Role of the Legionella pneumophila rtxA gene in amoebae

Suat L. G. Cirillo1, Ling Yan1, Michael Littman1, Mustapha M. Samrakandi1 and Jeffrey D. Cirillo1

Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, 203 VBS, Fair and East Campus Loop, Lincoln, NE 68583, USA1

Author for correspondence: Jeffrey D. Cirillo. Tel: +1 402 472 8587. Fax: +1 402 472 9690. e-mail: jcirillo1{at}unl.edu


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Legionella pneumophila infects humans, causing Legionnaires’ disease, from aerosols generated by domestic and environmental water sources. In aquatic environments L. pneumophila is thought to replicate primarily in protozoa. A ‘repeats in structural toxin’ (RTX) gene, rtxA, from L. pneumophila was identified recently that plays a role in entry and replication in human macrophages and also has the ability to infect mice. However, the role of this gene in the interaction of L. pneumophila with environmental protozoa and its distribution in different Legionella species has not been examined. Southern analyses demonstrated that rtxA is present in all L. pneumophila isolates tested and correlates with species that have been shown to cause disease in humans. To evaluate the importance of rtxA in the interaction with protozoa a series of studies was carried out in an environmental host for L. pneumophila, Acanthamoeba castellanii. The L. pneumophila rtxA gene plays a role in both adherence and entry into A. castellanii similar to that observed in human monocytic cells. Furthermore, it was found that rtxA is involved in intracellular survival and trafficking. In addition to demonstrating involvement of rtxA in the interaction of L. pneumophila with host cells, these data support a role for this gene both during disease in humans and in environmental reservoirs.

Keywords: phagocytosis, lysosomes, amoeba, pathogenesis, pneumonia

Abbreviations: LY, lucifer yellow; RhR, rhodamine red; RTX, repeats in structural toxin


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Legionnaires’ disease, a potentially lethal pneumonia, is the result of the ability of Legionella to enter, survive and replicate in macrophages (Davis et al., 1983 ; Nash et al., 1984 ; Winn & Myerowitz, 1981 ). There are not any known animal reservoirs for L. pneumophila; however, protozoa in the environment provide an important environmental source of the bacteria that infect humans (Anand et al., 1983 ; Henke & Seidel, 1986 ; Rowbotham, 1980 , 1986 ). Since person to person transmission is not thought to occur (Yu et al., 1983 ), it appears that the natural hosts for L. pneumophila are these aquatic protozoa. In addition, the interaction of L. pneumophila with protozoa in the environment appears to enhance the ability to infect mammalian cells (Cirillo et al., 1994 ) and cause disease in animals (Brieland et al., 1996 , 1997a , b ; Cirillo et al., 1999 ). Thus, it is likely that the virulence mechanisms used by L. pneumophila to cause disease in humans evolved for optimal parasitism of protozoa.

Acanthamoeba castellanii, one potential environmental host for L. pneumophila (Rowbotham, 1980 ), undergoes a respiratory burst (Davies et al., 1991 ), produces oxygen radicals (Davies & Edwards, 1991 ), expresses cell surface receptors (Allen & Dawidowicz, 1990a ) and has phagocytic mechanisms (Allen & Dawidowicz, 1990b ; Brown et al., 1975 ; Lock et al., 1987 ) similar to human macrophages. Furthermore, the mechanisms that provide a selective advantage in amoebae and macrophages are likely to be similar, since they enter by coiling phagocytosis (Bozue & Johnson, 1996 ; Horwitz, 1984 ), inhibit lysosomal fusion (Bozue & Johnson, 1996 ; Horwitz & Maxfield, 1984 ) and localize to a rough endoplasmic reticulum-lined compartment (Abu Kwaik, 1996 ; B. S. Fields et al., 1986 ; Horwitz, 1983 ; Newsome et al., 1985 ; Swanson & Isberg, 1995 ) in both cells. Certainly, a large number of potential virulence factors have been identified that affect the ability of L. pneumophila to survive in macrophages and amoebae (Cianciotto & Fields, 1992 ; Gao et al., 1997 ; Pruckler et al., 1995 ; Segal & Shuman, 1999 ). Although there are at least 42 Legionella species, approximately 90% of Legionnaires’ disease cases in humans is caused by Legionella pneumophila (Marston et al., 1994 , 1993 ; Reingold et al., 1984 ). Only 18 of the remaining species are infrequently associated with disease and the rest are primarily found in aquatic environments (Morris et al., 1979 ; Rowbotham, 1993 ). Even in these aquatic environments, L. pneumophila is the predominant species isolated (Fliermans et al., 1981 ).

These data suggest that L. pneumophila differs from other Legionella species in a manner that provides a selective advantage in both humans and the environment. Recently, our laboratory has identified three L. pneumophila loci that are involved in entry into human epithelial cells and macrophages (Cirillo et al., 2000 ). A gene present in one of these loci, rtxA, encodes a ‘repeats in structural toxin’ (RTX). RTX proteins are a large family of pore-forming cytolysins present in a number of different bacterial pathogens (Welch, 1991 ). The L. pneumophila rtxA gene has previously been shown to play a role in entry and replication in macrophages and virulence in mice (Cirillo et al., 2001 ). In the current study, we examined the distribution of the rtxA gene in different isolates of Legionella, including both environmental and clinical samples. Although the presence of rtxA correlates with the ability to cause disease in humans we found that it also affects the ability of L. pneumophila to infect, traffic and survive in amoebae. These data indicate that rtxA may affect both the severity of disease in humans and the ability of L. pneumophila to persist in environmental reservoirs.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
The L. pneumophila strains used for these studies were a streptomycin-resistant variant of AA100 transformed with the kanamycin-resistant vector pYUB289 (Cirillo et al., 2000 ) to control for effects of antibiotic resistance, the same strain containing an in-frame deletion of rtxA and transformed with pJDC35 ({Delta}rtxA::pJDC35) for complementation or pJDC40 ({Delta}rtxA::pJDC40) as a vector control, as well as environmental and clinical isolates as listed in Table 1. The rtxA mutant is an in-frame deletion that includes only the amino-terminal 6 aa and carboxy-terminal 124 aa of RtxA (Cirillo et al., 2001 ). This mutation can be complemented by transformation of the mutant with the wild-type rtxA gene (Cirillo et al., 2001 ). As described previously (Cirillo et al., 2001 ), pJDC35 is an integrating vector containing the complete rtxA gene and pJDC40 is the same vector with rtxA deleted. Strain {psi}lp55 is an AA100 clone that has been passed 14 times in the laboratory until it is sodium-resistant and unable to replicate in monocytic cells (data not shown). All L. pneumophila strains other than {psi}lp55, including the environmental and clinical isolates, were passaged no more than twice in our laboratory before use in these studies to prevent loss of virulence. L. pneumophila was grown on BCYE agar (Edelstein, 1981 ) for 3 days at 37 °C in 5% CO2. When necessary, kanamycin (Sigma) was added at a concentration of 25 µg ml-1 to bacterial growth medium.


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Table 1. Characteristics of Legionella strains

 
Amoeba.
The amoeba A. castellanii ATCC 30234 was grown in PYG broth in 75 cm2 tissue culture flasks in the dark at 23 °C (Cirillo et al., 1994 ; Moffat & Tompkins, 1992 ). Amoeba cells were brought into suspension by rapping the flask sharply and the number of viable cells was determined as described by Moffat & Tompkins (1992) .

Entry and adherence assays.
Entry assays were carried out as described previously (Cirillo et al., 1994 ). Amoebae were seeded in 24-well tissue culture dishes (Falcon) at a concentration of 1·5x105 cells per well and allowed to adhere overnight at 23 °C in the dark. The cells were then washed with Ac buffer (HS) (Moffat & Tompkins, 1992 ), 1 ml HS added and equilibrated at 37 °C for 1 h. The bacteria to be assayed were suspended and diluted in HS and added to the cells at an m.o.i. of 10. After the bacteria were allowed to interact with the cells for 30 min at 37  °C the wells were then washed with HS and incubated in HS plus 100 µg gentamicin ml-1 for 2 h. After antibiotic treatment the cells were washed with HS, then lysed by incubation for 10 min in 1 ml water followed by vigorous pipetting. After lysis, the number of intracellular bacteria was determined by plating for c.f.u. on BCYE. Entry levels were determined by calculating the percentage of the inoculum that became gentamicin-resistant over the course of the assay (i.e. entry=c.f.u. gentamicin-resistant/c.f.u. inoculum). To correct for variation in levels of uptake between experiments, entry is reported relative to AA100 [i.e. percentage entry=(entry test strain/entry AA100)x100]. Adhesion was tested in a similar manner to that for entry, except that bacteria were added to the cells, mixed and immediately washed three times to remove non-adherent bacteria prior to lysis.

Intracellular growth assays.
Intracellular growth assays were carried out as described previously (Cirillo et al., 1997 ). Bacteria were added to a monolayer of 1·5x105 amoebae in 24-well tissue culture dishes at an m.o.i. of 0·5 and incubated at 37 °C for 30 min. The cells were washed three times with HS, incubated in HS plus 100 µg gentamicin ml-1 for 2 h, placed in fresh HS and lysed at various time points with water. Survival is expressed as the percentage of c.f.u. present at each time point as compared to time zero (2·5 h).

Frequencies of lysosomal fusion.
Lysosomes were stained and the frequency of fusion with the bacterial phagosome was determined by transmission electron microscopy, as described previously (Cirillo et al., 1997 ). After pre-labelling the amoebae with thorium dioxide (Polysciences) they were infected with the bacteria for 30 min. The samples were then fixed and prepared for electron microscopy at various time points as described previously (Bowers & Korn, 1968 ; Cirillo et al., 1997 ; Niszl & Markus, 1989 ).

Lysosomes were stained for fluorescence microscopy with lucifer yellow (LY) as described by Bizal et al. (1991) , Heinzen et al. (1996) and Swanson (1989) . After pre-labelling the amoebae with 1 mg LY ml-1 for 2 h the cells were washed with HS and incubated in HS for 1 h at 37 °C. L. pneumophila cells were pre-stained with rhodamine red (RhR) prior to infection in a similar manner to that previously described using fluorescein (Francis et al., 1993 ; Wiater et al., 1998 ). Basically, the bacteria were suspended in PBS plus 5 µg RhR ml-1, incubated at 37 °C for 30 min and washed five times to remove unbound RhR. The RhR staining procedure did not affect bacterial viability or intracellular trafficking. Viability of the labelled bacteria was confirmed using the LIVE-DEAD assay (Molecular Probes) and by plating dilutions for c.f.u. on BCYE agar (Difco). The LY-labelled amoebae were infected with the bacteria for 30 min, washed with HS and incubated in HS for various periods of time. At each time point the monolayers were fixed in 4% (v/v) paraformaldehyde for 30 min and examined on a Nikon TE300 inverted microscope with fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate filters. Dual images were captured using an Optronics charge-coupled device video camera for subsequent manual quantification of lysosomal fusion frequencies. Images were merged and adjusted for brightness and contrast using Adobe Photoshop version 5.5.

Cytotoxicity and pore formation.
The standard lactate dehydrogenase (LDH) release cytotoxicity assay (Behl et al., 1994 ; Brander et al., 1993 ) was used in these studies as described previously (Cirillo et al., 2001 ). The procedure used was essentially as recommended by the manufacturer of the CytoTox96 Non-Radioactive Cytotoxicity Assay system (Promega). Serial dilutions were made of each bacterial strain at m.o.i.s of 500, 250, 100 and 10 in a final volume of 100 µl for each assay using 2x104 amoebae. Appropriate numbers of cells for CytoTox96 assays were determined as suggested by the manufacturer (Promega). As a positive control for 100% cytotoxicity and the utility of this assay in amoebae, the cells were lysed with 9% (v/v) Triton X-100 (Promega). The cells were incubated with the bacteria for 4 h at 37 °C with 5% (v/v) CO2. Cytotoxicity readings were taken using an ELISA plate reader at 450 nm. Percentage cytotoxicity was calculated as recommended by the manufacturer and corrected for small differences in the inocula used.

Formation of pores in host cells was assayed by ethidium bromide and acridine orange staining in exactly the same manner as described previously (Cirillo et al., 2001 ; Kirby et al., 1998 ; Zuckman et al., 1999 ) using A. castellanii cells. Stained coverslips were examined using a Nikon TE300 inverted microscope with fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate filters. Dual images of multiple fields were captured using an Optronics charge-coupled device video camera and analysed in the same manner as described by Zuckman et al. (1999) . Pore formation is expressed as the percentage of acridine-orange-stained cells that also stain with ethidium bromide resulting from incorporation of this dye into chromosomal DNA due to increased permeability of the host cell. All cells were stained with acridine orange since, unlike ethidium bromide, acridine orange readily crosses membranes of eukaryotic cells. As a positive control, amoebae were first fixed with formaldehyde and then permeabilized with methanol prior to staining. Under these conditions, all amoebae stained well with both ethidium bromide and acridine orange.

Southern analyses and colony hybridization.
Probes were labelled by PCR with digoxigenin using the methods suggested by the manufacturer of the Genius System (Boehringer Mannheim). Membranes were prepared by the suggested methods for both colony hybridization and Southern analysis. Hybridization and washes were carried out at several different stringencies to ensure that the data obtained were not due to sequence divergence between Legionella species. Experiments generating the data shown were carried out under low stringency hybridization conditions. The probe for the rtxA gene was prepared by PCR using oligo 1 (5'-CTGATGCTGCTACGGAACAC-3') and oligo 2 (5'-CCGCAGTCATTACACCTGCG-3'). This resulted in the production of a 542 bp fragment within the rtxA gene. The probe for the mip gene was also prepared by PCR using oligo 3 (5'-GCAGCTGTTATGGGGCTTGC-3') and oligo 4 (5'-GGCAATACAACAACGCCTGG-3'), producing a 352 bp fragment within the mip gene.

Statistical analyses.
All experiments were carried out in triplicate and repeated at least twice. Significance of the results was analysed using ANOVA. Values of P<0·05 were considered significant.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Correlation of rtxA with disease
The rtxA gene has previously been associated with the ability of Legionella to cause disease in mice (Cirillo et al., 2001 ). However, it remains unclear whether rtxA is of particular importance for pathogenesis in humans or is required for survival and replication of Legionella in the environment. Predominance in the environment is likely to correlate with the ability of different Legionella to interact positively with water-borne protozoa, including amoebae. To better evaluate the importance of the rtxA gene in human disease and survival in the environment, we examined whether there is a correlation between the presence of this gene and the strength of association of different Legionella isolates with human disease. The presence of the rtxA gene in 14 Legionella isolates was determined by Southern analysis (Fig. 1). The approximately 5 kbp fragment visible in L. pneumophila Allentown and Benidorm corresponds to the size of fragment observed in AA100 (data not shown). Under the same hybridization conditions, a probe from the mip gene hybridizes to chromosomal DNA from all of these species, suggesting that sequence divergence alone is not responsible for the inability to detect rtxA (data not shown). Differences in signal intensity by Southern analysis were observed in species other than L. pneumophila, suggesting that, as expected due to their more distant relationship to AA100, greater DNA sequence divergence exists within the rtxA gene in these species. Since nearly all Legionella species are thought to be able to cause disease at some frequency, we used stringent criteria to evaluate whether the isolates tested are strongly or weakly associated with disease in our studies. Thus, if an isolate was directly obtained from post-mortem tissue or sputum from a clinically ill individual or there was documented evidence that it is the same as that causing disease, the isolate was considered strongly associated with disease. If, however, the isolate was obtained from an environmental source, even if it was the same species as that observed to cause disease in humans, or the species of the isolate only rarely causes disease in humans, we considered the isolate to be only weakly associated with human disease. Using these criteria, eight of the isolates examined had a strong association with disease and, of these, seven (88%) also had the rtxA gene (Table 1). Furthermore, none of the isolates that are only weakly or rarely associated with disease in humans has the rtxA gene. Thus, the presence of the rtxA gene correlates (P<0·01) with the ability of Legionella to cause disease in humans.



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Fig. 1. Southern analysis of different Legionella isolates. All lanes are EcoRI digests of total chromosomal DNA probed with a 542 bp internal fragment of the Legionella rtxA gene. This blot was hybridized under low stringency conditions. The positions of {lambda} phage DNA size standards cut with HindIII are shown on the left. Lp, Legionella pneumophila.

 
L. pneumophila does not cause cytotoxicity or pore formation in amoebae
The prevalence of certain Legionella strains in human disease may be the result of higher virulence, a selective advantage in the environment or a combination of these features. As a measure of the importance of rtxA in the survival of L. pneumophila in the environment, we examined the effects of this gene on the interaction of L. pneumophila with A. castellanii. An in-frame deletion of the L. pneumophila rtxA gene transformed with a single-copy construct that complements the rtxA mutation (pJDC35) or the same construct without rtxA (pJDC40) were used for these studies. All of the strains used in the current study, as well as their phenotypes in mammalian cells, have been described previously (Cirillo et al., 2001 ). In contrast to previous results with murine and human macrophages (Cirillo et al., 2001 ; Kirby et al., 1998 ), no cytotoxicity or pore formation was observed after incubation of wild-type L. pneumophila or the rtxA mutant with A. castellanii (data not shown). To ensure that the conditions of the experiment were not responsible for this negative result, we examined bacteria to host-cell ratios as high as 1000:1, as well as co-incubation periods from 1 to 6 h. None of these conditions resulted in significant cytotoxicity or pore formation (data not shown). In addition, the positive controls of Triton-X-100-killed amoebae (cytotoxicity) and permeabilization with methanol after fixation followed by staining for fluorescent microscopy (pore formation) demonstrated that these assay methods work well in amoebae (data not shown). Thus, L. pneumophila does not cause cytotoxicity or pore formation in A. castellanii.

rtxA affects adherence and entry
Since the L. pneumophila rtxA gene plays a role in entry into human cells (Cirillo et al., 2001 , 2000 ), we examined its role in adherence and entry into A. castellanii (Fig. 2). The L. pneumophila rtxA mutation reduced adherence (P<0·03) and entry (P<0·02) to amoebae by approximately 50% compared to wild-type. Levels of adherence that were not significantly different than wild-type L. pneumophila were obtained in the rtxA mutant carrying a complementing construct, pJDC35. All strains were tested for sensitivity to assay conditions such as osmotic lysis, culture medium and serum. No significant differences in sensitivity to these conditions were observed with the strains used. These data suggest that, similar to its role in mammalian cells, rtxA is involved in adherence to and entry into amoebae in environmental reservoirs.



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Fig. 2. The ability of the wild-type L. pneumophila strain AA100, {Delta}rtxA mutant ({Delta}rtxA::pJDC40) and complemented clone ({Delta}rtxA::pJDC35) to adhere to (a) and enter into (b) Acanthamoeba castellanii. Data points and error bars represent the means of triplicate samples and their standard deviations, respectively. The adherence and entry levels of wild-type L. pneumophila strain AA100 are arbitrarily set to 100%. All experiments were performed at least three times.

 
rtxA plays a role in intracellular survival
Since rtxA plays a role in entry by L. pneumophila into amoebae it may also impact subsequent intracellular events. To evaluate this possibility, we compared the ability of the rtxA mutant to survive and replicate in amoebae with wild-type L. pneumophila (Fig. 3). The rtxA mutant did not survive as well as wild-type (P<0·01) for the majority of time points through the first 32 h after entry. Interestingly, the rtxA mutant complemented with a plasmid-borne copy of the gene displayed enhanced intracellular survival. Since rtxA does not affect growth in laboratory medium (Cirillo et al., 2001 ), its role in survival is specific to the intracellular growth environment. The rtxA mutant was killed (80%) much more efficiently than wild-type (62%) during the first 6 h in amoebae. The subsequent rate of intracellular replication was the same as wild-type, with the rtxA mutant increasing 50-fold and wild-type 45-fold by 32 h. These data suggest that rtxA is primarily involved in the early steps of the interaction of L. pneumophila with amoebae.



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Fig. 3. Growth of wild-type L. pneumophila strain AA100 ({square}), {Delta}rtxA mutant ({Delta}rtxA::pJDC40, {circ}) and complemented clone ({Delta}rtxA::pJDC35, {triangleup}) in amoebae. All data are expressed relative to time zero (t0=2·5 h). * (P<0·05) and ** (P<0·01) indicate data points significantly different from wild-type. Data points and error bars represent the means of quadruplicate samples and their standard deviations, respectively. All experiments were performed at least three times.

 
Role of rtxA in intracellular trafficking
One potential mechanism by which a gene that affects entry might impact initial intracellular survival would be through changes in trafficking of the bacterial vacuole resulting from signal transduction during phagocytosis. L. pneumophila normally has the ability to inhibit lysosomal fusion in macrophages (Horwitz & Maxfield, 1984 ) and amoebae (Bozue & Johnson, 1996 ). We examined the frequencies of lysosomal fusion with the wild-type and rtxA mutant bacterial vacuole by electron microscopy using thorium dioxide (Fig. 4), an electron-dense colloidal marker that is retained within secondary lysosomes (Armstrong & Hart, 1971 ). Vacuoles containing the rtxA mutant fused with lysosomes at a significantly higher rate (P<0·01) than wild-type L. pneumophila (Table 2). Although there was no significant difference in the frequencies of fusion between the rtxA mutant and wild-type at 15 min, the difference was nearly twofold by 1 h hour and more than twofold by 2 h after entry.



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Fig. 4. Fusion of lysosomes containing thorium (electron-dense granules) with the L. pneumophila strain AA100 (A, C) and {Delta}rtxA mutant (B, D) vacuole 120 min after entry. Examples of unfused vacuoles are shown for the wild-type (A) and rtxA mutant (B). Fused vacuoles are infrequently observed for the wild-type strain AA100 (C), whereas approximately half of the rtxA mutant vacuoles contain thoria (D). Bar, 1 µm.

 

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Table 2. Fusion of Legionella vacuole with lysosomes by electron microscopy

 
To confirm this result using a different method we pre-labelled the amoebal lysosomes with LY. This marker has been used extensively to label secondary lysosomes in macrophages and other eukaryotic cells (Bizal et al., 1991 ; Heinzen et al., 1996 ; McClure & Schiller, 1996 ; Straub et al., 1997 ; Swanson, 1989 ). This label allowed clear differentiation between fused and unfused bacterial vacuoles (Fig. 5). In addition, similar frequencies of lysosomal fusion were obtained for wild-type L. pneumophila to those by electron microscopy (Table 3). The laboratory-passaged avirulent AA100 mutant {psi}lp55 displayed the expected high frequencies of lysosomal fusion (78%) observed previously for similar mutants (Roy et al., 1998 ; Swanson & Isberg, 1996 ). We found that, even as early as 30 min after entry, the rtxA mutant fused with LY-labelled lysosomes at a significantly higher level (P<0·05) than wild-type L. pneumophila. The difference in the frequencies of lysosomal fusion between the rtxA mutant and wild-type was much more pronounced by 90 min (P<0·01). These data confirm that the L. pneumophila rtxA gene plays a role in intracellular trafficking in amoebae.



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Fig. 5. Fusion of lysosomes (green) containing LY with the L. pneumophila (red) vacuoles for strains AA100 (A–F), {Delta}rtxA (G–L) and the avirulent {psi}lp55 (M–R). All panels shown are 150 min after infection of the amoebae. Split and merged (Lp+LY) images are shown using RhR and LY filter sets. The red colour indicates RhR-stained bacteria, which appear as rods (A, D, G, J) or, when they are presumably partially degraded, as diffuse red-staining regions (M, P). The green colour indicates lysosomes stained with LY. When the bacterial vacuole co-localizes with lysosomes, a yellow colour results in the merged images (F, L, O, R). Fused vacuoles are infrequently observed for the wild-type strain AA100 (F), whereas approximately half of the rtxA mutant (L) and the majority of the {psi}lp55 (O, R) vacuoles co-localize with LY. Bar, 10 µm.

 

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Table 3. Fusion of Legionella vacuole with lysosomes by fluorescent microscopy

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We have found that the L. pneumophila rtxA gene is not present in all Legionella species and that its presence correlates with the ability to cause disease in humans. This observation, along with previous studies demonstrating that this gene plays an important role in virulence (Cirillo et al., 2001 ), has helped to fulfil ‘molecular Koch’s postulates’ for rtxA, supporting the conclusion that this gene plays a role in disease (Falkow, 1988 ). There are three criteria required to fulfil these postulates. First, specific inactivation of the rtxA gene leads to a measurable loss of virulence in mice (Cirillo et al., 2001 ). Second, complementation of the mutated rtxA gene leads to restoration of virulence in mice (Cirillo et al., 2001 ). Third, the rtxA gene is associated with pathogenic members of the genus, as demonstrated in the current study. This represents an important step in our understanding of the molecular aspects of Legionnaires’ disease. However, although rtxA fits the criteria for an important role in pathogenesis, the primary function(s) of this gene both during disease and in the environment are not fully understood.

Similar to data obtained in mammalian cells (Cirillo et al., 2001 , 2000 ), the rtxA gene affects adherence and entry into amoebae. The mechanism by which rtxA affects adherence and entry in mammalian cells is not known, though it may be the result of the ability of RTX proteins to bind to ß2 integrin receptors (Ambagala et al., 1999 ; Lally et al., 1997 ). Although receptors analogous to integrins have not yet been identified in Acanthamoeba, they have been identified in the amoebae Hartmannella (Venkataraman et al., 1997 ) and Entamoeba (Adams et al., 1993 ; Vines et al., 1998 ) as well as in coral and sponges (Brower et al., 1997 ). These observations suggest that integrin receptors are highly conserved throughout all eukaryotic cells. This conclusion is supported by evidence that the Entamoeba receptor shares an immunologically cross-reactive epitope with human ß2 integrins (Adams et al., 1993 ) and contains a ß2 integrin motif in its cytoplasmic domain (Vines et al., 1998 ). Thus, it remains possible that a similar receptor is available for binding to L. pneumophila via RtxA in both macrophages and amoebae. Considering that a similar receptor may be utilized, it is surprising that no pore formation or cytotoxicity is observed in Acanthamoeba, in contrast to macrophages. Since environmental amoebae are most likely the natural host for L. pneumophila, these data suggest that pore formation and cytotoxicity are not the primary roles of rtxA in bacterial–host-cell interactions. Several other potential roles in pathogenesis have been suggested for RTX proteins from other bacteria, including colonization (Goodwin & Weiss, 1990 ), immune modulation (Bhakdi et al., 1990 ; Scheffer et al., 1985 ) and inhibition of superoxide production (Keane et al., 1987 ). Therefore, closer examination of the role of the L. pneumophila rtxA gene in adherence, entry and intracellular survival are likely to provide a better understanding of its primary role in Legionnaires’ disease and may lead to insight into alternate functions of RTX proteins from other bacteria.

It is possible that the effects of the rtxA mutant on intracellular survival are directly related to the role of rtxA in adherence or entry, though the defect in trafficking is less pronounced than the salt-resistant mutant {psi}lp55. This concept would suggest that proper intracellular trafficking of L. pneumophila, and thereby survival, are the result of signalling events that occur during uptake. However, in our own and other studies (Joshi et al., 2001 ; Roy et al., 1998 ), little difference has been found between the trafficking of L. pneumophila mutants and wild-type during the first 5–10 min following entry. If signalling during uptake is involved, it has long-lasting effects upon trafficking of the bacterial phagosome. It is equally possible that RtxA is a multi-functional protein, binding a host-cell receptor affecting adherence and entry, as well as subsequently inhibiting lysosomal fusion. There are two primary mechanisms by which RtxA might inhibit lysosomal fusion independent of events associated with entry. First, it may be that the formation of pores caused by RtxA in the vacuole itself prevents proper docking of lysosomes, preventing fusion. Second, an as yet unidentified activity of RtxA modifies the vacuole or other host-cell component that is involved in trafficking. It remains to be determined whether RtxA is one of the proteins secreted by the dot/icm secretion apparatus previously implicated in the intracellular trafficking of L. pneumophila (Berger et al., 1994 ; Swanson & Isberg, 1996 ). Based on the frequencies of lysosomal fusion observed (40–50% with the rtxA mutant and 80–90% with a dotA mutant), there must be additional factors involved in the inhibition of lysosomal fusion other than RtxA. Our studies were carried out in amoebae rather than the human or murine monocytes used for the evaluation of dotA mutants. However, similar results were obtained by our laboratory for frequencies of lysosomal fusion using the rtxA mutant in murine macrophages (L. Yan & J. D. Cirillo, unpublished observations). The fact that other factors are likely to be involved in the prevention of lysosomal fusion by L. pneumophila suggests the importance of further studies in this area.

The rtxA gene plays a role in the ability of L. pneumophila to survive in A. castellanii. The impact of the rtxA mutation on intracellular survival is, however, relatively small. One possible explanation for this observation might be that the wild-type bacteria, when grown under standard laboratory conditions, do not express rtxA optimally and thus the absence of rtxA only results in a limited impact on survival. The fact that complementation of the rtxA mutant with a plasmid enhances intracellular survival supports this conclusion, since these bacteria are likely to express the rtxA gene at higher levels than wild-type due to copy number effects. Since several of the species that did not have this gene according to Southern analysis can still cause disease in animals and the rtxA mutant still replicates in human macrophages, albeit poorly (Cirillo et al., 2001 ), the rtxA gene is probably not absolutely required for pathogenesis. It is also likely that, similar to other intracellular pathogens, including Salmonella, Shigella and Listeria, the ability of L. pneumophila to survive and replicate intracellularly is multi-factorial. In the case of Salmonella, nearly 3% of the genome is thought to be required for the ability to survive intracellularly (P. I. Fields et al., 1986 ). Application of this model to L. pneumophila would suggest that between 100 and 150 genes could be involved in the ability to survive and replicate in macrophages. Thus, one would expect that few of these genes would completely debilitate L. pneumophila intracellularly. Mutations in most genes, other than those required for metabolism and biosynthesis, would result in only partial and potentially subtle intracellular defects.

These observations suggest that careful evaluation of potential virulence determinants using both laboratory and animal models of infection in both susceptible and resistant hosts is necessary to allow identification of the important factors involved in pathogenesis by L. pneumophila. The {Delta}rtxA mutant should greatly facilitate further investigation of the host-cell receptors, signalling pathways and intracellular trafficking mechanisms of L. pneumophila. Further studies are needed, in particular to determine whether the mechanism of entry involving RtxA is responsible for subsequent intracellular events or whether RtxA is a multi-functional protein having the ability to bind host-cell receptors as well as independently preventing lysosomal fusion. In addition to its role in virulence in mammals (Cirillo et al., 2001 ), the rtxA gene plays an important role in the ability of L. pneumophila to infect amoebae. These observations suggest that the rtxA gene is at least partially responsible for the high percentage of Legionnaires’ pneumonias caused by and the predominance in environmental isolates of L. pneumophila, as compared to other Legionella species.


   ACKNOWLEDGEMENTS
 
This work was supported by grant AI40165 from the National Institutes of Health.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 5 November 2001; revised 18 December 2001; accepted 6 February 2002.