severe, atypical pneumonia of humans, i.e., Legionnaires' disease. The capacity of this pathogen to cause disease is dependent on its ability to invade and to ...
INFECTION AND IMMUNITY, Apr. 1993, p. 1320-1329 0019-9567/93/041320-10$02.00/0 Copyright © 1993, American Society for Microbiology
Vol. 61, No. 4
Phenotypic Modulation by Legionella pneumophila Infection of Macrophages
upon
YOUSEF ABU KWAIK,1 BARRY I. EISENSTEIN,,"2t AND N. CARY ENGLEBERG'l2* Departments of Microbiology and Immunology' and Internal Medicine,2 University of Michigan Medical School, Ann Arbor, Michigan 48109-0620 Received 22 September 1992/Accepted 17 January 1993
Since many pathogenic bacteria manifest a coordinate regulation of gene expression in response to different environmental stimuli, we examined the phenotypic response of Legionella pneumophila to infection of macrophage-like U937 cells. Intracellular L. pneumophila was radiolabeled, and cell extracts were subjected to two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At least 35 Legionella proteins were selectively induced during infection of macrophages, and one of these proteins was not detected in organisms grown in vitro. Expression of at least 32 proteins was selectively repressed during infection of macrophages, and 9 of these proteins were undetectable in intracellularly grown organisms. Thirteen of the macrophage-induced proteins were also induced by one or more of several stress conditions in vitro, and two of these proteins were the heat shock GroEL- and GroES-like proteins. Nineteen of the macrophage-repressed proteins were also repressed by one or more of the stress conditions in vitro. Our data showed that intracellular L. pneumophila manifested a phenotypic modulation and a global stress response to the intracellular environment of the macrophage. The data suggested that multiple regulons are involved in this modulation, which may contribute to the survival of L. pneumophila within alveolar macrophages.
Legionella pneumophila is a ubiquitous bacterium in the natural aquatic environment where it survives as an intracellular parasite of freshwater protozoa (11, 12, 30, 36). When inhaled in aerosol form, L. pneumophila may cause a severe, atypical pneumonia of humans, i.e., Legionnaires' disease. The capacity of this pathogen to cause disease is dependent on its ability to invade and to multiply within host phagocytic cells (6, 36). Typically, L. pneumophila is taken up by alveolar macrophages and survives and multiplies within a ribosome-studded, membrane-bound vesicle (16). Events associated with normal phagocytosis, i.e., acidification of the phagosome and subsequent fusion with lysosomes, do not occur (17, 19). The bacterial factors that enable this intracellular pathogen to adapt to intracellular survival and to alter the normal phagocytic response of the macrophage are not known. As a general principle, pathogenic bacteria respond and adapt to the various local environmental conditions they encounter in a susceptible host by coordinate regulation of gene expression (22). For most pathogenic bacteria, the intracellular environment of the macrophage is a potentially hostile environment where the viability of the organism may be threatened by oxidative or nonoxidative microbicidal mechanisms. However, some bacteria, such as L. pneumophila and Salmonella typhimunum, are well adapted to survive within these cells. Buchmeier and Heffron have provided a global view of the genetic adaptation of S. typhimunum to the intracellular environment (3). These investigators showed that S. typhimurium increases the synthesis of over 30 proteins and represses the synthesis of 136 proteins upon infection of macrophages. They also concluded that this phenotypic modulation involves multiple regulons. *
In this study, we used a similar approach to examine the modulation of protein expression by L. pneumophila in response to the intracellular environment of the macrophage. We found that at least 35 proteins were induced and 32 proteins were repressed during exponential growth of the organism in U937 cells. The phenotypic modulation by intracellular L. pneumophila was partially induced by several stress conditions in vitro. Our data showed that the adaptation by L. pneumophila to grow within the macrophage is complex and reflects a response to a stressful microenvironment. As with S. typhimurium, the intracellular adaptation of L. pneumophila appears to involve multiple regulons.
MATERIALS AND METHODS Bacterial strains and in vitro culture conditions. L. pneumophila AA100 is a clinical isolate that was originally designated Wadsworth 130b (9). The organism was maintained on buffered charcoal-yeast extract-agar plates supplemented with a-ketoglutaric acid (BCYE-a) at 37°C (8). A complete defined medium (designated LCDM) for the growth of L. pneumophila was developed to facilitate radiolabeling of the organism in vitro (Table 1). All components were dissolved in water with the exceptions of L-CyStine and L-tyrosine, which were dissolved in 1 N HCI and 10 mM KOH, respectively. The pH of the medium was adjusted to 6.9 with KOH. The medium was made and stored at room temperature without cysteine and iron, which were added just prior to use. All amino acids were purchased from Sigma Chemical Co. (St. Louis, Mo.). For comparison of bacterial growth in liquid media, Casamino Acid (CAA) and buffered yeast extract (BYE) broths were prepared as described previously (10, 24). For radiolabeling of bacterial proteins in vitro, strain AA100 was inoculated from a 48-h BCYE-ot to the LCDM broth, grown to mid-log phase, and then pulsed with [35S]methionine (Amersham, Arlington Heights, Ill.) for 30
Corresponding author.
t Present address: Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285. 1320
VOL. 61, 1993
PHENOTYPIC MODULATION BY INTRACELLULAR L. PNEUMOPHILA 2.0 -
TABLE 1. Components of complete defined medium (LCDM) Media component
Concn
1.8
500 900 L-Arginine-HCl .................. 150 L-Asparagine .................. L-Aspartic acid .............. 1,000 400 L-Cysteine-HCl H20 .............. 75 L-Cystine .............. L-Glutamic acid .............. 1,650 250 L-Glutamine .......................
L-Alanine ..................
Glycine .......
L-Histidine-HCI ............................................. L-Isoleucine ................................................. L-Leucine ....................................................
L-Lysine-HCl
............................................ L-Methionine ........ ............................................ L-Proline .................................................... L-Serine .............................................. ...................................................... ............................................ ............................................ L-Valine ............................................. ............................................ ACES buffer ............................................ Ferric ............................................
L-Phenylalanine L-Threonine
L-Tryptophan L-Tyrosine
ct-Ketoglutarate
1321
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1.4
1,350
300 555 555 750 300 450 250 650 50 400 75 600
1.2
OD 550
1,000 10,000
250 pyrophosphate 316 NH4Cl ............................................ KOH ............................................. 3,000 4,000 K2HPO4 ............................................ 1,000 KH2PO4 .................................................. 70 MgSO4. 7H20 ........................................................... 50 NaCl ............................................ 40 CaCl2 .................................................... 0.0 0
min. Mid-log-phase bacteria were also labeled in vitro for 30 min after exposure to one of the following stress conditions: heat shock at 42°C, osmotic shock in 0.5 M NaCl, and oxidative stress in 0.05 mM H202. The radiolabeled bacteria were chilled on ice, harvested by centrifugation, and then washed once with cold water. Tissue culture infection and intracellular radiolabeling of bacterial proteins. Differentiated U937 cells were used to cultivate L. pneumophila as described previously (29, 32). Monolayers of differentiated U937 cells in flat-bottom flasks were infected with L. pneumophila AA100 resuspended in RPMI 1640 (GIBCO, Gaithersburg, Md.) at a bacterium/ U937 cell ratio of 10:1. After an incubation of 30 min, the monolayers were washed to remove the extracellular bacteria. The flasks were then incubated at 37°C in RPMI 1640 with gentamicin (50 ,ug/ml) to kill any adherent bacteria that had not been engulfed and with cycloheximide (200 ,ug/ml) to arrest macrophage protein synthesis. [35S]methionine (150 ,uCi/ml) was added after 20 h of infection, and the incubation was continued for an additional 2 h to permit incorporation of the label. Uninoculated U937 cell monolayers and monolayers infected with heat-killed L. pneumophila were also radiolabeled under the same experimental conditions to confirm that the inhibition of protein synthesis of the macrophage was complete. To harvest the intracellular, radiolabeled organisms, the infected monolayer was washed twice with phosphate-buffered saline (PBS) and then lysed osmotically with water. The bacterial cells were harvested by centrifugation and washed once with water, and the pellets were stored at -70°C. Without radiolabeling, the same procedures for the infection
20
40
60
80
Time (h) FIG. 1. Growth curves of L. pneumophila AA100 grown in CAA semidefined medium (El) and in LCDM (0). OD550, optical density at 550 nm.
followed to quantitate the number of intracellular bacteria at several time points during the infection. Resolution of radiolabeled proteins on two-dimensional gel electrophoresis (2DGE). Cell extracts of the radiolabeled bacterial pellet were prepared by the methods of O'Farrell (27) with modifications (33). Equal amounts of tricarboxylic acid-precipitated radiolabeled proteins (106 cpm) were subjected to equilibrium isoelectric focusing prior to separation in the second dimension on sodium dodecyl sulfate-11.5% polyacrylamide slab gels. After electrophoresis, the gels were dried and autoradiographed by using Kodak X-OMATAR film. Protein isolation and partial N-terminal sequence analysis. Selected protein spots from 2DGE gels were isolated for N-terminal sequencing. After electrophoresis of proteins from strain AA100 grown to mid-log phase in LCDM, gels were soaked in transfer buffer [10 mM 3-(cyclohexylamino)1-propanesulfonic acid, 10% methanol (pH 11.0)] for 10 min to reduce the amount of glycine and Tris contamination. Polyvinylidene difluoride (PVDF; Immobilon, Millipore) membranes were soaked first in 100% methanol and then in the transfer buffer. Proteins were transferred from gels to the were
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