SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The gene encoding the 25 kDa major outer membrane protein (MOMP) of Legionella pneumophila was transformed into Escherichia coli JM 83 and the resultant E. coli LP 116 clone expressed the Legionella-MOMP. Compared with the parent E. coli strain, the clone showed a fivefold increase in opsonin-independent binding to U-937 cells. Furthermore, this gene was incorporated by electroporation into a low virulence derivative of Leg. pneumophila which showed reduced expression of the MOMP but enhanced expression of a 31 kDa protein in the OMP profile. After electroporation, the attenuated strain showed an increased expression of the MOMP while the 31 kDa protein was eliminated and virulence for the chick embryo was re-established. The use of a monoclonal antibody specific for the MOMP abolished virulence and adherence. These studies suggest that the 25 kDa MOMP of Leg. pneumophila serves as an adhesive molecule for host cells and that this protein plays a major role in the virulence of the organism for the chick embryo.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Legionella pneumophila is a Gram-negative, facultative intracellular pathogen responsible for Legionnaires’ disease. The bacterium is capable of replicating in a variety of cell types but primarily parasitizes alveolar macrophages during human disease. The outer membrane content of Legionella has been explored to determine whether a single factor is responsible for uptake and intracellular survival of the organism ( Cianciotto et al. 1989  ; Kirby et al. 1998  ; Vogel et al. 1998 ) and several genes have been identified that are responsible for intracellular survival, evasion of endocytic pathways and killing of host cells ( Segal & Shuman 1997 ; Andrews et al. 1998 ). Although reports have varied as to the commonality of various proteins across strains of Leg. pneumophila and Legionella species, some Leg. pneumophila serogroups do contain a single major outer membrane protein (MOMP) that ranges in mass between 24 and 29 kDa ( Ehret et al. 1984  ; Butler et al. 1985  ; Hindahl & Iglewski 1986). Butler & Hoffman (1990) have also reported the presence of 28 and 31 kDa outer membrane proteins (OMP) in Leg. pneumophila.

Attachment of facultative intracellular bacteria to host cells forms a prelude to infection. Many pathogens possess pili, fimbriae, capsular mucopolysaccharide, flagella, lectin-like molecules and OMPs, all of which have been implicated in bacterial attachment to host cells ( Gibbons 1973 ; Beachy 1981 ; Rodgers 1983 ; Findlay & Falkow 1989 ; Small et al. 1994 ). Two mechanisms of attachment have been proposed for Leg. pneumophila : opsonin-dependent ( Horwitz 1993) and opsonin-independent systems ( Rodgers & Gibson 1993 ; Gibson et al. 1994 ). In the complement-mediated system, complement receptors CR1 and CR3 on human professional phagocytes that recognize complement fragments C3b and C3bi, respectively, mediate phagocytosis of Leg. pneumophila. The MOMP has been shown to fix selectively the complement component C3 and this facilitates uptake by complement-mediated phagocytosis ( Bellinger-Kawahara & Horowitz 1990 ). Gibson et al. (1994) showed that treatment of host cells with monoclonal antibody (MAb) specific to CR1 (CD35), CR3 (CD11b+CD18) and CR4 (CD11c+CD18) to block these receptors or their sub-units did not interrupt bacterial attachment or intracellular replication.

In previous studies, a recombinant plasmid containing a 750 base pair DNA fragment from the low-passage, fully virulent Leg. pneumophila N7 strain was constructed ( High et al. 1993 ). The plasmid, pLP 116, was shown to express the 25 kDa Leg. pneumophila MOMP in E. coli by Western blot analysis using an anti-Legionella MOMP-specific MAb. The amount of 25 kDa MOMP expressed in the attenuated strain was much reduced compared with that of the virulent strain. In addition, it was noted that a 31 kDa protein which dominated the profile of the attenuated strain did not appear in the profiles of the virulent strain. To determine whether or not the 25 and 31 kDa proteins were interrelated, the plasmid containing a DNA fragment encoding the 25 kDa MOMP was introduced into the attenuated strain using electroporation. The 25 kDa MOMP appeared in the OMP profiles only when the 31 kDa protein was absent. Furthermore, it was seen that the presence of the 25 kDa MOMP enhanced the ability of the organism to attach to host cells and increased virulence in chick embryos.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial cultivation

Legionella pneumophila strain N7 is a highly virulent clinical isolate from a patient who succumbed to Legionnaires’ disease. A low virulence derivative of this strain was produced by multiple passage on bacteriological media. These strains were grown for 72 h on buffered charcoal yeast extract agar supplemented with l-cysteine, ferric pyrophosphate and α-ketoglutarate (BCYE-α) in a humid atmosphere at 37 °C. Escherichia coli JM 83 LP clone 116 (LP 116) is a genetically-mutated derivative of E. coli JM 83 generated in this laboratory. This strain has been shown to express the MOMP of Leg. pneumophila, is resistant to ampicillin and was grown on bacteriological media containing 50 μg ml−1 ampicillin. All strains were stored frozen at −70 °C in serum sorbitol. The attenuated Leg. pneumophila strain was harvested and suspended in phosphate-buffered saline at pH 7·2 (PBS) to give a suspension containing 109 cfu ml−1. This was centrifuged at 5000 g for 10 min at 4 °C and the pellets were harvested for electroporation.

For adherence and virulence assays, Leg. pneumophila N7 was seeded at 1×106 cfu ml−1 into buffered yeast extract broth supplemented with l-cysteine, ferric pyrophosphate and α-ketoglutarate (BYE-α) and grown for 24 h with shaking at 37 °C. JM 83 and LP 116 were inoculated onto nutrient agar or nutrient agar with 50 μg ml−1 ampicillin, respectively ; they were allowed to grow for 24 h at 37 °C and then seeded at 5×105 cfu ml−1 in nutrient broth or nutrient broth containing 50 μg ml−1 ampicillin. These suspensions were incubated in a shaking incubator for 6 h (JM 83) or 8 h (LP 116) at 37 °C to give approximately 5×107 cfu ml−1 prior to adherence or virulence assays.

U-937 cell cultivation

U-937 cells are monocyte-like cells established from a human histiocytic lymphoma and express macrophage-like characteristics when treated with phorbol esters. The cells were grown to 1–2×106 cells ml−1 in T-75 flasks containing RPMI-1640 growth medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 3 mmol l−1l-glutamine. Cells were harvested by centrifugation at 250 g, resuspended in fresh medium, counted, and treated with 10−8 mmol l−1 phorbol myristate acetate (PMA) for 24 h at 37 °C in 5% CO2. Adherent cells were washed three times with Hank’s balanced salt solution, pH 7·2 (HBSS), scraped from the culture flasks and collected by centrifugation at 250 g. Cells were resuspended in fresh medium to give 5×105 cells ml−1, seeded into 6- or 24-well cell culture plates at 3 and 1 ml, respectively, and allowed to re-adhere for 24 h at 37 °C.

Adherence assay–VBCC counts

Monolayers of U-937 cells in 24-well plates were washed three times with HBSS to remove opsonic serum components from the medium, and treated with 5 μg ml−1 cytochalasin D in HBSS for 1 h. Bacterial cultures were centrifuged at 6000 g, washed three times with HBSS and resuspended with 1 ml fresh HBSS. Organisms were added to U-937 cells at a multiplicity of infection of 100 bacteria per host cell and incubated for 1 h at 37 °C in 5% CO2. The U-937 cells were washed three times with HBSS to remove unbounded bacteria and 1 ml sterile water was added to each well and incubated at room temperature for 30 min to lyse the host cells. U-937 cells were disrupted with vigorous aspiration and expulsion through a sterile Pasteur pipet. A 100 μl aliquot of each suspension was placed into 900 μl 1% peptone and serially 10-fold diluted. Duplicate 25 μl samples of each dilution were placed on either nutrient agar (JM 83), nutrient agar supplemented with 50 μg ml−1 ampicillin (LP 116) or BCYE-α (Leg. pneumophila). Organisms were enumerated by viable bacterial cell colony (VBCC) counts. In addition, samples of each strain were treated with either an anti-Legionella MOMP-specific MAb ( Gosting et al. 1984  ; High et al. 1993 ) or a non-immune serum for 1 h at 37 °C and the ability of each to adhere to U-937 cells was assessed.

The number of virulent N7 organisms binding to the U-937 cells was defined as the 100% adherence value and the binding potential of all other strains to host cells was compared with this. In this fashion, variations in attachment capabilities between strains were determined and increases or decreases greater than 50% were considered significant ( Gibson et al. 1994  ; Maganti et al. 1998 ). In addition, data were subjected to statistical analysis by the Mann-Whitney and Student t-tests using GraphPad InStat 2 on a Macintosh computer. Results were expressed as the average of three trials, each conducted in duplicate.

Adherence assay–IFA assay

Prior to addition of U-937 cells to the 6-well cell culture plates, 22 mm sterile glass coverslips were added to each well and the U-937 cells were allowed to adhere to the coverslips. Monolayers were inoculated with JM 83, LP 116 and Leg. pneumophila as described for VBCC counts. After 1 h of incubation, host cells were washed three times to remove non-adherent bacteria and fixed in 10% formalin in PBS, for 1 h at 25 °C. Cells were washed three times with PBS and treated with rabbit polyclonal anti-JM 83, anti-LP 116 or anti-Leg. pneumophila N7 serum for 1 h at 37 °C. Cells were washed three times to remove unbound globulin, and goat anti-rabbit fluorescein isothiocyanate-conjugated (FITC) antibody was added for 1 h at 37 °C. Cells were washed with PBS, stained with 0·01% propidium iodide in PBS for 30 min and mounted in glycerol containing 1% 1,4-diazobicyclo(2,2,2)octane. Blocking studies using both the MOMP-specific MAb and non-immune serum were also assessed by indirect immunofluorescent antibody (IFA) assay. Adherence data were evaluated in a similar fashion to the VBCC counts. Immunofluorescent antibody assay results were averages of three trials, each conducted in duplicate, enumerating the number of adherent bacteria on the first 200 cells per trial counted.

Virulence assay

Lethal dose50 (LD50) studies were performed using embryonate white Leghorn chicken eggs (UNH Poultry Farm). Bacteria that were either scraped from agar plates or grown in broth were suspended in PBS with organism densities ranging from 101 to 107 cfu ml−1. These were inoculated into the yolk sac of fertile chicken eggs using 0·1 ml of 10-fold dilutions ( Tzianabos & Rodgers 1989). The eggs were candled twice daily to determine embryo lethality. The LD50 for each sample and the controls were calculated by the method of Reed & Muench (1938). Experiments were performed using 10 embryonate eggs per dilution and the results were the average of three separate experiments. In addition, samples of the E. coli JM 83, clone LP 116 and Leg. pneumophila N7 strains were treated with anti-Legionella MOMP-specific MAb and virulence was evaluated for each.

Electroporation

Bacterial pellets were resuspended and washed twice in cold, filter-sterilized electroporation (Ep) buffer, containing 272 mmol l−1 sucrose, 1 mmol l−1 MgCl and 7 mmol l−1 NaHPO4, at pH 7·4, to remove PBS as this was found to be excessively conductive for these experiments. After centrifugation at 5000 g for 10 min at 4 °C, the cells were resuspended in Ep buffer and kept on ice for 15 min. Purified plasmids of pUC 19 or pLP 116 with the Leg. pneumophila DNA insert were suspended to give a final concentration of 5 μg μl−1 in 0·8 ml cell suspension in Tris-EDTA (TE) buffer containing 40 mmol l−1 Tris and 2 mmol l−1 disodium EDTA at pH 7·5. This mixture was placed in a chilled 0·4 cm Gene Pulser cuvette and exposed to a single pulse at 2·5 kV peak voltage and 25 μF capacitance in an electroporation unit (Bio-Rad, Richmond, CA, USA). The procedure generated a field strength of 6·25 kV cm−1 with a time constant range of 4·5–4·8 ms. Immediately after high voltage treatment, the cuvettes were placed on ice for 30 min.

Selection of transformants and outer membrane protein analysis

Legionellae are susceptible to ampicillin in vitro and the minimum inhibitory concentration (MIC) of this antibiotic for Leg. pneumophila N7 was previously established as 0·5 μg ml−1 ( Elliot & Rodgers 1985). A sample of the electroporated cell suspension (400 μl) was spread on BCYE-α agar and incubated overnight (18 h) at 37 °C. Growth was harvested and inoculated onto BCYE-α agar containing 50 μg ml−1 ampicillin to select for transformants. To ensure that growth on the ampicillin-supplemented media was the result of transformation and not spontaneous mutations, samples of non-electroporated virulent and attenuated Leg. pneumophila N7 strains, as well as similar samples which had been electroporated in the absence of added DNA, were inoculated onto antibiotic-containing media.

Transformants were screened by plasmid analysis using the isolation methods of Birnboim & Doly (1979). Outer membrane proteins for all Leg. pneumophila samples were isolated by methods previously described ( Wenman et al. 1985 ), assayed for protein content ( Lowry et al. 1951 ) and subjected to SDS-PAGE ( Laemmli 1970). Gels were silver-stained by the method described by Oakley et al. (1980) . Transformants were cured of the plasmids by replica plating ( Miller 1972) onto ampicillin-supplemented media from colonies grown on plates in the absence of ampicillin. No mutagenic agents were added to the media.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The role of the MOMP as an adhesion in the binding of Leg. pneumophila to macrophage-like U-937 cells was assessed. This was accomplished by comparing the attachment of the parent E. coli JM 83, and the genetically-mutated E. coli JM 83 clone LP 116 which expresses the MOMP gene of virulent Leg. pneumophila. Comparison of E. coli JM 83 and LP 116 showed that the presence of the MOMP gene facilitated organism binding in the absence of opsonins. LP 116 was five times more adherent than the parent strain by both VBCC counts and IFA (P < 0·0001 by both the Mann-Whitney and Student t tests) ; the treatment of the virulent N7 and the clone LP 116 with the MOMP-specific MAb abolished binding of these strains to U-937 cells ( Fig. 1), whereas treatment with non-immune serum had no effect on binding of virulent N7 or clone LP 116. These results suggest that the MOMP of Leg. pneumophila is an important factor involved in opsonin-independent binding of this organism to U-937 cells. Previously published LD50 data derived from the chicken embryo lethality assay showed that the E. coli JM 83 parent strain was of low virulence while that of the derived clone LP 116 was much enhanced ( High et al. 1993 ).

image

Figure 1. Adherence of Legionella pneumophila strain N7, Escherichia coli strain JM 83 and the E. coli clone LP 116 to macrophage-like U-937 cells assayed by VBCC counts. A, virulent N7 ; B, N7 treated with MOMP-specific MAb ; C, JM 83 ; D, clone LP 116 ; E, clone LP 116 treated with MOMP-specific MAb. Assays performed by IFA assays gave similar results. Error bars represent standard error of the means

Download figure to PowerPoint

Following electroporation of the attenuated Leg. pneumophila with either pLP 116 or pUC 19, OMP profiles were examined by SDS-PAGE. In these preparations it was seen that a 31 kDa protein band occurred in the attenuated strain but was not found in the original virulent isolate. After introduction of the pLP 116 plasmid into the attenuated strain, the OMP profiles showed an increase in the amount of the 25 kDa MOMP present in the transformant as a result of the plasmid with the Leg. pneumophila DNA insert ( Fig. 2). The 31 kDa protein could no longer be detected after transformation. The pLP 116 plasmid was cured from the transformant by growing it in media in the absence of ampicillin. As a consequence, the concentration of the 25 kDa MOMP in SDS-PAGE analysis decreased while the 31 kDa OMP returned to the protein profiles. Earlier studies on the virulent and attenuated Leg. pneumophila N7 strains showed no changes in the OMP banding patterns before or after electroporation in the absence of DNA. OMP samples of the attenuated strain electroporated with the vector only, also remain unchanged.

image

Figure 2.  SDS-PAGE OMP profiles. Treatments for OMP samples and polyacrylamide gels were as stated in the text. The molecular mass standards consisted of lysozyme (14 300), β-lactoglobin (18 400) and α-chymotrypsinogen (25 700). Lane 1 : molecular mass standard ; 2 : attenuated Legionella pneumophila N7 ; 3 : virulent Leg. pneumophila N7 ; 4 : electroporated attenuated Leg. pneumophila N7 with pUC 19 only ; 5 : electroporated attenuated Leg. pneumophila N7 with pLP 116 ; 6 : cured electroporated Leg. pneumophila pLP 116 transformants

Download figure to PowerPoint

LD50 studies demonstrated that the introduction of pLP 116 increased virulence of the attenuated Leg. pneumophila strain. However, there was no appreciable change in those transformants containing pUC 19 alone ( Table 1). In addition, the virulence of the attenuated samples electroporated in the absence of exogenous DNA was not affected, indicating that electroporation alone did not influence virulence. Treating samples of the virulent N7, the attenuated N7 containing the pLP 116 insert or the E. coli clone LP 116 with the MOMP-specific MAb abolished the virulence of these strains for the chick embryo ( Table 1). Treatment of these strains with non-immune serum had no influence on virulence.

Table 1.  Virulence of Legionella pneumophila N7, Escherichia coli JM 83 and clone LP 116 inoculated into the chicken embryo yolk sac in the presence or absence of anti-Leg. pneumophila MOMP antibody
StrainsLD50 endpoint *
  1. *Ten eggs per dilution were inoculated with 0·1 ml samples and LD 50 data were calculated from three experiments. Control eggs were inoculated with PBS and no deaths occurred up to 8 d post-inoculation. Legionella transformants cured of the plasmid or vector gave LD50 data similar to those obtained for the attenuated strain. Pre-incubation of strains with non-immune serum resulted in LD50 values similar to untreated samples.

Leg. pneumophila virulent N74·6×102
Virulent N7 treated with anti-MOMP MAb>107
Virulent N7 electroporated in the absence  of DNA3·3×102
Leg. pneumophila attenuated N71·1×106
Attenuated N7 electroporated in the absence  of DNA3·0×106
Attenuated N7 with pUC 195·2×106
Attenuated N7 with pLP 1169·2×102
Attenuated N7 with pLP 116 treated with  anti-MOMP MAb>107
E. coli JM 83 with pUC 19>107
JM 83 with pUC treated with anti-MOMP  MAb>107
Clone LP 1162·5×104
Clone LP 116 treated with anti-MOMP  MAb>107

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

As a facultative intracellular pathogen, Leg. pneumophila interacts with the cell membranes of alveolar macrophages prior to phagocytosis and, as a result, surface-located antigens are crucial to disease induction. The MOMP of Leg. pneumophila is known to be tightly associated with LPS ( Gabay et al. 1985  ; Hindahl & Iglewski 1986) and resists complete dissociation in SDS at 100 °C. As a consequence of difficulties associated with separating the MOMP completely from LPS, we used recombinant DNA techniques to obtain Legionella surface-expressed antigens. The genetically-mutated E. coli clone, LP 116, shown previously to express the MOMP of Leg. pneumophila ( High et al. 1993 ), was used to assess the role of this 25 kDa protein in the pathogen–host cell adherence phenomenon. The fivefold increase in binding to U-937 cells observed for the clone as compared with the parent E. coli suggested that the MOMP was, in part, responsible for this opsonin-independent attachment. It is possible that the high expression pUC 19 vector used in these studies resulted in an increase in the amount of MOMP on the surface of LP 116 organisms, and that this expression was responsible for the greater than twofold increased binding of the clone to U-937 cells compared with the virulent Leg. pneumophila N7 strain. Alternatively, the attachment potential of the clone may have been influenced by conformational changes in the expression of the MOMP in the E. coli clone. Furthermore, it is possible that the MOMP of Leg. pneumophila is not associated with LPS in the clone ; thus, steric factors may have contributed to the observed increase in adherence. Further studies on these strains will yield information on the nature of the MOMP adhesion–host cell receptor interaction.

Introduction of genetic material into the legionellae has proved problematic. Studies have shown that under limited conditions, Legionella can accept foreign DNA through conjugal transfer ( Vogel et al. 1998 ) and that this DNA can remain either in the cytoplasm, or in some instances, can be delivered into the chromosome ( Chen et al. 1984  ; Dreyfus & Iglewski 1985). Successful triparental matings using plasmids constructed with Leg. pneumophila DNA have yielded information on pathogenesis ( Engleberg et al. 1988  ; Cianciotto et al. 1989 ). However, transformation of Legionella has not been reported. The mechanisms involved in electroporation are not fully understood. It is likely that the charge across cell membranes forms openings allowing foreign DNA to enter ( Sowers & Lieber 1986). This transfer system has performed well with eukaryotic cells ( Potter et al. 1984  ; Chu et al. 1987 ) and has been applied successfully to various bacterial species including Leg. pneumophila ( Wirth et al. 1989  ; High & Rodgers 1991 ; Segal & Shuman 1997 ; Gao et al. 1998 ). Electroporation protocols have been maximized for use with E. coli but plasmids or plasmid constructs with DNA native to the organism being transformed have proved most effective. In our studies, it was shown that the small 2·7 kb pUC 19 vector and the 3·5 kb plasmid construct from Leg. pneumophila N7 were suitable for electroporation. After 10 d of incubation, pinpoint colonies appeared on media containing 50 μg ml−1 ampicillin, thus indicating that the plasmid was functional. The control samples showed no growth. The frequency of transformation for these experiments was 10−6. It appeared that this procedure may have selected for a small number of restriction and/or modification mutants that existed in the population, but these frequencies were similar to those found for other transfer methods used for Legionella ( Chen et al. 1984  ; Dreyfus & Iglewski 1985). After initial slow growth, the transformants grew well on ampicillin-containing media in 1–3 d.

Transformation of Leg. pneumophila N7 grown in broth-based media resulted in insufficient organism numbers for electroporation. The use of overnight platings on BCYE-α agar resulted in an adequate concentration of bacteria to produce transformants when transferred to BCYE-α agar containing ampicillin. Electroporation did not yield transformants when less than 108 cfu ml−1 were present in the original suspension. Conversely, suspensions exceeding 109 cfu ml−1 were not productive as the additional cells caused a decrease in the time constant, a critical factor in electroporation. It is likely that transformation frequencies for Leg. pneumophila N7 may be greater if concentrations of ampicillin lower than 100 times the MIC are used. Analysis of the plasmids by agarose gel electrophoresis showed that the vector and the recombinant plasmid, pLP 116, remained autonomous in the bacterial cell. Both the vector and the pLP 116 experienced curing rates at a frequency of 96% by growth on media without ampicillin present. As initial growth on media containing ampicillin was delayed for the transformants, these plates were incubated for 10 d to recover all organisms that resisted curing.

The 31 kDa protein band found in the SDS-PAGE OMP profiles of the attenuated strain, but not in the original clinical isolate, could no longer be detected after the introduction of the pLP 116 plasmid into the attenuated strain. At the same time, an increase in the amount of the 25 kDa MOMP occurred in the transformant as a result of the presence of the plasmid, and this coincided with an increased capacity for the organisms to bind to U-937 cells. Loss of pLP 116 from the attenuated Leg. pneumophila strain following growth on media without ampicillin resulted in a decrease in the concentration of the 25 kDa MOMP while the 31 kDa OMP returned to the profiles. The OMP profiles of the electroporated strains of virulent and attenuated Leg. pneumophila N7 in the absence of DNA showed no changes in the banding patterns of the proteins. OMP samples of the attenuated strain electroporated with the vector only also remained unchanged. These data demonstrated that increased production of the 25 kDa MOMP in the cell caused elimination of the 31 kDa protein from the profile. Butler & Hoffman (1990) previously reported that the 31 kDa OMP was an anchor protein involving attachment of peptidoglycan and was encoded by the same set of genes as the 28 kDa OMP more external to the cell ( Hoffman et al. 1992 ). However, the virulence characteristics of the strain used in those studies were not described.

It is well established that multiple passage on bacteriological media results in stable attenuated derivatives of Leg. pneumophila ( Catrenich & Johnson 1988). Our observation that under identical preparation conditions the presence of the 31 kDa OMP was inversely related to the occurrence of the 25 kDa MOMP in Legionella suggested a molecular rationale for these differences between virulent and attenuated strains. The LD50 data showed that the presence of the 25 kDa MOMP introduced via pLP 116 into either the attenuated Leg. pneumophila strain by electroporation, or the E. coli JM 83 strain by transformation, resulted in increased levels of virulence for the chick embryo and that virulence for all strains could be abolished using MAb specific to the MOMP.

A correlation between these two proteins in terms of bacterial opsonin-independent adherence to U-937 cells as well as virulence for chicken embryos seems evident. However, the precise nature of the interrelationship is not clear. Further investigations of this phenomenon are necessary to determine further the relationship between these two proteins and their function in virulence, and to define the part they play regarding survival mechanisms for this organism. Given the role of the 25 kDa MOMP in virulence and in binding the organism to host cells, it is possible that this protein may serve as a useful moiety for the development of molecular vaccines against Legionnaires’ disease.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors thank Dr Frank C. Gibson III for helpful discussion and Dr L. Gosting for donation of the anti-Legionella MOMP MAb. This work was supported by a Public Health Service grant AI-27929 from the National Institutes of Health and by CURF grants from the University of New Hampshire, Office of Sponsored Research.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Andrews, H.L., Vogel, J.P., Isberg, R.R. 1998 Identification of linked Legionella pneumophila genes essential for intracellular growth and evasion of the endocytic pathway. Infection and Immunity, 66, 950 958.
  • Beachy, E.H. 1981 Bacterial adherence: adhesion-receptor interactions mediating the attachment of bacteria to mucosal surfaces. Journal of Infectious Disease, 143, 325 345.
  • Bellinger-Kawahara, C.G. & Horowitz, M.A. 1990 Complement component C3 selectively fixes to the major outer membrane protein (MOMP) of Legionella pneumophila and mediates phagocytosis of liposome-MOMP complexes by human monocytes. Journal of Experimental Medicine, 172, 1201 1210.
  • Birnboim, H.C. & Doly, J. 1979 A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research, 7, 1513 1523.
  • Butler, C.A. & Hoffman, P.S. 1990 Characterization of a major 31-kilodalton peptidoglycan-bound protein of Legionella pneumophila. Journal of Bacteriology, 172, 2401 2407.
  • Butler, C.A., Street, E.D., Hatch, T.P., Hoffman, P.S. 1985 Disulfide-bonded outer membrane proteins in the genus Legionella. Infection and Immunity, 48, 14 18.
  • Catrenich, C.E. & Johnson, W. 1988 Virulence conversion of Legionella pneumophila: a one-way phenomenon. Infection and Immunity, 56, 3121 3125.
  • Chen, G.C.C., Lema, M., Brown, A. 1984 Plasmid transfer into the members of the family Legionellaceae. Journal of Infectious Disease, 150, 513 516.
  • Chu, G., Hayakawa, H., Berg, P. 1987 Electroportion for the efficient transfection of mammalian cells with DNA. Nucleic Acids Research, 15, 1311 1326.
  • Cianciotto, N.P., Eisenstein, B.I., Mody, C.H., Toews, G.B., Engleberg, N.C. 1989 A Legionella pneumophila gene encoding for species-specific surface protein potentiates initiation of intracellular infection. Infection and Immunity, 57, 1255 1262.
  • Dreyfus, L.A. & Iglewski, B.H. 1985 Conjugation-mediated genetic exchange in Legionella pneumophila. Journal of Bacteriology, 161, 80 84.
  • Ehret, W., Anding, G., Ruckdeschel, G. 1984 Characterization of membrane proteins from various strains and serogroups of Legionella pneumophila and other Legionella species. In: Proceedings of the Second International Symposium on Legionella (eds Thornsberry, C. Balows, A. Feeley, J.C. & Jakubowski, W), pp. 265 268. Washington, D.C. ASM.
  • Elliot, T.S.J. & Rodgers, F.G. 1985 Morphological response and growth characteristics of Legionella pneumophila exposed to ampicillin and erythromycin. Journal of Medical Microbiology, 19, 383 390.
  • Engleberg, N.C., Cianciotto, N., Smith, J., Eisenstein, B.I. 1988 Transfer and maintenance of small, mobilizable plasmids with ColE 1 replication origins in Legionella pneumophila. Plasmid, 20, 83 91.
  • Findlay, B.B. & Falkow, S. 1989 Common themes in microbial pathogenicity. Microbiological Reviews, 53, 210 230.
  • Gabay, J.E., Blake, M., Niles, W.D., Horwitz, M.A. 1985 Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin. Journal of Bacteriology, 162, 85 91.
  • Gao, L.Y., Harb, O.S., Kwaik, Y.A. 1998 Identification of macrophage-specific infectivity loci (mil) of Legionella pneumophila that are not required for infectivity of protozoa. Infection and Immunity, 66, 883 892.
  • Gibbons, R.J. 1973 Bacterial adherence in infection and immunity. Reviews in Microbiology, 4, 48 60.
  • Gibson, F.C., Tzianabos, A.O., Rodgers, F.G. 1994 Adherence of Legionella pneumophila to U-937 cells, guinea-pig alveolar macrophages, and MRC-5 cells by a novel, complement-independent binding mechanism. Canadian Journal of Microbiology, 40, 865 872.
  • Gosting, L.H., Cabrian, K., Sturge, J.C., Goldstein, L.C. 1984 Identification of a species-specific antigen in Legionella pneumophila by a monoclonal antibody. Journal of Clinical Microbiology, 20, 1031 1035.
  • High, A.S. & Rodgers, F.G. 1991 Electroporation of the major outer membrane protein-encoding genes from virulent to avirulent Legionella pneumophila. In: Abstracts of the 91st General Meeting of the American Society for Microbiology. Washington, D.C. ASM.
  • High, A.S., Torosian, S.D., Rodgers, F.G. 1993 Expression of a 25 kDa major outer membrane protein (MOMP) of Legionella pneumophila in Escherichia coli. Journal of General Microbiology, 139, 1715 1721.
  • Hindahl, M.S. & Iglewski, B.H. 1986 Outer membrane proteins from Legionella serogroups and other Legionella species. Infection and Immunity, 51, 94 101.
  • Hoffman, P.S., Ripley, M., Weeratna, R. 1992 Cloning and nucleotide sequence of a gene (ompS) encoding for the major outer membrane protein of Legionella pneumophila. Journal of Bacteriology, 174, 914 920.
  • Horwitz, M.A. 1993 Toward an understanding of host and bacterial molecules mediating Legionella pneumophila pathogenesis. In Legionella. Current Status and Emerging Perspectives (eds Barbaree, J.M. Breiman, R.F. & Dufour, A.P), pp. 55 62. Washington, D.C. ASM.
  • Kirby, J.E., Vogel, J.P., Andrews, H.L., Isberg, R.R. 1998 Evidence for pore-forming ability by Legionella pneumophila. Molecular Microbiology, 27, 323 336.
  • Laemmli, U.K. 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 117, 680 685.
  • Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J. 1951 Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265 275.
  • Maganti, S., Pierce, M.M., Hoffmaster, A., Rodgers, F.G. 1998 The role of sialic acid in opsonin-dependent and opsonin-independent adhesion of Listeria monocytogenes to murine peritoneal macrophages. Infection and Immunity, 66, 620 626.
  • Miller, J.H. 1972. Experiments in Molecular Genetics, pp. 56 59. Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press.
  • Oakley, B.R., Kirsh, D.R., Morris, N.R. 1980 A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Analytical Biochemistry, 105, 361 363.
  • Potter, H., Weir, L., Leder, P. 1984 Enhancer-dependent expression of human k immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proceedings of the National Academy of Science USA, 81, 7161 7165.
  • Reed, L.J. & Muench, H. 1938 A simple method for determining 50 percent endpoints. American Journal of Hygiene, 27, 493 497.
  • Rodgers, F.G. 1983 The role of structure and invasiveness on the pathogenicity of Legionella. Zentralblatt für Mikribiologie und Hygiene, A 255, 138 144.
  • Rodgers, F.G. & Gibson, F.C. 1993 Opsonin-independent adherence and intracellular development of Legionella pneumophila within U-937 cells. Canadian Journal of Microbiology, 39, 718 722.
  • Segal, G. & Shuman, H.A. 1997 Characterization of a new region required for macrophage killing by Legionella pneumophila. Infection and Immunity, 65, 5057 5066.
  • Small, P.L.C., Ramakrishnan, L., Falkow, S. 1994 Remodeling schemes of intracellular pathogens. Science, 263, 637 639.
  • Sowers, A.E. & Lieber, M.R. 1986 Electropore diameters, lifetimes, numbers and locations in individual erythrocyte ghosts. FEBS Letters, 205, 179 184.
  • Tzianabos, A.O. & Rodgers, F.G. 1989 Pathogenesis and chemotherapy of experimental Legionella pneumophila infection in the chick embryo. Zentralblatt für Mikribiologie und Hygiene, A 271, 293 303.
  • Vogel, J.P., Andrews, H.L., Wong, S.K., Isberg, R.R. 1998 Conjugative transfer by the virulence system of Legionella pneumophila. Science, 279, 873 876.
  • Wenman, W.M., Chai, J., Louie, T.J et al. 1985 Antigenic analysis of Campylobacter flagellar protein and other proteins. Journal of Clinical Microbiology, 21, 108 112.
  • Wirth, R., Friesenegger, A., Fiedler, S. 1989 Transformation of various species of Gram-negative bacteria belonging to 11 different genera by electroporation. Molecular and General Genetics, 216, 175 177.