of the Mycobacterium avium-intracellularae-scrofulaceum (MAIS) complex. In addition to lipid and sugarresidues, GPL contain a tetrapeptide sequence. The consensusstructure of GPL consists of a carboxy-terminal alaninolattached to a tetrapeptide, which is attached at the N-terminalgroup to a long chain fatty acid. The alaninol residue and theamino acids are glycosidically linked to various oligosaccha-rides. Although GPL are not expressed by virulent tuberclebacilli (M. tuberculosis, M. bovis or M. leprae), they areexpressed by M. avium, another virulent species. Studies ofinteractions between GPL and biological membranes indicatethat the degree of glycosylation dictates the ability of theseglycolipids to perturb membrane function.59,60

Intracellular trafficking of mycobacterial lipids

Lipids of internalized mycobacteria are not restricted tophagosomes; rather, these components are released into theendocytic network. Macrophage infections with M. avium,3

M. tuberculosis3 and M. bovis BCG61 are accompanied by theintracellular release of mycobacterial cell wall constituents,including lipids. As demonstrated by immunoelectronmicroscopy, multivesicular and multilamellar vesicles stainedwith antibodies against the cell wall glycolipids LAM andPIM (Fig. 2; Beatty et al., unpubl. data, 2000).3 These

vesicles were clearly distinct from phagosomes containingmycobacteria.

Released mycobacterial lipids access the endocytic–lysosomal network of infected macrophages. This has beenshown by fluorescently labelling the surface lipids of M. bovis BCG with a fluorophore-hydrazide tag.61 Fluores-cently labelled bacilli were shown to release cell wall lipidsinto the endocytic network and tubular lysosomes. Mycobac-terial lipids were also released from infected macrophagesinto the culture supernatant in the form of small, multi-lamellar vesicles, called exosomes. This event could be trig-gered by Ca2+ ionophore treatment, which suggests that theexosomes follow the Ca2+-dependent exocytic pathway oflysosomes. Exosomes contained MHC class II molecules andmature cathepsin D, suggesting a late endosomal/lysosomalorigin and a possible role in antigen presentation (Beattyet al., unpubl. data, 2000). Labelled cell wall material wasalso taken up by non-infected neighbouring macrophages,possibly in the form of exosomes, illustrating a mechanismfor the cell-to-cell transfer of mycobacterial lipids. In thisway, mycobacterial lipids could affect uninfected cells distantfrom the infection foci.61

The released lipids were heterogeneous and all lipids trafficked to the same endosomal compartments and to exosomes (Fig. 3). Retention factors (R

f) and acid charring

Phagosomal niche of Mycobacterium spp. 305

Figure 2 Mycobacteria shed cellwall lipoglycans into infectedmacrophages. Murine bonemarrow-derived macrophageswere infected with Mycobac-terium tuberculosis (CDC1551)and were subsequently fixed andprocessed for cryoimmunoelec-tron microscopy at 7 days post-infection. Sections were incubatedwith mouse antiphosphomanno-side (PIM) and rabbit anti-lipoara-binomannan (LAM) and probedwith gold-conjugated antibodies.Antibodies to PIM (12 nm gold)and LAM (18 nm gold) bind elec-tron-dense bacilli (B) and phago-somal contents as well as distinctendocytic vesicles and vesiclesclose to the Golgi (G).

properties on TLC plates indicated that the released materialincluded polar phospholipids, glycolipids and neutral waxes,the latter of which were only labelled by radioactive lipid pre-cursors. Similar classes of released lipids have been retrievedfrom M. tuberculosis-infected macrophages (ER Rhoades,unpubl. obs.). Immunoelectron microscopy observations byXu and colleagues3 have identified PIM in the released material. Precise identification of the rest of the releasedlipids is currently in progress.

Biological effects of lipids on host cell function

The ready release of surface lipids,61 along with their relativeresistance to degradation,62 makes mycobacterial lipidsattractive candidates for mediators that could affect eventswithin the phagosome. Evidence that mycobacterial lipidsinterfere with phagosome fusion has been demonstratedusing a sulfolipid, SL-I.35 Intracellular processing of yeastparticles is compromised by coating the yeast with SL-I fromM. tuberculosis. Whereas all uncoated yeast end up in ferritin-labelled lysosomal compartments of peritonealmacrophages, 33% of internalized SL-I-coated yeast particlesdo not colocalize with ferritin-labelled lysosomes in similarlyinfected macrophages. The other two-thirds of the coatedyeast particles exhibit minimal fusion with the ferritin-labelled compartments. The mechanism of the limited inter-action with lysosomes is unknown, but Goren et al. havespeculated that the glycolipid blocks phagosome maturationby directly interfering with the membrane fusion process.

Phagosome maturation is dictated by membrane-associated proteins and by the physical properties of the lipidbilayer. The hydrophobic nature of mycobacterial lipidsconfers solubility in biological membranes. These con-stituents can intercalate directly into membranes fromaqueous dispersions,63 thereby altering the lipid compositionand physical properties of the membrane. In addition, lipidsbearing oligosaccharides or peptides can interact with mem-brane-associated receptors, thereby mediating phagocytosis64

and cytokine secretion42 and suppressing macrophage activa-tion.65 In this segment, we will consider the evidence for theability of mycobacterial lipids to modify biological mem-branes and intracellular signalling cascades and discuss theimplications on phagosome biogenesis.

Physical interference with membrane function

Trehalose dimycolate and GPL insert into model membranes,such as liposomes and phospholipid monolayers,63,66 and alsointo the membranes of purified mitochondria.63,67 Physicalinteraction studies have suggested that these glycolipidsinsert into the lipid bilayers, with acyl moieties inserted amidthe fatty acyl chains of the phospholipids and the polar carbo-hydrates in the water phase or the air/water interface.59,60,68

Retzinger et al.68 have analysed the physical properties ofTDM in membranes and have proposed that the disaccharideresides in the water phase while the long mycolic acids arekinked and folded in the lipid bilayer, effectively inserting sixtightly packed acyl chains. Studies on GPL insertion haveindicated that the less extensive acyl moiety does not fold oroccupy as much space in the lipid bilayer.59,60 Less glyco-sylated tetrapeptide cores favour insertion into the air/waterinterface, whereas heavily glycosylated peptide cores favourextension into the water phase above the phospholipid heads.These models have been depicted nicely by Laneelle andDaffe.69

Differences in insertion may affect different membraneproperties. It has been proposed that TDM decreases mem-brane fluidity by ordering the surrounding phospholipids andcross-linking the leaflets,66,69 whereas insertion of the GPLpeptide core amid phospholipid head groups effectivelycreates spaces in the fatty acyl layer, thereby increasing

ER Rhoades and HJ Ullrich306

Figure 3 Mycobacterial lipids are released into the endocyticnetwork and from infected macrophages in the form of exocyticvesicles. Mycobacterium bovis Bacillus Calmette–Guérin (BCG)was metabolically labelled using 14C-acetate prior to infection ofmurine bone marrow-derived macrophages. At 48 h postinfection,mycobacteria-containing phagosomes were removed from endo-cytic vesicles on a sucrose gradient. The endocytic fraction wasfurther separated by density gradient electrophoresis into earlyendosomes and plasma membrane (I), middle endosomes andGolgi (II) and late endosomes and lysosomes (III). Exosomeswere isolated from the cell supernatant by differential centrifuga-tion. Lipids extracted from radiolabeled M. bovis BCG and radio-labelled uninfected macrophages served as positive and negativecontrols, respectively. Lipid extracts of all fractions were devel-oped on high performance thin-layer chromatography plates inchloroform/methanol/H

2O (65:25:4), which resolves polar phos-

pholipids (near origin, B1 and B2) and glycolipids (intermediateR

f, B3 and B4) from waxes (at front, B5). Reproduced with

permission from Beatty et al.,61 ©2000 Munksgaard InternationalPublishers Ltd. Copenhagen, Denmark.

membrane passive permeability to ions and water-solublemolecules. In all, insertion of mycobacterial lipids into bio-logical membranes could lead to: (i) disruption of bilayerlipid ordering; (ii) cross-linking of bilayer lipid leaflets; (iii)limited movement and formation of phospholipid domains;(iv) altered membrane permeability; (v) altered membranefluidity; and (vi) disrupted transmembrane protein movementand function.

Possible effects of TDM and GPL insertion have beenstudied in model membranes and purified mitochondria. Inliposomes, both glycolipids decrease the fluidity of the mem-branes,63 but TDM is more effective in doing so. Trehalosedimycolate and GPL also increase membrane passive perme-ability, as indicated by the leakage of dye, but GPL is morecapable of conferring this effect. Both glycolipids also disruptrespiration and oxidative phosphorylation functions in puri-fied mitochondria.63,67 The effects of GPL mimic the iono-phore dinitrophenol.59 It has been proposed that protons leakthrough the inner mitochondrial membrane as a consequenceof glycolipid integration. This would abolish the proton-motive force that is required for oxidative phosphorylation.

Although the in vitro models present a clear associationbetween glycolipid integration and altered membrane func-tions, care should be taken when considering the physiologi-cal significance of the mitochondrial data. It has beensuggested that similar effects occur in vivo. Evidence in themouse model shows changes in liver mitochondrial ultra-structure70 and loss of liver enzyme function71 followinginjection with TDM. Yet no direct evidence for in vivoincorporation of the glycolipids along with alterations inmembrane function have been reported.

Manipulation of signalling pathways

In addition to modifying the physical properties of host mem-branes, mycobacterial lipids could mediate their effects bymodulation of intracellular signalling cascades. Myco-bacterial infection suppresses subsequent IFN-γ-mediatedmacrophage activation. Tyrosine phosphorylation of Janus-associated kinases (JAK) and signal transducers and activators of transciption (STAT) kinases is deficient in M. avium-infected murine macrophages, which are refractoryto IFN-γ-mediated activation.72 STAT 1 exhibits impairedDNA binding, which correlates with diminished expressionof IFN-γ response genes. Heat-killed bacilli are equallypotent, consistent with the possible involvement of a cell wallcomponent. Mycobacterium tuberculosis infection of humanmonocyte-derived macrophages diminishes IFN-γ-mediatedupregulation of surface expression of FcγR1.46 Suppression isassociated with decreased association of STAT 1 with tran-scriptional coactivators, CREB-binding protein and p300.This effect can be duplicated using gamma-irradiated bacillior a cell wall fraction, suggesting that surface components of mycobacteria are responsible. Mycobacterium leprae-infected macrophages are also less responsive to subsequentIFN-γ-mediated activation65 and pretreatment of uninfectedmacrophages with manLAM reproduces refractoriness toIFN-γ.65

Protein kinase C (PKC) and mitogen-activated protein(MAP) kinase activities are diminished by LAM, thereby

suppressing kinase-mediated signalling events.73,74 Myco-bacterium tuberculosis-derived manLAM inhibits PKC activity, which coincides with a block of the respiratory burst and transcriptional activation of IFN-γ-inducible genesin human macrophages.73 In another report,74 LAM pretreat-ment of THP-1 macrophages has been shown to promotetyrosine dephosphorylation of multiple protein, and weaken phosphorylation-dependent activation of MAP kinase. Thiscoincided with LAM-mediated induction of SHP-1 phos-phatase activity, suggesting that phosphatase activity couldbe responsible. Lipomannan and PIM are less potent for theactivation of SHP-1 phosphatase activity, suggesting that the branched, mannose-capped structures of manLAM arerequired for inhibition events.

The molecular mechanisms of LAM-mediated inhibitionof IFN-γ activation are unknown, but it is likely related to theability of LAM to interact with the cell surface. Mannosecapping is involved in the immunosuppressive effects42,74 andmanLAM also mediates interactions with the cell surface.Terminal carbohydrates of LAM interact directly or indirectlyto surface protein receptors, including the macrophagemannose receptor,53,64 CD1475 and Toll-like receptor-2.76,77

The acyl groups of LAM are also required to inhibit PKCactivity in LAM-treated macrophages, illustrating the impor-tance of lipid-mediated LAM interactions. The GPI anchorand any additional acyl functions of LAM can integratedirectly into the plasmalemma, inserting non-specifically78 orinto domains rich in GPI-anchored proteins.79 In the latterstudy, it has also been shown that LAM integration decreasesthe lateral mobility of GPI-anchored Thy-1 on the surface ofa lymphoma cell line.

Concluding remarks

Our understanding of the intracellular life of mycobacteriahas grown immensely in the past 30 years. We have verydetailed pictures of the compartment in which Myco-bacterium resides and the interaction with the host’s endo-cytic network, yet the mediators and mechanisms underlyingthe establishment of this compartment are unknown.Mycobacterial cell wall lipids could be important mediatorsof this process, because they affect parameters that areinvolved in endocytic trafficking, namely: (i) membrane flu-idity and permeability; (ii) mobility of membrane-associatedproteins; and (iii) phosphorylation-mediated signalling cas-cades. There is no direct evidence that lipid-mediated effectsalter mycobacteria-containing phagosome maturation, but wecan speculate that the reported effects of mycobacterial lipidsextend to phagosomal membranes and endocytic traffickingevents. Perhaps the formation of highly curved fusion intermediates could be hindered by glycolipid-mediatedrigidification of phagosomal membranes. This hypothesis issupported by past analysis of Ca2+-mediated phospholipidvesicle fusion, which has shown that TDM inhibits fusion.80

Perhaps the assembly of fusion protein complexes that areinvolved in vesicle docking could be hindered if lateralmobility of these proteins was decreased. Alternatively,perhaps mycobacterial glycolipids, such as LAM, couldretard phosphorylation-dependent endocytic traffickingevents, because it is known that vesicular transport is affected

Phagosomal niche of Mycobacterium spp. 307

by phosphorylation and that some endosomal proteins aresorted by their phosphorylated targeting motifs.

Acknowledgements

We thank DG Russell and D Swenson for helpful suggestionsand WL Beatty for providing unpublished data.

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