Whereas amastigotes of the protozoan parasite Leishmania proliferate inside acidic phagolysosomal vacuoles of the macrophage, vacuoles induced by Leishmania donovani promastigotes during initiation of infection are poorly characterized. Here, evidence is presented that interaction of these parasitophorous vacuoles with endocytic organelles is very limited. In contrast, vacuoles formed around L. donovani mutants lacking the cell surface lipophosphoglycan (LPG) fuse extensively with endosomes and lysosomes. The role of LPG repeating units in the inhibition of phagosome–endosome fusion was demonstrated using two different approaches. First, genetic complementation of the LPG-defective C3PO mutant restored its ability to inhibit phagosome–endosome fusion to a degree similar to that of wild-type promastigotes. Second, opsonization of C3PO mutant cells with purified L. donovani LPG also conferred to this mutant the ability to inhibit phagosome–endosome fusion. Inasmuch as LPG is essential for infecting macrophages, these results suggest that inhibition of phagolysosomal biogenesis by LPG repeating units represents an intramacrophage survival strategy used by promastigotes to establish infection.

During their life cycle, protozoan parasites of the genus Leishmania alternate between the midgut of their insect vector, where they exist as promastigotes, and a macrophage phagolysosomal compartment, where they proliferate as amastigotes. Over the past several years, evidence was provided that Leishmania parasites succeed in avoiding host immune defenses and destruction by expressing specialized stage-specific molecules (1, 2). The dominant cell surface molecule of promastigotes is lipophosphoglycan (LPG)1, a glycosylinositolphospholipid (GPI)-anchored polymer consisting, in L. donovani, of the repeating disaccharide–phosphate unit [Gal(β1,4)Man(α1-PO4→ 6)] (between 16 to 30 units) (1). This molecule, particularly the repeating units moiety, is essential for the interaction of promastigotes with both the insect vector and the mammalian host (1, 311). The requirement for LPG in the establishment of infection inside the macrophage was evidenced by the demonstration that LPG repeating units–defective mutants are rapidly destroyed after phagocytosis, and that passive transfer of purified LPG significantly prolonged their survival (7, 8). This role for LPG repeating units for intramacrophage survival has also been genetically proven (11). Thus, without LPG repeating units promastigotes are unable to withstand the conditions prevailing inside the maturing parasitophorous vacuole. This is in contrast with the amastigotes, which proliferate inside acidic, hydrolase-rich vacuoles (1217), despite the fact that they synthesize little or no detectable LPG (1820). Thus, the role of this molecule in intramacrophage survival may be restricted to the establishment of infection, during promastigote-to-amastigote conversion (79, 11).

Different properties of LPG repeating units are consistent with their protective role during the establishment of infection, including efficient scavenging of toxic oxygen metabolites generated during the oxidative burst (8, 21), and modulation of the inducible nitric oxide synthase expression (22). In addition, the several million copies of LPG at the promastigote surface form a dense glycocalyx that may provide a physical barrier against the action of hydrolytic enzymes (10, 13). Recent in vitro studies demonstrated that incorporation of full-length LPG in lipid bilayers resulted in reduced fusogenic capacities (23). This finding raised the possibility that LPG repeating units may protect freshly phagocytized promastigotes by limiting the delivery of endosomal contents to the parasitophorous vacuoles, a process requiring multiple fusion events (24, 25). Here, we compared the ability of parasitophorous vacuoles induced by either wild-type L. donovani promastigotes or LPG repeating units–defective mutants to interact with endocytic organelles. We show that LPG repeating units enable Leishmania to inhibit the phagosome-endosome fusion process efficiently, thereby suggesting a survival strategy during their differentiation into amastigotes.

Materials And Methods

Cell Culture.

The murine macrophage cell line J774 was passaged in DMEM supplemented with 10% FCS, 1% glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a 5% CO2 atmosphere. J774 cells were plated in 3-cm dishes for electron microscopy studies. All experiments were done with subconfluent cultures.

Parasites and LPG.

Leishmania donovani wild-type strain 1S (WT), the LPG-deficient mutants C3PO and RT5 (26), C3PO overexpressing LPG2 (C3PO+LPG2) (27), and the LPG repeating unit–defective line created by targeted disruption of LPG2 (termed lpg2 knockout) (27) were all grown at 26°C in modified M199 medium as described (8). Opsonization of C3PO with purified L. donovani LPG (provided by S.J. Turco, University of Kentucky) was performed as described (8). All lines were used in stationary phase of growth.

Macrophage Infections, Electron Microscopy, and Morphological Studies.

Interactions and fusion between endocytic organelles and either L. donovani–containing phagosomes or latex bead–containing phagosomes were studied using a morphological approach at the electron microscope level. This in vivo fusion assay, based on the transfer of electron-dense tracers from endosomes to phagosomes, was used because of its simplicity over in vitro fusion assays and its sensitivity. Endocytic organelles were loaded by internalization of 5- and 16-nm gold particles coated with BSA (BSA– gold) using standard procedures (28). Phagosomes were formed by the addition of stationary-phase promastigotes at a parasiteto-host cell ratio of 10:1 in culture medium at 37°C for 60 min. Excess parasites were removed by four washes with cold PBS. Infected cells were then further incubated in culture medium for 60 min to allow complete internalization of bound promastigotes. Two types of loading procedures were used. In some cases, BSA– gold was internalized first, followed by incubation in culture medium to allow the marker to fill various endosome populations. Cells containing BSA–gold were then infected with L. donovani. This approach allowed us to study the interaction of early endosomes and lysosomes with newly formed phagosomes. The second procedure consisted in infecting macrophages with L. donovani first for 60 min, followed by incubation in culture medium for either 60 min or 4 h to form early or late phagosomes, respectively. These macrophages were then fed BSA–gold particles for 30 min and were either processed immediately or further incubated in culture medium for 60 min. This approach allowed us to study the interaction of preexisting phagosomes of various ages with maturing endosomes. For some experiments, promastigotes and 0.8-μm latex beads (Sigma, St. Louis, MO; diluted 1:200 in culture medium) were cointernalized, to study the interaction of endosomes with different phagosome populations. After all internalization steps, cells were washed thoroughly with cold PBS three times for 5 min. At the end of each experiment, cells were fixed in 1% glutaraldehyde, postfixed in OsO4, processed for embedding in Epon 812 resin as described previously (24), and observed by electron microscopy.

Intermixing of BSA–gold particles and L. donovani was then recorded for each combination of incubations performed. The presence of a single gold particle inside a phagosome was scored as a fusion event. For the analysis of fusion occurrence (Fig. 2), each experiment was done at least twice and a minimum of 25 Leishmania-containing phagosomes per timepoint were recorded, while extreme care was taken to avoid serial sections. For the quantitative analysis of the transfer of BSA–gold particles from endosomes to Leishmania-containing phagosomes (Table 1), the number of 5- and 16-nm gold particles was counted on at least 12–20 of the BSA–gold positive phagosome profiles at the electron microscope.

Results And Discussion

The transfer of BSA–gold particles from endosomes to phagosomes containing either wild-type promastigotes or the LPG repeating unit–defective C3PO mutant cells (26) was first used to evaluate the interaction occurring between these organelles. Very few gold particles were generally transferred to WT–containing phagosomes despite the close presence of BSA–gold particle–filled endosomes (Fig. 1,A). In contrast, most phagosomes containing the LPG repeating units–defective C3PO mutant were filled with numerous BSA–gold particles (Fig. 1,B). While only ∼40% of WTphagosomes received BSA–gold particles, >90% of phagosomes induced by C3PO mutant cells fused with endocytic organelles (Fig. 2,A). The difference in levels of fusion was more striking when the number of gold particles transferred from endosomes to phagosomes was quantified. Indeed, a sixfold increase in the BSA–gold particle content of C3PO phagosomes over WT-phagosomes was observed (Table 1). High levels of fusion were also observed with phagosomes induced by lpg2 null mutant cells, which are phenotypically identical to C3PO cells (27), and by RT5, a mutant that accumulates truncated forms of LPG containing 3–5 repeating units (26) (Fig. 2 A). Because assembly of the Gal(β1,4)Man(α1-PO4→ 6) repeating units is defective in these mutants, our observations indicated a role for this LPG moiety in the inhibition of phagosome–endosome fusion. Further, immunofluorescence analysis showed that LPGexpressing promastigotes are contained within a LAMP1negative compartment, whereas LPG-deficient mutants were found in LAMP1-positive phagosomes (data not shown), indicating their respective levels of fusion with endocytic organelles enriched for LAMP1 molecules.

To demonstrate directly a role for LPG in the inhibition of phagosome–endosome fusion, two distinct approaches were taken. First, we assessed the fusogenic properties of phagosomes induced by C3PO cells transfected with a LPG2 expression construct (C3PO+LPG2) (27). As expected, phagosomes containing C3PO+LPG2 cells behaved similarly to WT-phagosomes with respect to their ability to fuse with endocytic organelles (Fig. 2,A; and Table 1). Thus, restoration of full-length LPG repeating units synthesis by genetic complementation enabled C3PO to inhibit phagosome–endosome fusion. Because LPG2 expression in C3PO also restores repeating units addition on secreted molecules, including the secreted acid phosphatase (27), the possibility existed that such repeating unit addition could have accounted for the ability of C3PO+LPG2 cells to inhibit phagosome–endosome fusion. Therefore, in a second approach, C3PO cells were opsonized with purified L. donovani LPG (8) before infection of J774 macrophages. Effectiveness of the opsonization procedure was confirmed by immunofluorescence (data not shown) with the antirepeating units mAb CA7AE (29). Electron microscopy analysis revealed that phagosomes containing LPG-opsonized C3PO cells displayed low fusion properties similar to that of WT-phagosomes (Fig. 2,A; Fig. 3). This data provided a direct evidence that the sole presence of LPG repeating units at the promastigote surface is sufficient to inhibit phagosome–endosome fusion. Therefore, repeating unit modification of the secreted acid phosphatase, which is not required for both secretion and activity (27, 30), appears to be without apparent effect on phagosomal fusogenic properties.

We also assessed the ability of newly formed phagosomes to fuse with two distinct endosome populations. Here, BSA– gold particles were first internalized for 30 min, followed by either a 15-min or 4-h chase. The first population is considered as early endosomes and the second as lysosomes. At the end of the chase period, macrophages were infected with either WT promastigotes or C3PO mutant cells for 1 h. Increased fusion between early endosomes and C3PO/phagosomes with respect to WT-phagosomes was observed (see Fig. 2 B). Interestingly, WT-phagosomes consistently displayed lower fusogenicity with lysosomes (35% fusion) than with early endosomes (55% fusion), suggesting that, similar to Mycobacterium phagosomes (3134), fusion with the various endosomal populations might be selective, albeit to a lesser extent.

To evaluate the specificity of the LPG repeating units– induced alteration(s) of phagosome fusion properties, J774 macrophages were coinfected with latex beads and the various Leishmania lines. Latex beads and promastigotes were present in distinct phagosomal compartments (Fig. 4). Regardless of the cointernalized Leishmania line, the fusion rate between endocytic organelles and latex bead phagosomes was around 90% (see Fig. 2,C). Thus, in a given macrophage, fusion can occur between endosomes and latex bead phagosomes but not with WT-phagosomes. This observation indicated that (a) inhibition of fusion by LPG repeating unit is selective, (b) the general host cell fusion machinery remains operational during infection, and (c) LPG repeating units selectively alter local phagosomal fusogenic properties. From a mechanistic point of view, the following models can be considered. First, WT promastigotes and the repeating unit–defective mutants use distinct receptors for attachment and entry, resulting in the formation of phagosomes with different biochemical composition and fusion properties (3537). However, the observation that promastigote-to-amastigote transformation, which is paralleled by the loss of LPG, restores phagosome–endosome fusion argues against this idea (1220). In the second model, LPG may inhibit activation of phagosome-associated protein kinase C (PKC) (3840). Although the precise function of PKC in phagosome–endosome interaction remains speculative, it is known to phosphorylate MARCKS, a membrane protein associated with actin-based motility (41) and with membrane trafficking (42). In this regard, PKC-dependent phosphorylation of phagosome-associated MARCKS results in its displacement from the membrane to Lamp1positive lysosomes (42) and may therefore participate in the movement of both phagosomes and endosomes on microtubules. Inhibition of PKC-dependent MARCKS phosphorylation by LPG (4344) may block this movement. However, this model is not consistent with the inhibition of PKC-dependent processes by C3PO and amastigotes during infection (43, 45, 46), both of which induce phagosomes that fuse freely with endocytic organelles (see Figs. 1 and 2 A; references 1217). In a third model, LPG is transferred within minutes from the promastigote surface to the macrophage membrane at the immediate area of internalization (29). Insertion of LPG in lipid-bilayer membranes stabilizes the bilayer against the formation of an inverted hexagonal structure, resulting in reduced fusogenic properties (23). As a consequence, LPG would give rise to an effective steric repulsion between phagosomal and endosomal membranes or reduce the negative curvature strain in bilayers, increasing the energy barrier for forming highly curved fusion intermediates (23), thereby preventing fusion. Interestingly, truncated forms of LPG containing few repeating units are ineffective in modifying the fusogenic properties of membranes (23). This in vitro finding is in agreement with our observation that RT5, a mutant expressing truncated forms of LPG with three to five repeating units (26), is unable to inhibit phagosome–endosome fusion. Alternatively, the possibility exists that LPG inhibits phagosome–endosome fusion indirectly. Indeed, LPG may prevent parasite destruction by scavenging toxic oxygen radicals generated during the oxidative burst (8, 21), allowing for the production of a yet unidentified factor directly responsible for preventing fusion. Regardless of the nature of the exact underlying mechanism, our data have unequivocally demonstrated that expression of full-length LPG enables promastigotes to inhibit phagosome–endosome fusion. The extent to which inhibition of fusion by LPG contributes to promastigote survival remains to be determined.

In a study aimed at determining the position of L. mexicana parasitophorous vacuoles within the endocytic network of the host macrophage, Russell et al. (16) observed that the transfer of endosomal content to parasitophorous vacuoles induced by promastigotes increased in efficiency with respect to the age of the infection. To explain this phenomenon, the authors suggested that these vacuoles had functionally translocated from a lysosomal to a late endosomal compartment, consistent with their increased content in mannose-6-phosphate receptors. In the light of the findings reported herein, we suggest that the reduced fusogenicity of the parasitophorous vacuoles observed in early infections (day 2) by Russell et al. (16) is caused by the presence of promastigote-derived LPG. Indeed, LPG repeating units epitopes are maximally present in the macrophage membrane 1–2 d after infection, and by 5–6 d, these epitopes are no longer detectable (29). Thus, the transformation of promastigotes into amastigotes, associated with the loss of LPG expression, occurs concomitently with the release of phagosome–endosome fusion inhibition.

Inhibition of phagosome–endosome fusion is an intramacrophage survival strategy used by a variety of intracellular pathogens (3537). Although the molecular bases of this phenomenon are poorly understood, microbial surface components analogous to the Leishmania LPG may alter the fusion properties of the endocytic system (47). In this regard, in vitro studies have shown that Cord factor (α,α-trehalose 6,6′-dimycolate), a cell wall glycolipid of Mycobacteria, inhibits fusion between phospholipid vesicles (48). Although exclusion of the vacuolar proton–ATPase from mycobacteria–phagosomes appears to be closely related with their reduced fusogenicity, it is not known, however, whether this exclusion is the consequence or the cause of the altered fusion properties (31).

The mechanisms and molecules involved in the regulation of phagosome fusion properties remain largely unknown. While proteins of the rab family were found to associate and dissociate from maturing phagosomes, their role in phagosome fusion is still unclear (24). Phagosome maturation is also accompanied by the acquisition of Nramp1, a molecule conferring resistance to a variety of intracellular microbes, including L. donovani (49), the sequential phosphorylation and dephosphorylation of some of their proteins, as well as modifications of their phospholipid content (50, 51). Although no direct correlation between phagosome fusogenic properties and their lipid composition has been demonstrated, the finding that LPG modifies the molecular structure of lipid bilayers (23) suggests a role for lipids in the fine tuning of membrane fusion events. In this regard, we expect that molecular and biochemical characterization of Leishmania-containing phagosomes will yield novel insights on the mechanistic aspects of membrane fusion.

LPG repeating units are essential for the successful transition of Leishmania parasites from the sandfly midgut to the inside of a macrophage phagolysosome (311). In the macrophage, LPG repeating units may contribute to promastigote survival through their capacity to scavenge oxygen radicals (21), to inhibit PKC (40, 44), and to modulate nitric oxide synthase expression (22). Our data suggest a novel function for LPG repeating units during the early phase of macrophage infection, where inhibition of phagolysosomal biogenesis may protect invading promastigotes from hydrolytic degradation and provide an environment propitious for their differentiation into amastigotes. Finally, the anti-fusogenic properties of LPG repeating units provide a powerful probe to investigate the regulation of phagosome–endosome fusion during microbial invasion.

Acknowledgments

We acknowledge the technical assistance of C. Rondeau and J. Léveillé. We are grateful to S.J. Turco (University of Kentucky) and S.M. Beverley (Harvard Medical School) for authorizing the use of C3PO, C3PO+LPG2, and lpg2. We thank S.J. Turco for kindly providing RT-5 cells and LPG purified from Leishmania donovani and for suggestions, and R. Nabi, G. Matlashewski, and R. Epand for comments and critical reading of the manuscript.

This work was supported by grants from the Medical Research Council (MRC) of Canada to M. Desjardins (MT-12951) and A. Descoteaux (MT-12933). M. Desjardins is a Scholar from the Fonds de la recherche en santé du Québec and A. Descoteaux is a Scholar from the MRC.

References

1
Turco
SJ
,
Descoteaux
A
The lipophosphoglycan of Leishmaniaparasites
Annu Rev Microbiol
1992
46
65
94
[PubMed]
2
McConville
MJ
,
Ferguson
MAJ
The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes
Biochem J
1993
294
305
324
[PubMed]
3
Pimenta
PFP
,
Turco
SJ
,
McConville
MJ
,
Lawyer
P
,
Perkins
P
,
Sacks
DL
Stage-specific adhesion of Leishmaniapromastigotes to the sand fly midgut
Science (Wash DC)
1992
256
1812
1815
[PubMed]
4
Pimenta
PFP
,
Saraiva
EMB
,
Rowton
E
,
Modi
GB
,
Garraway
LA
,
Beverley
SM
,
Turco
SJ
,
Sacks
DL
Evidence that the vectorial competence of phlebotomine sand flies for different species of Leishmaniais controlled by structural polymorphisms in the surface lipophosphoglycan
Proc Natl Acad Sci USA
1994
91
9155
9159
[PubMed]
5
Butcher
BA
,
Turco
SJ
,
Hilty
BA
,
Pimenta
PF
,
Panunzio
M
,
Sacks
DL
Deficiency in β1,3-galactosyltransferase of a Leishmania major lipophosphoglycan mutant adversely influences the Leishmania–sand fly interaction
J Biol Chem
1996
271
20573
20579
[PubMed]
6
Culley
FJ
,
Harris
RA
,
Kaye
PM
,
McAdam
KPWJ
,
Raynes
JG
C-reactive protein binds to a novel ligand on Leishmania donovaniand increases uptake into human macrophages
J Immunol
1996
156
4691
1696
[PubMed]
7
Handman
E
,
Schnur
LF
,
Spithill
TW
,
Mitchell
GF
Passive transfer of Leishmanialipopolysaccharide confers parasite survival in macrophages
J Immunol
1986
137
3608
3613
[PubMed]
8
McNeely
TB
,
Turco
SJ
Requirement of lipophosphoglycan for intracellular survival of Leishmania donovaniwithin human monocytes
J Immunol
1990
144
2475
2750
9
Elhay
M
,
Kelleher
M
,
Bacic
A
,
McConville
MJ
,
Tolson
DL
,
Pearson
TW
,
Handman
E
Lipophosphoglycan expression and virulence in ricin-resistant variants of Leishmania major.
Mol Biochem Parasitol
1990
40
255
268
[PubMed]
10
Eilam
Y
,
El-On
J
,
Spira
DT
Leishmania major: excreted factor, calcium ions, and the survival of amastigotes
Exp Parasitol
1985
59
161
168
[PubMed]
11
Beverley, S.M., and S.J. Turco. 1995. Identification of genes mediating lipophosphoglycan biosynthesis by functional complementation of Leishmania donovani mutants. Ann. Trop. Med. Parasitol. 89 (Suppl.):11–17.
12
Alexander
J
,
Vickerman
K
Fusion of host cell secondary lysosomes with the parasitophorous vacuoles of Leishmania mexicana–infected macrophages
J Protozool
1975
22
502
508
[PubMed]
13
Chang
K-P
,
Dwyer
DM
Multiplication of a human parasite (Leishmania donovani)in phagolysosomes of hamster macrophages in vitro
Science (Wash DC)
1976
193
678
680
[PubMed]
14
Antoine
J-C
,
Prina
E
,
Jouanne
C
,
Bongrand
P
Parasitophorous vacuoles of Leishmania amazonensis–infected macrophages maintain an acidic pH
Infect Immun
1990
58
779
787
[PubMed]
15
Prina
E
,
Antoine
J-C
,
Wiederanders
B
,
Kirschke
H
Localization and activity of various lysosomal proteases in Leishmania amazonensis–infected macrophages
Infect Immun
1990
58
1730
1737
[PubMed]
16
Russell
DG
,
Xu
S
,
Chakraborty
P
Intracellular trafficking and the parasitophorous vacuole of Leishmania mexicana–infected macrophages
J Cell Science
1992
103
1193
1210
[PubMed]
17
Veras
PST
,
de Chastellier
C
,
Rabinovitch
M
Transfer of zymosan (yeast cell walls) to the parasitophorous vacuoles of macrophages infected with Leishmania amazonensis.
J Exp Med
1992
176
639
646
[PubMed]
18
McConville
MJ
,
Blackwell
JM
Developmental changes in the glycosylated phosphatidylinositols of Leishmania donovani: characterization of the promastigote and amastigote glycolipids
J Biol Chem
1991
266
15170
15179
[PubMed]
19
Turco
SJ
,
Sacks
DL
Expression of a stage-specific lipophosphoglycan in Leishmania majoramastigotes
Mol Biochem Parasitol
1991
45
91
100
[PubMed]
20
Moody
SF
,
Handman
E
,
McConville
MJ
,
Bacic
A
The structure of Leishmania majoramastigote lipophosphoglycan
J Biol Chem
1993
268
18457
18466
[PubMed]
21
Chan
J
,
Fujira
T
,
Brennan
P
,
McNeil
M
,
Turco
SJ
,
Sibille
J
,
Snapper
M
,
Aisen
P
,
Bloom
BR
Microbial glycolipids: possible virulence factors that scavenge oxygen radicals
Proc Natl Acad Sci USA
1989
86
2453
2457
[PubMed]
22
Proudfoot
L
,
Nikolaev
AV
,
Feng
G-J
,
Wei
X-Q
,
Ferguson
MAJ
,
Brimacombe
JS
,
Liew
FY
Regulation of the expression of nitric oxide synthase and leishmanicidal activity by glycoconjugates of Leishmanialipophosphoglycan in murine macrophages
Proc Natl Acad Sci USA
1996
93
10984
10989
[PubMed]
23
Miao
L
,
Stafford
A
,
Nir
S
,
Turco
SJ
,
Flanagan
TD
,
Epand
RM
Potent inhibition of viral fusion by the lipophosphoglycan of Leishmania donovani.
Biochemistry
1995
34
4676
4683
[PubMed]
24
Desjardins
M
,
Huber
LA
,
Parton
RG
,
Griffiths
G
Biogenesis of phagolysosomes proceeds through a sequential series of interactions with endocytic apparatus
J Cell Biol
1994
124
677
688
[PubMed]
25
Desjardins
M
Biogenesis of phagolysosomes: the ‘kiss and run' hypothesis
Trends Cell Biol
1995
5
183
186
[PubMed]
26
McNeely
TB
,
Tolson
DL
,
Pearson
TW
,
Turco
SJ
Characterization of Leishmania donovanivariant clones using anti-lipophosphoglycan monoclonal antibodies
Glycobiology
1990
1
63
69
[PubMed]
27
Descoteaux
A
,
Luo
Y
,
Turco
SJ
,
Beverley
SM
A specialized pathway affecting virulence glycoconjugates of Leishmania.
Science (Wash DC)
1995
269
1869
1872
[PubMed]
28
Rabinowitz
S
,
Horstmann
H
,
Gordon
S
,
Griffiths
G
Immunocytochemical characterization of the endocytic and phagolysosomal compartments in peritoneal macrophages
J Cell Biol
1992
116
95
112
[PubMed]
29
Tolson
DL
,
Turco
SJ
,
Pearson
TW
Expression of a repeating phosphorylated disaccharide lipophosphoglycan epitope on the surface of macrophages infected with Leishmania donovani.
Infect Immun
1990
58
3500
3507
[PubMed]
30
Bates
PA
,
Hermes
I
,
Dwyer
DM
Golgi-mediated post-translational processing of secretory acid phosphatase by Leishmania donovanipromastigotes
Mol Biochem Parasitol
1990
39
247
256
[PubMed]
31
Sturgill-Koszycki
S
,
Schlesinger
PH
,
Chakraborty
P
,
Haddix
PL
,
Collins
HL
,
Fok
AK
,
Allen
RD
,
Gluck
SL
,
Heuser
J
,
Russell
DG
Lack of acidification in Mycobacteriumphagosomes produced by exclusion of the vesicular proton–ATPase
Science (Wash DC)
1994
263
678
681
[PubMed]
32
Russell
DG
,
Dante
J
,
Sturgill-Koszycki
S
Mycobacterium avium– and Mycobacterium tuberculosis–containing vacuoles are dynamic, fusion competent vesicles that are accessible to glycosphingolipids from the host cell plasmalemma
J Immunol
1996
156
4764
4773
[PubMed]
33
Clemens
DL
,
Horwitz
MA
The Mycobacterium tuberculosisphagosome interacts with early endosomes and is accessible to exogenously administered transferrin
J Exp Med
1996
184
1349
1355
[PubMed]
34
de Chastellier
C
,
Lang
T
,
Thilo
L
Phagocytic processing of the macrophage endoparasite, Mycobacterium avium, in comparison to phagosomes which contain Bacillus subtilisor latex beads
Eur J Cell Biol
1995
68
167
182
[PubMed]
35
Joiner
KA
,
Fuhrman
SA
,
Miettinen
HM
,
Kasper
LH
,
Mellman
I
Toxoplasma gondii: fusion competence of parasitophorous vacuoles in Fc receptor-transfected fibroblasts
Science (Wash DC)
1990
249
641
646
[PubMed]
36
Small
PLC
,
Ramakrishnan
L
,
Falkow
S
Remodeling schemes of intracellular pathogens
Science (Wash DC)
1994
263
637
639
[PubMed]
37
Finlay
BB
,
Falkow
S
Common themes in microbial pathogenicity
Microbiol Rev
1989
53
210
230
[PubMed]
38
Descoteaux
A
,
Turco
SJ
The lipophosphoglycan of Leishmaniaand macrophage protein kinase C
Parasitol Today
1993
9
468
471
[PubMed]
39
Allen
L-AH
,
Aderem
A
A role for MARCKS, the α isoenzyme of protein kinase C and myosin I in zymosan phagocytosis by macrophages
J Exp Med
1995
182
829
840
[PubMed]
40
Giorgione
JR
,
Turco
SJ
,
Epand
RM
Transbilayer inhibition of protein kinase C by the lipophosphoglycan from Leishmania donovani.
Proc Natl Acad Sci USA
1996
93
11634
11639
[PubMed]
41
Rosen
A
,
Keenan
KF
,
Thelen
M
,
Nairn
AC
,
Aderem
AA
Activation of protein kinase C results in the displacement of its myristoylated, alanine-rich substrate from punctate structures in macrophage filopodia
J Exp Med
1990
172
1211
1215
[PubMed]
42
Allen
L-AH
,
Aderem
A
Protein kinase C regulates MARCKS cycling between the plasma membrane and lysosomes in fibroblasts
EMBO (Eur Mol Biol Organ) J
1995
14
1109
1121
[PubMed]
43
Descoteaux
A
,
Matlashewski
G
,
Turco
SJ
Inhibition of macrophage protein kinase C–mediated protein phosphorylation by Leishmania donovanilipophosphoglycan
J Immunol
1992
149
3008
3015
[PubMed]
44
Descoteaux
A
,
Turco
SJ
,
Sacks
DL
,
Matlashewski
G
Leishmania donovanilipophosphoglycan selectively inhibits signal transduction in macrophages
J Immunol
1991
146
2747
2753
[PubMed]
45
Descoteaux
A
,
Matlashewski
G
C-fos and TNF gene expression in Leishmania donovani–infected macrophages
Mol Cell Biol
1989
9
5223
5227
[PubMed]
46
Olivier
M
,
Brownsey
RW
,
Reiner
NE
Defective stimulus–response coupling in human monocytes infected with Leishmania donovaniis associated with altered activation and translocation of protein kinase C
Proc Natl Acad Sci USA
1992
89
7481
7485
[PubMed]
47
Goren
MB
,
D'Arcy
P
,
Hart
,
Young
MR
,
Armstrong
JA
Prevention of phagosome–lysosome fusion in cultured macrophages by sulfatides of Mycobacterium tuberculosis.
Proc Natl Acad Sci USA
1976
73
2510
2514
[PubMed]
48
Spargo
BJ
,
Crowe
LM
,
Ioneda
T
,
Beaman
BL
,
Crowe
JH
Cord factor (α,α-trehalose 6,6′-dimycolate) inhibits fusion between phospholipid vesicles
Proc Natl Acad Sci USA
1991
88
737
740
[PubMed]
49
Gruenheid
S
,
Pinner
E
,
Desjardins
M
,
Gros
P
Natural resistance to infection with intracellular parasites: the Nramp1 protein is recruited to the membrane of the phagosome
J Exp Med
1997
185
717
730
[PubMed]
50
Desjardins
M
,
Celis
JE
,
van Meer
G
,
Dieplinger
H
,
Jahraus
A
,
Griffiths
G
,
Huber
LA
Molecular characterization of phagosomes
J Biol Chem
1994
269
32194
32200
[PubMed]
51
Emans
N
,
Nzala
NN
,
Desjardins
M
Protein phosphorylation during phagosome maturation
FEBS Lett
1996
398
37
42
[PubMed]

1Abbreviations used in this paper: LPG, lipophosphogylcan; GPI, glycosylinositolphospholipid; PKC, protein kinase C; WT, wild-type.

Author notes

Address correspondence to A. Descoteaux at the Institut Armand-Frappier, Centre de recherche en immunologie, 531 boulevard des Prairies, CP100, Laval, Québec, Canada H7N 4Z3, Phone: 514-686-5332; FAX: 514-685-5501; E-mail: albert_descoteaux@iaf.uquebec.ac, or to M. Desjardins at the Département d'anatomie, Université de Montréal, CP6128 Succ. Centre Ville, Montréal, Québec, Canada H3C3J7, Phone: 514-343-7350; FAX: 514-343-2459; E-mail: desjarm@ere.umontreal.ca