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Infection and Immunity, September 1999, p. 4490-4498, Vol. 67, No. 9
Department of Medical Microbiology and
Hygiene, Institute for Microbiology and Immunology, University of
Ulm, Ulm, Germany
Received 19 March 1999/Returned for modification 7 May
1999/Accepted 7 June 1999
After uptake and intracellular multiplication of Legionella
pneumophila in MRC-5 lung fibroblasts, important cytoskeletal filament structures, like actin, tubulin, or vimentin, and a cell membrane-associated fibronectin were rearranged during early infection, resulting in a loss of cell adhesion and collapse of the cytoskeleton. Dysregulation of the cellular phosphorylation and dephosphorylation cascade may contribute to the observed changes and may support intracellular survival and multiplication of L. pneumophila. We therefore studied expression of phosphoproteins
during intracellular growth of L. pneumophila. By using an
anti-tyrosine phosphoprotein antibody we showed that proteins
phosphorylated on tyrosine residues accumulated progressively during
late infection exclusively around or in phagosomes filled with
bacteria. In contrast, expression of serine/threonine phosphoproteins
did not change. To discern the origin of phosphorylated proteins, the
host cells were treated with cycloheximide, an inhibitor of eukaryotic
protein synthesis. The newly synthesized proteins were labeled
metabolically with [35S]methionine-cysteine and
immunoprecipitated with a phosphotyrosine-specific antibody. Sodium
dodecyl sulfate gel electrophoresis gave evidence for synthesis of at
least three protein clusters (160 to 200, 35 to 60, and 19 to 28 kDa)
of Legionella origin that were phosphorylated on tyrosine
residues 24 h after infection. Treatment of infected host cells
with genistein, a tyrosine kinase inhibitor, revealed that tyrosine
protein phosphorylation was not important for bacterial uptake but
contributed to intracellular growth of L. pneumophila. Bacterial tyrosine phosphoproteins and the observed intracellular structural changes may be important to understanding the process involved in intracellular growth of L. pneumophila.
Intracellular microorganisms use the
intracellular environment of the host cell to avoid host defenses and
to survive and replicate. Microbial entry into mammalian cells is a
result of specific interactions between bacteria and host cells.
Bacteria and/or host cells respond to external events by transducing
information from outside into the cell. Signal transduction is based on
a phosphorylation cascade of signal-transducing proteins
(3).
In eukaryotes, the phosphorylation cascades generally employ serine,
threonine, and tyrosine phosphorylation. In contrast, bacterial
phosphorylation cascades are usually an internal histidine kinase-dependent transfer of a phosphate moiety to an aspartate carboxyl group on a responder protein (19, 35). However,
there is also an increasing number of studies reporting the utilization of a serine/threonine protein kinase in microbial signal transduction systems (2, 6, 8, 9).
Microbial pathogens may abuse host signal transduction enzymes as part
of their strategy to enter and survive within host cells. As shown for
Yersinia, Salmonella, and enteropathogenic Escherichia coli, the patterns of phosphorylation misuse are
tailored to the needs of the microorganism (3). Microbial
internalization factors of Salmonella typhimurium and
E. coli stimulate protein tyrosine kinase activity in the
host cells, while the microbial internalization factor of
Yersinia pseudotuberculosis is accompanied by the
downregulation of such activity (12, 15, 17, 31, 33).
Legionella pneumophila, a gram-negative, facultative
intracellular bacterium which is a parasite of protozoa in the aquatic environment (18) and a potential cause of pneumonia in
humans (26), leads to increased tyrosine phosphorylation
when infecting monocytes. As shown by Coxon et al. (7), 15 min after infection with L. pneumophila, protein bands of
75, 71, 59, 56, 53, and 52 kDa intensely stained with an
antiphosphotyrosine antibody had already appeared. The tyrosine
phosphorylation level returned to baseline during the following 60 min.
However, these changes were not pathogen specific. Coxon et al.
speculated that tyrosine phosphorylation patterns might be different
and perhaps pathogen specific when L. pneumophila infects
nonprofessional phagocytes, such as epithelial cells or MRC-5 cells
(7). We therefore investigated cytoskeleton changes and
phosphorylation in MRC-5 cells infected with virulent L. pneumophila.
Microorganism.
L. pneumophila serogroup 1 strain
Philadelphia I was obtained from the American Type Culture Collection,
Manassas, Va. (ATCC 33152). The bacteria were passaged twice on
buffered charcoal-yeast (BCYE) agar (Merck, Darmstadt, Germany),
harvested, and suspended to yield a concentration of 2 × 108 to 3 × 108 CFU per ml.
Cell line.
The human lung fibroblast line MRC-5 has been
described previously (ATCC CCL-171) (11, 28, 36). MRC-5
cells were cultured in Dulbecco modified Eagle medium (DMEM)
supplemented with 10% fetal calf serum and 2 mM glutamine (Gibco BRL,
Grand Island, N.Y.). Cells were passaged five times with a solution
containing 0.1% trypsin and 0.5 mM EDTA in complete medium. Prior to
infection, the MRC-5 cells were passaged twice.
Cell infection.
Bacteria (2 × 108 to
3 × 108/ml) were resuspended at appropriate
concentrations in serum-free DMEM and added to the cells at a
bacterium/cell ratio of 100:1 (in a typical experiment, we used 2 × 106 cells in a 100-cm2 petri dish to which
2 × 108 bacteria were added) at a time point defined
as time zero. The mixture of bacteria and cells was centrifuged at
400 × g for 10 min and incubated for an additional 50 min at 37°C in 5% CO2. The cells were then washed twice
to remove extracellular bacteria. The adherent bacteria that had not
been ingested by host cells were killed with an additional incubation
for 60 min at 37°C in cell culture medium containing 100 µg of
gentamicin per ml. For mock infection we used heat-inactivated L. pneumophila (60 min at 70°C) and nonpathogenic laboratory strain
E. coli HB101 (29). Each experiment was repeated
three to five times.
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Legionella pneumophila Invasion of MRC-5
Cells Induces Tyrosine Protein Phosphorylation
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Drug treatment. Host cell protein synthesis was inhibited by the addition of medium containing 100 µg of cycloheximide per ml to the cells 1 h prior to and during metabolic labeling with [35S]methionine-cysteine (5).
Inhibition of protein tyrosine phosphorylation was achieved by the addition of 100 µM genistein (Calbiochem, San Diego, Calif.). The concentration used was at least 10 times above the 50% inhibitory concentration (31). The MRC-5 cells were incubated with genistein for 4 and 24 h.Radioactive labeling and immunoprecipitation.
Biosynthetic
labeling of MRC-5 cells and intracellular growth of L. pneumophila were carried out as described previously
(36), with minor modifications. Semiconfluent MRC-5 cell
monolayers (100 cm2) were incubated for 30 min with 5 ml of
methionine-cysteine-free DMEM containing 10% dialyzed fetal calf serum
either at 4 or 24 h after infection, corresponding to time points
defined as early and late infection. Thereafter, the monolayers were
pulsed with 400 µCi of [35S]methionine-cysteine
(PRO-MIX; Amersham, Braunschweig, Germany) for an additional 30 or 120 min at 37°C. For immunoprecipitation the cells were rinsed three
times with cold phosphate-buffered saline (PBS) supplemented with 1 mM
Na3VO4. Newly synthesized cellular and
bacterial proteins were extracted in 3 ml of lysis buffer (50 mM
Tris-HCl [pH 8.3], 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 1 mM Na3VO4, 0.1 µg of
aprotinin/ml, 0.1 mg of ABTS [2,2'-azinobis 3-ethylbenzthiazoline
sulfonic acid], and 0.1 mg of leupeptin per ml). The lysates were
incubated for 30 min at 4°C on a cell mixer and then cleared by
centrifugation at 19,000 × g for 10 min. The
supernatants were either stored at 
70°C or directly used for immunoprecipitation.
Immunofluorescence staining and laser scanning confocal
microscopy.
For immunofluorescence, the MRC-5 cells were grown on
coverslips (2 cm2) to optimal cell density. At the time
points indicated below (early infection, 4 h postinfection
[p.i.]; late infection, 24 to 48 h p.i.), the uninfected,
mock-infected, or infected cells were washed twice with PBS containing
1 mM Na3VO4 and fixed with absolute methanol
for 20 min at 20°C. Subsequently, the coverslips were rinsed in
PBS, blocked with 2% goat serum in PBS, and incubated for 60 min at
room temperature with primary antibodies diluted as suggested by the
manufacturers. We used a mouse antiphosphotyrosine antibody (clone
PT-66) and mouse anti-phosphoserine/threonine monoclonal antibodies
(clones PTR-8 and PSR-45), all obtained from Sigma. The cell
cytoskeleton was assessed with a monoclonal mouse antiactin antibody
(clone AC-40; Sigma), mouse anti-
-tubulin antibodies (clone DM 1A;
Sigma), a mouse antibody to cell-associated fibronectin (clone FN-3E2;
Sigma), and a mouse antivimentin antibody (clone V9). Following
incubation with the primary antibodies, cells were washed three times
in PBS containing 2% goat serum and then incubated for 60 min with
affinity-purified fluorescein isothiocyanate (FITC)-conjugated goat
anti-rabbit (F 0382; Sigma) or goat anti-mouse (Southern Biotechnology,
Birmingham, Ala.) antibody diluted 1:70 in PBS. Subsequently, the
monolayers were washed three times in PBS. The coverslips were mounted
in Mowiol 4-88 (Calbiochem) containing 1%
1,4-diazobicyclo[2.2.2]octane to reduce fluorescence fading and 10 µg of propidium iodide per ml to stain intracellular bacteria and
host cell nuclei.
Statistical analysis. Efficiency of growth of L. pneumophila as determined by plating of Legionella on BCYE agar was calculated by analysis of covariance on rank-transformed data (34).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Modulation of intracellular events by intracellular growth of L. pneumophila. In order to obtain information on changes of important cell microfilaments, we analyzed the distribution and assembly of actin, tubulin, vimentin, and cell membrane-associated fibronectin during progressive infection of MRC-5 cells, which occurred in 40 to 50% of cells 48 h after infection. Figures 1 to 4 show representative pictures.
In uninfected cells immunofluorescence studies showed fine filamentous structures of F-actin. These structures were stretched longitudinally from one pole to the other in each cell (Fig. 1a). During the early-infection phase F-actin appeared in the form of stress fibers, but 48 h after infection, probably due to severe morphological changes of actin molecules, a collapse of the cytoskeleton occurred.
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Analysis of the expression of phosphoproteins during replication of L. pneumophila in MRC-5 cells. The cellular changes described above may be linked to the changes in phosphorylation of tyrosine or serine/threonine. Therefore, we analyzed the expression of different phosphoproteins and the kinetics of their phosphorylation.
In uninfected cells, the majority of the proteins which phosphorylated on serine/threonine residues were localized in or around nuclear areas and showed a fine granular expression pattern. A minor portion of the serine/threonine phosphoproteins appeared speckled diffusely in the cell cytoplasm (Fig. 2a). We did not see any marked changes in serine/threonine kinase activity, as measured by expression of the serine/threonine phosphoproteins, either 4 h after infection (early infection) or during intracellular replication of Legionella (48 h after infection [late infection]), but the micrographs suggest a slight redistribution of the serine/threonine phosphoproteins, especially at the areas of the nuclei. There was no obvious colocalization of serine/threonine phosphoprotein residues and L. pneumophila.
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Kinetics of the synthesis of tyrosine-phosphorylated proteins. In order to obtain data about the kinetics of the synthesis of phosphoproteins in broth-grown Legionella, uninfected and infected MRC-5 lung fibroblasts were metabolically labeled with [35S]methionine-cysteine. The phosphorylated proteins as well as Legionella-specific antigens were immunoprecipitated with antibodies listed above.
SDS-PAGE of immunoprecipitated antigens revealed that broth-grown L. pneumophila and uninfected fibroblasts had either an undetectable or a very low rate of endogenous phosphoprotein synthesis (Fig. 5a and b, respectively, lanes 3 and 4). However, during intracellular growth the biosynthesis of tyrosine phosphoproteins was heavily upregulated. The profile of proteins phosphorylated on tyrosine residues showed three clusters: a cluster of high-molecular-mass phosphoproteins (160 to 200 kDa), a middle-range cluster with 35- to 60-kDa proteins, and a low-weight cluster with three main phosphoproteins of apparently 19, 23, and 28 kDa (Fig. 5c and d, lanes 3).
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,
newly synthesized bacterial tyrosine phosphoproteins.
Effect of protein tyrosine inhibitors on intracellular growth of L. pneumophila. As a consequence of L. pneumophila infection, the tyrosine phosphorylation of different bacterial and host cell proteins occurred. Therefore, it was of interest to determine whether the observed protein phosphorylation was required for the effective initiation of infection and intracellular growth of L. pneumophila. To address this issue, we treated the MRC-5 cells with genistein, a potent tyrosine kinase inhibitor.
In the untreated cells the number of bacteria increased nearly 100 times within 24 h of infection. During the first 4 h after infection the number of ingested bacteria was not significantly affected by genistein (median value [range] at time zero, 5 × 104 CFU/ml [2 × 104 to 9 × 104 CFU/ml] without genistein versus 4 × 104 CFU/ml [2 × 104 to 4 × 104 CFU/ml] with genistein); however, prolonged incubation revealed slower multiplication of L. pneumophila (median value [range] at 24 h p.i., 730 × 104 CFU/ml [350 × 104 to 1,250 × 104 CFU/ml] without genistein versus 60 × 104 CFU/ml [10 × 104 to 160 × 104 CFU/ml] with genistein) (Table 1). Treatment of broth-grown L. pneumophila with genistein had no influence on its viability. Genistein at the applied dose did not significantly affect viability of MRC-5 cells, which was always in a range between 80 and 90%, as measured by trypan blue exclusion (7).| |
DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The present study shows that uptake of L. pneumophila by MRC-5 cells results in rearrangement of the cytoskeleton and phosphorylation of proteins at the tyrosine residue. As suggested by Coxon et al., MRC-5 cells as an example of nonprofessional phagocytes were used in order to find out if the mode of uptake influences intracellular phosphorylation (7). Further arguments for the use of the MRC-5 cell line were that epithelial cells or lung fibroblasts may also serve as Legionella host cells and that growth of Legionella in pneumocytes may even better reflect changes of virulence (16, 25, 37). In addition, the MRC-5 cell line has the advantage that it had already been used for Legionella infection experiments and that rearrangement of the cytoskeleton during infection can be well characterized (24, 37).
Some of the metabolically labeled protein bands which were stained with antiphosphotyrosine antibody resembled tyrosine-phosphorylated proteins of epithelial cells after invasion by Chlamydia trachomatis (64, 100, and 140 kDa) (2, 14), Mycoplasma penetrans (145 kDa) (1), or S. typhimurium (44 kDa) (32). It might be possible that one of the bands in the 50- to 60-kDa range corresponds to the p-53-Src tyrosine kinase (39). In contrast to Yamamoto et al., who investigated the phosphorylation pattern of Legionella-infected peritoneal macrophages (38), we did not find a tyrosine-phosphorylated 76-kDa protein. We were interested to find out whether one or more bands of the tyrosine-phosphorylated proteins were of Legionella origin and therefore treated the infected cells with cycloheximide, which shuts down protein synthesis of animal cells but does not modify protein synthesis by microbes or multiplication of Legionella. In spite of the cycloheximide pretreatment, six bands were stained with an antiphosphotyrosine antibody and could thus represent de novo synthesized Legionella proteins.
Several arguments suggest that some of the bands represent bacterial proteins. (i) They were synthesized in spite of cycloheximide treatment. (ii) Some bands have molecular masses similar to those of Legionella antigens (major outer member protein [28 kDa] [21], iron binding proteins of 90 kDa [27], and a heat shock protein of 60 kDa [20]). (iii) Tyrosine-phosphorylated proteins could be detected within the replicative vacuole. Residual eukaryotic protein synthesis, however, might also account for some of the bands detected. Nonspecific heavy staining by the antiphosphotyrosine antibody is rather improbable since the monoclonal antibody has been reported to be specific. It is impossible to determine if the observed effects are pathogen specific, since the control bacteria (live E. coli K-12 and heat-killed Legionella) do not enter the MRC-5 cells; however, nonspecific lipopolysaccharide-related effects can be excluded. An argument in favor of the specificity of the changes observed is that invasion of HeLa cells by C. trachomatis leads to tyrosine-phosphorylated proteins with molecular masses of 75 to 78 and 100 kDa, which are different from those detected in our host-pathogen system (14). Interestingly, in the experiments of Fawaz et al., phosphotyrosine-stained proteins could be seen within and in the vicinity of the Chlamydia-containing vacuoles (14).
It is not clear whether the Legionella proteins were phosphorylated by host or bacterial kinases. There are arguments for both hypotheses. Association of the phagosomes with the endoplasmic reticulum may enable kinases to obtain access to the bacterium. On the other hand, bacteria may also contain protein tyrosine kinases, as shown for Acinetobacter calcoaceticus (10, 13). Although uncommon, it is not unheard of that bacterial proteins might be translationally modified by phosphorylation. For Chlamydia psittaci it has been shown that IncA protein incorporated into the inclusion membrane is also phosphorylated (30). Listeria monocytogenes is an additional, well-studied example of a bacterium which produces a protein (ActA) which is phosphorylated following its secretion into the host cell (4). Similarly, during invasion by enteropathogenic E. coli, a 90-kDa bacterial protein was phosphorylated and then inserted into the host cell membrane (23). In addition, it has been shown that flagella of Pseudomonas aeruginosa can be phosphorylated (22).
The functions of the phosphorylation of bacterial proteins in the different host pathogen systems are clearly different. In enteropathogenic E. coli and Yersinia, the phosphorylation of a bacterial protein is a first step towards invasion and the bacterial protein is secreted by a type III secretion system into the host cell (23). As well, homologies to a type III secretion system have been found in chlamydiae during early infection, but it is not clear if antigens like IncA or IncB are secreted in a way similar to that of Enterobacteriaceae (30). In L. pneumophila, phosphorylation occurs late after invasion and a type III secretion system has not been identified.
Inhibition of tyrosine phosphorylation by genistein had a distinct
effect on intracellular bacterial multiplication, although the
bacterial counts for treated and untreated cells at 4 h p.i. appeared to be similar (Table 1). This is
in parallel to the more significant changes observed with
immunofluorescence and immunoprecipitation experiments. Different
mechanisms might be responsible for growth inhibition. Inhibition of
signal transduction could limit the uptake of nutrients via the
cellular membranes and thus limit growth of Legionella. On
the other hand, interruption of signal transduction might hinder the
ability of Legionella to misuse the intracellular
environment of MRC-5 cells for maximal intracellular multiplication. It
might be argued that genistein also had a toxic effect on the bacteria;
however, genistein-treated Legionella was as virulent in
cell culture assays as the control (untreated) Legionella.
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The experiments presented revealed that the intracellular environment changed dramatically after uptake and during intracellular multiplication of L. pneumophila and that for successful intracellular growth the phosphorylation of numerous proteins at tyrosine residues should occur.
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ACKNOWLEDGMENTS |
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We thank Steffanie Güntner and Sonja Weiß for excellent technical assistance.
This work was supported by University of Ulm Research Fund grant P374 and by the German Research Society (DFG).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Medical Microbiology and Hygiene, University of Ulm, Robert Koch Str. 8, 89081 Ulm, Germany. Phone: 49-731-502-4614. Fax: 49-731-502-4619. E-mail: milorad.susa{at}medizin.uni-ulm.de.
Editor: D. L. Burns
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Abstract
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Materials and Methods
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Discussion
References
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Infection and Immunity, September 1999, p. 4490-4498, Vol. 67, No. 9
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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