Department of Microbiology and Immunology, College of Medicine, University of Kentucky, Chandler Medical Center, Lexington, KY 40503, USA
Correspondence
Yousef Abu Kwaik
yabukw{at}uky.edu
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ABSTRACT |
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INTRODUCTION |
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There are at least 25 000 cases of pneumonia due to L. pneumophila reported annually that require hospitalization (Abu Kwaik, 1998). This number is thought to be underestimated due to difficulties in bacterial isolation and diagnosis from secretions (Abu Kwaik et al., 1998
; Jaulhac et al., 1992
; Koide & Saito, 1995
). The Centers for Disease Control and Prevention (CDC) figures between 1980 and 1998 show a general decline in mortality after reaching a peak in 1988 (Benin et al., 2002
). In England and Wales, the legionellae surveillance carried out showed a similar trend to what has been reported in the USA, reaching its peak in 1988 (Joseph et al., 1995
, 1997
). One major recent outbreak of Legionnaires' disease occurred in the Netherlands during which 188 individuals attending a flower show were hospitalized. This outbreak was linked to a whirlpool spa and a sprinkler system used to water the flowers (Den Boer et al., 2002
). People at risk of contracting Legionnaires' disease are elderly individuals, smokers, and people with underlying respiratory or immunocompromising conditions (Marston et al., 1994
).
Infection by legionellae occurs after inhalation of contaminated aerosol, after which the organism enters and multiplies within alveolar macrophages. Within the intracellular environment of mammalian phagocytic (Horwitz, 1983) and alveolar epithelial cells (Gao et al., 1998
), legionellae replicate within a rough endoplasmic reticulum (RER)-surrounded phagosome. The dot/icm loci are composed of 24 genes that are involved in the assembly of a type IV secretion apparatus that is central to pathogenesis. The dot/icm loci are essential for enhancement of phagocytosis by human-derived cells (Hilbi et al., 2001
), macropinocytic uptake by A/J mice-derived macrophages (Watarai et al., 2001
), evasion of lysosomal fusion and intracellular replication (Segal et al., 1998
; Vogel et al., 1998
), induction of apoptosis (Zink et al., 2002
), and pore-formation-mediated lysis of the host cell and bacterial egress upon termination of intracellular replication (Alli et al., 2000
; Molmeret et al., 2002b
). Biphasic killing of mammalian cells by L. pneumophila (Alli et al., 2000
; Gao & Abu Kwaik, 1999b
) has been proposed in which apoptosis is first initiated, followed by a temporal induction of necrosis and lysis of the host upon growth transition into the post-exponential phase (Alli et al., 2000
; Byrne & Swanson, 1998
; Kirby et al., 1998
). The pore-forming activity mediates lysis of the host cell, and mutants defective in pore-forming activity are defective in lysis of the host cell and are delayed in subsequent egress from mammalian (Alli et al., 2000
) and protozoan cells (Gao & Abu Kwaik, 2000b
). The C-terminus of IcmT has been shown to be essential for pore-formation-mediated bacterial egress from the host cell (Molmeret et al., 2002a
, b
). Importantly, pore-forming activity plays a major role in pulmonary cytotoxicity and inflammation in experimental animals (Alli et al., 2000
; Molmeret et al., 2002b
). These pathogenic mechanisms have been studied for L. pneumophila but little is known about their role in the pathogenesis of other species of legionellae.
Based on previous studies on L. dumoffii and L. micdadei, the general assumption is that replication within phagocytes is the hallmark of virulent legionellae (Levi et al., 1987; Moffat & Tompkins, 1992
; O'Connell et al., 1995
). The pathogenic traits of other Legionella spp. apart from L. pneumophila are not well defined. Previous studies have addressed the replication of legionellae within human macrophage cell lines (O'Connell et al., 1996
) and guinea pigs (Doyle et al., 2001
; Izu et al., 1999
). The growth of Legionella spp. in protozoan and human macrophage cell lines has also been compared (Neumeister et al., 1997
). However, these studies have examined a single virulence trait in the limited number of strains of legionellae studied. In this study, we aimed to define traits responsible for the pathogenesis of Legionella spp. in 27 strains belonging to 16 species, by examining five pathogenic traits: cytopathogenicity, intracellular multiplication in U937 macrophages, pore-formation-mediated cytolysis, the induction of apoptosis, and the presence of the dot/icm loci. We selected strains of legionellae that have been shown to cause disease along with strains that have not been associated with Legionnaires' disease with the hope of defining the virulence trait(s) that differentiate L. pneumophila strains from the rest. We found no virulence trait that can differentiate L. pneumophila from the remaining species of legionellae using the five aforementioned pathogenic traits.
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METHODS |
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Growth kinetics of Legionella spp. in U937 macrophages.
Infections of U937 macrophages by Legionella spp. were performed, in triplicate, in 96-well plates containing 105 cells per well at an m.o.i. of 1 for U937 macrophages as previously described (Alli et al., 2000). At the end of the infection period, the monolayers were treated with gentamicin (50 µg ml-1) for 1 h as described above. The number of bacteria in the monolayers at several time intervals after washing of the gentamicin was determined.
Contact-dependent pore formation assay.
Contact-dependent pore formation in the plasma membrane was determined by examining haemolysis of sheep red blood cells (sRBCs) by Legionella spp. at an m.o.i. of 25 following 2 h of bacterialsRBC contact at 37 °C, as previously described (Kirby et al., 1998). Briefly, sRBCs (Remel) were diluted in RPMI 1640 medium, and washed three times by centrifugation for 10 min at 2000 g until the supernatant did not show any sign of haemolysis; the cells were then counted with a haemocytometer. Reactions were set up in a final volume of 1 ml with final concentrations of 1x108 sRBC ml-1 and 2·5x109 bacteria ml-1 and incubated at 37 °C for 2 h. At the end of incubation period, the pellets were resuspended by vortexing and repelleted by centrifugation for 2 min at 17 000 g. Supernatants were transferred to cuvettes and the absorbance was read at 415 nm.
DNA fragmentation analysis and TUNEL assays.
DNA fragmentation analysis was carried out as previously described (Gao & Abu Kwaik, 1999b). Differentiated U937 macrophages were plated in six-well plates (1·5x106 cells per well) and infected with Legionella spp. at an m.o.i. of 50 for 1 h. At the end of the infection period, the monolayers were washed three times to remove unattached extracellular bacteria and maintained at 37 °C in culture medium. At 3 h post-infection, the cells in each well were lysed in 500 µl lysis buffer [10 mM Tris (pH 7·5), 20 mM EDTA (pH 8·0), 0·5 % Triton X-100] for 30 min on ice. The lysates were treated with 0·5 % SDS and 300 µg proteinase K ml-1 for 2 h and DNA extracted with phenol and chloroform before precipitation with ethanol. The precipitates were dissolved in 10 mM Tris (pH 8·0)/1 mM EDTA containing 0·5 µg RNase ml-1, electrophoresed in 1·8 % agarose gel, and stained with ethidium bromide; individual lanes were examined for the presence of DNA fragmentation. DNA fragmentation was scored as positive in each strain when compared with the negative control that was not infected with any bacteria.
Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) assays were performed exactly as we described previously (Gao & Abu Kwaik, 1999b). Briefly, cells attached to 96-well plates were infected for 1 h with the strains of bacteria at an m.o.i. of 50, and then extracellular bacteria were washed off. For labelling of apoptotic nuclei, the cells were subjected to FITC-conjugated TUNEL using an apoptosis detection kit, according to the manufacturer's instructions (Boehringer Mannheim). Cells were examined using an Axiovert S100 Zeiss fluorescence microscope. A minimum of 100 cells per sample was counted, and apoptosis was quantified as the percentage of apoptotic cells (TUNEL-positive nuclei). The ability of the bacteria to induce apoptosis was scored as positive if the percentage of apoptotic cells was greater or equal to 50 %. Multiple independent samples were examined.
DNA hybridization analysis.
The dot/icm genes have been shown to play an important role in the virulence of L. pneumophila (Segal et al., 1998; Vogel et al., 1998
). We screened all the Legionella spp. using Southern hybridization probed with the PCR products derived from all regions of the dot/icm loci from L. pneumophila AA100. A cocktail of probes that contained 2·1 kb PCR product of icmTSRQ, 1·6 kb PCR product of dotDCB, 1·8 kb PCR product of icmJB, 1·0 kb PCR product of icmWX and 1·0 kb PCR product of icmLK was used. The 2·1 kb PCR product of icmTSRQ was generated using primers P1 (5'-CACAGTTAAAACTTCAAGCTGAACC-3') and P2 (5'-CTGCTCAGAGCTATTTTT-3'). The 1·6 kb PCR product of dotDCB was generated using primers P3 (5'-CGATTGGTCTGGTCCGATTGA-3') and P4 (5'-TCTCGAATAATGGAAGCTAACAATGTC-3'). The 1·8 kb PCR product of icmJB was generated using primers P5 (5'-TGCCATGTTCTTTTTTGTGCTATTAC-3') and P6 (5'-GAGCGTAAACCAGATCAATCCAAGTAG-3'). The 1·0 kb PCR product of icmWX was generated using primers P7 (5'-TGGGTTGGTTCCTGAGGTATGA-3') and P8 (5'-TGGGGCGCTGAAATTTTGATAT-3'). The 1·0 kb PCR product of icmLK was generated using primers P9 (5'-CGGAAGGCTGGGACCAATT-3') and P10 (5'-CCACTCGATAATCCACGGCTTTC-3'). Labelling of DNA probes and Southern hybridizations were performed as described previously (Abu Kwaik et al., 1997
). High-stringency hybridization and washes were performed at 60 °C; low-stringency hybridization and washes that allowed for 20 % mismatch were performed at 42 °C.
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RESULTS |
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The pore-forming activity of Legionella spp.
The pore-forming activity of L. pneumophila has been shown to contribute to cytotoxicity (Kirby et al., 1998) and the ability of the organism to egress from the host cell after cessation of intracellular replication (Alli et al., 2000
; Gao & Abu Kwaik, 2000a
). Contact-dependent haemolysis of sRBC assay was performed to examine pore-forming activity, as previously described (Kirby et al., 1998
). The L. pneumophila rib mutant GR159, which is defective in pore-forming activity (Alli et al., 2000
) was used as a negative control in this assay. The group I and II strains demonstrated variable pore-forming activity (Fig. 3a, b, d
). Taken together, the data showed that there was no correlation between pore-forming activity and cytopathogenicity. The data also showed that only the L. pneumophila serogroup 1 (AA100, SG1-62, 65, 66 and 67) strains along with L. spiritensis had pore-forming activity, while L. pneumophila SG1-64 and Knoxville strains did not (Fig. 3
). All the other strains tested in this study did not show any significant pore-forming activities.
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DISCUSSION |
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L. pneumophila GR159, a rib mutant defective in pore-forming activity, has already been characterized extensively by our laboratory (Alli et al., 2000; Molmeret et al., 2002a
, b
). Pore-forming activity plays an important role in the cytopathogenicity of L. pneumophila since all the strains that possessed this phenotypic trait belong to group I. However, we found that this is not the only factor responsible for this high cytopathogenicity since L. dumoffii and L. micdadei Rivera strains did not possess this phenotypic trait but were still highly cytopathogenic. Pore-forming activity is a phenotypic trait that could aid in the identification and diagnosis of L. pneumophila in the laboratory, since all the strains of L. pneumophila possessed this particular phenotype with the exception of SG1-64 and SG1 Knoxville. Genetic characterization of these strains need to be carried out to ascertain the basis for lack of pore-forming activity. Mutation within the dot/icm loci cannot be ruled out, as we have shown in our previous studies that a base-pair deletion within icmT is sufficient to change the pore-forming activity phenotype from positive to negative (Molmeret et al., 2002a
, b
).
Classification of isolates of Legionella spp. has been carried out previously using mouse and guinea pig macrophages. Izu et al. (1999) grouped 20 reference strains into four groups. In contrast, our classification based on cytopathogenicity divided 27 strains of Legionella spp. into two broad main groups. Group I classification by Izu et al. (1999)
includes most of the group II strains in our study along with L. dumoffii. There is no direct correlation with the grouping in this study, which could be due to the differences in the strains and/or methods of classification. This is not surprising as Izu et al. (1999)
found no significant characteristics common to another grouping done by Neumeister et al. (1997)
, which was based on the bacterial doubling time in Mono Mac 6 cells and Acanthamoeba castellanii. Our data on L. cincinnatiensis are consistent with the finding of Izu et al. (1999)
that this species of Legionella does not grow within U937 cells despite the fact there might be some strain differences and different host cell lines used. However, the main objective of this study was not to provide a classification method for Legionella spp., but rather to provide a better understanding of the pathogenic trait(s) that could be responsible for variation in the virulence of these bacteria.
Interestingly, L. dumoffii and L. micdadei Rivera, which did not exhibit pore-forming activity, and did not induce apoptosis/DNA fragmentation in macrophages, belonged to high cytopathogenic group I. The lack of two pathogenic traits in these strains despite their ability to cause cell death indicates that other factors in addition to apoptosis and pore-forming activity are involved in cytopathogenicity of these species. Cytopathogenicity could be the result of unrestricted growth of L. micdadei (Rivera) and L. dumoffii. However, high levels of intracellular growth have also been demonstrated among the group II strains, suggesting that high growth rate within macrophages is not sufficient to cause cell death for these strains. It is possible that variation in the combination of the three phenotypic traits (induction of apoptosis in host cells, pore-forming activity, and high intracellular growth index) could be what determines the variability in the incidence of Legionella spp. in causing Legionnaires' disease. L. pneumophila and L. micdadei are the two most common species of legionellae that cause Legionnaires' disease, with L. pneumophila and L. micdadei responsible for 85 % and 5 % respectively, of cases (Dowling et al., 1983; Halberstam et al., 1992
; Hebert et al., 1980
; Pasculle et al., 1980
). Importantly, these two species belonged to group I in our classification scheme, suggesting that the grouping can discriminate between the Legionella spp. that are responsible for high and low incidence of infection. Cytopathogenicity is the only pathogenic trait that gives direct correlation with high incidence of a particular species to cause disease in a human population. The L. longbeachae strains used in this study failed to induce apoptosis/DNA fragmentation, which is in sharp contrast to a previous study that showed this particular species of legionellae did induce apoptosis (Arakaki et al., 2002
). The differences in results could be due to strain differences, as we have already shown heterogeneity in virulence trait(s) in L. micdadei and L. pneumophila.
Southern analyses revealed the presence of the dot/icm loci in all the Legionella spp., indicating the highly conservative nature of these loci in all the species of legionellae. Previous studies have shown the presence of dotA and icmX genes in L. micdadei Pittsburg, L. micdadei Tatlock, L. bozemanii, L. gratiana (Matthews & Roy, 2000), and of dotA, dotB, dotE and dotFG in L. micdadei 31B, Rivera, Camileri and D-2676 (Joshi & Swanson, 1999
). The IcmX protein has been demonstrated in L. micdadei and L. gratiana (Matthews & Roy, 2000
). In our studies, all strains of legionellae investigated harboured all the dot/icm loci. In spite of the presence of the dot/icm loci, two different groups of Legionella spp. based on the degree of cytopathogenicity can be demonstrated. It remains to be seen whether all the proteins of the dot/icm genes are expressed and functional in all the Legionella spp. The roles of the dot/icm loci in pathogenesis have been well established for L. pneumophila (Segal et al., 1998
; Vogel et al., 1998
), but the contributions of these loci to the pathogenesis of other species are yet to be determined. Our data suggest that the dot/icm regions of L. pneumophila may represent a sensitive and powerful molecular tool for definitive diagnosis and identification of legionellae in a clinical microbiology laboratory since all the strains of legionellae tested in this study harbour these loci. However, a large-scale screen is required to confirm the specificity of hybridization to all other strains of legionellae and not to other genera of bacteria.
In conclusion, most of the group II strains of legionellae, with low cytopathogenicity, are unable to replicate within macrophages and lack pore-forming activity, and the majority of them are unable to induce apoptosis; this could be a general feature of non-pathogenic strains of legionellae. In contrast, strains belonging to group I are able to replicate within macrophages, with the majority of them exhibiting pore-forming activity, and some of them can induce apoptosis/DNA fragmentation. No single virulence traits were found to correlate with the cytopathogenicity of this genus.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 6 September 2002;
revised 6 December 2002;
accepted 19 December 2002.