Legionella pneumophila feoAB promotes ferrous iron uptake and intracellular infection

Abstract

In order to determine the role of ferrous iron transport in Legionella pathogenesis, we identified and mutated the feoB gene in virulent Legionella pneumophila strain 130b. As it is in Escherichia coli, the L. pneumophila feoB gene was contained within a putative feoAB operon. L. pneumophila feoB insertion mutants exhibited decreased ferrous but not ferric iron uptake compared to the wild type. Growth on standard buffered charcoal yeast extract agar or buffered yeast extract broth was unaffected by the loss of L. pneumophila FeoB. However, the L. pneumophila feoB mutant had a reduced ability to grow on buffered charcoal yeast extract agar with a reduced amount of its usual iron supplementation, a phenotype that could be complemented by the addition of feoB in trans. In unsupplemented buffered yeast extract broth, the feoB mutant also had a growth defect, which was further exacerbated by the addition of the ferrous iron chelator, 2,2'-dipyridyl. The feoB mutant was also 2.5 logs more resistant to streptonigrin than wild-type 130b, confirming its decreased ability to acquire iron during extracellular growth. Decreased replication of the feoB mutant was noted within iron-depleted Hartmannella vermiformis amoebae and human U937 cell macrophages. The reduced intracellular infectivity of the feoB mutant was complemented by the introduction of a plasmid containing feoAB. The L. pneumophila feoB gene conferred a modest growth advantage for the wild type over the mutant in a competition assay within the lungs of A/J mice. Taken together, these results indicate that L. pneumophila FeoB is a ferrous iron transporter that is important for extracellular and intracellular growth, especially in iron-limited environments. These data represent the first evidence for the importance of ferrous iron transport for intracellular replication by a human pathogen.

FIG. 1.

Kinetics of ferrous iron uptake by L. pneumophila strains. ⁵⁵Fe²⁺ was supplied to wild-type 130b (solid squares) and feoB mutant NU269 (open squares) bacteria, and then, after various incubation times, the amounts of radioactive iron associated with 10⁶ CFU were determined. Data show the means and standard deviations from triplicate samples. The mutant exhibited significantly less iron uptake at 1 min (Student's t test, P < 0.05) and at 5, 10, and 20 min (Student's t test, P < 0.01). These experiments were performed in duplicate with similar results.

FIG. 2.

Growth of L. pneumophila in standard and iron-limited BYE broth. Wild-type 130b (solid squares) and feoB mutant NU269 (open squares) were grown in BYE broth (A) with standard iron supplementation, (B) without iron supplementation, (C) without iron supplementation in 35 μM DIP, (D) with 45 μM DIP, or (E) with 60 μM DIP. In panel F, NU269 was grown either in BYE broth that lacked its standard iron supplement (solid line) or in BYE broth that lacked its standard iron supplement but contained 60 μM DIP and 80 μM FeCl₃ (dotted line). Data represent the means and standard deviations from triplicate samples from a representative experiment. In panels B to E, significant differences were noted between the wild-type and the feoB mutant between 5 and 11 h (Student's t test, P < 0.05). Experiments were repeated in triplicate with similar results.

FIG. 3.

Coculture of L. pneumophila with H. vermiformis amoebae. Wild-type (solid bars) and NU269 (open bars) strains were mixed at an MOI of 0.1 to amoebae that were treated or not with 25 or 50 μM DIP, and then numbers of bacteria within the cocultures were sampled at 0 and 48 h postinoculation. Data represent the mean and standard deviation from triplicate samples. The experiment was repeated in triplicate with similar results. Significant differences between the wild-type and mutant strains were observed in the 48-h cultures containing 50 μM DIP (Student's t test, P < 0.005).

FIG. 4.

Intracellular growth of L. pneumophila in U937 macrophages. (A) Wild-type 130b- (solid squares) and mutant NU269- (open squares) were used to infect U937 macrophages at an MOI of 1 and then, at various times postinoculation, the numbers of bacteria were assessed. Panel B shows complementation performed with NU269(pMR20) (vertical striped bars) or NU269(pMR21) (horizontal striped bars) containing plasmids encoding the entire feoB or feoAB genes, respectively. Complemented strains were compared to wild-type and mutant bacteria containing the empty vector pMMB207 (solid and open bars, respectively). Data represent the mean and standard deviation from triplicate samples. Experiments were performed in duplicate or triplicate with similar results. Significant differences were observed between the wild-type and mutant strains between 24 and 72 h postinoculation in panel A and between NU269(pMMB207) and NU269(pMR20) or NU269(pMR21) at 72 h postinoculation (Student's t test, P < 0.01).

FIG. 5.

Cytopathicity of L. pneumophila for U937 macrophages. Wild-type (solid squares) and NU269 (open squares) strains were used to infect U937 macrophages at an MOI of (A) 1 or (B) 10, and host cell viability was assessed at various times postinoculation Cytopathicity data are presented as the percentage of surviving macrophages in infected monolayers compared to the numbers in uninfected monolayers and represent the mean and standard deviation from six wells. Experiments were performed in triplicate with similar results. Significant differences were observed between the wild-type and mutant strains at 48 and 72 h postinoculation (Student's t test, P < 0.05).

FIG. 6.

Intracellular growth of L. pneumophila in iron-depleted U937 cells. The numbers of wild-type (solid bars) and NU269 (open bars) cells were determined in U937 cell monolayers cultured in medium containing 0, 25, or 50 μM DIP. Data represent means and standard deviations recovered from triplicate wells. The experiment was performed in triplicate with similar results. Significant differences were observed between the wild-type and mutant strains at 25 and 50 μM DIP (Student's t test, P < 0.05).

FIG. 7.

Pulmonary infections of A/J mice with L. pneumophila. Wild-type 130b and mutant NU269 strains in a 1:1 ratio were introduced into the lungs of A/J mice by intratracheal inoculation. At days 1 and 3 postinoculation, the ratio of 130b to Km^r NU269 in infected lungs was determined. Data are representative of actual values obtained per mouse (n = 3 to 5), and a solid bar represents the mean value.