SEARCH

SEARCH BY CITATION

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Pathogenic mycobacteria prevent maturation of the phagosomes in which they reside inside macrophages and this is thought to be a major strategy allowing them to survive and multiply within macrophages. The molecular basis for this inhibition is only now beginning to emerge with the molecular characterization of the phagosome membrane enclosing these pathogens. We have used here several electron microscopy approaches in combination with counts of bacterial viability to analyse how expression of Nramp1 at the phagosomal membrane may influence survival of Mycobacterium avium and affect its ability to modulate the fusogenic properties of the phagosome in which it resides. The experiments were carried out in bone marrow-derived macrophages from wild-type 129sv (Nramp1G169) mice and from isogenic 129sv carrying a null mutation at Nramp1 (Nramp1–/–) following infection with a virulent strain of M. avium. We show here that Nramp1 expression has a bacteriostatic effect and that abrogation of Nramp1 restores the bacteria's capacity to replicate within macrophages. The combined analyses of the acquisition of endocytic contents markers delivered to early endosomes and/or lysosomes either prior to or after phagocytic uptake showed that in Nramp1-positive macrophages, M. avium was unable to prevent phagosome maturation and fusion with lysosomes but that in Nramp1-negative macrophages this capacity was restored. Several hypotheses are proposed to explain how Nramp1 could affect survival of M. avium. We also propose how the present observations could relate to the model according to which mycobacteria can prevent phagosome maturation by establishing a tight interaction with constituents of the phagosome membrane. Furthermore, these results show the importance of the choice of macrophages used as a model to study intracellular survival strategies of pathogens.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In inbred strains of mice, loss-of-function mutations at the Nramp1 gene (chromosome 1 locus previously known as Bcg/Lsh/Ity) cause susceptibility to infection with several species of Mycobacterium, Salmonella, and Leishmania (for reviews see Skamene et al., 1998; Forbes and Gros, 2001). Susceptibility to infection in vivo in mutant mice bearing naturally occurring or experimentally induced mutations at Nramp1 is characterized by rapid growth of these pathogens in spleen and liver, whereas in vitro it is associated with unrestricted intracellular replication of these unrelated pathogens in macrophages (for reviews see Skamene et al., 1998; Forbes and Gros, 2001). In humans, polymorphic variants at or near the Nramp1 locus are associated with differential susceptibility to tuberculosis and leprosy, both in endemic areas of disease and in first contact epidemics (Abel et al., 1998; Bellamy et al., 1998; Alcais et al., 2000; Greenwood et al., 2000).

The mechanism by which Nramp1 contributes to natural macrophage defences against intracellular infections is of considerable interest, and has been studied at the cellular and biochemical levels. Secondary structure predictions from primary sequence analysis (Cellier et al., 1995), together with direct biochemical characterization (Picard et al., 2000) indicate that Nramp1 is a 12 transmembrane domain integral membrane protein of 80–100 kDa extensively modified by glycosylation and phosphorylation (Vidal et al., 1996). Inbred strains of mice naturally susceptible to intracellular infections (Bcgs) display a glycine to aspartate substitution in the fourth predicted transmembrane domain of Nramp1 (Vidal et al., 1993; Malo et al., 1994). This mutation affects protein maturation and/or stability, such that no mature G169D protein can be found in macrophages from Bcgs strains (Vidal et al., 1996). Nramp1 mRNA and protein are expressed specifically in macrophages and neutrophils (for a review see Skamene et al., 1998). Subcellular localization studies by conventional immunofluorescence and confocal microscopy indicate that, in uninfected cells, Nramp1 is not expressed at the plasma membrane but is rather associated with compartments of the endocytic pathway and more particularly with lysosomes (Gruenheid et al., 1997; Searle et al., 1998). Upon phagocytosis of zymosan particles (Jabado et al., 2000) or hydrophobic latex beads (Gruenheid et al., 1997), Nramp1 is rapidly recruited to the membrane of phagosomes (Gruenheid et al., 1997). At this stage, hydrophobic latex-bead-containing phagosomes are still immature and they display membrane characteristics of early endosomes with which they intermingle contents and membrane (de Chastellier et al., 1995; Desjardins, 1995; de Chastellier and Thilo, 1997). Nramp1 then remains associated with hydrophobic latex bead-containing phagosomes throughout their processing into phagolysosomes (Gruenheid et al., 1997). Likewise, Nramp1 co-localizes with phagocytic vacuoles containing live bacteria such as Salmonella typhimurium or Yersinia enterocolitica (Govoni et al., 1999; Cuellar-Mata et al., 2002) and also Leishmania major-containing parasitophorous vacuoles (Searle et al., 1998). Nramp1-positive vesicles also migrate to converge towards, but not always fuse with, M. avium-containing phagosomes (Searle et al., 1998). It seems therefore that Nramp1 is associated with many types of phagocytic vacuoles whether they are immature, early endosome-like or whether they have acquired lysosomal characteristics.

The human and mouse genomes contain a second closely related protein Nramp2 (87% identity) which is ubiquitously expressed in many cell types (Gruenheid et al., 1995; Gunshin et al., 1997). Functional characterization in Xenopus leavis oocytes has established that Nramp2 (DCT1/DMT1) is a pH-dependent divalent metal transporter of broad substrate specificity (Gunshin et al., 1997; Picard et al., 2000). Studies in normal tissues (Gruenheid et al., 1999) and experiments with naturally occurring mouse (mk) and rat (Belgrade) Nramp2 mutants have identified Nramp2 as (1) the transferrin-independent iron uptake system of the brush border of the duodenum, and (2) the protein responsible for the transport of transferrin iron across the membrane of acidified endosomes and into the cytoplasm (for a review see Andrews, 2000). A parallel role of Nramp1 in transport of divalent cations at the phagosomal membrane was recently established (Jabado et al., 2000; Goswami et al., 2001). Using a metal-sensitive fluorescent probe (Fura-FF6) covalently attached to zymosan particles, microfluorescence imaging studies in intact cells showed that Nramp1 recruitment to the phagosomal membrane causes a marked reduction in accumulation of Mn2+ in the phagosomal space. Nramp1 was shown to function as a pH-dependent efflux pump at the phagosomal membrane, which can be abrogated by the vacuolar H+-ATPase inhibitor bafilomycin (Jabado et al., 2000). Others have suggested that Nramp1 can function as a H+/bivalent cation transporter at that site as well (Goswami et al., 2001). The mechanism by which Nramp1-dependent modulation of phagosomal divalent cations content affects bacteriostatic/bactericidal activities of the macrophage remains unclear. Several bacterial species including Mycobacterium and Salmonella possess structural Nramp homologues (MntH) that function as pH-dependent uptake systems for divalents cations (for a review see Cellier et al., 2001). It has been proposed that bacterial and mammalian Nramp homologues may compete for the same substrates in the microenvironment of the phagosome (Forbes and Gros, 2001).

After phagocytic uptake by macrophages, pathogenic mycobacteria such as M. tuberculosis and M. avium are able to prevent the phagosomes in which they reside from fusing with lysosomes (Armstrong and Hart, 1971; Fréhel et al., 1986). In this manner, intraphagosomal mycobacteria can avoid exposure to the harsh hydrolytic environment of lysosomes and this is thought to be a major strategy allowing them to survive and multiply inside macrophages. It is now widely accepted that mycobacteria do not interfere with phagosome-lysosome fusion directly but rather affect the preceding step of endocytic processing, namely that of phagosome maturation (for reviews see de Chastellier and Thilo, 1999; Deretic and Fratti, 1999; Russell, 2001) without which fusion with lysosomes cannot take place (de Chastellier et al., 1995; de Chastellier and Thilo, 1997). Mycobacterium-containing phagosomes clearly retain compositional and intermingling characteristics of early endosomes. They remain positive for rab5 (Via et al., 1997; Clemens et al., 2000), continue to exchange cell surface-derived glycoconjugates, transferrin receptor, and glycosphingolipids with early endosomes (de Chastellier et al., 1995; Clemens and Horwitz, 1995; 1996; Russell et al., 1996; Sturgill-Koszycki et al., 1996; de Chastellier and Thilo, 2002). Evidence for fusion of Mycobacterium-containing phagosomes with early endosomes, but not with lysosomes, was obtained directly on electron micrographs by studying the acquisition of newly internalized endocytic content markers either added at different times following infection or chased to lysosomes prior to phagocytic uptake (Clemens and Horwitz, 1995; de Chastellier et al., 1995; de Chastellier and Thilo, 1997). Additional studies of recruitment of cathepsin D to mycobacterial phagosomes showed that this enzyme was delivered to these phagosomes via the endocytic pathway (55 kDa isoform), but not through fusion with lysosomes (31 kDa + 17 kDa isoforms) (Ullrich et al., 1999). It was suggested (de Chastellier and Thilo, 1997) that this might be also the case for the lysosome-associated membrane protein, Lamp1, found in low amounts in the membrane of Mycobacterium-containing phagosomes (Sturgill-Koszycki et al., 1994; Xu et al., 1994; Clemens and Horwitz, 1995; Sturgill-Koszycki et al., 1996). Finally, it is well established that Mycobacterium-containing phagosomes show reduced acidification (Crowle et al., 1991; Sturgill-Koszycki et al., 1994; de Chastellier et al., 1995; Oh and Straubinger, 1996; Hackam et al., 1997) when compared with Leishmania amazonensis or latex bead-containing phagosomes (Sturgill-Koszycki et al., 1994), possibly as a result of reduced density of V-ATPases at the phagosomal membrane (Hackam et al., 1997; Schaible et al., 1998). Inhibition of phagosome maturation and fusion with lysosomes, as well as prevention of phagosome acidification, both require mycobacterial cell viability and strain virulence and are not observed when bacteria are avirulent and/or dead (for reviews see de Chastellier and Thilo, 1999; Russell, 2001).

In the present study, we have used several electron microscopy approaches in combination with counts of colony-forming units (CFU) to analyse how expression of Nramp1 at the phagosomal membrane may influence survival of a virulent strain of M. avium within bone marrow-derived mouse macrophages and affect its ability to modulate the fusogenic properties of the phagosome in which it resides.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Survival of M. avium within macrophages from 129–/– vs. 129sv mice

Bone marrow-derived macrophages from wild-type 129sv (Nramp1G169) mice and from isogenic 129sv carrying a null mutation at Nramp1 (Nramp1–/–, 129–/–) were prepared as described in Experimental procedures, and grown for 7 days prior to infection with M. avium TMC 724, a virulent strain that replicates within macrophages from Bcgs mice (de Chastellier et al., 1993; Fréhel et al., 1997), at a ratio of 1 : 1 viable mycobacteria per macrophage. At predetermined time intervals, between 0 and 10 days after infection, the cells were lysed, and the number of viable mycobacteria was determined by CFU counts on Middlebrook 7H10 agar (Fig. 1). During the first 4 days following infection, bacteria grew slowly in 129–/– macrophages, but replication increased afterwards, leading to a 1 log rise in CFU counts by day 10 when compared to T0 in that group (deduced generation time, 40 h). By contrast, little if any bacterial replication was observed in macrophages from wild-type 129sv macrophages during the same period (Fig. 1), highlighting the effect of Nramp1 on intracellular replication of M. avium.

image

Figure 1. Growth of M. avium in bone marrow-derived macrophages from 129sv (▪) and 129–/– mice (□). Macrophages were infected for 4 h with M. avium, washed and incubated in mycobacteria-free medium. At selected times after infection, cells were lysed with Triton and the number of viable mycobacteria was determined on Middlebrook 7H10 agar plates. Each value represents the mean ± SEM (three determinations per time point) of a typical experiment.

Download figure to PowerPoint

In parallel experiments, similarly infected macrophages were fixed and processed for electron microscopy (Fig. 2). Analysis of infected cells at day 0 and day 14 allowed us to confirm the results obtained by CFU counts in both groups, and, in addition, to discriminate between ultrastructurally intact or altered bacteria, that are both potentially live, vs. damaged (dead) cells according to morphological criteria we have previously described (Fréhel et al., 1997). Examination of 129sv and 129–/– cells immediately after the 4 h infection period (day 0) showed very similar mean numbers of bacteria per macrophage thin section (Fig. 2A shows a thin section of a 129–/– macrophage), indicating that macrophages from both types of mice can phagocytose M. avium with equal efficiency. Also at day 0, most intracellular bacteria were morphologically intact in both cell types, with only 5–7% showing signs of degradation. At day 14, the mean number of bacteria per thin section had increased slightly in 129sv macrophages (Fig. 2B) but up to 40% of intracellular bacteria showed signs of alteration, including an irregular shape and/or the presence of lipid granules (Fig. 3A). Only about 5% of the bacteria showed strong signs of degradation such as a disrupted cytoplasmic membrane and/or wall, thinning or disappearance of the electron translucent wall layer (capsule) or a disorganized cytoplasm. In contrast, many infected 129–/– cells were found to contain a large number of intracellular M. avium at day 7 and day 14 (Fig. 2C) and> 90% were morphologically intact throughout the 14 days following infection (Figs 2C and 3B). As observed before in macrophages from mice susceptible to mycobacterial infections (de Chastellier et al., 1995; de Chastellier and Thilo, 1997), each phagosome usually contained a single bacterium due to splitting up of the phagosomes upon bacterial replication. These results strongly suggest that Nramp1 expression is associated with inhibition of bacterial replication. However, bacteria can survive for at least 14 days but a large fraction of the bacterial population presents an altered morphology. Abrogation of Nramp1 function restores not only the normal morphological appearance of intracellular bacteria but also the capacity to replicate within the intracellular environment of a macrophage.

image

Figure 2. General views of macrophages from 129sv (B) or 129–/– (A,C) mice infected with M. avium. Macrophages were infected with M. avium. Cells were fixed and processed for electron microscopy at selected time points after infection.

A. 129–/– macrophage, day 1: cells contain a few intraphagosomal bacteria that are generally (95%) morphologically intact. Similar results (not shown) were obtained in 129sv cells.

B. 129sv macrophage, day 14: the number of bacteria has slightly increased.

C. 129–/– macrophage, day 14: M. avium replicates in Nramp1-negative cells. Note that each phagosome generally contains a single bacterium. Bar = 1 µm.

Download figure to PowerPoint

image

Figure 3. Differences in the morphological appearance of M. avium within 129sv (A) and 129–/– (B) macrophages. Cells were infected with M. avium as in Fig. 1 and fixed 14 days later.

A. 129sv macrophage showing the aspect of bacteria with an altered morphology. As depicted here, one of the major features is the presence of several lipid droplets (arrows) within the cytoplasm. The shape of altered bacteria is often irregular.

B. 129–/– macrophage: the bacteria shown here are morphologically intact. Note that they present very few, if any, lipid droplets. Bar = 0.5 µm.

Download figure to PowerPoint

Fusion of M. avium-containing phagosomes with endocytic organelles

It is now widely accepted that newly formed phagosomes first fuse and intermingle contents and membrane with early endosomes (for reviews see Berón et al., 1995; Alvarez-Dominguez et al., 1999; de Chastellier and Thilo, 1999). During this process, phagosomes mature to a state where they no longer fuse with early endosomes and only then do they become able to fuse with lysosomes (de Chastellier et al., 1995; de Chastellier and Thilo, 1997). By preventing maturation of the phagosomes in which they reside, pathogenic mycobacteria ensure that the phagosomes will not reach a state where they are ready to fuse with lysosomes (for reviews see de Chastellier and Thilo, 1999; Russell, 2001). Because immature and mature phagosomes cannot be distinguished directly, implicit parameters were used to determine the effect of Nramp1 on the state of maturation of individual phagosomes. These are based on direct morphological evidence of fusion with either early endosomes or lysosomes labelled with electron dense endocytic tracers and on the kinetics of acquisition, by phagosomes, of endocytic content markers added to macrophages either at selected intervals after infection with M. avium or chased to lysosomes prior to phagocytic uptake of M. avium. It has been previously established (for a review see de Chastellier and Thilo, 1997) that early endosomes can be distinguished from prelysosomes/lysosomes in their cytochemical staining pattern after uptake of the endocytic marker, horseradish peroxidase (HRP). In early endosomes, the HRP reaction product only rings the inner face of the membrane, while in lysosomes the entire lumen is filled with reaction product. Thus, using HRP, one can determine if phagosomes fuse with early endosomes (in which case they are still immature), or with lysosomes (in which case they have matured). Macrophages from 129sv and 129–/– mice were infected with M. avium for 4 h. At selected time intervals (0, 1 and 7 days) after infection, cells were exposed to HRP for 0–60 min, fixed and then examined for direct morphological evidence of fusion with either early endosomes or with lysosomes. In the case of 129sv macrophages (Fig. 4), M. avium -containing phagosomes showed evidence of fusion with early endosomes (Fig. 4A) but also with lysosomes (Fig. 4B) at 2 h, thereby indicating that part of the phagosomes had already matured. Starting from day 1, phagosomes fused only with lysosomes (Fig. 4C). Strikingly, in 129–/– macrophages M. avium-containing phagosomes fused with early endosomes (Fig. 5A and B) and generally not with lysosomes (Fig. 5C), and this at all times during infection, unless the phagosomes contained several bacteria in which case they rapidly matured and underwent fusion with lysosomes, as observed before in Bcgs macrophages (de Chastellier et al., 1995). This Nramp1-dependent difference was specific to M. avium-containing phagosomes. It was not seen with Bacillus subtilis-containing phagosomes which all matured and fused normally with lysosomes within 15 min after phagocytic uptake in both cell types (data not shown). Likewise, 2 h-old-phagosomes containing hydrophobic latex beads of 1 µm in diameter, that remain immature for at least 3 h after uptake in Bcgs macrophages (de Chastellier et al., 1995; de Chastellier and Thilo, 1997), uniformly failed to fuse with lysosomes while fusing with early endosomes in both cell types (data not shown).

image

Figure 4. Interaction of M. avium-containing phagosomes with HRP-filled endocytic organelles in macrophages from 129sv mice. After a 4 h infection with M. avium, cells were incubated in bacteria-free medium. Two hours or 1 day later, cells were exposed to HRP for 0–60 min, prior to processing for electron microscopy.

A, B. 2 h post infection, 60 min exposure to HRP: (A) part of the phagosomes display a staining pattern similar to that of early endosomes (E); (B) other phagosomes have already fused with lysosomes (L) and display large amounts of HRP reaction product.

C. day 1 post infection, 30 min exposure to HRP: the phagosome has fused with lysosomes. It displays the typical appearance of a phagolysosome, i.e. a loose membrane around the bacterium and bulges filled with HRP reaction product. The bacterium is still intact including the thick electron translucent layer surrounding the bacterium. HRP reaction product is indicated by arrowheads. Bar = 0.5 µm.

Download figure to PowerPoint

image

Figure 5. Interaction of M. avium-containing phagosomes with HRP-filled endocytic organelles in macrophages from 129–/– mice. Cells were infected with M. avium and exposed to HRP as described in Fig. 4.

A. 2 h post infection, 60 min exposure to HRP: fusion of a phagosome with an early endosome (E);

B. 1 day post infection, 45 min exposure to HRP: phagosome fusing with a large endosome (E).

C. 1 day post infection, 60 min exposure to HRP: the phagosome does not fuse with the lysosome (L). Bar = 0.5 µm.

Download figure to PowerPoint

To further test whether M. avium-containing phagosomes remained immature and therefore retained intermingling characteristics of early endosomes, or whether they were processed into phagolysosomes with fusion properties of lysosomes, we determined whether M. avium-containing phagosomes could acquire HRP from the medium immediately as it is the case for early endosomes or phagosomes with early endosome characteristics, or if they acquired HRP after a lag time of> 5 min as is typical for lysosomes or phagolysosomes (de Chastellier and Thilo, 1997). The results are depicted in Fig. 6. For 129sv cells, HRP acquisition by M. avium-containing phagosomes was immediate only at 2 h, and required a lag time of 10–20 min at both days 1 and 7 post infection. By contrast, 129–/– phagosomes acquired HRP immediately at both 2 h and day 1. When cells were exposed to HRP at day 7, the phagosomes became HRP-positive only after a lag of 5–10 min although they fused with early endosomes and not with lysosomes. The lag observed in this case is probably due to the disorganization of the actin filament network (Guérin and de Chastellier, 2000a) which delays fusion events between early endosomes and phagosomes (Guérin and de Chastellier, 2000b). Therefore, the direct morphological observations together with the kinetic studies of the acquisition of HRP suggest that in Nramp1-positive macrophages M. avium-containing phagosomes are processed into phagolysosomes, while in Nramp1-negative macrophages, phagosomes do not mature and instead continue to fuse with early endosomes.

image

Figure 6. Acquisition of endocytic tracer, HRP, by pre-existing M. avium-containing phagosomes in macrophages from 129sv (▪) vs. 129–/– (□) mice. Cells were infected for 4 h with M. avium and reincubated in fresh medium devoid of bacteria to allow for phagosome pro­cessing. This was followed by receptor-mediated uptake of HRP (at 25 µg ml−1) for the indicated time at days 0, 1 or 7 post infection. HRP-containing phagosomes (HRP+) were scored as a fraction of total phagosomes on electron microscope sections after cytochemical staining as indicated in the Experimental procedures.

Download figure to PowerPoint

Lysosomes can also be specifically labelled with gold-tagged bovine serum albumin (BSA-Au) by a 30-min long pulse of endocytic uptake, followed by a 2 h-long chase. After such a treatment, lysosomes are heavily labelled with BSA-Au whereas early endosomes are no longer labelled in uninfected macrophages (de Chastellier and Thilo, 1997). Delivery of this electron dense tracer to phagosomes will therefore be an indication of phagosome maturation and fusion with lysosomes. 129sv and 129–/– macrophages were therefore labelled with BSA-Au as described above. The cells were then allowed to phagocytose either M. avium, B. subtilis or hydrophobic latex beads and cells were fixed for EM analysis at different times after infection/phagocytosis. The different cases are illustrated in Figs 7 and 8. In the case of M. avium, BSA-Au was more often observed in Nramp1-positive (Fig. 7A) than in Nramp1-negative phagosomes (Fig. 7B) provided the phagosomes contained only one bacterium. When phagosomes contained instead several bacteria, all the phagosomes became BSA-Au-positive within the first 3 h following bacterial uptake in both cell types, as expected. A quantitative evaluation at 0 and 17 h after infection with M. avium (Fig. 9) showed that, the fraction of phagosomes that had matured and become able to fuse with lysosomes was significantly higher in 129sv cells than in 129–/– cells, with 62% (T0) and 59% (17 h) being positive for BSA-Au in 129sv cells, as compared to 42% (T0) and 40% (17 h) in 129–/– cells (Fig. 9). These results are in agreement with the above morphological and kinetic studies with the endocytic marker, HRP, added to cells after phagocytic uptake. They are also in good agreement with the observation that, at day 7 after infection, 60% of the M. avium-containing phagosomes were positive for acid phosphatase, in 129sv cells, compared to 26% for 129–/– cells (data not shown). This hydrolytic enzyme accumulates essentially, although not exclusively, in lysosomes (discussed in de Chastellier et al., 1995). As above, the Nramp1 difference was specific to M. avium. Control experiments indeed showed that when macrophages were exposed to B. subtilis, phagosomes rapidly matured and close to 90% of them showed signs of fusion with BSA-Au-containing lysosomes within 10 min after phagocytic uptake in both cell types (Fig. 8A and B; Fig. 9). Conversely, hydrophobic latex bead-containing phagosomes in either 129sv or 129–/– cells fused equally poorly with lysosomes (Fig. 8C and D) as only 5–10% of them had become positive at 2 h after phagocytic uptake (Fig. 9). These results suggest that differences in maturation of the M. avium-containing phagosome noted in 129sv and 129–/– macrophages are specific for this bacterium and not-linked to a non-specific effect of Nramp1 on fusogenic properties of this vacuole.

image

Figure 7. Interaction of M. avium-containing phagosomes with BSA-Au filled lysosomes in macrophages from 129sv (A) vs. 129–/– (B) mice. Cells were given a 30-min pulse of BSA-Au followed by a 2 h chase in BSA-Au-free medium to label lysosomes (L). Cells were then infected for 4 h with M. avium and fixed after 17 h of incubation in bacteria-free medium to allow for phagosome processing.

A. 129sv macrophage: phagosomes contain the lysosomal marker (arrowheads) which means that they have matured and fused with lysosomes.

B. 129–/– macrophage: phagosomes are usually devoid of label thereby indicating that they are still immature. Bar = 0.5 µm.

Download figure to PowerPoint

image

Figure 8. Interaction of B. subtilis (A, B) and hydrophobic latex bead (C, D) -containing phagosomes with BSA-Au filled lysosomes in macrophages from 129sv (A, C) vs. 129–/– (B, D) mice. Cells were exposed to BSA-Au as indicated in Fig. 7.

A, B. Cells exposed to B. subtilis for 30 min followed by a 10 min chase in bacteria-free medium prior to fixation for electron microscopy. In both 129sv (A) and 129–/– (B) macrophages all the phagosomes are labelled with BSA-Au (arrowheads), thereby indicating that they have matured and fused with lysosomes.

C, D. Cells exposed to latex beads for 45 min followed by a 2-h chase in particle-free medium prior to fixation. In both 129sv (C) and 129–/–(D) macrophages the phagosomes are not labelled with BSA-Au, thereby indicating that they are still immature and therefore unable to fuse with lysosomes. Bar = 0.5 µm.

Download figure to PowerPoint

image

Figure 9. Acquisition of the lysosomal marker BSA-Au by phagosomes containing either M. avium, B. subtilis or hydrophobic latex beads in macrophages from 129sv (▪) vs. 129–/–(□) mice. Lysosomes were labelled with BSA-Au as indicated in Fig. 7. Cells were then exposed to the phagocytic particles as indicated in Figs 7 and 8 and fixed for electron microscopy. BSA-Au-containing phagosomes (BSA-Au+) were scored as a fraction of total phagosomes on electron microscope thin sections (100–200 different phagosomes were analysed per time point). In the case of M. avium, the fraction of phagosomes that had acquired the lysosomal marker was substantially higher in 129sv than in 129–/– macrophages immediately after infection and 17 h later. In the case of B. subtilis, close to 90% of the phagosomes had fused with lysosomes in both cell types and, in the case of latex beads, only 5–10% of the phagosomes had fused with lysosomes also in both cell types.

Download figure to PowerPoint

Acidification of M. avium-containing phagosomes

The degree of acidification of M. avium-containing phagosomes was measured in 129sv and 129–/– cells, using DAMP (3-(2,4-dinitroanilo)-3′-amino-N-methylpropylamine), as we have previously described (de Chastellier et al., 1995). DAMP accumulates in acidic compartments, becomes covalently linked to proteins in the presence of aldehyde fixative which allows its retention in acidic organelles. Intraphagosomal acidification can then be quantitated after sequential immunogold labeling with the appropriate antibodies followed by protein A coupled to gold particles. The number of gold particles per phagosome was first determined. As illustrated in Fig. 10, the number of particles was quite heterogeneous from 0 to more than a 100. The distribution was, however, clearly shifted towards higher number in the case of phagosomes within 129sv macrophages, thereby indicating that phagosomes were more acidic in 129sv than in 129–/– macrophages. The number of gold particles per phagosome was then converted to values for intraphagosomal acidification by using the method described by Orci et al. (1994). Already at 2 h post infection, M. avium-containing phagosomes from 129sv macrophages were found to be more acidic than those formed in 129–/– cells, with a ΔpH of 0.4 for 129sv and 0.8 for 129–/– cells, when compared to the pH of prelysosomes/lysosomes used as an internal standard in the same sections (data not shown). These results suggest that expression of Nramp1 also affects phagosome acidification.

image

Figure 10. Acidification of phagosomes containing M. avium at 2 h post-infection of macrophages from 129sv vs. 129–/– mice. The data are based on quantification of gold particles associated with phagosomes. Fifty different phagosomes were analysed in each type of macrophage.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The mechanism by which Nramp1 contributes to macrophage function and to host defences against intracellular infections remains only partly understood. The recent demonstration that Nramp1 functions as a pH-dependent efflux pump for divalent cations at the phagosomal membrane (Jabado et al., 2000) has suggested that depleting the intraphagosomal space of divalent cations such as Fe2+, Mn2+, Co2+, can affect dramatically the bacteriostatic and/or bactericidal activity of these cells towards intracellular pathogens such as M. avium. A number of hypotheses may be considered to explain the deleterious effect of divalent cation depletion on intracellular survival and replication of pathogens as diverse as Mycobacterium, Salmonella and Leishmania (for a review see Forbes and Gros, 2001). First, divalent metals could be rate limiting for general metabolic activity of the bacteria, and depletion from the phagosomal space, in turn depleting bacteria within it, would thus prevent replication, compromise survival ultimately resulting in death of the ingested bacteria. Second, the effect could be through increased sensitivity to killing by oxygen radicals, as Zn2+, Cu2+, Fe2+ and Mn2+ are required cofactors for activity of superoxide dismutases (SOD) produced by these three pathogens (Zhang et al., 1991; Dey and Datta, 1994; Tsolis et al., 1995). SODs constitute major defence mechanisms against superoxide ions and hydroxyl radicals produced by the macrophage NADPH oxidase. Indeed, it has previously been shown that iron-replete M. smegmatis is considerably more resistant to killing by hydrogen peroxide than iron-deprived cells (Lundrigan et al., 1997). Third, Nramp1-mediated depletion of divalent metals from the phagosome could affect the expression and/or activity of virulence determinants that are normally expressed by the bacteria, and that may be involved in prevention of phagosome maturation, a well-established means of intracellular survival for Mycobacterium (for reviews see de Chastellier and Thilo, 1999; Russell, 2001), and/or maintenance of the phagosome in an immature state which involves remodelling of the phagosome membrane (Ullrich et al., 2000; de Chastellier and Thilo, 2002). It may be difficult to formally distinguish between these possible mechanisms as they may be non-exclusive and may take place simultaneously and/or at different stages of the same intracellular infection. However, an important role for efficient acquisition of divalent cations by bacteria during pathogenesis has been clearly established and is highlighted by the noted redundancy in divalent metal transporters (Cellier et al., 2001), including the presence of bacterial Nramp1 structural and functional homologues (MntH, Mramp) that may compete with macrophage Nramp1 for the same substrate in the microenvironment of the phagosome (Forbes and Gros, 2001).

In the present study, we have used electron microscopy approaches to study the intracellular behaviour and fate of M. avium after infection of wild-type (129sv) and Nramp1–/– (129–/–) macrophages, including possible modulation of phagosome maturation and fusion with lysosomes. A comparison of the number of viable bacteria that could be recovered from infected macrophage cultures at different times during infection, indicated that after an initial lag period of 2–4 days, there was active replication in permissive 129–/– cells, while this was not seen in wild-type macrophages. This is in agreement with previous studies of mycobacterial replication in macrophages from different inbred strains expressing either the Nramp1G169 or Nramp1D169 protein variants (de Chastellier et al., 1993). In addition, although there were no major differences in the proportion of altered or degraded bacteria soon after infection of either cell type, up to 40–50% of intracellular bacteria showed signs of alteration, as indicated by the accumulation of intracytoplasmic lipid droplets and distortion of the cell shape, at day 7 in wild-type cells, while this number stayed at around 5% in Nramp1-negative 129–/– cells. The seemingly constant number of CFUs recovered over time from infected wild-type macrophages, together with the significantly larger proportion of altered M. avium detected in these cells compared to 129–/– mutant macrophages, could suggest a certain degree of active replication and concomitant Nramp1-dependent killing in wild-type macrophages. However, because the number of degraded, and therefore dead, bacteria stayed at around 5% throughout the 14-day infection period even in wild-type cells, and that the altered bacteria, as described above, seem to correspond to live bacteria, unable to replicate (dormant bacteria) and not to dead bacteria (Fréhel et al., 1997), we favour a model in which expression of Nramp1 at the phagosomal membrane has a bacteriostatic effect.

The combined analyses of the acquisition of endocytic markers delivered either from endosomes or from lysosomes (morphological characteristics and kinetic data), including studies with BSA-Au, provided a clear result and indicated that Nramp1-positive and negative mycobacterial phagosomes have very different fusogenic properties. Indeed, while M. avium-containing phagosomes formed in wild-type macrophages readily fused with endosomes initially, and with lysosomes subsequently, most of the Nramp1-negative phagosomes fused with early endosomes indefinitely and only in rare instances with lysosomes. Only when Nramp1-negative phagosomes contained several bacteria (bacterial aggregates) did they mature rapidly and fuse extensively with lysosomes. Prevention of phagosome maturation and fusion with lysosomes was concomitant to a less acidic environment in Nramp1-negative phagosomes, possibly owing to reduced recruitment of vacuolar ATPases at that site, as we have previously observed in the case of M. bovis BCG-containing phagosomes (Hackam et al., 1998). At present, however, the precise relationship between acidification and phagosome maturation and fusion with lysosomes is unknown as far as whether acidification precedes maturation or conversely (for a review see de Chastellier and Thilo, 1999). Therefore, the Nramp1-negative more hospitable phagosome showed many characteristics of an immature phagosome, when compared to the phagolysosome formed in wild-type cells.

To conclude, we favour a model for Nramp1 action which is based on the ability of M. avium to modulate the fusogenic properties of the phagosome in which it resides (for reviews see de Chastellier and Thilo, 1999; Russell, 2001). Prevention of phagosome maturation, and maintenance of the phagosome in an immature state is only seen when mycobacteria are live (reviewed in de Chastellier and Thilo, 1999; Russell, 2001). It has been proposed that it requires an intimate contact and/or interaction between the bacterial surface and the membrane of the phagosome (de Chastellier and Thilo, 1997). This could possibly occur through (i) intercalation of mycobacterial cell wall lipids and/or proteins into the phagosomal membrane (Beatty et al., 2000a; Beatty and Russell, 2000b); (ii) remodelling of the phagosome membrane by deletion/addition of plasma membrane glycoconjugates (de Chastellier and Thilo, 2002); and/or (iii) expression of gene products by mycobacteria when they reside within macrophages, such as Mig (macrophage-induced gene) protein (Plum and Clark-Curtiss, 1994; Plum et al., 1997) which seems to be involved in the metabolism of a unique type of cell wall lipids (Morsczeck et al., 2001). Our results strongly suggest that bacterial cells must be replete with divalent cations in order to achieve expression/function of these virulence determinants, with Nramp1-dependent removal of such cations either directly or indirectly interfering with this process. This hypothesis is supported by the observation that Nramp1 recruitment to the phagosome membrane antagonizes the specific defence mechanisms or virulence determinants expressed by unrelated intracellular parasites, and that are essential for their intracellular survival. Indeed, the present study shows that Nramp1 antagonizes the ability of Mycobacteria to prevent phagosome maturation and, thereby, phagosome-lysosome fusion, as well as phagosome acidification by the vacuolar ATPase. Independently, and in a similar experimental setting (Cuellar-Mata et al., 2002), we have demonstrated that Nramp1 antagonizes the ability of Salmonella to become secluded in a compartment that no longer acquires extracellularly added markers and that remains negative for the mannose-6-phosphate receptor, while it has no effect on acidification or recruitment of lysosomal markers to the Salmonella-containing vacuole. This pathogen-specific effect of Nramp1 supports the contention that divalent cations are essential for expression of unique survival strategies utilized by each pathogen. Identifying such divalent cation-dependent virulence factors will be of great interest, as they may constitute novel targets for drug discovery and therapeutic intervention in mycobacterial diseases.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Cells and culture medium

Bone marrow cells were isolated from femurs of 6- to 8-week-old 129sv (Nramp1G169, wild-type) or isogenic 129sv female mice carrying a null mutation at Nramp1 (Nramp1–/–, 129–/–) and seeded onto tissue culture dishes of 35 mm in diameter (4 × 105 cells per dish) or 24-well tissue culture plates (1 × 105 per well) (Falcon, Beckton Dickinson Labware, Meylan, France). The culture medium was Dulbecco's modified Eagle's medium (DMEM) with low glucose (1 g l−1) and high carbonate (3.7 g l−1) concentrations supplemented with 10% heat-inactivated fetal calf serum (FCS), 10%l-cell-conditioned medium (a source of CSF-1), and 2 mM l-glutamine. At 4 days after seeding, the adherent cells were rinsed twice with Hanks' balanced salt solution (HBSS) containing 10 mM N-2-hydroxyethylpiperazine ethanesulphonic acid (HEPES). Medium was then renewed three times a week. No antibiotics were added.

Phagocytic particles

(i) Mycobacterium avium.  The transparent (Tr) colony variant TMC 724 (serovar 2) from the Trudeau Mycobacterial Culture Collection was kindly provided by Frank Collins. This strain is virulent for C57Bl/6 mice (Fréhel et al., 1991) and grows within macrophages from the same, or other mice, susceptible to mycobacterial infections (de Chastellier et al., 1993; Fréhel et al., 1997). Because M. avium strains tend to lose their virulence when passaged in liquid medium, we expanded bacteria of a first passage after isolation from mouse liver of C57Bl/6 mice, infected 8–10 weeks previously. Bacteria used for experiments were always of the first passage grown on Middlebrook 7H10 agar plates, supplemented with 0.05% Tween 80, 0.2% glycerol and 10% oleic acid-albumin-dextrose-catalase (OADC). Aliquots of bacterial suspensions were concentrated in Middlebrook 7H9 medium devoid of Tween and stored at − 80°C. When required, frozen samples were quickly thawed, vortexed and adjusted to the desired titre in complete cell culture medium. More than 95% of the bacteria were morphologically intact and viable at this stage.

(ii) Bacillus subtilis.  Strain MO 719 (trp, phe spoII AC xx Kanar) was kindly provided by Patrick Stragier, Institut de Chimie et Biophysique, Paris, France. This strain was grown in Luria–Bertani (LB) medium and stored at − 80°C with 10% glycerol until required. For each experiment a small aliquot of bacteria was first grown on LB agar at 37° overnight. Bacteria from one colony were then grown in 5 ml of LB liquid medium to a concentration of 5 × 108 bacteria per ml. At this stage bacteria became very mobile and the cellular chains fragmented into individual bacilli. More than 95% of the bacilli were morphologically intact and viable.

(iii) Native (hydrophobic) latex beads.  Beads, 1 µm in diameter, (Sigma, St Louis, MO, USA) were diluted in culture medium.

Phagocytic uptake.

Particles were added to 7-day-old macrophage cultures as follows: (i) cells were incubated for 4 h at a mycobacterium/macrophage ratio of 10 : 1, washed in four changes of ice-cold phosphate-buffered saline (PBS) to eliminate non-ingested bacteria, and further incubated in complete medium devoid of antibiotics. The medium was renewed twice a week; (ii) cells were infected for 30 min with the B. subtilis suspension adjusted to yield a bacteria/macrophage ratio of 25 : 1. Cells were then washed with PBS as above and reincubated in medium devoid of bacteria; (iii) cells were given latex beads for 30 min. The hydrophobic latex bead solution was diluted 1 000-fold in complete medium in order to obtain similar particle uptake as with B. subtilis. Cells were then washed with PBS as above and reincubated in complete medium devoid of antibiotics.

Evaluation of growth and degradation of intracellular mycobacteria.

(i) CFU counts.  At selected times after infection, the medium was removed. Cells were washed twice with PBS and lysed with 0.1% Triton X-100 (in distilled water). The number of viable bacteria per well was determined by plating 10-fold dilutions of macrophage lysate on Middlebrook 7H10 agar. Colonies were counted after incubation at 37°C for 14 days. For each time point, counts were made from three different wells. The number of macrophages per well was determined at the same time points using the naphtol blue staining method, as described (Fréhel et al., 1997).

(ii) Morphological assessment of degradation.  In parallel experiments, M. avium-infected macrophages were processed for electron microscopy as described below. On thin sections, intracellular bacteria can be sorted into three distinct categories on the basis of their morphological appearance (Fréhel et al., 1997): (i) intact bacteria that display a regular shape, an electron translucent zone (or capsule), a well-organized cytoplasm and nucleus, and an intact cell wall and cytoplasmic membrane; (ii) altered bacteria that are distorted and display several electron translucent lipid droplets in the cytoplasm but otherwise have retained normal morphological features; (iii) degraded bacteria that characteristically display breaks in the cytoplasmic membrane and/or cell wall, a disorganized cytoplasm and no electron translucent zone. The fraction of intact, altered and degraded bacteria was measured by examining 100–200 different bacteria per time point. Care was taken to avoid serial sections.

Acid phosphatase (AcPase) cytochemistry.

At selected time points following infection, macrophages were fixed for 1 h with 1.25% glutaraldehyde in cacodylate buffer, pH 7.2, containing 0.1 M sucrose, 5 mM CaCl2 and 5 mM MgCl2. The cells were washed overnight with the same buffer, rinsed once with 0.1 M acetate buffer, pH 5.0, and incubated for 30 min in pre-warmed Gomori reaction medium, as described (Fréhel et al., 1986; de Chastellier et al., 1993). Macrophages were rinsed twice with acetate buffer and once with cacodylate buffer and then processed for electron microscopy as described below. The presence of electron dense deposits within phagosomes identified transfer of the hydrolytic enzyme AcPase to phagosomes. The fraction of AcPase-positive phagosomes was measured by examining 100–200 different phagosomes per time point. Care was taken to avoid serial sections.

Acquisition of lysosomal marker by phagosomes.

To label lysosomes, macrophages were rinsed in serum-free medium and incubated for 30 min at 37°C with colloidal gold particles of 10 nm in diameter conjugated with bovine serum albumin (BSA-Au) diluted in serum-free DMEM. Cells were washed three times with serum-free medium and further incubated for 2 h at 37°C in complete medium devoid of BSA-Au. BSA-Au treated cells were infected with M. avium as indicated above. Cells were washed four times with ice-cold PBS and further incubated for 2 h at 37°C in complete medium devoid of mycobacteria. Cells were then fixed at selected time and processed for electron microscopy. In all cases, 100–200 different phagosomes per sample were examined for the presence or absence of BSA-Au. Care was taken to avoid serial sections.

Horseradish peroxidase (HRP) uptake and quantification

At selected time points after phagocytic uptake of M. avium, B. subtilis or hydrophobic latex beads, cells were incubated at 37°C in complete medium containing 25 µg ml−1 HRP. Under these conditions, HRP was essentially endocytosed via the mannose receptor (Lang and de Chastellier, 1985). At selected intervals between 0 and 60 min, endocytosis was stopped by fixing the cells. Cells were then processed for HRP cytochemistry and electron microscopy. The fraction of HRP-positive phagosomes was measured by examining 200–500 different phagosomes per time point. Care was taken to avoid serial sections. Each value was obtained as the mean (± SEM) from four or five different determinations per time point.

HRP cytochemistry

Cells exposed to HRP were fixed for 1 h at room temperature with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, containing 0.1 M sucrose, 5 mM CaCl2 and 5 mM MgCl2. Cells were washed overnight at 4°C with sucrose-containing cacodylate buffer and then incubated with 3,3′-diaminobenzidine tetrachlorhydrate (DAB)-H2O2 for 15 min in the dark as described (de Chastellier et al., 1995). After three washes in cacodylate buffer, cells were postfixed for 1 h at room temperature with 1% osmium tetroxide in the same buffer and processed for electron microscopy as described below.

Processing for conventional electron microscopy

Glutaraldehyde-fixed cells were washed with cacodylate buffer, and post-fixed for 1 h at room temperature with 1% osmium tetroxide in the same buffer. They were scraped off the dishes, concentrated in 2% agar in cacodylate buffer and treated for 1 h at room temperature with 1% uranyl acetate in Veronal buffer. Samples were dehydrated in a graded series of acetone (or alcohol when they contained latex beads), and embedded in Epon. Thin sections were stained with 2% uranyl acetate in distilled water then with lead citrate.

DAMP treatment

DAMP (3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine), synthesized at the Pasteur Institute (Paris, France) according to the method described by Anderson et al. (1984), was stored at − 20°C in ethanol at a concentration of 30 mM. At 2 h following infection with M. avium, macrophages were incubated for 30 min at 37°C with 100 µM DAMP diluted in pre-warmed complete medium. After three rinses in DMEM containing 10% FCS, cells were fixed for 1 h at room temperature with 1.25% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, containing 0.1 M sucrose and then overnight at 4°C with 1% paraformaldehyde in the same buffer. Fixed cells were washed three times with 0.1 M cacodylate buffer containing 50 mM NH4Cl to quench any remaining aldehyde activity. After three brief rinses in cacodylate buffer devoid of sucrose and NH4Cl, the cells were scraped off the culture dishes, concentrated in 2% agar in cacodylate buffer and postfixed for 30 min at 4°C with 0.5% uranyl acetate in Veronal buffer. The samples were subsequently dehydrated in alcohol and embedded in LRWhite resin according to Ozawa et al. (1994).

DAMP immunolocalization

LR White thin sections, picked up on formvar and carbon-coated nickel grids, were sequentially incubated for 60 min at room temperature with 2.5 µg ml−1 each of rabbit anti-dinitrophenol (anti-DNP) antibodies and goat anti-rabbit antibodies coupled to gold particles of 10 nm in diameter (GAR10). Antibodies and conjugate were diluted in PBS containing 1% bovine serum albumin (BSA). Sections were washed five times rapidly with PBS-BSA between the incubations and after treatment with conjugate. After three washes with distilled water, sections were stained with 2% uranyl acetate and lead citrate. As a control, thin sections were incubated either with rabbit anti-ovalbumin antibodies followed by GAR10 or only with GAR10. Both controls were negative. The number of gold particles per phagosome was determined on 40–60 different phagosomes per time point. The number of gold particles in the late endocytic compartments was also determined on the same micrographs. The surface area of the phagosome and late endocytic compartments was determined morphometrically by counting the number of points of the Weibel grid (Weibel et al., 1966) that fall within these structures. The density of gold particles was determined as the ratio of total number of gold particles/total number of grid points obtained for a given structure. These data could be used to obtain estimates for intraphagosomal pH, using the method proposed by Orci et al. (1994).

Chemicals

DMEM, FCS, BSA (fraction V), HRP grade II, H2O2, Hepes, LR White, glutaraldehyde grade I and osmium tetroxide were purchased from Sigma (St Louis, MO, USA). PBS powder, HBSS and glutamine were from Seromed (distributed by PolyLabo-Paul Block, Paris, France). DAB was from Polysciences (distributed by Fisher, Elancourt, France). PAO was purchased from the Utrecht University School of Medicine, Department of Cell Biology (Utrecht, the Netherlands). BSA-Au was a kind gift from Janice Griffith, Utrecht University School of Medicine, Department of Cell Biology (Utrecht, the Netherlands). Polyclonal anti-DNP and antiovalbumin antibodies were kindly provided by Jean Claude Antoine, Institut Pasteur, Paris, France.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The authors thank Bernard Bruneau and Céline Symphorien (Inserm U411) for expert technical assistance, Janice Griffith (University of Utrecht, the Netherlands) for the kind gift of BSA-Au and Jean Claude Antoine (Institut Pasteur, Paris, France) for providing the antidinitrophenol antibodies. C.F. and C.C. received financial support from the Institut National de la Santé et de la Recherche Médicale (Inserm funding to unit 411), P.G. was supported by a research grant (number AI355237) from the National Institutes of Health (N.I.A.I.D.), F.C.H. was supported by a postdoctoral fellowship from and by Milestone Medica Corporation. C.C. and P.G. were also funded through a joint research agreement between INSERM (France) and the Fondation pour la Recherche Scientifique du Québec (R.S.Q., Québec). P.G. is a Distinguished Scientist of the Canadian Institutes for Health Research.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  • Abel, L., Sanchez, F.O., Oberti, J., Thuc, N.V., Hoa, L.V., Lap, V.D. et al. (1998) Susceptibility to leprosy is linked to the human NRAMP1 gene. J Infect Dis 177: 133145.
  • Alcais, A., Sanchez, F.O., Thuc, N.V., Lap, V.D., Oberti, J., Lagrange, P.H. et al. (2000) Granulomatous reaction to intradermal injection of lepromin (Mitsuda reaction) is linked to the human NRAMP1 gene in Vietnamese leprosy sibships. J Infect Dis 181: 302308.
  • Alvarez-Dominguez, C., Mayorga, L., and Stahl, P.D. (1999) Sequential maturation of phagosomes provides unique targets for pathogens. In: Advances in Cell and Molecular Biology of Membranes and Organelles. Gordon, S, (ed). Stamford, CT: JAI Press, 5: 285–297.
  • Anderson, R.G.W., Falck, J.R., Goldstein, J.L., and Brown, M.S. (1984) Visualization of acidic organelles in intact cells by electron microscopy. Proc Nat Acad Sci USA 81: 48384842.
  • Andrews, N.C. (2000) Iron homeostasis: Insights from genetics and animal models. Nat Rev Genet 1: 208217.
  • Armstrong, J.A., and D'Arcy Hart, P. (1971) Response of cultured macrophages to Mycobacterium tuberculosis with observations on fusion of lysosomes with phagosomes. J Exp Med 134: 713740.
  • Beatty, W.L., Rhoades, E.R., Ullrich, H.J., Chatterjee, D., Heuser, J., and Russell, D.G. (2000a) Trafficking and release of mycobacterial lipids from infected macrophages. Traffic 1: 235247.
  • Beatty, W.L., and Russell, D.G. (2000b) Identification of mycobacterial surface proteins released into subcellular compartments of infected macrophages. Infect Immun 68: 69977002.
  • Bellamy, R., Ruwende, C., Corrah, T., McAdam, K.P.W.J., Whittle, H.C., and Hill, A.V.S. (1998) Variations in the NRAMP1 gene and susceptibility to tuberculosis in West Africans. N Engl J Med 338: 640644.
  • Berón, W., Alvarez-Dominguez, C., Mayorga, L., and Stahl, P.D. (1995) Membrane traffic along the phagocytic pathway. Trends Cell Biol 5: 100104.
  • Cellier, M., Bergevin, I., Boyer, E., and Richer, E. (2001) Polyphyletic origins of bacterial Nramp gene transporters. Trends Genet 17: 365370.
  • Cellier, M., Prive, G., Belouchi, A., Kwan, T., Rodrigues, V., Chia, W., and Gros, P. (1995) The natural resistance associated macrophage protein (Nramp) defines a new family of membrane proteins conserved throughout evolution. Proc Nal Acad Sci USA 92: 1008910094.
  • De Chastellier, C., and Thilo, L. (1997) Phagosome maturation and fusion with lysosomes in relation to surface property and size of the phagocytic particle. Eur J Cell Biol 74: 4962.
  • De Chastellier, C., and Thilo, L. (1999) Mycobacteria and the endocytic pathway. In: Advances in Cell and Molecular Biology of Membranes and Organelles. Gordon, S, (ed). Stamford, Ct: JAI Press, 6: 107135.
  • De Chastellier, C., and Thilo, L. (2002) Pathogenic Mycobacterium avium remodels the phagosome membrane in macrophages within days after infection. Eur J Cell Biol 81: 1725.
  • De Chastellier, C., Fréhel, C., Offredo, C., and Skamene, E. (1993) Implication of phagosome-lysosome fusion in restriction of Mycobacterium avium growth in bone marrow macrophages from genetically resistant mice. Infect Immun 61: 37753784.
  • De Chastellier, C., Lang, T., and Thilo, L. (1995) Phagocytic processing of the macrophage endoparasite, Mycobacterium avium, in comparison to phagosomes which contain Bacillus subtilis or latex beads. Eur J Cell Biol 68: 167182.
  • Clemens, D.L., and Horwitz, M.A. (1995) Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited. J Exp Med 181: 257270.
  • Clemens, D.L., and Horwitz, M.A. (1996) The Mycobacterium tuberculosis phagosome interacts with early endosomes and is accessible to exogenously administered transferrin. J Exp Med 184: 13491355.
  • Clemens, D.L., Lee, B.-Y., and Horwitz, M.A. (2000) Deviant expression of Rab5 on phagosomes containing the intracellular pathogens Mycobacterium tuberculosis and Legionella pneumophila is associated with altered phagosomal fate. Infect Immun 68: 26712684.
  • Crowle, A., Dahl, R., Ross, E., and May, M. (1991) Evidence that vesicles containing live virulent M. tuberculosis or M. avium in cultured human macrophages are not acidic. Infect Immun 59: 18231831.
  • Cuellar-Mata, P., Jabado, N., Liu, J., Finlay, B.B., Gros, P., and Grinstein, S. (2002) Nramp1 modifies the fusion of Salmonella typhimurium-containing vacuoles with cellular endomembranes in macrophages. J Biol Chem 277: 22582265.
  • Deretic, V., and Fratti, R.A. (1999) Mycobacterium tuberculosis phagosome. Mol Microbiol 31: 16031609.
  • Desjardins, M. (1995) Biogenesis of phagolysosomes: the ‘kiss and run’ hypothesis. Trends Cell Biol 5: 183186.
  • Dey, R., and Datta, S.C. (1994) Leishmanial glycosomes contain superoxide dismutase. Biochem J 301: 317319.
  • Forbes, J.R., and Gros, P. (2001) Divalent metal transport by NRAMP proteins at the interface of host–parasite interactions. Trends Microbiol 9: 397403.
  • Fréhel, C., De Chastellier, C., Lang, T., and Rastogi, N. (1986) Evidence for inhibition of fusion of lysosomal and prelysosomal compartments with phagosomes in macrophages infected with pathogenic Mycobacterium avium. Infect Immun 52: 252262.
  • Fréhel, C., De Chastellier, C., Offredo, C., and Berche, P. (1991) Intramacrophage growth of Mycobacterium avium during infection of mice. Infect Immun 59: 22072214.
  • Fréhel, C., Offredo, C., and De Chastellier, C. (1997) The phagosomal environment protects virulent Mycobacterium avium from killing and destruction by clarithromycin. Infect Immun 65: 27922802.
  • Goswami, T., Bhattacharjee, A., Babal, P., Searle, S., Moore, E., Li, M., and Blackwell, J.M. (2001) Natural-resistance-associated macrophage protein 1 is an H+/bivalent cation antiporter. Biochem J 354: 511519.
  • Govoni, G., Canonne-Hergaux, F., Pfeifer, C.G., Marcus, S.L., Mills, S.D., Hackam, D.J., Grinstein, S. et al. (1999) Functional expression of Nramp1 in vitro in the murine macrophage line RAW267.4. Infect Immun 67: 22252232.
  • Greenwood, C.M., Fujiwara, T.M., Boothroyd, L.J., Miller, M.A., Frappier, D., Fanning, E.A. et al. (2000) Linkage of tuberculosis to chromosome 2q35 loci, including NRAMP1, in a large aboriginal Canadian family. Am J Hum Genet 67: 405416.
  • Gruenheid, S., Cellier, M., Vidal, S., and Gros, P. (1995) Identification and characterization of a second mouse Nramp gene. Genomics 25: 514521.
  • Gruenheid, S., Pinner, E., Desjardins, M., and Gros, P. (1997) Natural resistance to infection with intracellular pathogens: The Nramp1 protein is recruited to the membrane of the phagosome. J Exp Med 185: 717730.
  • Gruenheid, S., Canonne-Hergaux, F., Gauthier, S., Hackam, D.J., Grinstein, S., and Gros, P. (1999) The iron transport protein Nramp2 is an integral membrane protein that co-localizes with transferrin in recycling endosomes. J Exp Med 189: 831841.
  • Guérin, I., and De Chastellier, C. (2000a) Pathogenic mycobacteria disrupt the macrophage actin filament network. Infect Immun 68: 26552662.
  • Guérin, I., and De Chastellier, C. (2000b) Disruption of the actin filament network affects delivery of endocytic contents marker to phagosomes with early endosome characteristics: The case of phagosomes with pathogenic mycobacteria. Eur J Cell Biol 79: 735749.
  • Gunshin, H., Mackenzie, B., Berger, U.V., Gunshin, Y., Romero, M.F., Boron, W.F., Nussberger, S. et al. (1997) Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 388: 482488.
  • Hackam, D.J., Rotstein, O.D., Zhang, W., Demaurex, N., Woodside, M., Tsai, O., and Grinstein, S. (1997) Regulation of phagosomal acidification. J Biol Chem 272: 2981029820.
  • Hackam, D.J., Rotstein, O.D., Zhang, W.J., Gruenheid, S., Gros, P., and Grinstein, S. (1998) Host resistance to intracellular infections: Mutation of Nramp1 impairs phagosomal acidification. J Exp Med 188: 351364.
  • Jabado, N., Jankowsky, A., Dougaparsad, S., Picard, V., Grinstein, S., and Gros, P. (2000) Natural resistance to intracellular infections: Nramp1 functions as a pH-dependent manganese transporter at the phagosomal membrane. J Exp Med 192: 12371248.
  • Lang, T., and De Chastellier, C. (1985) Fluid phase and mannose receptor-mediated uptake of horseradish peroxidase in mouse bone marrow-derived macrophages. Biochemical and ultrastructural study. Biol Cell 53: 149154.
  • Lundrigan, M.D., Arceneaux, J.E., Zhu, W., and Byers, B.R. (1997) Enhanced hydrogen peroxide sensitivity and altered protein expression in iron-starved Mycobacterium smegmatis. Biometals 10: 215225.
  • Malo, D., Vogan, K., Vidal, S., Hu, J., Cellier, M., Schurr, E., Fuks, A. et al. (1994) Haplotype mapping and sequence analysis of the mouse Nramp gene predict susceptibility to infection with intracellular parasites. Genomics 23: 5161.
  • Morsczeck, C., Berger, S., and Plum, G. (2001) The macrophage-induced gene (mig) of Mycobacterium avium encodes a medium-chain acyl-coenzyme A synthetase. Biochim Biophys Acta 1521: 5965.
  • Oh, Y.-K., and Straubinger, R.M. (1996) Intracellular fate of Mycobacterium avium: use of dual-label spectrofluorometry to investigate the influence of bacterial viability and opsonization on phagosomal pH and phagosome–lysosome interaction. Infect Immun 64: 319325.
  • Orci, L., Halban, P., Perrelet, A., Amherdt, M., Ravazzola, M., and Anderson, R.G.W. (1994) PH-independent and-dependent cleavage of proinsulin in the same secretory vesicle. J Cell Biol 126: 11491156.
  • Ozawa, H., Picart, R., Barret, A., and Tougard, C. (1994) Heterogeneity in the pattern of distribution of the specific hormonal product and secretogranins within the secretory granules of rat prolactin cells. J Histochem Cytochem 42: 10971107.
  • Picard, V., Govoni, G., Jabado, N., and Gros, P. (2000) Functional analysis of mammalian Nramp2 in intact cells by a calcein quenching assay. J Biol Chem 275: 3573835745.
  • Plum, G., and Clark-Curtiss, J.E. (1994) Induction of Mycobacterium avium gene expression following phagocytosis by human macrophages. Infect Immun 62: 476483.
  • Plum, G., Brenden, M., Clark-Curtiss, J.E., and Pulverer, G. (1997) Cloning, sequencing, and expression of the mig gene of Mycobacterium avium, which codes for a secreted macrophage-induced protein. Infect Immun 65: 45484557.
  • Russell, D.G. (2001) Mycobacterium tuberculosis: here today, and here tomorrow. Nature Rev Molec Cell Biol 2: 19.
  • Russell, D.G., Dant, J., and Sturgill-Koszycki, S. (1996) Mycobacterium avium and Mycobacterium tuberculosis-containing vacuoles are dynamic, fusion-competent vesicles that are accessible to glycosphingolipids from the host cell plasmalemma. J Immunol 156: 47644773.
  • Schaible, U.E., Sturgill-Koszycki, S., Schlesinger, P.H., and Russell, D.G. (1998) Cytokine activation leads to acidification and increases maturation of Mycobacterium avium-containing phagosomes in murine macrophages. J Immunol 160: 12901296.
  • Searle, S., Bright, N.A., Roach, T.I.A., Atkinson, P.G.P., Barton, H., Meloen, R.H., and Blackwell, J.M. (1998) Localisation of Nramp1 in macrophages: modulation with activation and infection. J Cell Sci 111: 28552866.
  • Skamene, E., Schurr, E., and Gros, P. (1998) Infection genomics: Nramp1 as a major determinant of natural resistance to intracellular infections. Annu Rev Med 49: 275287.
  • Sturgill-Koszycki, S., Schlesinger, P.H., Chakraborty, P., Haddix, P.L., Collins, H.L., Fok, A.K., Allen, R.D. et al. (1994) Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 263: 678681.
  • Sturgill-Koszycki, S., Schaible, N.E., and Russell, D.G. (1996) Mycobacterium-containing phagosomes are accessible to early endosomes and reflect a transitional state in normal phagosome biogenesis. EMBO J 15: 69606968.
  • Tsolis, R.M., Baumler, A.J., and Heffron, F. (1995) Role of Salmonella typhimurium Mn-superoxide dismutase (SodA) in protection against early killing by J774A macrophages. Infect Immun 63: 17391744.
  • Ullrich, H.J., Beatty, W.L., and Russell, D.G. (1999) Direct delivery of pro-cathepsin D to phagosomes: implications for phagosome biogenesis and parasitisim by Mycobacterium. Eur J Cell Biol 78: 739748.
  • Ullrich, H.-J., Beatty, W.L., and Russell, D.G. (2000) Interaction of Mycobacterium avium – containing phagosomes with the antigen presentation pathway. J Immunol 165: 60736080.
  • Via, L.E., Deretic, D., Ulmer, R.J., Hibler, N.S., Huber, L.A., and Deretic, V. (1997) Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab 5 and rab7. J Biol Chem 272: 1332613331.
  • Vidal, S.M., Malo, D., Vogan, K., Skamene, E., and Gros, P. (1993) Natural resistance to infection with intracellular parasites: isolation of a candidate for Bcg. Cell 73: 469485.
  • Vidal, S., Pinner, E., Lepage, P., Gauthier, S., and Gros, P. (1996) Natural resistance to intracellular infections: Nramp1 encodes a membrane phosphoglycoprotein absent in macrophages from susceptible (Nramp1D169) mouse strains. J Immunol 157: 35593568.
  • Weibel, E.R., Kistler, G.S., and Scherle, W.F. (1966) Practical stereological methods for morphometric cytology. J Cell Biol 30: 2338.
  • Xu, S., Cooper, A., Sturgill-Koszycki, S., Van Heyningen, T., Chatterjee, D., Orme, I., Allen, P. et al. (1994) Intracellular trafficking in Mycobacterium tuberculosis and Mycobacterium avium-infected macrophages. J Immunol 153: 25682578.
  • Zhang, Y., Lathigra, R., Garbe, T., Catty, D., and Young, D. (1991) Genetic analysis of superoxide dismutase, the 23kilodalton antigen of Mycobacterium tuberculosis. Mol Microbiol 5: 381391.