APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2008, p. 2187–2199 Vol. 74, No. 70099-2240/08/$08.00�0 doi:10.1128/AEM.01214-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Packaging of Live Legionella pneumophila into Pellets Expelled byTetrahymena spp. Does Not Require Bacterial Replication and

Depends on a Dot/Icm-Mediated Survival Mechanism�

Sharon G. Berk,1 Gary Faulkner,2 Elizabeth Garduno,2 Mark C. Joy,1Marco A. Ortiz-Jimenez,3 and Rafael A. Garduno2,4*

Center for the Management, Utilization and Protection of Water Resources, Tennessee Technological University, Cookeville,Tennessee1; Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada2;

Instituto de Investigaciones Biomedicas, Universidad Autonoma de Mexico, Mexico City, D.F., Mexico3; andDepartment of Medicine-Division of Infectious Diseases, Dalhousie University, Halifax, Nova Scotia, Canada4

Received 31 May 2007/Accepted 25 January 2008

The freshwater ciliate Tetrahymena sp. efficiently ingested, but poorly digested, virulent strains of thegram-negative intracellular pathogen Legionella pneumophila. Ciliates expelled live legionellae packaged in freespherical pellets. The ingested legionellae showed no ultrastructural indicators of cell division either withinintracellular food vacuoles or in the expelled pellets, while the number of CFU consistently decreased as afunction of time postinoculation, suggesting a lack of L. pneumophila replication inside Tetrahymena. Pulse-chase feeding experiments with fluorescent L. pneumophila and Escherichia coli indicated that actively feedingciliates maintain a rapid and steady turnover of food vacuoles, so that the intravacuolar residence of theingested bacteria was as short as 1 to 2 h. L. pneumophila mutants with a defective Dot/Icm virulence systemwere efficiently digested by Tetrahymena sp. In contrast to pellets of virulent L. pneumophila, the pelletsproduced by ciliates feeding on dot mutants contained very few bacterial cells but abundant membrane whorls.The whorls became labeled with a specific antibody against L. pneumophila OmpS, indicating that they wereouter membrane remnants of digested legionellae. Ciliates that fed on genetically complemented dot mutantsproduced numerous pellets containing live legionellae, establishing the importance of the Dot/Icm system toresist digestion. We thus concluded that production of pellets containing live virulent L. pneumophila dependson bacterial survival (mediated by the Dot/Icm system) and occurs in the absence of bacterial replication.Pellets of virulent L. pneumophila may contribute to the transmission of Legionnaires’ disease, an issuecurrently under investigation.

Legionella pneumophila (the causative agent of Legion-naires’ disease in susceptible humans) is a gram-negative fresh-water bacterium that has evolved as an intracellular pathogenof amoebae (17, 53). Rowbotham first recognized L. pneumo-phila to be an intracellular parasite of amoebae shortly afterthe isolation and identification of the “Legionnaires’ diseasebacterium” from human patients by McDade et al. (reviewedin reference 45). Rowbotham (44) also described in detail thegrowth cycle of L. pneumophila in Acanthamoeba polyphaga,and his careful observations suggested that L. pneumophila iswell adapted to infect amoebae. To grow inside amoebae L.pneumophila requires a functional Dot/Icm system (21, 48),a type IV secretion apparatus that allegedly delivers effectorproteins required for phagocytosis-invasion, recruitment ofphosphatidylinositol-4 phosphate, and initiation of intra-amoebal growth, as determined in Dictyostelium discoideum(27, 33, 52, 57).

In contrast to amoebae, the role that ciliates play as naturalhosts of L. pneumophila is far from clear and remains contro-versial. On the one hand, it has been experimentally shown

that the freshwater ciliate Tetrahymena pyriformis supports themultiplication of L. pneumophila at 35°C (3, 18, 19), and in factthe ability to grow in T pyriformis at 35°C was considered amarker of L. pneumophila virulence (18). On the other hand,L. pneumophila growth was restricted at 25°C in the same T.pyriformis strain (19) that was permissive at 30 to 35°C. An-other study (54) showed that only Legionella longbeachae con-sistently grew in T. pyriformis at 30°C, whereas several strainsof L. pneumophila (among several other Legionella species)showed inconsistent growth. In addition, L. pneumophila didnot replicate in the ciliate Cyclidium in axenic culture (3).Finally, Tetrahymena vorax did not support the intracellulargrowth of L. pneumophila at 20 to 22°C (50). However, in allcases (even when no growth was observed) L. pneumophilaingested by ciliates survived within food vacuoles. For instance,whereas E. coli was digested in T. vorax, ingested L. pneumo-phila survived at 20 to 22°C, and food vacuoles containing livelegionellae were retained for an extended period of severalhours (50). In contrast, other Tetrahymena species readily ex-pelled the live undigested legionellae into the extracellularmilieu, generating a large number of free legionella-laden ves-icles that accumulated as aggregates (38).

Rowbotham originally proposed the idea that legionella-laden vesicles produced by amoebae could constitute a largeinfectious unit that may be important for the transmission ofLegionnaires’ disease (44). To date, legionella-laden vesicles,

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, Dalhousie University, Sir Charles TupperBuilding, 7th floor, 5850 College Street, Halifax, Nova Scotia B3H-1X5, Canada. Phone: (902) 494-6575. Fax: (902) 494-5125. E-mail:rafael.garduno@dal.ca.

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as well as legionella-infected amoebae, continue to be consid-ered important epidemiological elements, either as infectiousparticles or as complex units that offer enhanced environmen-tal survival (7, 8, 10). Given the apparent efficiency at whichTetrahymena sp. generates legionella-laden vesicles (38) andthe potentially important role that these vesicles may play inthe transmission of Legionnaires’ disease, the ciliate-mediatedprocess of vesicle production was investigated in detail. Wereport here that the previously named vesicles are insteadclusters of bacteria surrounded by bacterial debris and werethus renamed as pellets. The massive production of legionella-laden pellets by Tetrahymena sp. feeding on virulent L. pneu-mophila is a rapid process that occurs in the absence of bac-terial replication and depends on a Dot/Icm system-mediatedmechanism used by L. pneumophila to avoid digestion while intransit through the ciliate. Tetrahymena sp. is thus presentedhere as an efficient packager of virulent L. pneumophila infreshwater, and an experimental model to study the Dot/Icm-mediated mechanisms used by L. pneumophila to resist diges-tion in ciliates, in the absence of bacterial replication.

MATERIALS AND METHODS

Bacterial strains and culture conditions. The L. pneumophila and E. colistrains used are listed in Table 1. All L. pneumophila strains were kept as frozenstocks at �80°C. In particular, frozen stocks of Lp02, Lp1-SVir, and JR-32 weremade from crude lysates of infected HeLa cells after intracellular growth hadtaken place (24, 25). Strain ATCC 33216 was grown on buffered charcoal-yeastextract agar (BCYE [42]), whereas stocks of Lp1-SVir and JR-32 were grown onBCYE supplemented with 100 �g of streptomycin/ml. Strain Lp02 and its de-rivatives JV303, JV309, and JV918 were grown on BCYE containing streptomy-cin and thymidine (both at 100 �g per ml), and JV1133 and JV1170 were grownon BCYE containing streptomycin but no thymidine. All L. pneumophila strainsand mutants from frozen stocks were grown on agar plates for 3 to 5 days at 37°Cin a humid incubator. Bacteria from this primary growth on agar plates (mostlyin stationary phase [24]) were used in feeding experiments. E. coli strain JM109,used in control feeding experiments, was grown on Luria-Bertani (LB) agarplates (46) at 37°C.

Bacterial transformation and complementation of the dotA mutant JV309. Asuspension of Lp02 (1011 bacteria/ml in distilled-deionized water [ddH2O]) wasprepared from thin lawns grown overnight at 30°C on BCYE plates, washed twicein ddH2O, and resuspended to the same concentration in 15% (wt/wt) glycerolsolution in ddH2O to produce electrocompetent cells. Plasmid pBH6119::htpAB(10 �g) was added to 400 �l of the Lp02 suspension in a 2-mm electroporationcuvette, and electroporated in a GenePulser II (Bio-Rad Laboratories, Missis-

sauga, Ontario, Canada). Plasmid pBH6119::htpAB is a variant of plasmid pflaG(26) in which the flaA promoter was replaced with the L. pneumophila htpABpromoter and was constructed by K. Brassinga (Dalhousie University) after theprocedure reported by Hammer and Swanson (26) on the backbone plasmidpBH6119 (provided by M. S. Swanson, University of Michigan–Ann Arbor). Thecuvette was placed on ice for 20 min before inoculating a series of BCYE agarplates containing 100 �g streptomycin/ml (but no thymidine), with 10, 100, and200 �l of the electroporated suspension, to select for transformants expressinggreen fluorescent protein.

Calcium chloride-treated, competent E. coli DH5� cells (Table 1) were trans-formed with plasmid pDsRed2 (Clontech), and transformants constitutively ex-pressing the red fluorescent protein were selected and grown at 37°C on LB agarcontaining 50 �g of ampicillin/ml.

Electrocompetent JV309 cells (prepared as described above for Lp02) weretransformed by electroporation (as described above) with 10 �g of the comple-menting plasmid pKB9 (obtained from R. R. Isberg, Tufts University) carryingdotA (4, 5). The electroporated suspension was placed on ice for 20 min beforeinoculating 200 �l on a monolayer of L929 cells in a 10-cm-diameter tissueculture dish, to allow the formation of plaques (16). The bacterial inoculum wasleft overnight on the L929 cells (at 37°C in a humid CO2 incubator) beforewashing and overlaying the cell monolayer with 10 ml of agarose-solidifiedminimal essential medium. Three days after the first overlay, a second minimalessential medium overlay containing gentamicin (20 �g/ml) and neutral red (toa final concentration of 0.003%) was added. Two days after the second overlay,plugs of agarose containing bacteria from individual visible plaques were cut withglass Pasteur pipettes and then transferred to wells containing fresh HeLa cellmonolayers to confirm the ability to grow intracellularly in the absence of thy-midine. Colonies from BCYE plates inoculated with infected HeLa cells wereconfirmed to carry the pKB9 plasmid by the method of Kado and Liu (31).

Ciliate culture, maintenance, and identification. Tetrahymena sp. was inten-tionally isolated from a cooling tower biofilm to test a nonamoebic protozoanmodel relevant to the ecology of L. pneumophila in man-made aquatic environ-ments. The sampled biofilm was dispersed and cultured in a sterile petri dishcontaining cereal leaves medium, prepared by boiling 1 g of dehydrated cerealleaves (Sigma [St. Louis, MO] catalogue no. C7141) in 1 liter of distilled waterfor 15 min, followed by filtration through a 0.45-�m-pore-size membrane andautoclaving. All biofilm protozoa were then grown and maintained in 25-cm2 cellculture flasks (Falcon Plastics) containing 10 ml of cereal leaves medium fromwhich individual cells were captured with fine-bore Pasteur pipettes with the aidof a microscope. Individual cells were successively transferred in depressionslides until a single ciliate cell was observed per slide. A clonal isolate of Tetra-hymena sp. was expanded and maintained in cereal leaf medium and subse-quently made axenic by repeated subculture (three times a week for 2 weeks) inmedium supplemented with 200 U of penicillin/ml, 200 �g of streptomycin/ml,and 25 �g of gentamicin/ml. Axenic Tetrahymena sp. was initially maintained incereal leaves medium without antibiotics at 25°C before adopting the mainte-nance procedures outlined by Elliot (14) as follows. Line A was maintained at18°C in tubes containing a biphasic medium consisting of 5 ml of a slanted solidphase (in g/liter: dextrin, 8; sodium acetate trihydrate, 0.6; Autolized Yeast

TABLE 1. Bacterial strains used in this study

Designation Characteristics Source (reference)a

L. pneumophilaLp1-SVir Philadelphia-1, virulent, streptomycin resistant P. S. Hoffman (28)JR-32 Philadelphia-1, virulent derivative of the streptomycin-resistant strain AM511 H. A. Shuman (37)Lp02 Philadelphia-1, virulent, thymidine auxotroph, streptomycin resistant R. R. Isberg (4)JV309 Salt-tolerant, dotA mutant derivative of Lp02 R. R. Isberg (5)JV303 Salt-tolerant, dotB mutant derivative of Lp02 J. P. Vogel (56)JV918 �dotB derivative of Lp02 J. P. Vogel (49)JV1133 JV918 carrying the empty plasmid vector pJB908 J. P. Vogel (49)JV1170 JV918 carrying the complementing plasmid pJB1153 with a wild-type copy of dotB J. P. Vogel (49)33216 Dallas 1E, serogroup 5, subsp. fraseri ATCC

E. coliJM109 Rec� K-12 derivative, F� �traD36 proAB� lacIq lacZ�M15� endA1 recA1 hsdR17

supE44 thi gyrA96 �(lac-proAB), shows the rK� phenotype

P. S. Hoffman (46)

DH5� F� �80 lacZ�M15� �lacU169 supE44 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 Clontech (46)

a Paul S. Hoffman is at the University of Virginia, Howard A. Shuman is at Columbia University, Ralph R. Isberg is at Tufts University Medical School, and JosephP. Vogel is at the Washington University School of Medicine.

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[Difco], 5; liver concentrate [Sigma], 0.6; Bacto Casitone [Difco], 3; CasaminoAcids [Difco], 2; agar, 16; pH 7.3) covered with 3 ml of sterile ddH2O. This linewas subcultured every 2 to 3 months. Line B was maintained in the describedbiphasic medium, but was kept at room temperature (22 to 24°C) and subcul-tured every 2 to 3 weeks. Line C (the working culture) was kept in plate countbroth (Difco) at 30°C and subcultured weekly. Every 4 to 6 months (dependingon the robustness of its growth) line C was discarded and a new one started fromline B. Line A served as a reserve culture of low manipulation and to start newline B cultures.

To confirm the identity of the ciliate as Tetrahymena sp., we opted to sequencethe gene encoding the small ribosomal subunit RNA. Total Tetrahymenagenomic DNA was purified by using the standard phenol-chloroform extractionmethod reported by Arroyo et al. for Trichomonas vaginalis (2). The final DNApellet obtained after cold ethanol precipitation was resuspended in TAE buffer(46) and used as a template for amplification of the 18S rRNA gene by PCR ina Biometra T-Personal instrument using Medlin’s universal eukaryotic forwardprimer 5�-ACCTGGTTGATCCTGCCAGT-3� (39), and the reverse primer re-ported by Jerome et al., 5�-TTGGTCCGTGTTTCAAGACG-3� (30). The am-plification product was sequenced in-house in a Beckman-Coulter CEQ 8000genetic analysis system (Dalgen; Dalhousie University), in both directions, usingthe above primers. The obtained sequence was then compared against the NCBIdatabase using BLAST, and the most similar sequences were retrieved. Ourisolate clustered in the T. tropicalis-T. mobilis clade, which is evolutionarilydistanced from the T. pyriformis and T. vorax clade (the two Tetrahymena speciesused in previous studies with L. pneumophila [19, 50, 54]), as indicated by thephylogenetic tree constructed by Brandl et al. based on a subset of Tetrahymena18S rRNA gene sequences (9).

Tetrahymena pyriformis ATCC 30202 was used as a reference ciliate in a fewfeeding experiments. T. pyriformis was cultured and maintained as describedabove for Tetrahymena sp.

Feeding experiments. Before use in feeding experiments, Tetrahymena sp. cellswere gradually transferred from their plate count broth growth medium intoeither raw cooling tower water filtered through a 0.2-�m-pore-size membrane(Millipore) or Tris-buffered Osterhout’s solution (41, 51). That is, ciliates weresequentially pelleted at 700 � g for 10 min and resuspended in increasingconcentrations (30, 60, and 100%) of filtered cooling tower water at roomtemperature or Osterhout’s solution at 30°C. The use of raw cooling tower waterwas adopted to initially mimic the environment of isolation and was collectedfrom the basin of the cooling tower from which the Tetrahymena sp. isolate wasobtained, during the period of lowest biocide concentration. Due to confidenti-ality issues, the chemistry of the cooling tower water remained undefined; there-fore, the adoption of Osterhout’s solution facilitated the standardization ofsubsequent feeding experiments. Osterhout’s solution contained (in mg/liter):NaCl, 420; KCl, 9.2; CaCl2, 4; MgSO4 � 7H2O, 16; MgCl2 � 6H2O, 34; and Trisbase, 121 (pH 7.0). The solution was sterilized by filtration in a bottle-top filterof 0.45-�m pores (Nalgene). Based on the recorded in situ temperature range ofcooling tower waters (6), our feeding experiments were initially set at 25°C incooling tower water and later standardized to 30°C in Osterhout’s solution.Bacteria were also washed and resuspended to an optical density at 620 nm of 1unit, in either filter-sterilized cooling tower water or Osterhout’s solution, beforebeing used as inoculum for feeding experiments.

Plate-grown L. pneumophila strain 33216 and Tetrahymena sp. cells werewashed and resuspended in cooling tower water to achieve various bacterium/ciliate ratios. The ciliate concentration (determined by direct microscopy ofsamples of known volume fixed with Lugol’s iodine [1]) was kept constant at104/ml, and different numbers of bacteria were added to achieve bacterium/ciliate ratios of 100, 1,000, and 10,000. After a 24-h incubation at 25°C, pelletsproduced were enumerated with a Brightline hemacytometer. Feeding experi-ments standardized at 30°C in Osterhout’s solution were run for 48 h with variousL. pneumophila Philadelphia-1 strains in either six-well plates or 25-cm2 cellculture flasks. Samples were taken at different times to microscopically assess thenumbers of live ciliates and free bacteria. Typically, the concentration of ciliateswas 5 � 104/ml of Osterhout’s solution, but feeding experiments with Lp02 dotmutants were done at a concentration of 104 Tetrahymena sp. cells per ml and abacterium/ciliate ratio of 10,000.

Pulse-chase experiments with fluorescent bacteria. L. pneumophila Lp02 car-rying plasmid pBH6119::htpAB (displaying green fluorescence) and E. coli DH5�carrying plasmid pDsRed2 (displaying red fluorescence) were added to ciliatesuspensions according to the following schemes: (i) a 1-h pulse of red fluorescentE. coli DH5�, followed by a chase with nonfluorescent E. coli DH5�; (ii) a 1-hpulse of red fluorescent E. coli, followed by a 3-h chase with green fluorescent L.pneumophila; and (iii) a 1-h pulse of green fluorescent L. pneumophila, followedby a chase with nonfluorescent L. pneumophila Lp02. The total bacterium/ciliate

ratio for these experiments was kept at 10,000. The ciliates were separatedfrom free bacteria (between pulse and chase changes) by centrifugation at a lowspeed (700 � g for 10 min), leaving most bacteria in the supernatant. Thesefeeding experiments were set at 30°C in six-well plates (Falcon Plastics) with0.5 million ciliates per well in 3 ml of Osterhout’s solution.

Production of pellets for morphological characterization. Tetrahymena cells(1.5 � 106) resuspended in 3 ml of Osterhout’s solution were fed with 5 � 108

bacteria (bacterium/ciliate ratio of 333). These ciliate feeding experiments wereset in six-well plates (Falcon Plastics) at 30°C. To obtain a sample enriched inpellets for microscopic observations, after an overnight incubation at room tem-perature (usually 16 to 18 h) the mixture of ciliates, free bacteria, and aggregatedpellets was centrifuged at 700 � g for 10 min in 15-ml conical tubes (FalconPlastics), and the live ciliates were allowed to swim back into suspension beforeremoving the supernatant (containing live ciliates and free bacteria). The recov-ered pellets were resuspended in fresh Osterhout’s solution. This operation wasrepeated three times before the pellet-enriched samples were prepared for var-ious microscopy observations.

Enumeration of bacterial CFU in ciliate cultures. A total of 106 ciliates in25-cm2 cell culture flasks (Falcon) containing 30 ml of Osterhout’s solution werefed with 3 � 1010 bacteria (obtained from 40 ml of either Lp1-SVir, Lp02, orJR-32 suspensions with an optical density at 620 nm of 1 unit) to provide abacterium/ciliate ratio of 30,000. After 3 h at 30°C, the mixed suspension wastreated with gentamicin (100 �g/ml) for 30 min, and then the ciliates wereseparated from the free bacteria and gentamicin by gentle filtration-washing(with Osterhout’s solution) through Millipore membranes of 8.0-�m pores. Theciliates remaining on the filter (carrying ingested legionellae) were resuspendedin 30 ml of Osterhout’s solution by allowing them to freely swim for a fewminutes at room temperature, followed by a very gentle agitation, and incubatedat 30°C. Then, three 1-ml samples were taken at various times to perform CFUcounts. Each 1-ml sample was placed in a 1.5-ml microcentrifuge tube andcentrifuged at 10,000 � g for 1 min. The centrifugation pellet was resuspendedwith vigorous pipetting into 50 �l of 0.5% Triton X-100 in sterile ddH2O, and 450�l of ddH2O was added, followed by vigorous vortexing for 1 min. The samplewas then brought to 1 ml with ddH2O, before 10-fold serial dilutions wereperformed. Aliquots (100 �l) of the 102 to 106 dilutions were spotted onto BCYEplates and incubated at 37°C for 4 to 5 days before the colonies were counted.

Light microscopy. Ciliate cultures were routinely monitored by using a CK2Olympus inverted microscope. Wet mounts of ciliates with ingested legionellae

FIG. 1. Early kinetics of L. pneumophila ingestion and pellet pro-duction. (A to D) Images of green fluorescent Lp02 overlaid on theircorresponding DIC images of Tetrahymena cells. Samples were takenat the indicated times after addition of the bacterial inoculum to showthe progressive formation and accumulation of food vacuoles. Thearrowhead in panel A points to the clearly seen vestibulum of thecytopharynx, at the end of which a food vacuole seems to be forming.The arrowhead in panel D points to a pellet being expelled. The sizebar in panel A represents 10 �m and applies to all panels.

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and fluorescent bacteria were observed in an Olympus BX6 upright microscopeequipped with differential interference contrast (DIC), epifluorescence, and anEvolution QEi Monochrome digital camera (Media Cibernetics, San Diego,CA). Image capture and analysis (TIFF files) was performed with ImageProsoftware v.5.0 (Media Cibernetics). Viability of the legionellae within pellets wasassessed by the BacLight LIVE/DEAD stain (Molecular Probes, Inc., Eugene,OR) in a Leica TCS-SL confocal microscope with an excitation argon laser of 488nm and a barrier emission filter for 520 nm. Direct microscopy counts of freebacteria were done by using an internal standard (a suspension of 0.8-�m latexbeads at a concentration of 3 � 109 per ml), following the procedures of Mallette(35).

Transmission electron microscopy (TEM). Samples of Tetrahymena cells andpellets (prepared as described above) were taken at different times postinocu-lation to be fixed in glutaraldehyde, postfixed in osmium tetroxide, and embed-ded in epoxy resin for thin sectioning, followed by standard staining in uraniumand lead salts, as described previously (15). Stained thin sections were observedin a JEOL JEM-1230 transmission electron microscope equipped with aHamamatsu ORCA-HR high-resolution (2K by 2K) digital camera, and imageswere captured as TIFF files.

Immunogold labeling. Pellets produced by Tetrahymena sp. fed with theLp02 strain, and its derivatives JV303 (dotB mutant) and JV309 (dotA mu-tant) were fixed in freshly depolymerized paraformaldehyde, and embeddedin LR-White resin as previously reported (23). Thin sections mounted onnickel grids were then immunostained with rabbit polyclonal sera (see below)and goat anti-rabbit secondary antibodies conjugated to 10-nm gold spheres(Sigma Immunochemicals), as follows. Grids were sequentially floated for 10min on drops of sodium borohydride (1 mg/ml) freshly dissolved in ddH2Oand then 10 mM glycine dissolved in 100 mM sodium borate (pH 9). Blocking

was done for 1 h on drops of labeling buffer (100 mM Tris, 0.2 M NaCl [pH8]) containing 1% bovine serum albumin (BSA) and 1% skimmed milk. Thegrids were then sequentially floated for 1 h on drops of rabbit hyperimmunesera diluted 1:400 in labeling buffer containing 0.2% BSA and on drops ofgold-conjugate diluted 1:100 in labeling buffer containing 0.2% BSA. Aftereach antibody incubation, grids were washed three times by floating androcking them for 10 min on 1-ml aliquots of wash buffer (100 mM Tris, 0.3 MNaCl [pH 8]) in 24-well plates. Finally, the labeled sections were floated on2.5% glutaraldehyde in wash buffer, thoroughly rinsed in ddH2O, and stainedwith uranium and lead salts (15). Rabbit sera raised against the major outermembrane protein of L. pneumophila, OmpS, which is highly resistant toproteases (11, 29) or against the Hsp60 chaperonin (28), were obtained fromPaul Hoffman (Dalhousie University).

RESULTS

Ingestion of legionellae by Tetrahymena sp. and pellet pro-duction. Ciliates began to form and accumulate food vacuolesimmediately after the addition of legionellae (Fig. 1). In the24-h feeding experiments performed in filtered cooling towerwater at 25°C, Tetrahymena sp. efficiently packaged L. pneu-mophila 33216 into pellets that were expelled to the extracel-lular milieu, but no pellets were produced in unfed ciliates. Theaverage numbers of pellets/ciliate/h calculated for the com-plete 24-h period were 1, 2, and 5 at bacterium/ciliate ratios of

FIG. 2. Examples of ultrastructural similarity between Tetrahymena sp. food vacuoles at different times postinoculation. Electron micrographsof single food vacuoles showing virtually identical features in ciliates fixed after 30 min (A), 4 h (B), 8 h (C), and 13 h (D) of feeding on L.pneumophila strain Lp1-SVir. The arrow in panel B points to a region with a marked warping of the vacuolar membrane (which follows the contourof the contained bacteria), and the arrowhead indicates a mitochondrion in tight apposition to the vacuolar membrane. The arrow in panel C pointsto a structurally degraded bacterial cell, and that in panel D points to the membranous material present in all food vacuoles. All size bars represent0.5 �m.

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100, 1,000, and 10,000, respectively. Pellet formation was notdependent on the use of cooling tower water, since Tetrahy-mena sp. also produced pellets in subsequent 48-h experimentsperformed in Osterhout’s solution at 30°C. In these conditions,ciliates were stable and maintained their high motility. InOsterhout’s solution at 30°C, L. pneumophila strain Lp02 wasingested at the average rates of 1.5, 17, and 389 bacteria/ciliate/h, at bacterium/ciliate ratios of 100, 1,000, and 10,000,respectively. However, all of the above rates of ingestion andpellet production may not have been uniform over the full 48-or 24-h periods. These rates were likely higher in the earlystages of feeding than later when the ciliates were bacterium-laden and the concentration of free bacteria had decreased.The shortest interval between bacterial addition to ciliates andthe appearance of the first free pellets was 50 min, as deter-mined by light and fluorescence microscopy (Fig. 1). We alsodetermined that pellets were not exclusively produced by Tet-rahymena sp., since T. pyriformis produced pellets in Oster-hout’s solution at 30°C in a manner similar to that describedabove for Tetrahymena sp. (data not shown).

After 30 to 50 min of feeding, ciliates assumed a round shapeand maintained a relatively constant number of food vacuolesas they began to produce pellets, suggesting that the rates offood vacuole formation and discharge were balanced, as pre-viously reported for other Tetrahymena species (12, 14). Bac-terium/ciliate ratios of �100 were not studied, while legionel-lae concentrations of �106/ml were not conducive to pelletformation, regardless of the bacterium/ciliate ratio. It shouldbe noted that pellet production at 35 to 37°C could not betested because neither T. pyriformis ATCC 30202 nor our Tet-rahymena isolate survived an overnight incubation at theseelevated temperatures.

When ciliates were fed with E. coli strains JM109 or DH5�,no pellets were produced at bacterium/ciliate ratios of �1,000,but a few dispersed pellets were produced at ratios of 1,000,and no pellets were observed in unfed ciliates.

Morphological characterization of food vacuoles. Tetrahy-mena sp. samples fixed 0.5, 1, 2.5, 4, 8, 13, or 24 h afterinoculation with virulent Philadelphia-1 strains Lp1-Svir orLp02 depicted similar ultrastructural features (Fig. 2). The

FIG. 3. Expelled free pellets show one of several morphologies. (A) Low-magnification electron micrograph showing a group of sectionedpellets depicting different ultrastructural features. Features: 1, pellets containing tightly packed Lp02 cells held together by an amorphous materialand membrane fragments; 2, pellets with membrane fragments between bacteria and wrapped around the pellet’s surface; 3, pellet containing afew bacteria and abundant vesicular and membranous material; 4, pellet with no obvious peripheral or interbacterial binding material. Barrepresents 2 �m. (B to D) High-magnification electron micrographs showing ultrastructural detail of a tightly packaged pellet (B), a pellet wrappedin membrane fragments (C), and a pellet lacking any apparent binding material (D). Bars represent 1.0 �m.

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number of food vacuoles per ciliate and the number of bacteriaper vacuole were similar in all TEM samples, and all vacuolescontained membranous material (e.g., arrow in Fig. 2D). Al-though the appearance of food vacuoles varied (not all mor-phologies are shown), vacuoles with virtually identical features(like those shown in Fig. 2) were always found at all samplingtimes, and except for the 30-min sample, all other ciliate TEMsamples depicted the different vacuole morphologies in similarproportions. Noticeably, ultrastructural features typical of bac-terial cell division were absent in L. pneumophila cells con-tained in food vacuoles of different morphology and in samplestaken at different feeding times. Collectively, these ultrastruc-tural observations confirmed that a steady state of food vacuoleformation and trafficking had been established as early as 0.5 to1.0 h after inoculation. Unique structural features of the legio-nella-containing food vacuoles included the juxtaposition

and/or warping of the vacuolar membrane to follow the outlineof the contained bacteria (e.g., arrow in Fig. 2B) and the tightapposition of mitochondria (e.g., arrowhead in Fig. 2B), twofeatures previously observed in L. pneumophila-infected mam-malian cells (15) and in Tetrahymena vorax (50). Althoughpresent, these features were not obvious in the 0.5-h sample,confirming that their appearance was time dependent.

Morphological characterization of expelled pellets. The av-erage pellet diameter, measured by light microscopy of 100randomly selected pellets, was 4.2 � 0.1 �m, with an estimatedvolume of 38.8 �m3. The smallest pellets had a diameter of 1�m, and the largest had a diameter of 8 �m. Only 5% of thepellets were larger than 5 �m, 28% were 5 �m, 46% were 3 to5 �m, and 21% were 1 to 3 �m in diameter, a size distributionthat did not change significantly in pellets produced at differentbacterium/ciliate ratios or with different L. pneumophila

FIG. 4. Pulse-chase experiments suggest a steady and rapid turnover of food vacuoles in feeding ciliates. (A) Overlay images of red fluorescentE. coli DH5� and DIC images of Tetrahymena cells showing the chase phase of fluorescent E. coli with nonfluorescent E. coli at the times shown.Notice the polarized displacement of fluorescent vacuoles. The bar in the 0 h overlay represents 10 �m and applies to all images in panel A.(B) Overlay images of red fluorescent E. coli DH5�, chased by green fluorescent L. pneumophila Lp02. Only the chase phase is shown at the timesindicated, where the O/N indicates an overnight incubation (16 h). T, Tetrahymena-associated fluorescence; P, pellet-associated fluorescence.DIC images of Tetrahymena cells or pellets were omitted (except for the O/N-T overlay) for visual clarity. Red fluorescent E. coli was not packagedinto pellets, except for a few cells apparently copackaged with L. pneumophila (O/N-P). Size bars represent 10 �m. (C) Overlay images of greenfluorescent L. pneumophila Lp02 and DIC images of Tetrahymena cells showing the chase phase of fluorescent L. pneumophila with nonfluorescentL. pneumophila at the times shown. The polarized displacement of vacuoles and the transfer of fluorescence to expelled pellets should be noted.The bar in the 20-h overlay represents 10 �m and applies to all images in panel C.

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strains. Inferring a homogeneous width of 0.5 �m and a lengthof 2.0 �m (with an estimated bacterial cell volume of 0.36�m3), the average pellet could theoretically accommodate100 densely packed legionellae. Pellets depicted one of sev-eral ultrastructural morphologies (Fig. 3A). Some pellets con-sisted of tightly packed legionellae with an electron-translucentamorphous material and membrane fragments filling thespaces between bacterial cells (Fig. 3B), whereas other pelletscontained fewer bacteria and a more abundant portion ofinterbacterial membranous material (Fig. 3C). It was observedthat none of the pellets had a continuous membrane aroundthem (to suggest that they were vesicles). Instead, pellets eitherlacked any defined boundary (Fig. 3D) or were bound by stacksof noncontinuous membrane fragments and/or an amorphousmaterial (Fig. 3). The differences observed in pellet ultrastruc-ture suggested that L. pneumophila had followed different in-travacuolar fates, but regardless of their fate no TEM evidenceof cell division of the legionellae found in the different pelletswas forthcoming.

Food vacuole turnover in pulse-chase experiments. To de-termine whether all food vacuoles followed a similar turnoverrate, pulse-chase feeding experiments were performed withgreen fluorescent L. pneumophila and red fluorescent E. coli.When a 1-h pulse of red fluorescent E. coli was chased withnonfluorescent E. coli, a gradual polarization and decrease inthe number of vacuoles containing red fluorescent bacteria wasobserved (Fig. 4A). After an overnight incubation, only a smallnumber of ciliates (�10%) depicted a single spot of red fluo-rescence (Fig. 4A, panel O/N), and no expelled pellets con-taining red fluorescent bacteria were found, suggesting com-plete digestion. TEM confirmed the structural degradation ofE. coli in food vacuoles and the presence of expelled membra-nous pellets (Fig. 5). When a 1-h pulse of red fluorescent E.coli was chased for 3 h with green fluorescent L. pneumophila,vacuoles containing red fluorescent E. coli were rapidly dis-placed by numerous vacuoles containing green fluorescent le-gionellae (Fig. 4B). After the 3-h chase with green fluorescentlegionellae, all free bacteria were removed, and ciliates wereincubated overnight in the absence of further feeding. Underthese conditions most green fluorescent legionellae were ex-pelled in pellets during the overnight incubation (Fig. 4B,panels O/N-T and O/N-P). Finally, when a 1-h pulse of greenfluorescent Lp02 was chased with nonfluorescent Lp02, a rapidpolarization and decrease in the number of fluorescent vacu-oles was observed (Fig. 4C), as in the case of E. coli (Fig. 4A).However, in contrast to E. coli, a few intracellular vacuolescontaining green-fluorescent legionellae remained after 20 h offeeding (Fig. 4C, 20 h). As previously suggested for Tetrahy-mena vorax (50), it is possible that Tetrahymena sp. retained L.pneumophila-laden food vacuoles longer than those containingE. coli, but the possibility of reingestion of free fluorescentlegionellae (released from expelled pellets) cannot be ignored.These results suggest that feeding ciliates do not selectivelyretain vacuoles and that vacuole turnover remained steady.

Pellet formation is not associated with replication of L.pneumophila. A lack of L. pneumophila replication in Tetrahy-mena sp. was strongly suggested by our TEM observations (Fig.2) and previous reports indicating that Tetrahymena does notsupport the intracellular growth of L. pneumophila at temper-atures of �30°C (19, 50). Therefore, the number of L. pneu-

mophila CFU associated with ciliates that fed for 3 h on viru-lent legionellae was investigated. The initial number ofCFU/ml (set after the 3-h feeding period followed by a 1-hgentamicin treatment to kill free extracellular bacteria) de-creased 100-fold in 24 h for the SVir strain and 1,000-foldfor the Lp02 strain (Fig. 6A and B). When this experiment wasrepeated with the L. pneumophila strain JR-32, we also ob-served a 100-fold reduction in total CFU/ml, and the graphs(not shown) followed a shape similar to that of Fig. 6A. Re-ductions in CFU counts were also observed for L. pneumophilaATCC 33216 in the presence of Tetrahymena sp. at 25°C (notshown). Because we estimated that the average-size pelletcould contain up to a hundred L. pneumophila cells (seeabove), it is possible that the 100-fold decrease in CFUnumbers observed for SVir and JR-32 simply reflected theeffect of packaging. In fact, light microscopy indicated thatmost pellets (70%) were not disrupted by the Triton X-100treatment incorporated into the CFU counts protocol. Otherexplanations for the low CFU counts (which surpassed a 100-fold decrease for Lp02) include the possibility that some of theingested legionellae were killed and digested (as shown below)

FIG. 5. Tetrahymena efficiently digests E. coli cells. Transmissionelectron micrographs of intravacuolar E. coli JM109 showing signs ofstructural degradation (A) and a single dispersed pellet expelled byTetrahymena feeding on E. coli DH5� showing no surviving bacterialcells and abundant membranous whorls (B). The size bars in panels Aand B represent 500 nm.

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or entered a viable but nonculturable state (43, 55) (nottested).

It is important to note that 100% of the pellets examinedcontained viable L. pneumophila cells as indicated by theBacLight LIVE/DEAD staining kit (Fig. 6C and D). However,the viable/dead legionella ratio varied between pellets, as pre-viously determined with a different vital stain (38). TEM con-firmed that some cells of the two virulent L. pneumophilastrains Lp02 and Lp1-Svir (TEM of strain JR-32 was not per-formed) were structurally degraded inside food vacuoles (e.g.,Fig. 2C and see Fig. 8C). Nevertheless, regardless of the degreeof bacterial killing (or survival) inside food vacuoles, or themechanism by which legionellae may survive (see below), weconcluded that virulent legionellae does not show a net growthin Tetrahymena sp.

Formation of legionella-laden pellets depends on a Dot/Icm-mediated survival mechanism. When ciliates were fed with thedot mutants JV303 (dotB) or JV309 (dotA), few dispersedpellets were expelled as determined by light microscopy, whichwas similar to the low number of dispersed pellets produced byciliates feeding on E. coli. The pellets expelled by ciliates feed-ing on dot mutants (as those shown in Fig. 5B for E. coli)typically depicted abundant membranous material wrappedaround a few bacterial cells (Fig. 7A and B). Within intracel-lular food vacuoles, the mutants often appeared structurally

degraded and associated with abundant membranous and ve-sicular material (Fig. 7C and D). The membrane of food vacu-oles containing ingested dot mutants neither had tightly ap-posed mitochondria, nor did it follow the outline of thecontained mutants (Fig. 7C and D).

Because membrane whorls have been traditionally regardedas the remains of digested bacteria (12, 14), it was surmisedthat dot mutants were being effectively digested by Tetrahy-mena sp. Using immunogold labeling with an OmpS-specificantibody, the membrane material of dot mutant pellets wasclearly immunostained, strongly suggesting that this materialconsisted of undigested outer membranes from the dot mu-tants (Fig. 8). The anti-OmpS serum and colloidal gold conju-gate also labeled membranous material present in Lp02 pellets(Fig. 8C). The anti-Hsp60 antibody and colloidal gold conju-gate labeled the cytoplasm and envelope of intact bacterialcells (not shown) but not the membrane material of the ex-pelled pellets, suggesting that, in contrast to OmpS that isresistant to proteases, the free or membrane-associated chap-eronin had been digested.

Genetic complementation of the dot mutants restored theirability to remain morphologically intact inside food vacuoles(not shown), and numerous pellets containing viable bacteria(as determined by their ability to form colonies when spottedon BCYE agar plates) were produced by Tetrahymena sp. feed-

FIG. 6. The interaction of L. pneumophila with ciliates is associated with a loss in bacterial viability or culturability. Graphs of two independentfeeding experiments, each sampled in triplicate, for strains Lp1-SVir (A) and Lp02 (B), show a decrease in total L. pneumophila CFU per milliliterof Tetrahymena culture. Control curves represent L. pneumophila alone suspended in Osterhout’s solution. Means � standard deviations (n � 3)for each experiment are shown. A group of pellets expelled during feeding experiments with L. pneumophila strain 33216 stained with the BacLightLIVE/DEAD kit, as observed in DIC (C) or confocal fluorescence microscopy to detect live green fluorescent bacteria (shown here in grayscale)(D).

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ing on the complemented �dotB mutant (Fig. 9) and the com-plemented dotA mutant (not shown). Collectively, these resultssuggest that the Dot/Icm system is required for L. pneumophilato resist digestion in the ciliate Tetrahymena sp. and that re-sistance to digestion is, in turn, a requirement for the forma-tion of numerous legionella-laden pellets.

DISCUSSION

Four different strains of L. pneumophila were unable to shownet growth in Tetrahymena sp. at 25 and 30°C, signifying agree-ment with existing evidence (3, 18, 19, 50, 54) that ciliates donot consistently favor the replication of L. pneumophila. How-ever, the four L. pneumophila strains survived inside food vacu-oles during their short intracellular residence and were subse-quently expelled by the host Tetrahymena sp. in what wepreviously referred to as legionella-laden vesicles (38). Be-cause we have now demonstrated that a continuous singlemembrane does not surround the expelled legionellae and thatthe vesicle-associated membrane fragments are of bacterialorigin, we have chosen to replace the previously used term

“vesicle” with the term “pellet.” In contrast to L. pneumophila-infected amoebae that often release free legionellae after lysis(20, 44), Tetrahymena sp. appeared to exclusively release nu-merous legionella-laden pellets. Therefore, this ciliate couldplay an important role as an efficient packager of free legio-nellae in nature, but particularly in man-made aquatic envi-ronments (e.g., cooling towers), where the coexistence of cili-ates and elevated L. pneumophila numbers is favored. Giventhe potential ecological and epidemiological importance oflegionella-laden pellets (7, 8, 22), and the high efficiency atwhich Tetrahymena sp. apparently produced such pellets (38),we investigated the pellet production process in detail anddetermined that it both occurs in the absence of L. pneumo-phila replication and depends on the presence of a functionalDot/Icm virulence system in the packaged legionellae, which inturn mediates bacterial resistance to digestion by the ciliate.

The packaging of L. pneumophila into pellets appeared tocorrelate with a fractional loss of bacterial viability or cultur-ability, as collectively suggested by a decrease in CFU, our vitalfluorescent staining results, and TEM results that demon-strated in-vacuole degradation of L. pneumophila. While the

FIG. 7. Electron micrographs showing expelled pellets (A and B) or food vacuoles (C and D) produced by Tetrahymena cells feeding on dotAmutant JV309 (A and C) or dotB mutant JV303 (B and D). Pellet samples were fixed at 24 h postinoculation, whereas food vacuole samples werefixed at 4 h postinoculation. The arrows in panels C and D point at structurally degraded bacteria. Notice the abundance of membranous whorlsin pellets and membranous and vesicular material in food vacuoles. All size bars represent 0.5 �m.

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absence of bacterial replication was experimentally addressedhere, determination of the proportion of ingested legionellaeactually killed by Tetrahymena sp. remained a difficult task.Further analysis should consider that some of the structurallydegraded virulent L. pneumophila cells identified by TEMcould have been already dead when ingested and that TEM isunable to distinguish live from dead morphologically intactbacteria. As indicated by the BacLight LIVE/DEAD stain, 5 to10% of the bacterial inoculum typically used in feeding exper-

iments consisted of dead bacterial cells, a likely source of themembranous material always found in food vacuoles and pel-lets of virulent legionellae. In addition, the distinction of cul-turable from nonculturable forms among the viable legionellae(showing a positive vital stain) remains to be determined. How-ever, it is important to emphasize that regardless of how manylegionellae lose their viability or culturability, or the mecha-nism by which this happens, any given pellet always containedviable L. pneumophila cells and could therefore act as a com-plex infectious particle as discussed below.

The ability of L. pneumophila to survive inside food vacuolesand resist intravacuolar digestion was shown to require a func-tional Dot/Icm system. This type IV secretion system is key inresisting digestion in amoeba and mammalian macrophages,where effectors translocated to the host cell cytoplasm by theDot/Icm system mediate the establishment of a specializedmembrane-bound compartment (known as the Legionella-con-taining vacuole) where L. pneumophila replicates (13, 27, 33,34, 40, 48, 57). It seems logical that such effectors are alsotranslocated by the Dot/Icm system of virulent L. pneumophilainto the cytoplasm of Tetrahymena, across the membrane liningfood vacuoles. It is tempting to speculate that the juxtapositionof the vacuolar membrane and L. pneumophila cells, as well asthe close apposition of mitochondria, could be a host responseto (or the result of) translocated L. pneumophila effectors. Thefact that the dot mutants did not induce membrane warpingand mitochondria apposition indeed suggested that these areDot/Icm-mediated effects.

The presence of membranous remnants of digested legio-nellae in food vacuoles (e.g., Fig. 2) or in expelled pellets (Fig.3 and 8) side by side with morphologically intact undigestedbacteria implies that the Dot/Icm-dependent mechanism uti-lized to resist digestion did not protect all of the bacteriacontained in a given food vacuole. Furthermore, the differentamounts of bacterial outer membrane remnants present infood vacuoles and their resulting pellets (Fig. 3) suggested thatthe levels of resistance to digestion could vary between foodvacuoles. For instance, a vacuole where most of the containedlegionellae were digested would result in a membranous pelletwith few apparently intact bacteria (e.g., Fig. 3A, pellet 3).Alternatively, it is possible that ciliates have the ability to sortparticulate food into different vacuoles and thus were capableof forming vacuoles enriched in dead legionellae that upondigestion would give rise to membranous pellets. Notwith-standing, our results indicate that the production of pelletscontaining numerous live legionellae depends on the presenceof a functional Dot/Icm system in the ingested L. pneumophilacells. Here we are not suggesting that packaging of L. pneu-mophila into pellets is a process actively driven by the Dot/Icmsystem. Instead, to get packaged the ingested legionellae musthave a functional Dot/Icm system, which in turn mediatesbacterial survival in the ciliate.

A related issue is whether the maintenance of pellet shape(as spheres) is defined by a ciliate-mediated process or requiresthe presence of bacteria. We conducted preliminary experi-ments with albumin-coated, 1-�m-diameter, fluorescent beadsand observed that these beads trafficked undigested throughthe ciliate and were expelled as free particles (not in the formof pellets). Therefore, it seems that Tetrahymena sp. alone doesnot contribute to the maintenance of pellet shape in expelled

FIG. 8. Electron micrographs of sections labeled with OmpS-spe-cific polyclonal antibodies and a secondary antibody conjugated to10-nm gold particles, confirming the bacterial origin of the abundantmembranous material present in pellets and food vacuoles. (A) Pelletof dotA mutants. (B) Portion of a food vacuole containing some ap-parently intact dotB mutants and a degraded mutant (arrow).(C) Small pellet of virulent Lp02 cells. Notice the specific labeling ofthe membrane fragments and the outer membrane of structurally pre-served bacterial cells. All specimens were fixed 24 h postinoculation.Size bars represent 0.5 �m.

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undigested particles. Although it seems that this process re-quires the presence of bacteria, the mechanism by which shapeis maintained, and the possible role that the Dot/Icm system (ifany) may play in it, remains to be determined.

In contrast to amoebae and mammalian macrophages,where the early effects of the Dot/Icm system lead to intracel-lular replication of L. pneumophila, the Dot/Icm-mediated sur-vival in Tetrahymena food vacuoles did not result in intracel-lular replication at 30°C. It is not clear why L. pneumophilagrows in T. pyriformis at 30 to 35°C and not below 30°C (3, 18,19), but the fact that Steele and McLennan (54) reported that

their T. pyriformis strain did not survive at 35°C (as we ob-served for T. pyriformis 30202 and Tetrahymena sp.) and thatManasherob et al. (36) indicated that T. pyriformis was lysed at35°C suggests the possibility that a heat-tolerant culture ofaltered physiology was used by Fields et al. (19). Because L.pneumophila grows in amoebae at temperatures below 30°Cand even at 20°C as indicated by Lee and West (32), we pro-pose that physiological and/or biochemical host factors and notsimply temperature are responsible for restricting the intracel-lular growth of L. pneumophila in Tetrahymena sp. The factthat the ingested L. pneumophila cells did not show any ultra-

FIG. 9. The resistance to digestion and, consequently, production of numerous pellets containing live legionellae is Dot/Icm system dependent.(A to C) Electron micrographs of the pellets produced by ciliates feeding on the �dotB mutant JV918 (A), the genetically complemented �dotBmutant JV1170 (B), and the mock-complemented �dotB mutant JV1133 (C). (D to F) Low-magnification phase-contrast micrographs showingTetrahymena sp. cells and pellets in a live culture fed with the �dotB mutant JV918 (D), the genetically complemented �dotB mutant JV1170 (E),and the mock-complemented �dotB mutant JV1133 (F). Only ciliates feeding on the genetically complemented �dotB mutant often acquired around shape (arrowhead in panel E) and produced massive aggregative pellets (arrow in panel E) that contained numerous bacterial cells (B).Cytoplasmic inclusions that were not properly infiltrated with epoxy resin appear bubbled and enlarged (A and B). Ciliates feeding on Dot/Icm-defective L. pneumophila looked slender, swam very actively, and produced a few dispersed pellets (D and F). The size bars in panels A to Crepresent 0.5 �m. The length of the arrow in panel E represents 33 �m and applies to panels D and F.

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structural indicators of cell division at any sampling time sug-gests that a growth restriction mechanism (which the Dot/Icmsystem was unable to overcome) was in effect very early afteringestion. It is possible that the rapid turnover of food vacuolesalone did not allow L. pneumophila enough time to initiatereplication. However, the observations of Smith-Somerville etal. in Tetrahymena vorax (50), where L. pneumophila did notreplicate in spite of food vacuoles being retained for severalhours, argues against such a possibility. Alternatively, Tetrahy-mena sp. may fail to respond to some L. pneumophila effectors,or perhaps L. pneumophila is unable to translocate all of itseffectors into the ciliate. Tetrahymena sp. may be able to se-lectively degrade some Dot/Icm effectors or rely on uniquetrafficking mechanisms (not present in amoebae or mammaliancells) to mobilize its food vacuoles. Finally, it is possible thatthe ingested legionellae may not receive a “germination” signal(47) to allow differentiation of L. pneumophila into replicativeforms that can grow in the ciliate. Tetrahymena sp. may thusconstitute a useful experimental model to investigate some ofthe molecular mechanisms used by host cells to restrict theintracellular growth of L. pneumophila.

In conclusion, we propose that the interaction of L. pneu-mophila with Tetrahymena species that efficiently package le-gionellae into pellets may have important epidemiological andecological implications. One example would be the enhancedsurvival of pelleted legionellae (7, 8, 22), which is likely topromote a wide distribution of L. pneumophila in aquatic en-vironments and thereby favor contact with humans and thetransmission of Legionnaires’ disease. Our observation thatonly virulent legionellae that carry a functional Dot/Icm systemand resist the intravacuolar digestive mechanisms of ciliatesare selectively packaged (which may constitute the most infec-tious legionellae to humans [18]) adds relevance to the poten-tial role that pellets may play as complex infectious units.

ACKNOWLEDGMENTS

The technical support of Mary Ann Trevors in the preparation ofsamples for TEM is gratefully acknowledged, as is the technical assis-tance of Kim Jefferies and Poornima Gourabathini in conducting someof the experiments reported here. We thank Paul S. Hoffman (cur-rently at the University of Virginia, Charlottesville) for the hyperim-mune rabbit sera raised against L. pneumophila OmpS or Hsp60 andfor bacterial strains and A. K. C. Brassinga (currently at the Universityof Virginia) for plasmid pBH6119::htpAB. We thank Howard A. Shu-man and Joe Vogel for strains, Ralph Isberg for strains and plasmidpKB9, and Michele Swanson for plasmid pBH6119. We acknowledgeAlistair Simpson (Dalhousie University) for his contribution to theanalysis of the small ribosomal subunit rRNA gene sequence. Finally,we thank the anonymous reviewers who made excellent suggestions forthe improvement of the manuscript.

This study was supported by the Canadian Institutes of HealthResearch through both operating grant ROP-83334 and equipmentmaintenance grant PRG-80150 (R.A.G.); the Center for the Manage-ment, Utilization and Protection of Water Resources, TennesseeTechnological University (S.G.B.); and by grant R825352-01 (S.G.B.)from the U.S. Environmental Protection Agency.

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