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Howard Hughes Medical Institute,1 Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 021112
Received 20 December 2002/ Returned for modification 31 January 2003/ Accepted 20 March 2003
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ABSTRACT |
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INTRODUCTION |
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Some bacterial pathogens, including chlamydiae, mycobacteria, and legionellae, obtain nutrients and evade the immune system by growing within host cells (1). This has spawned interest in using host cells to perform genetic screens for host factors involved in intracellular pathogen infections. Here, we extended this analysis to the free-living unicellular soil amoebae Dictyostelium discoideum as a host for the intracellular bacterial pathogen Mycobacterium marinum.
M. marinum is closely related to Mycobacterium tuberculosis, one of the leading causes of infectious disease-related death (54). M. marinum causes systemic tuberculosis-like infections in fish, amphibians, and other ectotherms, involving persistent growth within macrophages (15, 40). M. marinum also causes human skin infections such as fish tank granuloma (20). The organism has several attractive features. It has a relatively short doubling time; its growth is limited by high temperature, reducing the risk of systemic infection in humans; and construction of mutations by homologous recombination is relatively easy (39). Like M. tuberculosis, M. marinum can evade the endocytic pathway after uptake by host cells (3), and factors associated with intracellular growth of this organism also appear to play a role in M. tuberculosis pathogenesis (10, 38, 48). Long-term infections of leopard frogs by M. marinum produce granulomas that have similarities to those seen in human tuberculosis infections (7). These data suggest that M. marinum is an excellent model for mycobacterial diseases.
Intracellular growth of mycobacterial species involves a unique trafficking pathway that blocks maturation of the phagolysosome and establishes a replicative compartment that maintains vesicular contact with the plasma membrane surface (23, 49). Thus far, few host factors that are necessary to support the establishment of this compartment have been identified. One host protein suggested to play a role in maintenance of this compartment is the phagosome-associated protein TACO, also known as coronin 1 in the mouse (16, 35, 44). Coronin isoforms are believed to be involved in promoting cytoskeletal remodeling of plasma membrane surfaces and phagosomal biogenesis in mammalian cells (13, 33). Mutants lacking the only characterized D. discoideum isoform of coronin have been isolated, and these strains show defects in particle phagocytosis, fluid phase uptake, migration, and cytokinesis (29, 41).
D. discoideum is a free-living soil amoeba which feeds on bacteria. The cells are easily maintained in the laboratory, and axenic strains of D. discoideum can be grown in the absence of bacteria in a rich medium (6). D. discoideum is highly motile and phagocytic, and many aspects of cytoskeletal function have been characterized (34, 53). Recently, D. discoideum has been established as a host cell for infection with several bacterial pathogens, including Legionella pneumophila (22, 46), Pseudomonas aeruginosa (37), and Mycobacterium avium (45). The attractions of the organism are that it is amenable to genetic analysis and the genome is being sequenced (26, 28, 30).
In this report, we demonstrate that M. marinum can infect and grow within D. discoideum cells. We show that M. marinum mutants that are defective in intracellular growth in macrophages are also defective for growth in D. discoideum cells. We also show that M. marinum growth is stimulated by the absence of the only characterized D. discoideum isoform of coronin.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M. marinum strains were kind gifts from L. Ramakrishnan (University of Washington Medical School). The msp12::gfp strain constitutively expresses GFP and grows efficiently in macrophages (10). GFP expression was maintained by adding the antibiotic apramycin to 20 µg/ml. Strains L1D and P59D contain kanamycin resistance insertions in adjacent genes (named mag24-1 and mag24-2, respectively) that encode members of the PE-PGRS family (38). The growth rate of P59D in macrophages is indistinguishable from that of isogenic wild-type controls, whereas strain L1D is impaired for growth in macrophages due to inactivation of mag24-1 (38). Both strains were grown in liquid medium and on plates containing kanamycin (20 µg/ml). All M. marinum strains were grown in Middlebrook 7H9 medium supplemented with Middlebrook OADC (10% oleic acid-albumin-dextrose complex; Becton Dickinson, Cockeysville, Md.) and 0.5% glycerol. Plates were solidified with 2% agar. Cultures of M. marinum were prepared by inoculating 10 ml of medium in a tissue culture flask from the freezer stock, incubating at 30°C for 1 week, and storing at 4°C for several weeks. To produce rapidly growing M. marinum for all experiments, the stored culture was diluted 1/10 in fresh medium and incubated at 30°C for 3 days prior to infections.
Live infection of D. discoideum with GFP-expressing M. marinum. To prepare D. discoideum for M. marinum infections, amoebae grown axenically in tissue culture dishes containing HL-5 medium (46) were harvested and washed in phosphate-buffered saline (PBS) as follows. Cells were pelleted by a 5-min spin at 600 x g, the medium was aspirated and replaced with an equal volume of PBS, the cells were pelleted again by a 5-min spin at 600 x g, and the PBS was aspirated. The cells were resuspended to 106 cells/ml in HL-5 medium containing 20 µg of apramycin/ml, disbursed to a glass-bottom 96-well plate (Corning), and allowed to equilibrate for at least 1 h at 25.5°C. M. marinum for infections was prepared by transferring the 3-day culture to a 50-ml conical tube and vortexing for 2 min at top speed with a tabletop vortexer. The approximate concentration of bacteria was determined at A600 by using a 1/10 dilution of culture, assuming that an A600 of 1.0 is equivalent to 7.7 x 107 bacterial/ml. M. marinum clump extensively, so the actual multiplicity of infection (MOI) can vary up to fivefold from this approximation. Viable counts were then performed on the dispersed culture.
D. discoideum were infected at a MOI of approximately 0.5. Infection was initiated by aspirating the medium from the wells and replacing it with HL-5 medium containing 20 µg of apramycin per ml and approximately 5 x 105 bacteria of the msp12::gfp strain per ml. The cells and bacteria were incubated for 3 h at 25.5°C. Non-cell-associated bacteria were removed by washing the cells twice with HL-5. Growth of extracellular bacteria was inhibited by addition of 5 µg of streptomycin per ml plus 20 µg of apramycin per ml in the final growth medium.
At various times the live cells were observed with a Nikon TE300 inverted phase microscope. The number of bacteria per cell was determined by direct observation, and images were captured by a Princeton Micromax slow-scan cooled charge-coupled device camera. The experiment was performed twice, counting 500 amoebae for each time point from multiple wells. No more than 20 cells were counted per field, so more than 25 fields were read for each time point.
Growth of P59D and L1D strains in D. discoideum. D. discoideum was infected as described above except that cells were plated in 24-well tissue culture plates, the MOI was 0.2, and cells were infected with either M. marinum strain L1D (mag24-1) or strain P59D (mag24-2).
Viable intracellular M. marinum cells were harvested and counted in the following fashion. Medium from a well was aspirated and replaced with 0.5 ml of PBS in order to remove streptomycin from the well. The PBS was aspirated, and the cells were lysed by adding 0.5 ml of PBS containing 0.1% Triton X-100 to release the intracellular bacteria. Dilutions of M. marinum were also performed in PBS containing 0.1% Triton X-100 to minimize bacterial clumping. The number of CFU of M. marinum was determined by plating dilutions of harvested bacteria on 7H9 containing 20 µg of kanamycin per ml. Plates were incubated for 7 days at 30°C before counting.
Transmission electron microscopy. D. discoideum amoebae were infected with M. marinum strains P59D and L1D as described above (MOI = 0.2). On day 8, cells from six wells of a 24-well plate were harvested and processed for electron microscopy exactly as described previously (46). Samples were analyzed on a Philips CM-10 transmission electron microscope.
Growth of M. marinum in AX2 and a D. discoideum strain lacking coronin. D. discoideum was infected as described above except that the MOI was 0.02. Both strains of D. discoideum were infected with the same mixture of M. marinum strain P59D and medium and thus were exposed to identical numbers of bacteria.
Viable intracellular M. marinum cells were counted by harvesting infected cells, using repetitive pipetting with a 1,000-µl automatic pipettor, and transferring to a microcentrifuge tube. Cells were pelleted in an Eppendorf microcentrifuge for 3.5 min at 8,000 rpm. The supernatant was aspirated and the pellet resuspended in 0.5 ml of PBS containing 0.1% Triton X-100. Dilutions and plating were as described above.
Polyclonal antibody directed against M. marinum. Exponentially growing M. marinum cells were harvested, pelleted, and washed twice with PBS. Cells were resuspended in PBS and fixed overnight at 4°C by adding paraformaldehyde to a final concentration of 3%. Fixed bacteria were washed three times in PBS and concentrated fivefold (approximately 3.5 x 108 bacteria/ml). Rats were immunized (Pocono Rabbit Farm and Laboratory, Canadensis, Pa.) with 40 µl of fixed M. marinum and complete Freund's adjuvant on day 0, 20 µl of fixed M. marinum and incomplete Freund's adjuvant on day 14, and 5 µl of fixed M. marinum and incomplete Freund's adjuvant on day 28. The rats were given boosters of 5 µl of fixed M. marinum every 28 days until a robust signal was seen by immunofluorescence, which took approximately 6 months of immunization.
Association of coronin-GFP with M. marinum phagosomes. The D. discoideum coronin-GFP strain expressing a coronin-GFP fusion was plated on poly-L-lysine-coated coverslips and infected with wild-type M. marinum at an MOI of two bacteria per amoeba as described above. After an overnight incubation (19 h), the cells were fixed for 1 h at room temperature in freshly made 4% paraformaldehyde containing 40 mM HEPES (pH = 7.4) and 6.5% sucrose. Samples were permeabilized by incubation with ice-cold methanol for 30 s, washed five times in PBS, and blocked in 4% goat serum (Gibco-BRL)-PBS for 1 h. M. marinum was visualized with a 1/100 dilution of polyclonal rat anti-M. marinum. After 1 h of incubation with primary antibody, the samples were washed five times in PBS, and anti-rat immunoglobulin G-Texas red was added at a 1/500 dilution (Molecular Probes). Samples were washed and processed as described elsewhere (51). Fixed samples were analyzed with a Nikon TE300 microscope, and images were captured by a Princeton Micromax slow-scan cooled charge-coupled device camera.
Analysis of sequence data. Comparison of ORFs with previously identified genes was performed by using the Basic Local Alignment Search Tool (BLASTP or tBLASTN) in the public National Center for Biotechnology Information databases. Searches for paralogs to the D. discoideum coronin protein were performed with the BLAST server for the unfinished D. discoideum sequencing project (http://dicty.sdsc.edu/). Sequence data for D. discoideum were obtained from the Genome Sequencing Centre Jena website at http://genome.imb-jena.de/dictyostelium/. These sequence data were also produced by the D. discoideum Sequencing Group at the Sanger Institute.
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RESULTS |
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When bacteria were introduced onto D. discoideum monolayers at an MOI of 0.5 and 25.5°C, luxuriant intracellular replication could be observed (Fig. 1). On day 1, the infected D. discoideum amoebae had few bacteria, with most cells having fewer than two bacteria per cell (Fig. 1A to C). The amoebae having more than one M. marinum cell probably internalized small clumps of two to eight bacteria (Fig. 1D to F). In contrast, at day 5, many of the infected amoebae appeared to be filled with M. marinum, and there were far too many bacteria in each cell to obtain an accurate count of intracellular bacteria (Fig. 1G to L). M. marinum did not grow in HL-5 medium with 5 µg of streptomycin per ml in the absence of cells (data not shown), indicating that the growth of M. marinum observed in this experiment was intracellular growth within D. discoideum.
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M. marinum was introduced into cultures of the wild-type control strain AX2 and the coronin mutant HG1569, by using procedures identical to those described above, and viable counts were determined at various times after infection (Fig. 5). The most striking phenotype was that within 3 h after infection, the absence of coronin enhanced the yield of intracellular bacteria compared to the wild-type control (Fig. 5A). This could be due to either increased uptake of the bacteria or enhanced survival of M. marinum after phagocytosis by the coronin mutant. Furthermore, the rate of intracellular replication also appeared to be enhanced (Fig. 5B), particularly during the first 3 days after initial infection. Both phenotypes were entirely reproducible, with some experiments showing that the rate of replication within the coronin mutant was sufficiently enhanced that by 7 days after initial infection, amoebae lysed and exposed the bacteria to the streptomycin in the medium, as visualized by a decrease in viable counts between 5 and 7 days postinfection (Fig. 5C). Coronin appears to limit the intracellular growth of M. marinum.
As the loss of coronin appeared to have the opposite effect of that predicted from previous work (16), we wanted to verify that the behavior of coronin in D. discoideum was similar to the behavior of TACO/coronin 1 during M. bovis BCG infections of mouse macrophages. Furthermore, although M. marinum is similar to M. bovis, the determinants of intracellular survival of these two organisms could be quite different. One key observation of previous work is that TACO/coronin 1 is sequestered and retained around the M. bovis BCG vacuole in mouse macrophages for extended periods (16), although in another study this persistence was not observed after infection of human macrophages (44). To analyze coronin sequestration in D. discoideum, a strain that expresses a coronin-GFP fusion as its only functional copy of coronin was challenged with M. marinum for 20 h, and the samples were fixed and immunoprobed for the bacteria. At this time point, the heat-killed control M. marinum was completely digested by the D. discoideum amoebae (data not shown). Analysis of amoebae challenged with the live bacteria showed that a fraction had associated coronin-GFP staining about the vacuole harboring M. marinum, although amoeba showing dense coronin staining about the phagosome were difficult to find. Figure 6 displays four typical examples of coronin-GFP sequestration around M. marinum phagosomes, showing the range of morphologies observed. Most of the phagosomes had faintly enhanced staining of coronin-GFP relative to the pools of protein found in the cell, making it difficult to quantitate the phagosomes having associated coronin (Fig. 6A to F). Occasionally, intense association of coronin-GFP with the compartment harboring the bacteria could be observed; however, the staining did not appear to be entirely circumferential (Fig. 6G to I). In addition, examples of compartments in which there was no clear association of coronin-GFP were found (Fig. 6J to L). We conclude that the D. discoideum compartment harboring M. marinum is heterogeneous with regard to coronin localization, with clear colocalization of the protein found in interspersed areas about the phagosome.
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DISCUSSION |
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We think it likely that our studies have underestimated the efficiency of intracellular replication of M. marinum, at least in a population of infected cells. Examination of infected D. discoideum cells by fluorescence microscopy indicated that many amoebae supporting intracellular growth had far too many bacteria to count, although we rarely observed more than a 10- to 20-fold increase in viable counts of the entire population over a 7-day incubation period. There are two explanations for this discordance. First, all incubations were performed in the presence of antibiotics to suppress extracellular growth, and heavily infected cells may have become permeable to the antibiotic. Second, there may be some heterogeneity and asynchrony in the replication of the bacteria within the amoebae (Fig. 2), with some D. discoideum cells lysing and exposing bacteria to the antibiotic long before the replication cycle is completed in other amoebae. This complication is also reflected in the fact that in these studies, the lag between initial infection of the amoebae and initiation of intracellular growth varied considerably between experiments, indicating there may have been some variation in the physiological states of the bacterial or amoebic cultures prior to infection.
Two other examples of replication center heterogeneity within D. discoideum were found. First, a pool of amoebae containing three to eight bacteria was observed in which the number of intracellular M. marinum cells did not appear to change over the course of the experiment (Fig. 2). It is possible that small clumps of bacteria internalized by a single amoeba are incapable of initiating intracellular growth and remain in either a dormant or an undigested state. Alternatively, infection of a single amoeba with multiple bacteria may be toxic for D. discoideum. M. marinum was found to cause considerable cytotoxicity to D. discoideum, even at relatively low MOIs (4:1). Perhaps the uptake of several M. marinum cells is enough to irreparably injure the host cell. Whatever the explanation, the presence of such heterogeneity was to be expected, as this has been observed with other mycobacteria in a variety of cell types and found to be difficult to eliminate (2, 11). Second, once replication was established within D. discoideum, we observed that the morphology of the phagosomes was heterogeneous. In some cases, phagosomes with large groups of bacteria could be observed, whereas in other cases, bacteria were found in their own individual phagosomes. Similar heterogeneity was observed during M. marinum granuloma formation in the frog (7). It is possible that M. marinum may use more than one tactic to establish and maintain replication within host cells and that this property is reflected in the different phagosome morphologies that we observed. This explanation implies that there may be more than one set of bacterial proteins capable of promoting intracellular growth, each set leading to formation of a compartment having a distinct morphology. Alternatively, it may be that there is one trafficking pattern for all vacuoles, but one of the morphologically distinct compartments is a precursor for the other.
The M. marinum mag24-1 mutant (strain L1D) has reduced growth in both macrophages and D. discoideum, indicating that intracellular replication in the two hosts may proceed via similar mechanisms. The mag24-1 mutant showed modest intracellular growth in the plating assay for viable bacteria (Fig. 3). When such infections were analyzed in more detail by electron microscopy, it appeared that most of the D. discoideum cells were infected with one or a few bacteria, with occasional cells showing small clusters of bacteria. Examples of luxuriant intracellular growth of this mutant, on the other hand, could not be found. This suggests that the mag24-1 mutation causes an early block in bacterial replication that can occasionally be bypassed. As the occasional bypass does not appear to result in efficient intracellular growth, the mutation may cause defects in both establishment and maintenance of replication within host cells.
The most surprising result from these studies was that the absence of coronin enhanced the intracellular growth of M. marinum. Previous work with M. bovis BCG strains has shown that mouse TACO/coronin 1 persists on the phagosome, and phagocytes that fail to sequester the protein cannot support intracellular survival of the organism (16). In a conflicting study in human macrophages, TACO/coronin 1 was reported to associate with the BCG-containing phagosome, but continued persistence was not observed (44). Those authors found persistence of TACO/coronin 1 about phagosomes only in cells that internalized clumps of bacteria. The latter study proposed that TACO/coronin 1 plays a role in the early steps involved in establishing the mycobacterial vacuole. We found a similar ambiguity in the localization of D. discoideum coronin about mycobacterial vacuoles, with intense colocalization being only rarely observed. Coronin in D. discoideum, however, cannot play a role in supporting either establishment or maintenance of a replication vacuole in D. discoideum. In fact, the improved survival and growth of bacteria in such a mutant indicates that the protein interferes with mycobacterial growth in D. discoideum.
That D. discoideum coronin mutants exhibit a variety of defects in phagocytosis and motility seems inconsistent with the observed increase in intracellular growth (29, 41), but we have observed similar stimulation of intracellular growth of L. pneumophila in D. discoideum mutants defective for phagocytosis (46). The simplest explanation for this phenomenon is that for many pathogens that grow within vacuoles of host cells, there is a unique uptake pathway that leads to formation of a replication compartment distinct from the default pathway of phagocytosis. As the default pathway traffics the microorganism into a compartment that is either degradative or incapable of supporting intracellular growth, it potentially competes with the route that leads to intracellular growth. Mutations that eliminate proteins necessary for default phagocytosis should remove this competition and enhance the efficiency of establishing a replication compartment. Since earlier work showed that D. discoideum coronin clearly is involved in the default pathway (29), it might have been predicted based on this model that the coronin mutant would be more proficient than wild-type strains at promoting intracellular growth of M. marinum. It should be noted that previous results indicating that the absence of coronin causes defective phagocytosis were based on assays using D. discoideum incubated in suspension culture. Many mutations affecting phagocytosis in suspension culture have little effect when analyzed with D. discoideum grown on solid supports, and all the assays performed with M. marinum in this study were done with amoebic monolayers, which may suppress the phagocytosis defect that is caused by the absence of coronin.
The involvement of the D. discoideum coronin protein in the default pathway of phagocytosis appears to contradict the proposal that establishment of an M. bovis BCG replication compartment requires an isoform of this protein. One explanation for these conflicting observations is that among the multiple members of the coronin protein family in mammals, some isoforms participate in phagocytosis of nonpathogens, whereas others are involved in establishment of intravacuolar growth. For instance, coronin 2 in mouse macrophages has been found on phagosomes harboring particles coated with complement component C3, indicating a role for coronin 2 in phagocytosis of nonpathogens, whereas there has been no report of TACO/coronin 1 on similar phagosomes harboring inert particles (33). The function of mammalian coronin 2 may be closely related to that of D. discoideum coronin, whereas TACO/coronin 1 may play a role in cellular physiology that is promoted by some other D. discoideum protein. The biochemical functions of members of this family are relatively obscure, and different isoforms could have very different activities or interacting partners. Comparison of the D. discoideum coronin sequence with its most closely related orthologs in mammalian cells does not give any hints as to whether it would be predicted to have a function corresponding to a particular mammalian isoform. Most of the amino acid changes that differentiate the mouse coronin isoforms are localized in the carboxyl termini of these proteins (47). Even though this particular region shows low sequence similarity among isoforms, the different mouse isoforms appear to be more highly similar to each other than to the D. discoideum coronin in this region. The other notable feature is that there are residues present in several isoforms that are missing in both TACO/coronin 1 and the D. discoideum coronin. This is the only link observed between these two proteins that potentially differentiates them from other family members.
Our results show that if TACO/coronin 1 plays a critical role in mycobacterial intracellular growth, its function can be replaced by factors other than coronin in D. discoideum, or else M. marinum and M. bovis BCG have very different requirements for establishing and maintaining a replication compartment within host cells. It is possible that the corresponding factors could be other uncharacterized coronin isoforms. D. discoideum has at least two other predicted ORFs that appear to be part of the coronin family, although they are rather divergent from mammalian coronins and are predicted to encode significantly larger polypeptides than other known members of the coronin family (Materials and Methods). Future work on the details of the molecular interactions promoted by different isoforms of coronin in both mammalian cells and D. discoideum should clarify this issue.
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ACKNOWLEDGMENTS |
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J.J.S. was supported by an NRSA fellowship. R.R.I. is an investigator of the Howard Hughes Medical Institute.
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FOOTNOTES |
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