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INFECTION AND IMMUNITY, JUlY 1991, p. 2232-2238
0019-9567/91/072232-07$02.00/0 Copyright © 1991, American Society for Microbiology
Inhibition of Macrophage Phagosome-Lysosome Fusion by Salmonella typhimurium NANCY A. BUCHMEIERt* AND FRED HEFFRONt Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037 Received 13 March 1991/Accepted 1 April 1991 SalmoneUa typhimurium-infected macrophages were examined by electron microscopy to determine whether intracellular survival of S. typhimurium is associated with failure of bacteria containing phagosomes to fuse with secondary lysosomes. S. typhimurium 14028 actively inhibited phagosome-lysosome fusion and appeared to preferentially divide within unfused phagocytic vesicles. In comparison with Escherichia coli, S. typhimurium inhibited phagosome-lysosome fusion in peritoneal macrophages, J774 macrophages, and bone marrowderived macrophages from both BALB/c (ityS) and SWR/J (it') mice. The mechanism responsible for Salmonella inhibition of phagosome-lysosome fusion is unknown but requires viable salmonellae, is not blocked by opsonization with fresh normal mouse serum, and is not due to lipopolysaccharide. Inhibition of phagosome-lysosome fusion may play a critical role in survival of salmonellae within macrophages and in virulence. MATERIALS AND METHODS
Electron microscopic studies of macrophages infected with microbes have revealed that intracellular pathogens employ diverse survival strategies. For example, Mycobacterium tuberculosis, M. leprae, Legionella pneumophila, and Toxoplasma gondii inhibit phagosome fusion with lysosomes, thereby preventing exposure to toxic lysosomal contents (1, 10, 14, 18, 25). In contrast, Trypanosoma cruzi, Listeria monocytogenes, and Shigella flexneri lyse the phagosomal membrane and escape into the cytoplasm (7, 23, 28). A third group of microorganisms (Leishmania spp. and Mycobacterium lepraemurium) is found within the macrophage phagolysosomal compartment, where they apparently resist inactivation by lysosomal factors (22). Because Salmonella typhimurium LT2 has been observed within fused phagolysosomes (P/L) (3, 6), S. typhimurium has been placed in this third group. However, intracellular survival of S. typhimurium in macrophages has not been directly compared with fusion levels of phagocytic vesicles with lysosomes. Using a highly virulent S. typhimurium strain, 14028, which survives well in macrophages, we have reexamined P/L fusion in salmonella-infected macrophages in order to correlate P/L fusion levels with intracellular survival. Virulent S. typhimurium infections were compared with avirulent Escherichia coli infections in four different populations of macrophages with different susceptibilities to salmonella replication. In comparison with two avirulent E. coli strains, S. typhimurium was able to inhibit macrophage P/L fusion in all of the macrophage populations that we examined. Salmonella inhibition of P/L fusion was not dependent on either opsonization or the 0 side chains of lipopolysaccharide (LPS) but required viable salmonellae. The data also suggests that salmonellae may preferentially divide in unfused phagosomes.
Bacterial strains. Growth of virulent S. typhimurium ATCC 14028s and its rough variant has been described elsewhere (4). 14028s has a 50% lethal dose of less than 10 by intraperitoneal infection of susceptible mice. Avirulent E. coli strains HS (21) and JM103 (29) were grown in LB broth under conditions identical to those used for S. typhimurium. Macrophages. The culture and infection of bone marrowderived macrophages, resident peritoneal macrophages, and the J774 macrophagelike cell line have previously been described (4). Macrophages were derived from BALB/c mice (itys), which are susceptible to Salmonella infection, and SWRIJ mice (ity'), which are resistant. The in vitro assay for bacterial survival within macrophages was performed as previously described (4). Results are expressed as means and standard errors of counts from triplicate wells. Phagocytosis. Electron microscopic examination of the uptake of salmonellae by macrophages was performed as described by Horowitz (15). Salmonellae (109 CFU) were mixed with 6 x 106 bone marrow-derived macrophages in small conical tubes on ice. The tubes were spun, quickly warmed, and sampled by removing the supernatant and adding cold Karnovsky fixative to the pellet. Samples were postfixed with 1% OS04, dehydrated with ethanol, and embedded in Epon 812. Sections were double stained with uranyl acetate and lead citrate and examined under a Hitachi HU 12A electron microscope. P/L fusion. P/L fusion was detected by using electrondense thorium dioxide (Thoria Sol; Polysciences, Warrington, Pa.) to label secondary lysosomes. Thoria Sol was prepared as described by Straley and Harmon (27) and loaded into macrophages as described by Horowitz (14). A 0.17% solution of Thoria Sol was added to confluent monolayers of macrophages in 24-well plates. Cells were incubated for 3 h, washed twice with phosphate-buffered saline, and incubated for an additional 3 h. Macrophages were then washed again and infected at a multiplicity of 10 with either unopsonized bacteria incubated in 1% bovine serum albumin (BSA) or bacteria opsonized with fresh normal mouse serum (4). Unbound bacteria were removed by washing the macrophages four times, and cultures were further incubated for
Corresponding author. t Present address: Department of Pathology, University of California, San Diego, CA 92093. t Present address: Department of Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201. *
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B
FIG. 1. Transmission electron micrographs of phagocytosis of S. typhimurium by bone marrow-derived macrophages, 6.5 min after warming. Arrow points to a coated pit. Bars represent 1 ,um.
the times indicated. Samples were fixed and embedded in the 24-well plates and processed for electron microscopy as described above. Phagosomes containing both bacteria and granules of Thoria Sol were scored as P/L fused. For each sample, 250 to 350 intracellular bacteria were counted and the percentage of bacteria in fused phagosomes was calculated. Statistical analysis was performed by using the twotailed paired t test. RESULTS
Early events in phagocytosis. Salmonella-infected macrophages were examined immediately after infection to identify unique features involved in the internalization process. Bone marrow-derived macrophages were used since they support salmonella growth at levels similar to those supported by macrophages from the spleen, a major site of salmonella infection in vivo (4). Salmonella internalization was rapid. At 6.5 min, bacteria were present within membrane-bound vacuoles. Cellular processes were observed surrounding individual salmonellae (Fig. 1A) and fusing with the cell membrane to form membrane-enclosed phagosomes (Fig. 1B). Coated pits were visible in several phagosomes during formation (Fig. 1A, arrow). There was no difference between the appearance of unopsonized and opsonized bacteria during internalization. Coiling phagocytosis, which has been described with L. pneumophila (15), was not observed with S. typhimurium. Comparison of S. typhimurium and E. coli P/L fusion. The fusion of secondary lysosomes (labeled with thorium diox-
ide) to phagosomes containing either S. typhimurium or E. coli was examined after initial P/L fusion had occurred (4 to 4.5 h) and after intracellular salmonellae had been allowed to multiply (14 to 20 h). Salmonellae were observed in both fused and nonfused phagosomes at all times examined (Fig. 2A to C). Most E. coli, however, were found in thorium dioxide-containing phagosomes (Fig. 2D). Six independent direct comparisons between the P/L fusion rates of S. typhimurium and E. coli were made by using different macrophage populations. In all cases, P/L fusion was significantly lower for S. typhimurium than for E. coli (P < 0.0001) (Tables 1 to 3). In three experiments using bone marrowderived macrophages from BALB/c mice, 64, 87, and 87% of E. coli were in fused P/L, compared with 39, 46, and 49% for S. typhimurium. S. typhimurium also exhibited reduced P/L fusion in J774 macrophages and bone marrow-derived macrophages from SWRIJ mice. Failure of the salmonellaTABLE 1. P/L fusion in bacterially infected macrophages % in fused phagolysosomes
Type ofSamnle Salmonellae macrophagea Bone marrow derived J774
Viable
Heat killed
39 (201/512)b 41 (176/425)
77 (86/111) 80 (41/51)
E. coli HS
64 (94/147) 75 (68/91)
a Assayed after 4 h of incubation. Given in parentheses is the number bacteria in fused phagolysosomes/ total number of intracellular bacteria. b
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FIG. 2. Electron micrographs of bacterially infected macrophages. Secondary lysosomes were loaded with thorium dioxide (dark granules) to detect P/L fusion. Bacteria are present in both clear unfused vacuoles and fused P/L containing thorium dioxide. (A) S. typhimurium 14028s-infected bone marrow-derived macrophages (BALB/c), 4 h postinfection; (B) S. typhimurium 14028s-infected bone marrow-derived macrophages (SWR/J), 4 h postinfection; (C) S. typhimurium 14028s-infected J774 macrophages, 14 h postinfection; (D) E. coli JM103-infected bone marrow-derived macrophages (BALB/c), 4 h postinfection; (E) heat-killed S. typhimurium 14028s-infected bone marrow-derived macrophages (BALB/c), 17 h postinfection; (F) S. typhimurium 14028r-infected bone marrow-derived macrophages (SWR/J), 20 h postinfection. All bacteria were opsonized with fresh normal mouse serum. Bars represent 1 ,um.
containing phagosomes to fuse with lysosomes was not due to lack of identifiable lysosomes, since numerous thorium dioxide-containing vacuoles were evident within each section. The large clear zone surrounding each bacterium was probably due to bacterial shrinkage during fixation as has been described for Shigella flexneri (24). Do killed salmonellae inhibit P/L fusion? P/L fusion levels observed with viable salmonellae were compared with those observed with salmonellae killed by either heating for 10 min at 65TC or fixation with gluteraldehyde for 1 h on ice. Heat-killed salmonellae failed to inhibit P/L fusion and more closely resembled E. coli than did viable salmonellae (Fig. 2E). In bone marrow-derived macrophages, 77% of heatkilled salmonellae were in fused P/L, compared with 39% of viable salmonellae; in J774 macrophages, 80% of the heatkilled salmonellae were in fused P/L, compared with 41% of viable salmonellae (P < 0.008) (Table 1). Salmonellae inactivated by gluteraldehyde also failed to inhibit P/L fusion (data not shown). Effect of opsonization of P/L fusion. The previous experiments were performed with bacteria opsonized with fresh normal mouse serum which contains low levels of antisalmonella antibody and complement. To determine whether opsonization of S. typhimurium or E. coli altered P/L fusion rates, we compared fusion of bacteria opsonized with fresh normal serum with that of bacteria preincubated with 1% BSA. At 4.5 h, after initial P/L fusion had taken place, opsonization had no effect on P/L fusion in peritoneal and J774 macrophages and caused a slight but insignificant reduction of P/L fusion in bone marrow-derived macrophages infected with either S. typhimurium or E. coli (P < 0.3) (Table 2). At 14 h, after bacterial division had occurred, considerably more opsonized salmonellae were observed in nonfused vacuoles in two of three types of macrophages examined. Opsonization therefore appeared responsible for
increased numbers of salmonellae in nonfused phagosomes in the long term but had no effect on E. coli P/L fusion. Effect of LPS on P/L fusion. To examine whether the outer layer of salmonella LPS is responsible for reduced P/L fusion, we compared S. typhimurium 14028s with a rough isogenic strain (14028r) which lacks 0 side chains. 14028r was found to inhibit P/L fusion as well as did 14028s (Fig. 2F). Differences in P/L fusion rates between 14028r and 14028s were statistically insignificant (P < 0.5) and were considerably lower than for E. coli (Table 3). Comparison of salmonella inhibition of P/L fusion in different macrophage populations. Macrophages from varying sources differ in microbicidal activity against salmonellae (4). For example, S. typhimurium grows best in J774 macrophages, survives moderately well in bone marrow-derived macrophages, and is killed most efficiently by peritoneal macrophages. We wanted to determine whether P/L fusion levels would reflect the differential survival of salmonellae in these macrophage populations. P/L levels of bone marrowderived macrophages infected with E. coli served as an avirulent comparison. Salmonella survival assays were performed as part of the same experiment in order to directly compare intracellular survival and P/L fusion. Initial salmonella P/L fusion levels (4.5 h) were very similar in the three populations of macrophages (P < 0.6) (Table 2). Significant differences in P/L fusion levels were observed only at 14 h with opsonized bacteria. In J774 macrophages, only 8% of intracellular salmonellae were in fused vacuoles. This low P/L fusion level was observed at the same time as a 10-fold increase in the numbers of intracellular salmonellae had taken place (Fig. 3). In bone marrow-derived macrophages, the salmonella P/L fusion rate was 46% at 14 h. This was associated with moderate salmonella survival. Salmonella P/L fusion levels in peritoneal macrophages was 31% at 14 h, while salmonella survival in these cells was low. P/L fusion
TABLE 2. Effect of opsonization and source of macrophages on P/L fusion % in fused phagolysosomes
Type of macrophage and time (h)
Bone marrow derived (BALB/c) 4.5 14 J774 4.5 14 Peritoneal (BALB/c) 4.5 14
E. coli JM103 E._coli_JM103
Salmonellae Salmonellae
Opsonizeda
Unopsonizedb
Opsonized
Unopsonized
33 (83/250)c 46 (160/350)
52 (186/354) 52 (132/254)
42 (125/298)
66 (109/164) 80 (24/30)
29 (99/341) 8 (23/293)
27 (37/135) 68 (84/123)
23 (59/259) 31 (59/192)
25 (40/162) 77 (105/136)
Opsonized with fresh normal mouse serum. b Preincubated with 1% BSA. Given in parentheses is the number bacteria in fused phagolysosomes/total number of intracellular bacteria.
a
c
87 (35/40)
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TABLE 3. Effect of LPS and mouse strain on P/L fusion in bone marrow-derived macrophages Strain and time (h)
BALB/c 4 20 SWR/J
% in fused phagolysosomes
S. typhimurium
E. coli JM103
14028s
14028r
31 (121/395)49 (112/228)
56 (100/179) 35 (90/255)
82 (360/436) 87 (332/380)
24 (71/294) 79 (357/452) 4 34 (105/308) 95 (238/250) 23 (56/239) 20 52 (146/282) a Given in parentheses is the number bacteria in fused phagolysosomes/ total number of intracellular bacteria.
in E. coli-infected bone marrow-derived macrophages was 87%, which was markedly greater than for any of the salmonella-infected macrophages at 14 h and corresponded with low numbers of viable intracellular E. coli. Appearance of dividing salmonellae inside macrophages. We observed three Salmonella bacteria in the process of division (Fig. 4). Each of these dividing bacteria was in an unfused phagosome. A pinching inward of the phagosome membrane at the point where bacterial septation is occurring is clearly visible in Fig. 4A and B and partially visible in Fig. 4C, suggesting that the phagocytic vacuole is also dividing. If
the vacuole surrounding individual salmonellae divides with each bacterium, this might explain why we usually observed bacteria occurring singly in vacuoles, even at later stages of infection. Notice the salmonellae in individual vacuoles in J774 macrophages 14 h after infection when several rounds of salmonella division had taken place (Fig. 2C). This is in contrast to what has been observed in Salmonella choleraesuis-infected epithelial cells. S. cholerae-suis replicates within MDCK cells in large vacuoles filled with multiple
106
IL
1o4
Hours
FIG. 3. Viable intracellular bacteria from infected macrophages. Each point is the mean of three samples plus the standard error of the mean. Percentage of bacteria observed in fused P/L is given beside each point. P/L fusion was detected by using thorium dioxide to label lysosomes; 250 to 350 intracellular bacteria for each point were counted. Symbols: 0, salmonella-infected J774 macrophages; *, salmonella-infected bone marrow-derived macrophages; O, salmonella-infected resident peritoneal macrophages; O, E. coli-infected bone marrow-derived macrophages.
FIG. 4. S. typhimurium 14028s dividing inside macrophages. (A) Bone marrow-derived macrophages (BALB/c), 14 h postinfection; (B) bone marrow-derived macrophages (SWR/J), 20 h postinfection; (C) J774 macrophages, 4 h postinfection. Bars represent 1 ,um.
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bacteria (9). This finding suggests major differences in bacterial requirements for intracellular survival within macrophages and epithelial cells, a result also suggested by studies using macrophage-sensitive mutants (11). DISCUSSION S. typhimurium 14028 inhibits fusion of phagosomes with secondary lysosomes in murine macrophages. In six direct comparisons, the level of P/L fusion for salmonella-containing phagosomes was approximately 55% of that seen with E. coli. S. typhimurium 14028 was used to relate P/L fusion levels to intracellular survival because it is a virulent strain and survives well inside macrophages. Although we have not used other Salmonella strains, inhibition of P/L fusion may be a common theme for S. typhimurium. In another direct comparison with E. coli, S. typhimurium LT2 was reported to inhibit P/L fusion in mouse peritoneal macrophages (16). Kagaya et al. (19) have also detected large numbers of S. typhimurium LT2 in unfused phagosomes. The long-term culture of peritoneal macrophages before infection is reported to greatly enhance P/L fusion levels (20) and may be responsible for the high levels of S. typhimurium P/L fusion initially reported by others (6). Inhibition of P/L fusion requires viable salmonellae. Neither heat-killed nor gluteraldehyde-fixed salmonellae were able to inhibit P/L fusion. The suggestion that inhibition of P/L fusion by S. typhimurium is the result of an active bacterial process is further supported by the studies of Ishibashi and Arai using UV-inactivated or streptomycintreated LT2 (16). S. typhimurium inhibition of P/L fusion does not require a complete LPS, nor is it affected by opsonization with normal mouse serum. A rough isogenic strain of 14028 was as capable as the smooth 14028 strain of inhibiting P/L fusion. Inhibition of P/L fusion in LT2 is also independent of LPS phenotype; in fact, LPS mutants of LT2 inhibit P/L fusion slightly better than does the wild-type parent (16). Our studies with 14028s showed that opsonization with normal mouse serum did not influence the initial rate of P/L fusion. Joiner and coworkers (17) have shown that coating salmonellae with C3 or immunoglobulin G influences fusion with neutrophil-specific granules but not azurophilic granules. Ligand-receptor interactions may influence the fusion of salmonella-containing phagosomes with macrophage vesicles apart from secondary lysosomes, and this may contribute to the enhanced intracellular survival that we have observed with opsonized salmonellae (5a). Our data suggest, however, that the type of receptor (Fc, CR1, CR3, or other) that S. typhimurium uses for entry is not responsible for inhibition of fusion of phagosomes with secondary lysosomes. Inhibition of P/L fusion is associated with enhanced intracellular survival. Comparison of S. typhimurium with E. coli showed a strong correlation between inhibition of P/L fusion and survival of these two organisms within macrophages. There were 100 times more viable salmonellae than E. coli at 14 h in bone marrow-derived macrophages, while there were approximately half the number of salmonellae in fused phagolysosomes (Fig. 3). Differences in P/L fusion, however, can not fully explain the differential survival of salmonellae in the three macrophage populations that we examined. Salmonellae grow best in J774 macrophages, and P/L fusion was greatly reduced in these cells, especially at 14 h. More subtle differences, such as type and amount of granule contents, are probably responsible for the differences in
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salmonella survival observed between peritoneal and bone marrow-derived macrophages. Two points of data suggest that salmonellae may preferentially divide in nonfused phagosomes. First, an increase in viable salmonellae within J774 cells (Fig. 3, 14 h) occurred at the same time as an increase in the percentage of bacteria in unfused phagosomes. One possible explanation for this increase in the number of unfused phagosomes between 4 and 14 h is that salmonellae were dividing more frequently in the unfused phagosomes. The observed increase in unfused phagosomes took place in the macrophage population (J774) in which salmonellae actively grow. Second, while admittedly only a small number, the only intracellularly dividing salmonellae that we observed (Fig. 4) were in unfused phagosomes. S. typhimurium joins the group of intracellular microorganisms that are capable of inhibiting phagosome fusion with lysosomes. Although these microorganisms may use similar methods to protect themselves from exposure to toxic lysosomal contents, there may not be a common mechanism for inhibition of P/L fusion. This conclusion is suggested by differences associated with P/L fusion inhibition. For example, inactivated Nocardia asteroides retains its ability to inhibit P/L fusion (8), while T. gondii (17), M. leprae (25), M. microti (13), and now S. typhimurium do not. Similarly, coating with antibody is reported to have no effect on M. leprae P/L fusion inhibition (25), while opsonization partially blocks the inhibition of P/L fusion for L. pneumophila (14), T. gondii (18), and M. tuberculosis (2). M. microti may prevent P/L fusion by preventing the movement of lysosomes within the cell (13). T. gondii may modify the structure of the phagosome during phagocytosis, thereby preventing P/L fusion (17). Surface components of M. leprae and M. tuberculosis are thought to be responsible for inhibition of P/L fusion (10, 12). Cord factor, a cell wall glycolipid of Mycobacterium, Nocardia, and Corynebacterium spp., inhibits fusion between phospholipid vesicles (26). While we do not know how S. typhimurium inhibits P/L fusion, it appears to be an active process requiring viable salmonellae. We have recently demonstrated that salmonellae respond to the intracellular environment by the enhanced synthesis of over 30 proteins (5). Some of these induced proteins may be involved in modifying the phagosome membrane to prevent fusion with secondary lysosomes. Although resistance of S. typhimurium to lysosomal contents may play a role in intracellular survival (6), our observations demonstrate that S. typhimurium is capable of inhibiting P/L fusion, and this ability has the potential for contributing to the organism's intracellular survival and virulence. ACKNOWLEDGMENTS We thank C. Chang for skillful assistance with the electron microscopy, D. Hone for the gift of E. coli HS, and F. Bowe and S. Libby for critical review of the manuscript. This research was supported by NIH grant Al 22933.
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wild-type Salmonella typhimurium and macrophage-sensitive mutants in diverse population of macrophages. Infect. Immun. 57:1-7. 5. Buchmeier, N. A., and F. Heffron. 1990. Induction of Salmonella stress proteins upon infection of macrophages. Science 248:730732. 5a.Buchmeier, N. A., and F. Heffron. Unpublished data. 6. Carrol, M. E. W., P. S. Jackett, V. R. Aber, and D. B. Lowrie. 1979. Phagolysosome formation, cyclic adenosine 3':5'-monophosphate and the fate of Salmonella typhimurium within mouse peritoneal macrophages. J. Gen. Microbiol. 110:421-429. 7. Clerc, P. L., A. Ryter, J. Mounier, and P. Sansonetti. 1987. Plasmid-mediated early killing of eucaryotic cells by Shigella flexneri as studied by infection of J774 macrophages. Infect. Immun. 55:521-527. 8. Davis-Scibienski, C., and B. L. Beaman. 1980. Interaction of Nocardia asteroides with rabbit alveolar macrophages: association of virulence, viability, ultrastructural damage, and phagosome-lysosome fusion. Infect. Immun. 28:610-619. 9. Finlay, B. B., and S. Falkow. 1988. Comparison of the invasion strategies used by Salmonella cholerae-suis, Shigells flexneri and Yersinia enterocolitica to enter cultured cells. Biochimie 70:1089-1099. 10. Frehel, C., and N. Rastogi. 1987. Mycobacterium leprae surface components intervene in the early phagosome-lysosome fusion inhibition event. Infect. Immun. 55:2916-2921. 11. Gahring, L., F. Heffron, B. Finlay, and S. Falkow. 1990. Invasion and replication of Salmonella typhimurium in animal cells. Infect. Immun. 58:443-448. 12. Goren, M., P. D. Hart, M. Young, and J. Armstrong. 1976. Prevention of phagosome-lysosome fusion in cultured macrophages by sulfatides of Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 73:2510-2514. 13. Hart, P. D., M. R. Young, A. H. Gordon, and K. H. Sullivan. 1987. Inhibition of phagosome-lysosome fusion in macrophages by certain Mycobacteria can be explained by inhibition of lysosomal movements observed after phagocytosis. J. Exp. Med. 166:933-946. 14. Horwitz, M. 1983. The legionnaires' disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J. Exp. Med. 158:2108-2126. 15. Horwitz, M. 1984. Phagocytosis of the legionnaires' disease bacterium (Legionella pneumophila) occurs by a novel mechanism: engulfment within a pseudopod coil. Cell 36:27-33. 16. Ishibashi, Y., and T. Arai. 1990. Specific inhibition of phagosome-lysosome fusion in murine macrophages mediated by
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Inhibition of Macrophage Phagosome-Lysosome Fusion by Salmonella typhimurium NANCY A. BUCHMEIERt* AND FRED HEFFRONt Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037 Received 13 March 1991/Accepted 1 April 1991 SalmoneUa typhimurium-infected macrophages were examined by electron microscopy to determine whether intracellular survival of S. typhimurium is associated with failure of bacteria containing phagosomes to fuse with secondary lysosomes. S. typhimurium 14028 actively inhibited phagosome-lysosome fusion and appeared to preferentially divide within unfused phagocytic vesicles. In comparison with Escherichia coli, S. typhimurium inhibited phagosome-lysosome fusion in peritoneal macrophages, J774 macrophages, and bone marrowderived macrophages from both BALB/c (ityS) and SWR/J (it') mice. The mechanism responsible for Salmonella inhibition of phagosome-lysosome fusion is unknown but requires viable salmonellae, is not blocked by opsonization with fresh normal mouse serum, and is not due to lipopolysaccharide. Inhibition of phagosome-lysosome fusion may play a critical role in survival of salmonellae within macrophages and in virulence. MATERIALS AND METHODS
Electron microscopic studies of macrophages infected with microbes have revealed that intracellular pathogens employ diverse survival strategies. For example, Mycobacterium tuberculosis, M. leprae, Legionella pneumophila, and Toxoplasma gondii inhibit phagosome fusion with lysosomes, thereby preventing exposure to toxic lysosomal contents (1, 10, 14, 18, 25). In contrast, Trypanosoma cruzi, Listeria monocytogenes, and Shigella flexneri lyse the phagosomal membrane and escape into the cytoplasm (7, 23, 28). A third group of microorganisms (Leishmania spp. and Mycobacterium lepraemurium) is found within the macrophage phagolysosomal compartment, where they apparently resist inactivation by lysosomal factors (22). Because Salmonella typhimurium LT2 has been observed within fused phagolysosomes (P/L) (3, 6), S. typhimurium has been placed in this third group. However, intracellular survival of S. typhimurium in macrophages has not been directly compared with fusion levels of phagocytic vesicles with lysosomes. Using a highly virulent S. typhimurium strain, 14028, which survives well in macrophages, we have reexamined P/L fusion in salmonella-infected macrophages in order to correlate P/L fusion levels with intracellular survival. Virulent S. typhimurium infections were compared with avirulent Escherichia coli infections in four different populations of macrophages with different susceptibilities to salmonella replication. In comparison with two avirulent E. coli strains, S. typhimurium was able to inhibit macrophage P/L fusion in all of the macrophage populations that we examined. Salmonella inhibition of P/L fusion was not dependent on either opsonization or the 0 side chains of lipopolysaccharide (LPS) but required viable salmonellae. The data also suggests that salmonellae may preferentially divide in unfused phagosomes.
Bacterial strains. Growth of virulent S. typhimurium ATCC 14028s and its rough variant has been described elsewhere (4). 14028s has a 50% lethal dose of less than 10 by intraperitoneal infection of susceptible mice. Avirulent E. coli strains HS (21) and JM103 (29) were grown in LB broth under conditions identical to those used for S. typhimurium. Macrophages. The culture and infection of bone marrowderived macrophages, resident peritoneal macrophages, and the J774 macrophagelike cell line have previously been described (4). Macrophages were derived from BALB/c mice (itys), which are susceptible to Salmonella infection, and SWRIJ mice (ity'), which are resistant. The in vitro assay for bacterial survival within macrophages was performed as previously described (4). Results are expressed as means and standard errors of counts from triplicate wells. Phagocytosis. Electron microscopic examination of the uptake of salmonellae by macrophages was performed as described by Horowitz (15). Salmonellae (109 CFU) were mixed with 6 x 106 bone marrow-derived macrophages in small conical tubes on ice. The tubes were spun, quickly warmed, and sampled by removing the supernatant and adding cold Karnovsky fixative to the pellet. Samples were postfixed with 1% OS04, dehydrated with ethanol, and embedded in Epon 812. Sections were double stained with uranyl acetate and lead citrate and examined under a Hitachi HU 12A electron microscope. P/L fusion. P/L fusion was detected by using electrondense thorium dioxide (Thoria Sol; Polysciences, Warrington, Pa.) to label secondary lysosomes. Thoria Sol was prepared as described by Straley and Harmon (27) and loaded into macrophages as described by Horowitz (14). A 0.17% solution of Thoria Sol was added to confluent monolayers of macrophages in 24-well plates. Cells were incubated for 3 h, washed twice with phosphate-buffered saline, and incubated for an additional 3 h. Macrophages were then washed again and infected at a multiplicity of 10 with either unopsonized bacteria incubated in 1% bovine serum albumin (BSA) or bacteria opsonized with fresh normal mouse serum (4). Unbound bacteria were removed by washing the macrophages four times, and cultures were further incubated for
Corresponding author. t Present address: Department of Pathology, University of California, San Diego, CA 92093. t Present address: Department of Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201. *
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B
FIG. 1. Transmission electron micrographs of phagocytosis of S. typhimurium by bone marrow-derived macrophages, 6.5 min after warming. Arrow points to a coated pit. Bars represent 1 ,um.
the times indicated. Samples were fixed and embedded in the 24-well plates and processed for electron microscopy as described above. Phagosomes containing both bacteria and granules of Thoria Sol were scored as P/L fused. For each sample, 250 to 350 intracellular bacteria were counted and the percentage of bacteria in fused phagosomes was calculated. Statistical analysis was performed by using the twotailed paired t test. RESULTS
Early events in phagocytosis. Salmonella-infected macrophages were examined immediately after infection to identify unique features involved in the internalization process. Bone marrow-derived macrophages were used since they support salmonella growth at levels similar to those supported by macrophages from the spleen, a major site of salmonella infection in vivo (4). Salmonella internalization was rapid. At 6.5 min, bacteria were present within membrane-bound vacuoles. Cellular processes were observed surrounding individual salmonellae (Fig. 1A) and fusing with the cell membrane to form membrane-enclosed phagosomes (Fig. 1B). Coated pits were visible in several phagosomes during formation (Fig. 1A, arrow). There was no difference between the appearance of unopsonized and opsonized bacteria during internalization. Coiling phagocytosis, which has been described with L. pneumophila (15), was not observed with S. typhimurium. Comparison of S. typhimurium and E. coli P/L fusion. The fusion of secondary lysosomes (labeled with thorium diox-
ide) to phagosomes containing either S. typhimurium or E. coli was examined after initial P/L fusion had occurred (4 to 4.5 h) and after intracellular salmonellae had been allowed to multiply (14 to 20 h). Salmonellae were observed in both fused and nonfused phagosomes at all times examined (Fig. 2A to C). Most E. coli, however, were found in thorium dioxide-containing phagosomes (Fig. 2D). Six independent direct comparisons between the P/L fusion rates of S. typhimurium and E. coli were made by using different macrophage populations. In all cases, P/L fusion was significantly lower for S. typhimurium than for E. coli (P < 0.0001) (Tables 1 to 3). In three experiments using bone marrowderived macrophages from BALB/c mice, 64, 87, and 87% of E. coli were in fused P/L, compared with 39, 46, and 49% for S. typhimurium. S. typhimurium also exhibited reduced P/L fusion in J774 macrophages and bone marrow-derived macrophages from SWRIJ mice. Failure of the salmonellaTABLE 1. P/L fusion in bacterially infected macrophages % in fused phagolysosomes
Type ofSamnle Salmonellae macrophagea Bone marrow derived J774
Viable
Heat killed
39 (201/512)b 41 (176/425)
77 (86/111) 80 (41/51)
E. coli HS
64 (94/147) 75 (68/91)
a Assayed after 4 h of incubation. Given in parentheses is the number bacteria in fused phagolysosomes/ total number of intracellular bacteria. b
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FIG. 2. Electron micrographs of bacterially infected macrophages. Secondary lysosomes were loaded with thorium dioxide (dark granules) to detect P/L fusion. Bacteria are present in both clear unfused vacuoles and fused P/L containing thorium dioxide. (A) S. typhimurium 14028s-infected bone marrow-derived macrophages (BALB/c), 4 h postinfection; (B) S. typhimurium 14028s-infected bone marrow-derived macrophages (SWR/J), 4 h postinfection; (C) S. typhimurium 14028s-infected J774 macrophages, 14 h postinfection; (D) E. coli JM103-infected bone marrow-derived macrophages (BALB/c), 4 h postinfection; (E) heat-killed S. typhimurium 14028s-infected bone marrow-derived macrophages (BALB/c), 17 h postinfection; (F) S. typhimurium 14028r-infected bone marrow-derived macrophages (SWR/J), 20 h postinfection. All bacteria were opsonized with fresh normal mouse serum. Bars represent 1 ,um.
containing phagosomes to fuse with lysosomes was not due to lack of identifiable lysosomes, since numerous thorium dioxide-containing vacuoles were evident within each section. The large clear zone surrounding each bacterium was probably due to bacterial shrinkage during fixation as has been described for Shigella flexneri (24). Do killed salmonellae inhibit P/L fusion? P/L fusion levels observed with viable salmonellae were compared with those observed with salmonellae killed by either heating for 10 min at 65TC or fixation with gluteraldehyde for 1 h on ice. Heat-killed salmonellae failed to inhibit P/L fusion and more closely resembled E. coli than did viable salmonellae (Fig. 2E). In bone marrow-derived macrophages, 77% of heatkilled salmonellae were in fused P/L, compared with 39% of viable salmonellae; in J774 macrophages, 80% of the heatkilled salmonellae were in fused P/L, compared with 41% of viable salmonellae (P < 0.008) (Table 1). Salmonellae inactivated by gluteraldehyde also failed to inhibit P/L fusion (data not shown). Effect of opsonization of P/L fusion. The previous experiments were performed with bacteria opsonized with fresh normal mouse serum which contains low levels of antisalmonella antibody and complement. To determine whether opsonization of S. typhimurium or E. coli altered P/L fusion rates, we compared fusion of bacteria opsonized with fresh normal serum with that of bacteria preincubated with 1% BSA. At 4.5 h, after initial P/L fusion had taken place, opsonization had no effect on P/L fusion in peritoneal and J774 macrophages and caused a slight but insignificant reduction of P/L fusion in bone marrow-derived macrophages infected with either S. typhimurium or E. coli (P < 0.3) (Table 2). At 14 h, after bacterial division had occurred, considerably more opsonized salmonellae were observed in nonfused vacuoles in two of three types of macrophages examined. Opsonization therefore appeared responsible for
increased numbers of salmonellae in nonfused phagosomes in the long term but had no effect on E. coli P/L fusion. Effect of LPS on P/L fusion. To examine whether the outer layer of salmonella LPS is responsible for reduced P/L fusion, we compared S. typhimurium 14028s with a rough isogenic strain (14028r) which lacks 0 side chains. 14028r was found to inhibit P/L fusion as well as did 14028s (Fig. 2F). Differences in P/L fusion rates between 14028r and 14028s were statistically insignificant (P < 0.5) and were considerably lower than for E. coli (Table 3). Comparison of salmonella inhibition of P/L fusion in different macrophage populations. Macrophages from varying sources differ in microbicidal activity against salmonellae (4). For example, S. typhimurium grows best in J774 macrophages, survives moderately well in bone marrow-derived macrophages, and is killed most efficiently by peritoneal macrophages. We wanted to determine whether P/L fusion levels would reflect the differential survival of salmonellae in these macrophage populations. P/L levels of bone marrowderived macrophages infected with E. coli served as an avirulent comparison. Salmonella survival assays were performed as part of the same experiment in order to directly compare intracellular survival and P/L fusion. Initial salmonella P/L fusion levels (4.5 h) were very similar in the three populations of macrophages (P < 0.6) (Table 2). Significant differences in P/L fusion levels were observed only at 14 h with opsonized bacteria. In J774 macrophages, only 8% of intracellular salmonellae were in fused vacuoles. This low P/L fusion level was observed at the same time as a 10-fold increase in the numbers of intracellular salmonellae had taken place (Fig. 3). In bone marrow-derived macrophages, the salmonella P/L fusion rate was 46% at 14 h. This was associated with moderate salmonella survival. Salmonella P/L fusion levels in peritoneal macrophages was 31% at 14 h, while salmonella survival in these cells was low. P/L fusion
TABLE 2. Effect of opsonization and source of macrophages on P/L fusion % in fused phagolysosomes
Type of macrophage and time (h)
Bone marrow derived (BALB/c) 4.5 14 J774 4.5 14 Peritoneal (BALB/c) 4.5 14
E. coli JM103 E._coli_JM103
Salmonellae Salmonellae
Opsonizeda
Unopsonizedb
Opsonized
Unopsonized
33 (83/250)c 46 (160/350)
52 (186/354) 52 (132/254)
42 (125/298)
66 (109/164) 80 (24/30)
29 (99/341) 8 (23/293)
27 (37/135) 68 (84/123)
23 (59/259) 31 (59/192)
25 (40/162) 77 (105/136)
Opsonized with fresh normal mouse serum. b Preincubated with 1% BSA. Given in parentheses is the number bacteria in fused phagolysosomes/total number of intracellular bacteria.
a
c
87 (35/40)
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TABLE 3. Effect of LPS and mouse strain on P/L fusion in bone marrow-derived macrophages Strain and time (h)
BALB/c 4 20 SWR/J
% in fused phagolysosomes
S. typhimurium
E. coli JM103
14028s
14028r
31 (121/395)49 (112/228)
56 (100/179) 35 (90/255)
82 (360/436) 87 (332/380)
24 (71/294) 79 (357/452) 4 34 (105/308) 95 (238/250) 23 (56/239) 20 52 (146/282) a Given in parentheses is the number bacteria in fused phagolysosomes/ total number of intracellular bacteria.
in E. coli-infected bone marrow-derived macrophages was 87%, which was markedly greater than for any of the salmonella-infected macrophages at 14 h and corresponded with low numbers of viable intracellular E. coli. Appearance of dividing salmonellae inside macrophages. We observed three Salmonella bacteria in the process of division (Fig. 4). Each of these dividing bacteria was in an unfused phagosome. A pinching inward of the phagosome membrane at the point where bacterial septation is occurring is clearly visible in Fig. 4A and B and partially visible in Fig. 4C, suggesting that the phagocytic vacuole is also dividing. If
the vacuole surrounding individual salmonellae divides with each bacterium, this might explain why we usually observed bacteria occurring singly in vacuoles, even at later stages of infection. Notice the salmonellae in individual vacuoles in J774 macrophages 14 h after infection when several rounds of salmonella division had taken place (Fig. 2C). This is in contrast to what has been observed in Salmonella choleraesuis-infected epithelial cells. S. cholerae-suis replicates within MDCK cells in large vacuoles filled with multiple
106
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1o4
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FIG. 3. Viable intracellular bacteria from infected macrophages. Each point is the mean of three samples plus the standard error of the mean. Percentage of bacteria observed in fused P/L is given beside each point. P/L fusion was detected by using thorium dioxide to label lysosomes; 250 to 350 intracellular bacteria for each point were counted. Symbols: 0, salmonella-infected J774 macrophages; *, salmonella-infected bone marrow-derived macrophages; O, salmonella-infected resident peritoneal macrophages; O, E. coli-infected bone marrow-derived macrophages.
FIG. 4. S. typhimurium 14028s dividing inside macrophages. (A) Bone marrow-derived macrophages (BALB/c), 14 h postinfection; (B) bone marrow-derived macrophages (SWR/J), 20 h postinfection; (C) J774 macrophages, 4 h postinfection. Bars represent 1 ,um.
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bacteria (9). This finding suggests major differences in bacterial requirements for intracellular survival within macrophages and epithelial cells, a result also suggested by studies using macrophage-sensitive mutants (11). DISCUSSION S. typhimurium 14028 inhibits fusion of phagosomes with secondary lysosomes in murine macrophages. In six direct comparisons, the level of P/L fusion for salmonella-containing phagosomes was approximately 55% of that seen with E. coli. S. typhimurium 14028 was used to relate P/L fusion levels to intracellular survival because it is a virulent strain and survives well inside macrophages. Although we have not used other Salmonella strains, inhibition of P/L fusion may be a common theme for S. typhimurium. In another direct comparison with E. coli, S. typhimurium LT2 was reported to inhibit P/L fusion in mouse peritoneal macrophages (16). Kagaya et al. (19) have also detected large numbers of S. typhimurium LT2 in unfused phagosomes. The long-term culture of peritoneal macrophages before infection is reported to greatly enhance P/L fusion levels (20) and may be responsible for the high levels of S. typhimurium P/L fusion initially reported by others (6). Inhibition of P/L fusion requires viable salmonellae. Neither heat-killed nor gluteraldehyde-fixed salmonellae were able to inhibit P/L fusion. The suggestion that inhibition of P/L fusion by S. typhimurium is the result of an active bacterial process is further supported by the studies of Ishibashi and Arai using UV-inactivated or streptomycintreated LT2 (16). S. typhimurium inhibition of P/L fusion does not require a complete LPS, nor is it affected by opsonization with normal mouse serum. A rough isogenic strain of 14028 was as capable as the smooth 14028 strain of inhibiting P/L fusion. Inhibition of P/L fusion in LT2 is also independent of LPS phenotype; in fact, LPS mutants of LT2 inhibit P/L fusion slightly better than does the wild-type parent (16). Our studies with 14028s showed that opsonization with normal mouse serum did not influence the initial rate of P/L fusion. Joiner and coworkers (17) have shown that coating salmonellae with C3 or immunoglobulin G influences fusion with neutrophil-specific granules but not azurophilic granules. Ligand-receptor interactions may influence the fusion of salmonella-containing phagosomes with macrophage vesicles apart from secondary lysosomes, and this may contribute to the enhanced intracellular survival that we have observed with opsonized salmonellae (5a). Our data suggest, however, that the type of receptor (Fc, CR1, CR3, or other) that S. typhimurium uses for entry is not responsible for inhibition of fusion of phagosomes with secondary lysosomes. Inhibition of P/L fusion is associated with enhanced intracellular survival. Comparison of S. typhimurium with E. coli showed a strong correlation between inhibition of P/L fusion and survival of these two organisms within macrophages. There were 100 times more viable salmonellae than E. coli at 14 h in bone marrow-derived macrophages, while there were approximately half the number of salmonellae in fused phagolysosomes (Fig. 3). Differences in P/L fusion, however, can not fully explain the differential survival of salmonellae in the three macrophage populations that we examined. Salmonellae grow best in J774 macrophages, and P/L fusion was greatly reduced in these cells, especially at 14 h. More subtle differences, such as type and amount of granule contents, are probably responsible for the differences in
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salmonella survival observed between peritoneal and bone marrow-derived macrophages. Two points of data suggest that salmonellae may preferentially divide in nonfused phagosomes. First, an increase in viable salmonellae within J774 cells (Fig. 3, 14 h) occurred at the same time as an increase in the percentage of bacteria in unfused phagosomes. One possible explanation for this increase in the number of unfused phagosomes between 4 and 14 h is that salmonellae were dividing more frequently in the unfused phagosomes. The observed increase in unfused phagosomes took place in the macrophage population (J774) in which salmonellae actively grow. Second, while admittedly only a small number, the only intracellularly dividing salmonellae that we observed (Fig. 4) were in unfused phagosomes. S. typhimurium joins the group of intracellular microorganisms that are capable of inhibiting phagosome fusion with lysosomes. Although these microorganisms may use similar methods to protect themselves from exposure to toxic lysosomal contents, there may not be a common mechanism for inhibition of P/L fusion. This conclusion is suggested by differences associated with P/L fusion inhibition. For example, inactivated Nocardia asteroides retains its ability to inhibit P/L fusion (8), while T. gondii (17), M. leprae (25), M. microti (13), and now S. typhimurium do not. Similarly, coating with antibody is reported to have no effect on M. leprae P/L fusion inhibition (25), while opsonization partially blocks the inhibition of P/L fusion for L. pneumophila (14), T. gondii (18), and M. tuberculosis (2). M. microti may prevent P/L fusion by preventing the movement of lysosomes within the cell (13). T. gondii may modify the structure of the phagosome during phagocytosis, thereby preventing P/L fusion (17). Surface components of M. leprae and M. tuberculosis are thought to be responsible for inhibition of P/L fusion (10, 12). Cord factor, a cell wall glycolipid of Mycobacterium, Nocardia, and Corynebacterium spp., inhibits fusion between phospholipid vesicles (26). While we do not know how S. typhimurium inhibits P/L fusion, it appears to be an active process requiring viable salmonellae. We have recently demonstrated that salmonellae respond to the intracellular environment by the enhanced synthesis of over 30 proteins (5). Some of these induced proteins may be involved in modifying the phagosome membrane to prevent fusion with secondary lysosomes. Although resistance of S. typhimurium to lysosomal contents may play a role in intracellular survival (6), our observations demonstrate that S. typhimurium is capable of inhibiting P/L fusion, and this ability has the potential for contributing to the organism's intracellular survival and virulence. ACKNOWLEDGMENTS We thank C. Chang for skillful assistance with the electron microscopy, D. Hone for the gift of E. coli HS, and F. Bowe and S. Libby for critical review of the manuscript. This research was supported by NIH grant Al 22933.
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