INFECTION AND IMMUNITY, Aug. 1994, p. 3254-3261 Vol. 62, No. 8 0019-9567/94/$04.00+0 Copyright C 1994, American Society for Microbiology Growth of Legionella pneumophila in Acanthamoeba castellanii Enhances Invasion JEFFREY D. CIRILLO, STANLEY FALKOW, AND LUCY S. TOMPKINS* Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, Califomia 94305 Received 4 March 1994/Returned for modification 12 April 1994/Accepted 29 April 1994 Legionella pneumophila is considered to be a facultative intracellular parasite. Therefore, the ability of these bacteria to enter, i.e., invade, eukaryotic cells is expected to be a key pathogenic determinant. We compared the invasive ability of bacteria grown under standard laboratory conditions with that of bacteria grown in Acanthamoeba castellanii, one of the protozoan species that serves as a natural host for L. pneumophila in the environment. Amoeba-grown L. pneumophila cells were found to be at least 100-fold more invasive for epithelial cells and 10-fold more invasive for macrophages and A. castellanii than were L. pneumophila cells grown on agar. Comparison of agar- and amoeba-grown L. pneumophila cells by light and electron microscopy demonstrated dramatic differences in the morphology and structure of the bacteria. Analyses of protein expression in the two strains of bacteria suggest that these phenotypic differences may be due to the expression of new proteins in amoeba-grown L. pneumophila cells. In addition, the amoeba-grown bacteria were found to enter macrophages via coiling phagocytosis at a higher frequency than agar-grown bacteria did. Replication of L. pneumophila in protozoans present in domestic water supplies may be necessary to produce bacteria that are competent to enter mammalian cells and produce human disease. Legionella pneumophila is the causative agent of the poten- sols from these contaminated sources (2, 3, 18). Furthermore, tially lethal pneumonia commonly known as Legionnaires' it has been amply shown that multiple passage of fresh clinical disease (13, 31). It is estimated that 6.1 cases of Legionella isolates of L. pneumophila on agar causes a loss of virulence as infection occur for every 100,000 adults annually in the United measured by the ability to replicate in tissue culture cells and States alone (28). Often the mortality rate of Legionnaires' cause pneumonitis in guinea pigs (4, 8, 30). This evidence may disease is greater than 25% (14, 27). L. pneumophila is thought indicate that there is an important link between Legionnaires' to replicate intracellularly in humans (6, 47) and has been disease and protozoans present in domestic water supplies. demonstrated to grow extracellularly only under laboratory These observations led us to examine the effects upon conditions (7). The cell types that L. pneumophila has been invasion of growth of L. pneumophila in one of its environ- shown to enter and replicate within include epithelial cells (5, mental hosts, Acanthamoeba castellanii (18, 40). Previous 37), macrophages (20, 26), fibroblasts (37, 48), and a number studies in our laboratory have shown that Legionella cells of protozoan species (11, 35, 40, 43). However, in human replicate efficiently within A. castellanii (35). In the current infections (47) as well as the guinea pig model of L. pneumo- study, we examined L. pneumophila cells grown on laboratory phila pneumonitis (6), the majority of bacteria are seen media (BCYE grown [BG]) and in the amoebae (amoeba intracellularly in macrophages. grown [AG]) for their ability to enter a number of cell types, Although entry into host cells is likely to be a critical step in including epithelial cells (HEp-2), macrophages (RAW 264.7 Legionella pathogenesis, little is understood concerning the and THP-1), and amoebae (A. castellanii). The ultrastructure, mechanisms involved. The initial studies on the Legionella morphology, and protein expression of AG and BG bacteria entry mechanisms were carried out with macrophages in vitro were compared. These results further support a role for (19). This work led to the observation that entry may occur protozoans in the production of Legionnaires' disease and through a complement-mediated mechanism involving com- have significant implications for studies on Legionella patho- plement receptors (38). In addition, phagocytosis was shown to genesis. occur through a novel mechanism termed "coiling phagocyto- sis" (19). The possibility that other cell types, particularly epithelial cells, are involved in Legionella pathogenesis has MATERIALS AND METHODS been suggested recently (24, The entry 33). mechanism of into Strains and A epithelial cells has not been studied, although they have been growth conditions. spontaneous streptomy- cin-resistant mutant of L. pneumophila serogroup 1 (130b) (9), shown to express complement receptors (12, 32). shown to be virulent in guinea pigs and amoebae (34), was used Previous studies on Legionella invasion have focused on bacteria cultured in laboratory media (19, 38). However, for all procedures. L. pneumophila cells were either grown on BCYE agar manner or protozoans present in domestic water supplies have been (BG) in the standard (7) harvested immediately after growth in amoebae (AG) as follows. First, a shown to provide an environmental reservoir for Legionella species (1, 18, 40). In addition, epidemiological studies have large-scale invasion assay was carried out in a 75-cm2 tissue culture flask (Falcon) at 37°C. In this assay a monolayer of suggested that human infections occur by inhalation of aero- approximately 107 amoebae was infected with L. pneumophila at a multiplicity of infection of 100 for 30 min. Then the monolayer was washed once with 10 ml of A.c. buffer (35) and * Corresponding author. Phone: (415) 723-6384. Fax: (415) 725- incubated for 2 h in 10 ml of A.c. buffer containing 100 ig of 5671. gentamicin per ml. It was then washed as before and incubated 3254 VOL. 62, 1994 INVASIVE LEGIONELLA PNEUMOPHILA 3255 in 25 ml of A.c. buffer at 37°C with 5% CO2 until the complete tially as described previously (19). The samples were then fixed amoeba monolayer was destroyed (-72 to 104 h). The result- in 2% glutaraldehyde and stained with 1% OSO4 for 2 h and ing culture was harvested and centrifuged for 10 min at 350 x 0.5% uranyl acetate overnight at 4°C. g to pellet the bacteria and amoebae. The pellet was suspended HEp-2 cells to be used for light microscopy were seeded in in 1 ml of distilled water for 10 min and passed through a 24-well dishes as described above except that the wells had 27-gauge syringe three times to lyse remaining amoebae as glass coverslips. These coverslips were then prepared for light described previously (35). To remove any remaining amoebae microscopy by fixation in methanol and staining by a modifi- or amoebic cysts, we centrifuged this preparation for 1 min at cation of the technique used by Gimenez (15). In this proce- 150 x g and transferred the supernatant to a new tube. The dure the coverslips are incubated in hot (50°C) 0.4% carbol resulting bacterial suspension, containing approximately 107 to basic fuchsin for 10 min, 0.8% malachite green for 5 to 10 s, 108 bacteria, was found to be free of intact amoebae by light and 0.004% methylene blue for 60 to 90 s, being washed with microscopy. water between each step. Bacteria were examined for motility L. pneumophila cells that were grown on BCYE agar but had by light microscopy without fixation or staining. come in contact with amoebae (AG/30) were prepared by Samples were prepared for fluorescence microscopy essen- carrying out an invasion in a 75-cm2 flask as described above tially as described previously (25), except that propinium but lysing the amoebae by incubation with water and passage iodide was excluded during the secondary-antibody incubation through a syringe immediately after the 30-min invasion. and the primary antibody used was raised against either AG or BCYE-passaged AG bacteria (AG/B) were prepared by har- BG bacteria. Primary antibodies were used at a dilution of vesting AG bacteria as described above, plating them on 1:500, and the secondary fluorescein isothiocyanate-conju- BCYE agar, and harvesting AG/B bacteria that grew as a lawn gated sheep anti-rabbit antibody (Sigma) was used at a dilution on this plate. Escherichia coli K-12 strain HB101 (ara-14 leuB6 of 1:3,000. Antibodies against each strain were produced in proA2 lacYl ginV44 galK2 recA13 rpsL20 xyl-5 mtl-i thi-1 New Zealand White rabbits by immunization with 108 forma- hsdS20) (Promega) grown in Lennox broth or agar (GIBCO lin-killed bacteria once every 7 days for 3 weeks. The resulting BRL) was used as a noninvasive control. antibody titers were greater than 1:20,000 by enzyme-linked A. castellanii ATCC 30234 was grown to confluence at 23°C immunosorbent assay (10). in 75-cm2 tissue culture flasks containing PYG broth (35). [35S]methionine labeling of LegioneUa proteins. L. pneumo- Amoebae were harvested before use by rapping the flask phila cells were first allowed to invade the amoebae under sharply to bring them into suspension, and the number of standard conditions. After gentamicin treatment the flask was viable cells was determined as described previously (35). washed once with 5 ml of A.c. buffer and incubated for various Cell lines and culture conditions. HEp-2 cells (ATCC periods in 25 ml of A.c. buffer at 37°C and with 5% CO2. The CCL23), established from a human epidermoid carcinoma, amoebae, some of which contained bacteria, were then har- were grown in RPMI 1640 plus 5% fetal calf serum (GIBCO). vested from the flask, pelleted at 185 x g for 10 min, and RAW 264.7 cells (39), a mouse macrophage cell line, were suspended in 1 ml of A.c.