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
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ABSTRACT |
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Keywords: phagocytosis, lysosomes, amoeba, pathogenesis, pneumonia
Abbreviations: LY, lucifer yellow; RhR, rhodamine red; RTX, repeats in structural toxin
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INTRODUCTION |
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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.
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METHODS |
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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.
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RESULTS |
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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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DISCUSSION |
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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 bacterialhost-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 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 510 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 (4050% with the rtxA mutant and 8090% 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 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.
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ACKNOWLEDGEMENTS |
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Received 5 November 2001;
revised 18 December 2001;
accepted 6 February 2002.