INFECTION AND IMMUNITY, JUlY 1991, p. 2376-2381 0019-9567/91/072376-06$02.00/0 Copyright © 1991, American Society for Microbiology
Vol. 59, No. 7
Acquisition of Iron by Legionella pneumophila: Role of Iron Reductase WILLIAM JOHNSON,* LAURIE VARNER, AND MARK POCH
Department of Microbiology, University of Iowa,
City, Iowa 52242
Received 7 January 1991/Accepted 9 April 1991
Legionella pneumophila has been shown to survive and multiply in a variety of intracellular environments, including protozoa and human mononuclear phagocytes. However, the mechanism by which this organism acquires iron in the intracellular environment has not been studied. Since L. pneumophila does not produce siderophores, alternative methods of iron acquisition were investigated. Virulent strains of L. pneumophila were able to grow in media containing as little as 3 p,M iron, whereas avirulent cells required a minimum of 13 ,uM iron for growth. Neither virulent nor avirulent cells were able to utilize 55Fe bound to transferrin. When incubated in the presence of 5sFe in the form of ferric chloride, both virulent and avirulent cells accumulated equal amounts of iron. The uptake of iron was energy dependent as indicated by inhibition of 55Fe uptake at 4°C and preincubation of the cells with KCN. Treatment of virulent cells with pronase or trypsin had no effect on iron uptake. In contrast, pronase or trypsin treatment of avirulent cells resulted in increased uptake of iron. Iron reductase activity in both virulent and avirulent cells was demonstrated, with the highest specific activity associated with the periplasmic fraction. Maximum reductase activity of virulent cells occurred with NADH as the reductant. In contrast, avirulent cells showed a twofold increase in enzyme activity when NADPH was used as the reductant. These results suggest that an iron reductase is important in iron acquisition by L. pneumophila.
Legionella pneumophila is a fastidious gram-negative bacterium that has been isolated from rivers, streams, cooling towers, and potable water supplies (4, 12, 21). Several studies have suggested that L. pneumophila has adapted to survival within these aquatic environments by evolving mechanisms for intracellular multiplication within amoebae (14, 28). In humans, L. pneumophila is associated with sporatic and epidemic outbreaks of both nosocomial and community-acquired pneumonia. Mounting evidence suggests that the organism is a facultative intracellular parasite and that the pathogenesis of legionella infections is associated with intracellular survival and multiplication within human mononuclear phagocytes (6, 15). However, the mechanisms by which this organism is able to evade the cellular defense mechanisms of the human host and multiply intracellularly are not entirely clear. Early studies on the nutritional requirements of L. pneumophila led to the development of both complex and chemically defined liquid media (22, 25, 26), all of which have included both cysteine and iron in their formulation. Iron is required for a number of cellular functions. However, in the human host, iron appears to be a limiting factor for the growth of many bacteria because iron is bound to transferrin or lactoferrin or is associated with insoluble ferritin complexes and therefore is not available in a form suitable for use by the bacterial cell. Many bacterial pathogens have evolved mechanisms to circumvent this limitation. These include either production of siderophores designed to directly compete with transferrin for iron or the direct binding of transferrin to the surface of the bacterial cell (1, 18, 29, 30). Although L. pneumophila requires relatively large amounts of iron for optimum growth in vitro (26), little is known about its mechanism of iron acquisition, particularly in the intracellular environment. Reeves et al. (24) have *
shown that L. pneumophila does not produce siderophores, and other studies have suggested that transferrin and lactoferrin are inhibitory to the growth of L. pneumophila (3, 23). In this study, we investigated the iron requirements of virulent and avirulent cultures of L. pneumophila and the mechanism of iron acquisition. Avirulent cells were shown to require significantly more iron for growth in vitro than virulent cells. While the uptakes of iron by both virulent and avirulent cells appear to take place at approximately the same rate, treatment of avirulent cells with pronase or trypsin increased iron uptake in contrast to results with virulent cells, in which treatment with proteolytic enzymes had no effect on iron uptake. Iron acquisition by both virulent and avirulent cells appears to involve an iron reductase, with the highest specific activity located in the periplasm of the cell. Neither virulent nor avirulent cells were able to utilize iron bound to transferrin. The results of these studies in relation to iron acquisition in vivo are discussed. MATERIALS AND METHODS
Organisms. L. pneumophila serogroup 1 strains Philadelphia 2 and Knoxville were obtained from the Centers for Disease Control, Atlanta, Ga. An environmental isolate of L. pneumophila, Lp 59, was obtained from the University Hygienic Laboratory, University of Iowa. Virulent cultures were maintained by passage in guinea pigs as previously described (7). An avirulent culture was derived by passaging the virulent culture several times on charcoal-yeast extract medium (13) and then sequentially passaging on supplemented Mueller-Hinton agar (8). Media. Virulent and avirulent cultures of L. pneumophila were maintained on charcoal-yeast extract agar. Virulent cultures were streaked for isolation and tested for absence of growth on supplemented Mueller-Hinton agar (8). The me-
Corresponding author. 2376
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dium used for determination of the minimal iron requirements for virulent and avirulent cells was cyclodextrin yeast extract (CDYE), a modification of the medium described by Imaizumi et al. (17), which contained the following: 1% Gelritegellan gum (Kal Co., a division of Merck), 1% yeast extract, 0.025% heptakis(2,6-O-dimethyl),B-cyclodextrin (Sigma), 0.03 M K2HPO4, and 0.03 M KH2PO4. Various dilutions of ferric citrate, ranging from a final concentration of 1 mM to 0.1 ,uM, were filter sterilized prior to addition to the medium. Water used in all experiments was distilled, deionized water from a Milli-Q water purification system (Millipore Corp.). The medium was autoclaved at 121°C for 20 min and cooled for 5 min in a 50°C water bath before the addition of MgSO4 to a final concentration of 2 mM. After an additional 5 min, 0.025% of L-cysteine was filter sterilized into the medium. For iron uptake experiments, cells were grown in CDYE medium devoid of iron supplementation to deplete endogenous iron supplies in the cells. Iron uptake assay. Cultures of virulent or avirulent cells grown on CDYE without iron were suspended to an A6. of 0.4 in 0.1% N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer, pH 7, containing 0.6 mM MgSO4 and 0.6 mM (NH4)2HP04. A 3-ml volume of the cell suspension was added to 2.9 ml of distilled water. The iron uptake assay was carried out according to the method of Cox (10) with the following modification: a solution of amino acids (MEM amino acids, 5Ox concentration; Gibco Laboratories) added to a final concentration of 1.5x was used in place of sodium succinate. At various time intervals, cells were trapped on 0.45-,um-pore-size filters (Schleicher & Schuell) and washed with a 5-ml volume of thioglycolate to ensure the reduction of any insoluble iron precipitates (10). Stock solutions of 20 ,uCi of "FeCl3 per ml (New England Nuclear) were used in all experiments. The final concentration of ferric chloride was 320 pM. Control samples without cells were included to account for nonspecific binding of radiolabeled iron to the filters. All binding assays represent the average of three separate experiments run in triplicate and are corrected for background. Samples were counted in a Beckman LS 5000TD scintillation counter. In one set of experiments, the temperature of the incubation was maintained at 4°C to assess the effect of temperature on iron uptake. The effect of potassium cyanide on iron uptake was determined by the addition of 10 mM KCN to the suspending medium and incubation at 37°C for 20 min prior to the addition of radiolabeled iron. Samples were removed at 1, 10, 30, 60, 120, and 180 min and processed for counting. The effect of proteolytic enzymes on iron uptake was determined by treatment of cell suspensions with protease (type VI; Sigma) or trypsin (type I; Sigma) at a concentration of 1 U per 3 ml of cell suspension. The cells were incubated for 30 min at 37°C and washed two times with ACES buffer prior to the addition of radiolabeled iron. Samples were removed at 1, 5, 10, 20, 30, and 60 min and processed for counting.
tetraacetic acid (EGTA) and 5"Fe-transferrin (22% saturation) were kindly supplied by C. D. Cox, University of Iowa. Samples were removed at 1, 60, and 180 min. Preparation of cell fractions. To release the periplasmic contents, the method of Cox (9) was used with two modifications. After an initial wash with 0.01 M Tris-hydrochloride (pH 7.6) containing 1 mM MgCl2 (TM buffer), the supernatant was saved as the periplasmic fraction. Also, before disruption of the cells by sonication, 1 mg each of DNase I (Sigma) and RNase A (Sigma) was added to the cell suspension. The cytoplasmic fraction was isolated by disruption of
the cells by sonication (cell disruptor, model W-375; Ultrasonics Inc.). The disrupted cell suspension was centrifuged at 3,000 x g at 4°C to remove whole cells and cellular debris. The supernatant was centrifuged at 140,000 x g for 2 h to obtain the membrane (pellet) and cytoplasmic (supernatant) fractions. The membrane fraction was suspended in a minimal volume of 0.01 M Tris-hydrochloride, pH 7.6. All three fractions (periplasmic, cytoplasmic, and membrane) were dialyzed against distilled water, lyophilized, and stored at 40C. Enzyme assay. Iron reductase activity was measured by the method of Dailey and Lascelles (11) by using the ferrous iron chelator ferrozine [3-(2-pyridyl)-5,6-bis-(4-phenylsulfonic acid)-1,2,4-triazine] (Sigma). The reaction mixture contained 0.4 ml of cell extract, 5 ,uM ferrozine in 0.01 M Tris-hydrochloride (pH 7.6), 100 ,ug of ferric citrate (pH 7), and 0.01 M Tris-hydrochloride (pH 7.6) in a final volume of 2.0 ml. The reactions were performed in polystyrene cuvettes at room temperature, and the change in optical density was monitored at 562 nm in a Varian DMS 90 double-beam spectrophotometer. The reference cuvettes contained all of the reaction components except reductant. Spontaneous reduction of iron by endogenous reductants was measured for 10 min before the addition of reductant. NADPH or NADH was then added to the reaction mixture at a concentration of 2 ,uM. The reductase activity was recorded as the change in optical density over time. Specific activity of the cell fractions was calculated as the amount of product formed per milligram of protein per 30 min. Protein concentrations were determined with the Bio-Rad protein assay kit with bovine serum albumin as the standard. RESULTS
The amount of iron required for the growth of virulent and avirulent cells was ascertained by growth of cells on ironlimiting CDYE medium. To ensure that growth was not due to the presence of residual iron present from the charcoalyeast extract agar used to maintain stock cultures, colonies from the CDYE plates were subcultured a minimum of three times on CDYE medium containing the same amount of iron present in the original CDYE plate. The minimum amount of iron required to support repeated subculture of virulent cells was 3 ,uM. In contrast, avirulent cells required the addition of 13 ,uM iron to support repeated passage. As shown in Fig. 1, both virulent and avirulent cells accumulated iron at a rapid rate over the first 30 min of incubation and began to reach a steady state after 60 min. The initial uptake was somewhat lower with virulent cells, but by the end of the 3-h incubation period, both virulent and avirulent cells had accumulated approximately the same amount of iron: 30 and 34 pM iron for the virulent and avirulent cells, respectively. At 4°C, there was little iron bound to avirulent cells. Preincubation of both virulent and avirulent cells with KCN reduced the level of bound iron by over 95%. Similar results were obtained with the Knoxville strain of L. pneumophila and the environmental isolate Lp 59 (data not shown). The addition of 0.64 nM unlabeled FeCl3 to avirulent cells resulted in a 60% decrease in the incorporation of "Fe into the cell after 60 min of incubation, and increasing the final concentration of unlabeled iron to 3.2 nM resulted in an 83% inhibition of "Fe uptake. Similar values were obtained for the virulent cells when unlabeled iron was added to the medium (data not shown). The data in Fig. 2 show the effect of treatment of cells with
JOHNSON ET AL.
60 z 0
TIME (MIN) FIG. 1. Uptake of "5Fe by virulent and avirulent L. pneumophila cells. 0, avirulent control; 0, avirulent cells incubated with KCN; A, avirulent cells at 4°C; 0, virulent control; *, virulent cells incubated with KCN.
proteolytic enzymes on the uptake of iron. Treatment of virulent cells with either trypsin or pronase had no effect on the amount of iron bound. In contrast, treatment of avirulent cells with either pronase or trypsin resulted in an enhanced uptake of iron starting at the earliest time period of the assay. At 1 min after the addition of iron, the uptakes of iron in pronase-treated and trypsin-treated cells were three and five times, respectively, greater than that of the controls. After 1 h of incubation, pronase-treated avirulent cells had accumulated twice the amount of iron as controls and the trypsin-treated cells had 1.5 times the amount of bound iron. Some bacterial pathogens are able to utilize iron bound to transferrin by directly binding transferrin and reducing the iron to a form that is soluble and can be transported into the cell. Therefore, experiments to determine the ability of virulent and avirulent cells to acquire iron from two sources, EGTA and transferrin, were performed. As shown in Fig. 3, neither virulent nor avirulent cells were able to acquire iron from either EGTA or transferrin, suggesting that L. pneumophila must acquire iron from sources other than proteinbound iron. In the infected monocyte, L. pneumophila must survive within the phagocytic vesicle at a pH that is lower than that found in the surrounding environment (6). To determine the effect of pH on iron uptake by virulent and avirulent cells, the pH of the suspending medium was varied and the amount of uptake of iron was determined. The results are shown in Table 1. At pH 7, the amounts of iron in virulent and avirulent cells were 54 and 58 pM, respectively. While the levels of iron in virulent and avirulent cells decreased to 24 and 25 pM, respectively, at pH 4, no significant differences
FIG. 2. The effect of pronase or trypsin treatment on the uptake of iron by virulent and avirulent L. pneumophila cells. 0, avirulent control; A, avirulent pronase-treated cells; 0, avirulent trypsintreated cells; 0, virulent control; A, virulent pronase-treated cells; *, virulent trypsin-treated cells.
in the levels of iron bound to virulent or avirulent cells at any of the pH levels tested were observed. Iron reductases have been shown to be important in several bacterial pathogens for their ability to reduce siderophore-bound iron or to acquire iron from soluble iron complexes, including ferric citrate (5, 9, 16, 19, 20). As shown in Table 2, both virulent and avirulent cultures of L. pneumophila produce an iron reductase. When NADH was used as the reductant, similar enzyme activity was present in each of the three fractions isolated from virulent and avirulent cells, with the highest specific activity associated with the periplasmic fraction (20.5 and -23.64 nM, respectively). When NADPH was used as the reductant, the activity of fractions isolated from virulent cells was unchanged from the values obtained with NADH, as the reductant and the highest specific activity was still associated with the periplasmic fraction, 20.5 nM for NADH and 23.1 nM for NADPH. In contrast, the specific activity of all three fractions isolated from avirulent cells incubated with NADPH was increased approximately 1.5 to 2 times over the values obtained for fractions isolated from virulent cells incubated with NADPH. In addition, the specific activity in the periplasmic fraction isolated from avirulent cells incubated with NADPH was twofold over that observed with NADH, 44.34 nM for NADPH versus 23.64 nM for NADH. DISCUSSION L. pneumophila has been shown to be present in a number of aquatic environments. In its natural habitat, the organism
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TABLE 2. Specific activity of iron reductasea in cell fractions isolated from virulent and avirulent L. pneumophila cells with NADH or NADPH as the reductant Cell
V A nLi
M A 15D14
Virulent cells Avirulent cells Virulent cells Avirulent cells
Cytoplasm 8.23 + 1.05 12.25 + 3.7 12.71 + 2.49 28.24 ± 6.36 Membrane 12.33 ± 3.46 16.05 ± 0.23 12.81 ± 4.26 27.6 ± 2.58 Periplasm 20.50 ± 1.4 23.64 ± 4.85 23.31 ± 3.87 44.34 ± 6.69 Lu
a Expressed as the nM Fe2+ formed per milligram of protein per 30 min.
140 160 180
TIME (MIN) FIG. 3. Uptake of iron from 5Fe-transferrin and 5Fe-EGTA by virulent and avirulent L. pneumophila cells. 0, avirulent control; A, avirulent cells incubated with EGTA; 0, avirulent cells incubated with transferrin; 0, virulent control; A, virulent cells incubated with EGTA; E, virulent cells incubated with transferrin.
appears to have evolved mechanisms for survival which include an intracellular existence in amoebae (14, 28). In humans, the pathogenesis of legionella infections appears to be directly related to the organism's ability to survive and multiply within mononuclear phagocytes (15). While the mechanism of survival of L. pneumophila in amoebae and protozoa is not understood, recent studies have suggested that intracellular multiplication of L. pneumophila in human mononuclear cells is iron dependent (6). Thus, the availability of iron and the ability of L. pneumophila to successfully compete with the host for sources of iron are critical in the pathogenesis of infections caused by L. pneumophila. Iron is an important nutritional factor required for several critical enzymes and for the functioning of ferrodoxins and cytochromes. Its importance is related to its ability to exist
TABLE 1. Effect of pH on iron uptake by virulent and avirulent L. pneumophila cells pM "Fe uptakea
4 5 6 7 a
24 + 3.7 34+2.4 38 + 4.6 54 + 7.3
25 + 1.8 40+3.5 42 + 2.8 58 ± 5.2
Expressed as the pM 5sFe uptake per
106 cells after 2 h of incubation.
in two redox states, with a large range of redox potentials. Previous studies on the nutritional requirements of L. pneumophila have demonstrated that the addition of iron at a concentration of 250 mg/liter is required for optimum growth (26). However, no attempt was made to distinguish differences in the iron requirements of virulent and avirulent cells. In our studies, we have shown that virulent cells require 3 ,uM iron, while avirulent cells require 13 ,uM iron. The acquisition of iron by both virulent and avirulent cells was energy dependent, since no uptake was seen when cells were preincubated with KCN and incubation of the reaction at 4°C abolished uptake. Given the limitations of iron availability in the intracellular environment and the documented effects of iron deprivation on intracellular growth of L. pneumophila (6), the inability of avirulent cells to survive within the phagocytic cell may be in part a result of the higher iron requirement of avirulent cells. These results also support previous studies which have suggested that activated macrophages may limit the growth of L. pneumophila by limiting the availability of iron (6). An unexpected finding of these studies was the differences observed in the effects of treatment with proteolytic enzymes on the binding of iron by virulent and avirulent cells. These results suggest that the cell surface receptors associated with iron binding by virulent L. pneumophila cells are either not proteins or are proteins buried in the cell surface and not susceptible to degradation by proteolytic enzymes. The increase in binding observed in avirulent cells after treatment with pronase and trypsin may reflect differences in the cell surface components surrounding avirulent cells. Whether these differences are related to the differential susceptibility of avirulent cells to high salt concentrations or the filamentous appearance of avirulent cells is currently under study (7, 8). The studies of Reeves et al. (24) have shown that siderophores are not produced by L. pneumophila and therefore play no role in iron acquisition by this organism. An alternative mechanism involves the direct binding of transferrin to the surface of the cell, as has been observed with Neisseria meningitidis and Neisseria gonorrhoeae (1, 18, 29, 30). Previous studies (3, 23) have demonstrated that lactoferrin and transferrin are inhibitory to the growth of L. pneumophila when the cells are grown in a broth medium. However, Bortner et al. (2) have shown that lactoferrin binds to and does not inhibit the growth of L. pneumophila when the cells are grown in an agar medium. These results suggested that agar-grown cells, as used in the present study, may also provide the optimum conditions for the binding of transferrin-bound iron to L. pneumophila. However, as shown in Fig. 3, no uptake of iron was observed when the cells were incubated with 55Fe-transferrin. The data in Fig. 3 also show that iron is not acquired from EGTA, a compound which binds iron with less affinity than transferrin. Thus, the
JOHNSON ET AL.
mechanism of iron acquisition by L. pneumophila does not appear to involve either siderophores or transferrin. Iron reductase enzymes have been shown to be important in the ability of several organisms to acquire iron from siderophores (16, 19, 20). The reductase reduces the iron in the ferrisiderophore chelate from Fe3" to Fe2 . Since Fe2+ has a lower affinity for the siderophore, the Fe2+ is released and available for utilization by the cell. There is also evidence that iron reductase enzymes are involved in the acquisition of iron from ferric citrate complexes and that these enzymes are distinct from those involved in the reduction of ferrisiderophores (9, 16). The results of our studies suggest that a ferric citrate reductase is important in the acquisition of iron by L. pneumophila. Both virulent and avirulent cells have ferric citrate reductase activity, with the highest specific activity associated with the periplasmic fraction. These results are in contrast to results with other bacteria, in which the reductase activity is associated with the cytoplasm (9, 16). In addition, preliminary data suggest that production of the reductase by virulent cells is constitutive and is therefore independent of the iron concentration in the medium (data not shown). The most significant difference between the reductase activity in virulent and avirulent L. pneumophila cells is the requirement for reductant. Virulent cells show maximum activity with NADH, while avirulent cells show maximum activity with NADPH. During phagocytosis, there is an eightfold increase in the activity of NADPH oxidase and a concomitant decrease in the amount of intracellular NADPH. In contrast, the concentration of NADH, which is required for optimum activity of virulent cells, remains constant (27). While there are several mechanisms that may lead to the inhibition of intracellular growth of avirulent L. pneumophila cells, it is interesting to speculate that the lowered levels of NADPH, which is required for maximum activity of the iron reductase of avirulent cells, may result in restrictions of the ability of these cells to acquire iron in the phagocytic cell and thus contribute to limiting the intracellular growth of avirulent cells. Additional studies are in progress to compare the kinetics of purified iron reductase isolated from virulent and avirulent L. pneumophila to elucidate the potential role of these enzymes and ironbinding proteins in the growth of L. pneumophila. ACKNOWLEDGMENTS
We thank C. D. Cox, Department of Microbiology, for his cooperation and providing several of the reagents used in these studies. This work was supported in part by Public Health Service grant Al 26523 from the National Institutes of Health. REFERENCES 1. Blanton, K. J., G. D. Biswas, J. Tsai, J. Adams, D. W. Dyer, S. M. Davis, G. D. Koch, P. K. Sen, and P. F. Sparling. 1990.
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