1 Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany
2 Institut für Zellbiologie, Ludwig-Maximilians-Universität, Schillerstr. 42, D-80336 München, Germany
3 Zentrum Biochemie, Medizinische Fakultät der Universität zu Köln, Joseph-Stelzmann-Str. 52, D-50931 Köln, Germany
4 Department of Clinical and Biological Sciences, University Turin, Ospedale S. Luigi, Orbassano 10043, Italy
Correspondence
Michael Steinert
michael.steinert{at}mail.uni-wuerzburg.de
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ABSTRACT |
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INTRODUCTION |
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In human macrophages, phagocytosis of L. pneumophila occurs through binding to complement receptors CR1, CR3 and other host-cell receptors. The coating of L. pneumophila with specific antibodies results in FcR-mediated phagocytosis (Jacob et al., 1994; Payne & Horwitz, 1987
). In the host amoeba Hartmannella vermiformis the bacteria attach to a 170 kDa galactose/N-acetylgalactosamine-inhibitable lectin which results in the dephosphorylation of paxillin, vinculin and pp125FAK (Venkataraman et al., 1998
). In addition, it was found that 33 amoebal proteins were induced and 11 other amoebal proteins were repressed upon contact with L. pneumophila (Abu Kwaik et al., 1994
). The resulting phagosomes which sequentially associate with vesicles and other organelles undergo neither acidification nor phagolysosomal fusion. Within this reprogrammed and maturation-blocked phagosome intracellular legionellae down-regulate a number of virulence traits including those that prevent the fusion with the lysosomal compartment. Evidence that the vacuole in which L. pneumophila replicates has characteristics of the endoplasmic reticulum (ER) came from electron microscopy and the detection of the ER protein BiP (Abu Kwaik, 1996
; Swanson & Isberg, 1995
; Roy, 2002
). During later phases of infection the bacteria-containing vacuoles merge with the lysosomal compartment (Swanson & Hammer, 2000
).
In recent years much progress has been made toward the characterization of how L. pneumophila modifies the phagosome maturation. Since the reprogramming of the phagocytic pathway occurs between the initial attachment and the replicative phase of the bacteria, it is highly likely that cell-surface components and secreted factors are involved in this process. Accordingly it has been shown that the Dot/Icm secretion system plays a pivotal role in macrophages and protozoa (Hilbi et al., 2001). Factors which are exported via this secretion system obviously separate the L. pneumophila-containing phagosome from the endosomal pathway (Segal et al., 1999
).
The molecular analysis of host-cell functions during L. pneumophila infection is much less understood. So far it has been shown that early L. pneumophila-containing phagosomes of macrophages lack major histocompatibility complex (MHC) class I and class II molecules, alkaline phosphatases and other membrane proteins. Further markers which are excluded from the phagosome during the course of intravacuolar growth of L. pneumophila are CD63, LAMP-1, LAMP-2, lysosomal cathepsin D, transferrin receptors and Rab7 (Hammer et al., 2002; Swanson & Hammer, 2000
). During the uptake by H. vermiformis, tyrosine-phosphorylated proteins including a 170 kDa receptor and various cytoskeleton-associated proteins are dephosphorylated (Abu Kwaik, 1998
; Venkataraman et al., 1998
). In macrophages, wild-type L. pneumophila induce phosphorylation of a 76 kDa protein (Yamamoto et al., 1992
). Also the caspase 3-dependent apoptosis in mammalian cells has been reported to be induced during early stages of infection (Müller et al., 1996
; Zink et al., 2002
). Nevertheless, an integrated view of how bacterial and host proteins interact and how the signalling cascade of the host is initiated and manipulated is still missing.
Due to its experimental versatility, Dictyostelium discoideum has proven to be a representative host-model system to analyse cellular aspects of L. pneumophila infection (Eichinger et al., 1999; Hägele et al., 2000
; Skriwan et al., 2002
; Solomon et al., 2000
). It has been reported that the sequence of events involved in particle uptake by D. discoideum resembles that of macrophages. The initial signal transduction to the actin system requires a heterotrimeric G-protein (Peracino et al., 1998
) and, as in mammalian cells, the phospholipase C (PLC) pathway is involved (Cardelli, 2001
; Seastone et al., 1999
). The phagosome matures by sequential transient fusion events with early and late endosomal compartments. A recent study demonstrated that macroautophagy is dispensable for the intracellular replication of L. pneumophila in D. discoideum. The L. pneumophila replicative vacuole is rather transformed by other means to resemble the rough ER (Otto et al., 2004
).
In this study we focused on the role of calcium, specific cytoskeleton-associated proteins and the two calcium-binding proteins calnexin and calreticulin during L. pneumophila infection of D. discoideum. Calreticulin is an ER luminal protein supplied with a K(H)DEL recognition signal and calnexin is an ER-specific type I transmembrane protein. Both proteins have been characterized as calcium storage proteins and several studies have indicated that changes in the concentration of calcium affect ER functions. Since L. pneumophila replicates in an ER-derived organelle, we set out to define the involved cell-signalling processes by the use of specific cellular inhibitors in phagocytosis assays. To examine host functions required for uptake and intracellular growth of L. pneumophila, a collection of well-defined D. discoideum mutants was investigated. Furthermore, by using green fluorescent protein (GFP)-transformed host cells we were able to localize the calcium-binding proteins calreticulin and calnexin during the infection process.
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METHODS |
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D. discoideum strains and growth conditions.
The Dictyostelium discoideum wild-type strain AX2, the mutant strains G (Peracino et al., 1998
), coronin (Maniak et al., 1995
),
-actinin/ABP120 AGHR2 (Rivero et al., 1996
), DAip1 (Konzok et al., 1999
), LimC, LimD, LimC/D (Khurana et al., 2002
), villidin (Gloss et al., 2003
), synexin (Döring et al., 1995
), calnexin, calreticulin and the gfp-transformed cells HG1738 and HG1767 (Müller-Taubenberger et al., 2001
) were grown at 23 °C either in shaking culture or in 75 cm2 cell-culture flasks with 10 ml HL5 medium [5 g yeast extract, 10 g glucose, 10 g proteose peptone (Oxoid), 0·64 g Na2HPO4, 0·48 g KH2PO4 dissolved in 1 l H2O, pH 7·5]. G418 (20 µg ml1) and/or blasticidin S (10 µg ml1) was added to the mutants and GFP-transformed cells. Generation of spores and storage of D. discoideum cells were performed as described previously (Hägele et al., 2000
).
Infection of D. discoideum cells and effect of cellular inhibitors on phagocytosis.
For infection, D. discoideum cells were harvested and resuspended in a 1 : 1 solution of HL5 medium and Sörensen phosphate buffer, pH 6·0, as described recently (Skriwan et al., 2002). The experiments were performed at 24·5 °C in 25 cm2 cell-culture flasks. In order to determine the effect of cellular inhibitors and host-cell mutations on bacterial uptake 1 ml of 10x106 host cells ml1 were co-incubated with L. pneumophila Corby at a m.o.i. of 10. For the inhibitor studies, D. discoideum AX2 cells were pretreated with various drugs (Sigma, ICN Biochemicals, Calbiochem) for 30 min (see Table 1
). According to previous publications, a broad range of different drug concentrations was used for each inhibitor (Peracino et al., 1998
): neomycin sulfate (0·1100 µM, IC50 110 µm), U73122 (0·550 µM, IC50 12·1 µM), U73343 (0·550 µM), EDTA (11000 µM), EGTA (11000 µM), BAPTA AM (501000 µM), thapsigargin (1500 nM). The drugs were tested for antibiotic effects and maintained during the entire experiment. Following an invasion period of 2 h, the remaining extracellular bacteria were killed by a gentamicin treatment (100 µg ml1); the D. discoideum cells were washed three times with Sörensen buffer and finally resuspended in a 1 : 1 solution of HL5 medium and Sörensen buffer. After this treatment, D. discoideum cells were removed from the culture flasks, and cell lysates were plated on BCYE agar.
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Transmission electron microscopy, in vivo microscopy and fluorescence imaging.
Transmission electron microscopy was performed as described previously (Hägele et al., 2000). Confocal imaging with or without the agar overlay technique (Köhler et al., 2000
, Yumura et al., 1984
) was performed with a Zeiss LSM 510 laser scanning microscope equipped with a 63x/1·4 Plan-Neofluar objective. GFP-transformed D. discoideum cells were harvested and washed twice with Sörensen buffer to remove antibiotics from the medium. The cells were allowed to adhere to glass coverslips for 5 min. For in vivo monitoring, bacteria were added at a m.o.i. of 10. For labelling of L. pneumophila and E. coli K-12 DH5
, bacteria were washed three times with PBS, pH 7·8, and resuspended in a solution of 0·3 mM 5- (and 6-) carboxytetramethylrhodamine, succinimidyl ester [5(6) TAMRA, SE] (Molecular Probes) in PBS. Bacteria were rotated slowly in the dark at room temperature for 30 min, washed four times with Tris/HCl (Applichem), pH 7·5, and finally resuspended in Sörensen buffer. Rhodamine-labelled L. pneumophila and E. coli were then added to the cells and incubated for 024 h and 06 h at 25 °C, respectively.
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RESULTS |
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The activation of PLC during L. pneumophila uptake was investigated by the inhibitors neomycin sulfate (inhibits PLC via binding to inositol phospholipids), U73122 (inhibits agonist-induced PLC activation) and U73343 (negative control for U73122). This analysis revealed that neomycin and U73122 reduced the uptake of L. pneumophila in a dose-dependent manner. Control experiments showed that the effect of neomycin sulfate was partly due to killing of the bacteria (16 %). Nevertheless, the PLC-related effect of neomycin sulfate remained significant (Table 1).
The role of calcium was determined by adding EDTA (extracellular chelator of calcium and magnesium), EGTA (extracellular chelator of calcium), BAPTA AM (intracellular calcium chelator) and thapsigargin (induces release of intracellular calcium by inhibition of the ER calcium ATPase). EDTA and EGTA exhibited an inhibitory effect on bacterial uptake at concentrations above 500 µM (84·2 % and 63·7 % phagocytosis of control, respectively). The differences between EDTA and EGTA can be explained by the lower affinity of EDTA for calcium (Bers, 1982; Shelling & Sykes, 1985
). BAPTA AM inhibited the uptake of L. pneumophila in a dose-dependent manner. The depletion of intracellular calcium stores by the addition of calcium ATPase inhibitor thapsigargin also resulted in a decrease of bacterial uptake. In summary, these results suggest that L. pneumophila uptake involves the PLC pathway and is modulated by changes of cytosolic calcium levels.
Effect of host-cell mutations on bacterial uptake and growth
A number of different proteins are known to regulate the phagocytic process. However, many interactions remain to be tested as to how cell signalling ultimately results in spatial and temporal changes of the cytoskeleton and membrane trafficking. In order to develop a roadmap of host-cell factors leading to the ER-associated replicative vacuole of L. pneumophila, we analysed the effects of three different classes of host-cell mutants: (i) G subunit of heterotrimeric G-proteins, (ii) cytoskeleton-associated proteins, as well as (iii) calcium-binding proteins. In order to differentiate between uptake and growth, we analysed intracellular bacterial numbers after 2 h post-infection (% of wild-type) and compared the growth rates during the exponential growth phase (2448 h post-infection) with the wild-type phenotype (Table 2
).
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The third group of mutants were lacking the calcium-binding proteins synexin/annexin C1, calnexin and calreticulin, respectively. D. discoideum synexin/annexin C1 is characterized by its ability to bind certain phospholipids in a calcium-dependent manner. The synexin-minus mutant showed a slight reduction in uptake of L. pneumophila (86·1 %) in our phagocytosis assay. In the gentamicin infection assay, L. pneumophila exhibited wild-type growth rates in synexin-minus host cells. Calreticulin and calnexin are calcium-binding proteins with chaperone activity in the ER (Müller-Taubenberger et al., 2001). The phagocytosis of L. pneumophila into calnexin-minus cells was reduced (66·4 %) and the intracellular growth rate of the bacteria was significantly lower (Fig. 1
). The calreticulin-minus cells showed an even more severe reduction in L. pneumophila uptake (43·3 %). The intracellular growth rate in this mutant was also reduced compared to wild-type host cells. Confocal laser scanning microscopy after different time points confirmed that the reduced bacterial numbers in the mutants were not only due to a defect in phagocytosis. Calreticulin- and calnexin-minus cells contained fewer bacteria within a single phagosome compared to phagosomes of wild-type cells. This indicates an intracellular growth defect of the bacteria within the phagosomes of the mutants. Taken together, these data suggest that both proteins have modulatory functions during phagocytic cup formation and the establishment of the replicative vacuole.
Intracellular distribution of GFP-tagged calnexin and calreticulin during L. pneumophila infection
Transmission electron microscopy of calnexin-minus and calreticulin-minus host cells demonstrated the association of the L. pneumophila-containing phagosomes with the rough ER (data not shown). This observation indicates that cellular trafficking of the bacteria in the mutant host cells is similar to the wild-type host cells (Solomon & Isberg, 2000). Since the intracellular calcium homeostasis is maintained primarily by the ER, we further analysed the distribution of calreticulin and calnexin upon infection. The previous visualization of GFP-tagged calnexin and calreticulin in D. discoideum revealed a physical connection of these proteins to the phagocytic cup during uptake of yeast particles and E. coli (Müller-Taubenberger et al., 2001
). To visualize the spatial and temporal distribution of both proteins during L. pneumophila uptake, we recorded phagocytosis in a confocal time series of wild-type cells using GFP-tagged calnexin or calreticulin. The phase-contrast sequence in Fig. 2
(a) (upper panel) shows the first engagement of the cell with L. pneumophila. The corresponding fluorescence images (lower panel) reveal a specific accumulation of GFP-tagged calnexin in the phagocytic cup which embraces the bacterium. A similar result with green fluorescence present in the protrusions on both sites of the cup was observed with GFP-tagged calreticulin (Fig. 2b
).
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DISCUSSION |
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In the present study we focused on early processes of L. pneumophila infection by using the LegionellaDictyostelium infection model (Hägele et al., 2000; Skriwan et al., 2002
; Solomon et al., 2000
). Phagocytosis in D. discoideum and macrophages is known to be regulated by a heterotrimeric G-protein-linked signal transduction. Inactivation of the gene encoding the G
subunit inhibits signal-induced actin polymerization (Damiani & Colombo, 2001
; Garin et al., 2001
). We were able to demonstrate that L. pneumophila uptake into D. discoideum cells involves the
-subunit of heterotrimeric G-proteins and the PLC pathway (Fig. 5
). These results suggest a conventional uptake of L. pneumophila by D. discoideum. This finding does not exclude the possibility that phagocytosis may be stimulated by secreted bacterial effectors (Hilbi et al., 2001
). In addition, our data also explain previous findings of increased bacterial growth of L. pneumophila in profilin-minus D. discoideum cells (Hägele et al., 2000
; Haugwitz et al., 1994
). Profilin up-regulates macropinocytosis and inhibits the PLC-regulated phagocytosis. In profilin-minus cells this regulation is shifted towards phagocytosis, which results in a higher rate of L. pneumophila uptake via phagocytosis (Cardelli, 2001
).
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Calcium is one of the most important signalling molecules of the cell. Changes in calcium levels appear to be essential for the induction of certain genes as well as for the activation of cytoskeletal components and enzymes. In our study the general role of calcium during L. pneumophila infection was determined by adding extracellular and intracellular chelators of calcium to phagocytosis assays. The extracellular chelators EDTA and EGTA exhibited inhibitory effects on bacterial uptake. BAPTA AM, a chelator of intracellular calcium, inhibited the uptake of L. pneumophila in a dose-dependent manner. The depletion of intracellular calcium stores by the addition of calcium ATPase inhibitor thapsigargin resulted in a moderate decrease of bacterial uptake. These results suggest that L. pneumophila uptake is modulated by changes of cytosolic calcium levels. Since calcium-mediated signal transduction is known to be essential for the activation of the phagocytic respiratory burst, production of nitric oxide, secretion of microbicidal granule constituents, and synthesis of proinflammatory mediators, it will be important to analyse the spatial alterations of cellular calcium concentrations (Malik et al., 2000). To obtain more specific insights into the role of calcium we chose different calcium-binding proteins for further investigations.
It was hypothesized that the calcium-buffering capacity of calreticulin and calnexin in the ER directly influences the calcium concentration in the narrow cytoplasmic zone of the phagocytic cup (Müller-Taubenberger et al., 2001). The release and reuptake of calcium between the cytosol and the ER also affect functions of the ER itself, including protein synthesis, protein secretion and chaperone activity. Since the L. pneumophila phagosome associates with the ER, the changes in calcium levels may be involved in this interaction. Our infection assays with calreticulin- and calnexin-minus mutants and the in vivo monitoring of GFP-tagged calreticulin and calnexin are a first indication in this respect. Another possibility is that calnexin and calreticulin play a role in the proper folding of proteins involved in infection. The firm association of the L. pneumophila phagosome and the GFP-tagged proteins extended beyond the uptake phase and was observed throughout the entire infection. The reasons as to why only the L. pneumophila-containing phagosome remained enveloped by calnexin and calreticulin, while the association of these proteins with the phagocytic cup of E. coli was transient remains to be elucidated. A recent study with bone marrow-derived macrophages from A/J mice suggests that L. pneumophila creates an ER-derived organelle by a process that involves intercepting early secretory vesicles exiting from transitional ER (Kagan & Roy, 2002
; Roy & Tilney, 2002
). The authors state that transport of L. pneumophila to the ER depends on ARF1 function and is distinct from ER-mediated phagocytosis.
Taken together our results show that L. pneumophila is internalized by phagocytosis which involves heterotrimeric G-proteins, the PLC pathway, and specific cytoskeleton-associated and calcium-binding proteins (Fig. 5). The observed effects of (i) the intracellular calcium concentrations on bacterial uptake, (ii) the reduced phagocytosis of L. pneumophila into calnexin- and calreticulin-minus cells, (iii) the impaired growth of L. pneumophila in both mutant host cells, and (iv) the permanent decoration of the replicative vacuole with calnexin and calreticulin suggest a strong modulatory function of these factors on L. pneumophila infection. The roadmap presented here lays the foundation for understanding the link of calcium signals, phagocytic cup formation and the establishment of the L. pneumophila-specific vacuole. Clearly, further work will be required to identify how L. pneumophila determines this intracellular route.
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ACKNOWLEDGEMENTS |
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Received 22 February 2004;
revised 7 June 2004;
accepted 17 June 2004.
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