Intracellular Growth Of Legionella Pneumophila Research Articles

Evaluation of impermeant, DNA-binding dye fluorescence as a real-time readout of eukaryotic cell toxicity in a high throughput screening format.

Interpretation of high throughput screening (HTS) data in cell-based assays may be confounded by cytotoxic properties of screening compounds. Therefore, assessing cell toxicity in real time during the HTS process itself would be highly advantageous. Here, we investigate the potential of putatively impermeant, fluorescent, DNA-binding dyes to give cell toxicity readout during HTS. Amongst 19 DNA-binding dyes examined, three classes were identified that were (1) permeant, (2) cytotoxic, or (3) neither permeant nor cytotoxic during 3-day incubation with a macrophage cell line. In the last class, four dyes (SYTOX Green, CellTox Green, GelGreen, and EvaGreen) gave highly robust cytotoxicity data in 384-well screening plates. As proof of principle, successful combination with a luminescence-based assay in HTS format was demonstrated. Here, both intracellular growth of Legionella pneumophila (luminescence) and host cell viability (SYTOX Green exclusion) were assayed in the same screening well. Incorporation of membrane-impermeant, DNA-binding, fluorescent dyes in HTS assays should prove useful by allowing evaluation of cytotoxicity in real time, eliminating reagent addition steps and effort associated with endpoint cell viability analysis, and reducing the need for follow-up cytotoxicity screening.

Defective lysosome maturation and Legionella pneumophila replication in Dictyostelium cells mutant for the Arf GAP ACAP-A.

Dictyostelium discoideum ACAP-A is an Arf GTPase-activating protein (GAP) involved in cytokinesis, cell migration and actin cytoskeleton dynamics. In mammalian cells, ACAP family members regulate endocytic protein trafficking. Here, we explored the function of ACAP-A in the endocytic pathway of D. discoideum. In the absence of ACAP-A, the efficiency of fusion between post-lysosomes and the plasma membrane was reduced, resulting in the accumulation of post-lysosomes. Moreover, internalized fluid-phase markers showed extended intracellular transit times, and the transfer kinetics of phagocyted particles from lysosomes to post-lysosomes was reduced. Neutralization of lysosomal pH, one essential step in lysosome maturation, was also delayed. Whereas expression of ACAP-A-GFP in acapA(-) cells restored normal particle transport kinetics, a mutant ACAP-A protein with no GAP activity towards the small GTPase ArfA failed to complement this defect. Taken together, these data support a role for ACAP-A in maturation of lysosomes into post-lysosomes through an ArfA-dependent mechanism. In addition, we reveal that ACAP-A is required for efficient intracellular growth of Legionella pneumophila, a pathogen known to subvert the endocytic host cell machinery for replication. This further emphasizes the role of ACAP-A in the endocytic pathway.

NMR structure note: the ferrous iron transport protein C (FeoC) from Klebsiella pneumoniae

Iron is an essential nutrient involved in many important biological processes ranging from electron transfer, oxygen transport, gene regulation, photosynthesis, N2 fixation to DNA biosynthesis (Andrews et al. 2003). Thus, the ability to acquire sufficient quantities of iron from the environment is vital for the survival of living organisms. This is particularly true for pathogens that must compete with the host iron-withdrawal response for their iron supplies. However, iron is also toxic and excess iron can be detrimental for the living system (Touati 2000). It is, therefore, essential for almost all life forms to maintain proper iron homeostasis (Andrews et al. 2003). Feo is a unique ferrous iron transporter commonly utilized by bacterial systems for acquiring ferrous iron from the environment (Cartron et al. 2006). It was first discovered in Escherichia coli K-12 (Hantke 1987). Subsequently the feo locus was cloned and sequenced (Kammler et al. 1993). The importance of the Feo system has been confirmed in several systems. Both E. coli and Salmonella feoB mutants are attenuated in their abilities to colonize the mouse intestine presumably due to their inability to transport ferrous iron within the anaerobic environment of the mouse intestine (Stojiljkovic et al. 1993; Tsolis et al. 1996). In H. pylori, FeoB appears to provide the main route of iron uptake. It is required for H. pylori colonization of mouse gastric mucosa, as well as for normal growth and iron-uptake under iron restricted conditions (Velayudhan et al. 2000). FeoB is also required for intracellular growth of Legionella pneumophila (Robey and Cianciotto 2002). Thus, various studies have clearly established an in vivo role for Feo in colonization of the gut and in virulence. The feo operon of enterobacteria encodes three proteins: FeoA, FeoB and FeoC. FeoA is a small SH3-like protein predicted to act as GTPase activating protein (GAP) and/or Fe-dependent repressor (Cartron et al. 2006). FeoB is a large protein (773 a.a.) composing of a 270-residue cytosolic N-terminal domain (NFeoB, residues 1–270) that contains a G-protein domain (a.a. 1–170) and a helical bundle S-domain (a.a. 171–270) presumed to be a GDP-dissociation inhibitor (GDI). The C-terminal region of FeoB is a helical transmembrane domain which is likely to act as the ferrous iron permease and essential for ferrous iron transport activity. FeoC (also called yhgG) is a small 78-residue, hydrophilic winged-helix domain (WHD) found only in c-proteobacteria. The feoC gene was found preceding the feoB gene in the feo operon. Multiple sequence alignment of FeoC proteins shows that they possess four conserved cysteine residues (CxxGxCKxCPx4-7C) that are likely to provide a binding site for an [Fe–S]-cluster. Thus, FeoC is presumed to be a [Fe–S]dependent transcriptional regulator directly controlling the expression of the feo operon (Cartron et al. 2006). Given its importance and novelty, there has had increasing interest in dissecting the molecular basis of the Feo system. In particular, the presence of a G-protein motif in the intracellular domain of FeoB (NFeoB) has spurred considerable interest in understanding its molecular basis and the structures of NFeoB from several species have K.-W. Hung T. Juan Y. Hsu T. H. Huang (&) Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC e-mail: bmthh@ibms.sinica.edu.tw

Gemfibrozil inhibits Legionella pneumophila and Mycobacterium tuberculosis enoyl coenzyme A reductases and blocks intracellular growth of these bacteria in macrophages.

We report here that gemfibrozil (GFZ) inhibits axenic and intracellular growth of Legionella pneumophila and of 27 strains of wild-type and multidrug-resistant Mycobacterium tuberculosis in bacteriological medium and in human and mouse macrophages, respectively. At a concentration of 0.4 mM, GFZ completely inhibited L. pneumophila fatty acid synthesis, while at 0.12 mM it promoted cytoplasmic accumulation of polyhydroxybutyrate. To assess the mechanism(s) of these effects, we cloned an L. pneumophila FabI enoyl reductase homolog that complemented for growth an Escherichia coli strain carrying a temperature-sensitive enoyl reductase and rendered the complemented E. coli strain sensitive to GFZ at the nonpermissive temperature. GFZ noncompetitively inhibited this L. pneumophila FabI homolog, as well as M. tuberculosis InhA and E. coli FabI.

The phagosomal nutrient transporter (Pht) family.

Phagosomal transporters (Phts), required for intracellular growth of Legionella pneumophila, comprise a novel family of multispanning alpha-helical proteins within the major facilitator superfamily (MFS). The members of this family derive exclusively from bacteria. Multiple paralogues are present in a restricted group of Alpha- and Gammaproteobacteria, but single members were also found in Chlamydia and Cyanobacteria. Their protein sequences were aligned, yielding a phylogenetic tree showing the relations of the proteins to each other. Topological analyses revealed a probable 12 alpha-helical transmembrane segment (TMS) topology. Motif identification and statistical analyses provided convincing evidence that these proteins arose from a six TMS precursor by intragenic duplication. The phylogenetic tree revealed some potential orthologous relationships, suggestive of common function. However, several probable examples of lateral transfer of the encoding genetic material between bacteria were identified and analysed. The Pht family most closely resembles a smaller MFS family (the UMF9 family) with no functionally characterized members. However, the UMF9 family occurs in a broader range of prokaryotic organism types, including Archaea. These two families differ in that organisms bearing members of the Pht family often have numerous paralogues, whereas organisms bearing members of the UMF9 family never have more than two. This work serves to characterize two novel families within the MFS and provides compelling evidence for horizontal transfer of some of the family members.

Restriction of Legionella pneumophila growth in macrophages requires the concerted action of cytokine and Naip5/Ipaf signalling pathways

Macrophages from the C57BL/6 (B6) mouse strain restrict intracellular growth of Legionella pneumophila, whereas A/J macrophages are highly permissive. The mechanism by which B6 macrophages restrict Legionella growth remains poorly understood, but is known to require the cytosolic microbe sensors Naip5 (Birc1e) and Ipaf. We hypothesized that Naip5 and Ipaf may act in partnership with other antimicrobial signalling pathways in macrophages. Indeed, we found that macrophages lacking either tumour necrosis factor (TNF)-alpha or type I interferon (IFN) signalling are permissive for growth of L. pneumophila, even in the presence of functional Naip5 and Ipaf alleles. Similarly, macrophages lacking Naip5 and/or Ipaf signalling were permissive even though we found that Naip5 or Ipaf were not required for induction of TNF-alpha and type I IFN. Therefore, our data suggest that the mechanism by which B6 macrophages restrict intracellular replication of L. pneumophila is more complex than previously appreciated, and involves the concerted action of cytokine and intracellular microbe sensor signalling pathways.

Differences in protein synthesis between wild type and intracellular growth-deficient strains of Legionella pneumophila in U937 and Acanthamoeba polyphaga

An important aspect of Legionnaires' disease is the growth of the causative agent, Legionella pneumophila, within infected host cells. Many proteins including stress proteins of L. pneumophila were strongly induced in a wild type strain that had been used to infect U937 human macrophage-like cells. In contrast, the expression of the proteins was much weaker within a protozoan host, Acanthamoeba polyphaga. The results suggested that active bacterial protein synthesis is required more within macrophages than within protozoa for adaptation of L. pneumophila to intracellular environments. The synthesis of these proteins was not observed in intracellular growth-deficient strains after infection in either type of host cells. The inability of protein synthesis in these strains is correlated with their inability of intracellular growth. Furthermore, on U937 infection, the synthesis of β-galactosidase encoded in an inducible reporter construct immediately ceased in the in intracellular growth-deficient strains after infection, while the wild type strain was able to synthesize it during the course of infection. These results suggested that the intracellular growth of Legionella pneumophila within macrophages requires active protein synthesis from an earlier stage of bacterial infection.

Function and Mechanism of Action of Dictyostelium Nramp1 (Slc11a1) in Bacterial Infection

Dictyostelium amoebae are professional phagocytes, which ingest bacteria as the principal source of food. We have cloned the Dictyostelium homologue of human natural resistance-associated membrane protein 1 (Nramp1) [solute carrier family 11 member 1 (Slc11a1)], an endo-lysosomal membrane protein that confers on macrophages resistance to infection by a variety of intracellular bacteria and protozoa. The Dictyostelium Nramp1 gene encodes a protein of 53 kDa with 11 putative transmembrane domains. The Nramp1 gene is transcribed during the growth-phase and downregulated to barely detectable levels upon starvation. To gain insights into their intracellular localization, we fused Nramp1 or the vatB subunit of the V-H(+)ATPase with green fluorescent protein and expressed in cells. Green fluorescent protein-vatB was inserted in membranes of all acidic compartments and the contractile vacuole network and decorated macropinosomes and phagosomes. Green fluorescent protein-Nramp1 decorated macropinosomes and phagosomes, in addition to intracellular vesicular compartments positive for endosomal SNARE protein Vti1 or vacuolin, a marker of the exocytic pathway. Nramp1 disruption generated mutants that were more permissive hosts than wild-type cells for intracellular growth of Legionella pneumophila and Micobacterium avium. Nramp1 overexpression protected cells from L. pneumophila infection. Evidence is provided that Nramp1 transports metal cations out of the phagolysosome in an ATP-dependent process and that L. pneumophila and M. avium use different mechanisms to neutralize Nramp1 activity.

Caenorhabditis elegans is a model host for Salmonella typhimurium.

The idea of using simple, genetically tractable host organisms to study the virulence mechanisms of pathogens dates back at least to the work of Darmon and Depraitère [1]. They proposed using the predatory amoeba Dictyostelium discoideum as a model host, an approach that has proved to be valid in the case of the intracellular pathogen Legionella pneumophila [2]. Research from the Ausubel laboratory has clearly established the nematode Caenorhabditis elegans as an attractive model host for the study of Pseudomonas aeruginosa pathogenesis [3]. P. aeruginosa is a bacterium that is capable of infecting plants, insects and mammals. Other pathogens with a similarly broad host range have also been shown to infect C. elegans [3,4]. Nevertheless, the need to determine the universality of C. elegans as a model host, especially with regards pathogens that have a naturally restricted host specificity, has rightly been expressed [5]. We report here that the enterobacterium Salmonella typhimurium, generally considered to be a highly adapted pathogen with a narrow range of target hosts [6], is capable of infecting and killing C. elegans. Furthermore, mutant strains that exhibit a reduced virulence in mammals were also attenuated for their virulence in C. elegans, showing that the nematode may constitute a useful model system for the study of this important human pathogen.

Intracellular growth of Legionella pneumophila in Dictyostelium discoideum, a system for genetic analysis of host-pathogen interactions.

Conditions were established in which Legionella pneumophila, an intracellular bacterial pathogen, could replicate within the unicellular organism Dictyostelium discoideum. By several criteria, L. pneumophila grew by the same mechanism within D. discoideum as it does in amoebae and macrophages. Bacteria grew within membrane-bound vesicles associated with rough endoplasmic reticulum, and L. pneumophila dot/icm mutants, blocked for growth in macrophages and amoebae, also did not grow in D. discoideum. Internalized L. pneumophila avoided degradation by D. discoideum and showed evidence of reduced fusion with endocytic compartments. The ability of L. pneumophila to grow within D. discoideum depended on the growth state of the cells. D. discoideum grown as adherent monolayers was susceptible to L. pneumophila infection and to contact-dependent cytotoxicity during high-multiplicity infections, whereas D. discoideum grown in suspension was relatively resistant to cytotoxicity and did not support intracellular growth. Some known D. discoideum mutants were examined for their effect on growth of L. pneumophila. The coronin mutant and the myoA/B double myosin I mutant were more permissive than wild-type strains for intracellular growth. Growth of L. pneumophila in a G(beta) mutant was slightly reduced compared to the parent strain. This work demonstrates the usefulness of the L. pneumophila-D. discoideum system for genetic analysis of host-pathogen interactions.

Effects of interferon-alpha, beta, and gamma on the function of differentiated leukemic HL-60 cells induced by 1,25-dihydroxyvitamin D3.

The differentiation of HL-60, a human leukemic cell line, into monocyte-like cells (D3-HL-60 cells) is induced by 1,25-dihydroxyvitamin D3 (D3). We examined the effects of interferon (IFN) treatment of D3-HL-60 cells on the expression of cell surface antigens, the phagocytic activity for fluorescent beads, production of oxygen radicals, and intracellular growth of Legionella pneumophila. Activation of D3-HL-60 cells with IFN-gamma, Beta, and alpha for 24 h significantly increased expression of CD16, CD36, CD71, and HLA-DR antigens. IFN-gamma markedly enhanced the phagocytic activity of beads in D3-HL-60 cells. There was no significant difference in phagocytic activity between cells exposed to IFN-alpha or beta and untreated D3-HL-60 cells. IFN-alpha, beta, and gamma enhanced production of oxygen radicals, including superoxide, by D3-HL-60 cells. Superoxide production was enhanced to the greatest degree by IFN-gamma, followed by IFN-beta and then IFN-gamma. Intracellular growth of L. pneumophila in D3-HL-60 cells was inhibited by interferons (IFN-gamma > beta > gamma). Similar results were obtained in human mononuclear cells. These data indicate that interferons can act as biologic response modifiers not only in human mononuclear cells but also in differentiated leukemic cells. Our results may have implications for the development of differentiation therapy for treatment of leukemia.

Association of flagellum expression and intracellular growth of Legionella pneumophila.

We examined the role of the flagella of Legionella pneumophila in the infection of amoebae and human monocyte-like cells. Insertional mutants were constructed with mini-Tn10. Ten mutants (F-) which did not react with polyclonal L. pneumophila antiflagellar antisera were identified. Ten randomly selected mutants (F+) that did react with the polyclonal antiflagellar antiserum were also identified. The infectivity of these 20 mutants in Hartmannella vermiformis and human U937 cells was characterized. Seven of the 10 F- mutants were attenuated in their ability to multiply in the amoebae during the first 3 days of coincubation and failed to multiply in U937 cells. Three of the 10 F- mutants multiplied as well as the wild-type parent strain did in amoebae and to a limited degree in U937 cells. None of the 10 F+ mutants were attenuated in either the amoebae or U937 cells. While the flagellar structure is not essential for virulence, the ability of L. pneumophila to infect amoebae and human phagocytic cells appears to be linked to flagellar expression. We believe that the attenuated F- mutants contain insertions in genes critical to both flagellum expression and the infection process.

A quantitative model of intracellular growth of Legionella pneumophila in Acanthamoeba castellanii

A model of intracellular growth for Legionella pneumophila in Acanthamoeba castellanii has been developed and provides a quantitative measure of survival and replication after entry. In this model, Acanthamoeba monolayers were incubated with bacteria in tissue culture plates under nutrient-limiting conditions. Gentamicin was used to kill extracellular bacteria following the period of incubation, and the number of intracellular bacteria was determined following lysis of amebae. Intracellular growth of virulent L. pneumophila and other wild-type Legionella species was observed when the assay was performed at 37 degrees C. At room temperature, none of the Legionella strains tested grew intracellularly, while an avirulent L. pneumophila strain was unable to replicate in this assay at either temperature. The effect of nutrient limitation on A. castellanii during the assay prevented multiplication of the amebae and increased the level of infection by Legionella spp. The level of infection of the amebae was directly proportional to the multiplicity of infection with bacteria; at an inoculum of 1.03 x 10(7) bacteria added to wells containing 1.10 x 10(5) amebae (multiplicity of infection of 100), approximately 4.4% of A. castellanii cells became infected. Cytochalasin D reduced the uptake of bacteria by the amebae primarily by causing amebae to lift off the culture dish, reducing the number of target hosts; methylamine also reduced the level of initial infection, yet neither inhibitor was able to prevent intracellular replication of Legionella spp. Consequently, once the bacteria entered the cell, only lowered temperature could restrict replication. This model of intracellular growth provides a one-step growth curve and should be useful to study the molecular basis of the host-parasite interaction.

Association of Lps gene with natural resistance of mouse macrophages against Legionella pneumophila

Peritoneal macrophages obtained from lipopolysaccharide (LPS)-low responder C3H/HeJ mice (J) permitted the intracellular growth of Legionella pneumophila after in vitro phagocytosis, while macrophages of LPS-high responder C3H/HeN mice (N) did not. Intracellular growth of the bacterium in macrophages of (J × N) F1 progeny was between the parent strains, showing that the traits were co-dominantly expressed. Correlation between intracellular bacterial growth in macrophages and LPS response of spleen cells was examined. Negative correlation was found between the two factors in F2, (J × F1) backcross and (N × F1) backcross progeny. This result implies that Lps gene controls the innate resistance of murine macrophages against the bacteria. Although macrophages of A/J strain also permit intracellular growth of L. pneumophila, gene complementation analysis of A/J and C3H/HeJ mice made clear that the gene control in C3H/HeJ differs from that of A/J strain. Macrophages of C57BL/10ScN, which is LPS-low responder line obtained from C57BL/10, were also defective in controlling the bacterial growth when compared to C57BL/10 mice. We suggest that the Lps gene also controls the natural resistance of murine macrophages against L. pneumophila.

Effects of cytochalasin D and methylamine on intracellular growth of Legionella pneumophila in amoebae and human monocyte-like cells

A cloned and axenically cultured strain of Hartmannella vermiformis was used as a model to study intracellular multiplication of Legionella pneumophila in amoebae. The growth of L. pneumophilia in both H. vermiformis and a human monocyte-like cell line (U937) was investigated with cytoskeletal and metabolic inhibitors. L. pneumophila replicated only intracellularly in these cellular models, and electron microscopy showed ultrastructural similarities in the initial phase of multiplication. Treatment of amoebae with an inhibitor of microfilament-dependent phagocytosis (cytochalasin D, 0.5 or 1.0 micrograms/ml) did not inhibit intracellular growth of L. pneumophila; however, intracellular multiplication was inhibited by treatment of U937 monocytes with the same concentrations of cytochalasin D. Methylamine (10 to 100 mM), an inhibitor of adsorptive pinocytosis, inhibited the replication of L. pneumophila in amoebae in a dose-dependent manner. All doses of methylamine tested (10 to 50 mM) inhibited growth of L. pneumophila in U937 monocytes. Cytochalasin D and methylamine had no effect on the multiplication of L. pneumophila in culture medium or on the viability of amoebae or U937 monocytes. Intracellular replication of L. pneumophila in H. vermiformis may be accomplished by a cytochalasin D-independent mechanism, such as adsorptive pinocytosis. In contrast, both cytochalasin D- and methylamine-sensitive mechanisms may be essential for the intracellular multiplication of L. pneumophila in U937 monocytes.

Activity of water biocide chemicals and contact lens disinfectants on pathogenic free-living amoebae

Free-living amoebae (FLA) are found in most soil and aquatic environments. They claim our attention not only because they are intrinsically interesting microbes about which little is known but also because of the capacity for certain species to cause severe and often fatal disease in man. Naegleria fowleri produces acute meningoencephalitis, whilst Acanthamoeba spp. cause chronic encephalitis in immunocompromised individuals and also a destructive ocular infection associated with contact lens wear. FLA can also support the intracellular growth of Legionella pneumophila, the bacterium causing Legionnaires' disease in man. The ability to regulate the presence of pathogenic and nonpathogenic FLA in the environment throufh the use of chemical disinfection is therefore relevant in the prevention of human disease. In this paper the activity of water biocide chemicals and contact lens disinfectants against pathogenic FLA is presented.

Legionella pneumophila intracellular growth in normal vs. Immune guinea pig macrophage cultures

Intracellular growth ofLegionella pneumophila, an opportunistic intracellular bacterium considered the cause of legionellosis, was assessed in peritoneal macrophages from normal and immunized guinea pigs. These bacteria grew exceedingly well in the normal guinea pig macrophages. Uptake of these bacteria was about the same by macrophages from either normal or immune guinea pigs, but their growth in immune macrophages was completely inhibited. Macrophages from normal guinea pigs stimulated with mezerin, a compound similar to diterpene ester, a known nonspecific stimulator of macrophages, or with specificLegionella vaccine released moderate or only small amounts of hydrogen peroxide, an indicator of macrophage activation to antimicrobicidal activity. In contrast, macrophages from immune guinea pigs produced much higher levels of hydrogen peroxide when stimulated with mezerein or theLegionella vaccine, and also showed a heightened response when cultured without a stimulator. These results indicate that macrophage activation related to the immune status of the host appears to have an important role in initial resistance toLegionella growth in susceptible individuals.

The effect of ofloxacin on the intracellular growth of Legionella pneumophila in guinea pig alveolar phagocytes.

Ofloxacin was evaluated as an antibiotic for possible use in the therapy of Legionnaires' disease in relation to its ability to penetrate alveolar phagocytes and inhibit Legionella pneumophila intracellular replication. A comparison with two other antibiotics used in the treatment of Legionnaires' disease, ciprofloxacin and erythromycin, was also made. Ofloxacin was found to be the most effective antibiotic, eliminating viable L. pneumophila from alveolar phagocytes at 0.001 mg/l. This was followed by ciprofloxacin, eliminating intracellular organisms at 0.01 mg/l. Erythromycin was shown to be much less effective, requiring a much higher concentration, of 0.1 mg/l. All three antibiotics had approximately similar MIC values and the considerable differences in intracellular penetration shown by these antibiotics indicate how discrepancies between in-vitro and in-vivo estimates of efficacy can occur.

Intracellular growth of Legionella pneumophila within Acanthamoeba castellanii Neff.

Acanthamoeba castellanii Neff supports the intracellular growth of Legionella pneumophila. When acanthamoebae were exposed to L. pneumophila for 1 h and then washed free of unassociated bacteria and placed in liquid culture, levels of viable amoeba-associated legionellae and legionellae free in the culture medium increased by three to four orders of magnitude in 48 to 72 h. However, most of the legionellae remained amoeba-associated and could be cultured only after disruption of the amoebae. Furthermore, legionella viability declined rapidly in amoeba culture medium alone or when bacteria and amoebae were separated by a microporous membrane. Therefore, direct amoeba-legionella contact is required for this growth. Infected acanthamoebae treated with cold acetone to permeabilize them to fluorescent-labeled anti-L. pneumophila antibody appeared to contain far more legionellae than amoebae fixed with glutaraldehyde so as to prevent antibody penetration. Electron micrographs of infected A. castellanii showed numerous bacteria, including some dividing forms, within vacuoles in the cytoplasm. These results together show that A. castellanii is able to provide an intracellular niche for the growth of L. pneumophila.