Genetic approaches to study Legionella pneumophila pathogenicity

Ott, Manfred

doi:10.1016/0168-6445(94)90004-3

Cited by 8 publications

(10 citation statements)

References 39 publications

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“…Similarly, the pathogenesis of legionellosis is absolutely dependent on the ability of the microbe to replicate within alveolar macrophages (28). To date, research has identified only a handful of the factors required for optimal intracellular infection (14,39). For example, the major outer membrane porin OmpS mediates attachment to CR1 and CR3 on monocytes (2,27).…”

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Legionella pneumophila mutants that are defective for iron acquisition and assimilation and intracellular infection

1996

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Legionella pneumophila, a parasite of macrophages and protozoa, requires iron for optimal extracellular and intracellular growth. However, its mechanisms of iron acquisition remain uncharacterized. Using mini-Tn10 mutagenesis, we isolated 17 unique L. pneumophila strains which appeared to be defective for iron acquisition and assimilation. Eleven of these mutants were both sensitive to the iron chelator ethylenediamine di(ohydroxyphenylacetic acid) and resistant to streptonigrin, an antibiotic whose lethal effect requires high levels of intracellular iron. Six mutants were also defective for the infection of macrophage-like U937 cells. Although none were altered in entry, mutants generally exhibited prolonged lag phases and in some cases replicated at slower rates. Overall, the reduced recoveries of mutants, relative to that of the wild type, ranged from 3-to 1,000-fold. Strain NU216, the mutant displaying the most severe lag phase and the slowest rate of replication, was studied further. Importantly, within U937 cells, NU216 was approximately 100-fold more sensitive than the wild type was to treatment with the Fe 3؉ chelator deferoxamine, indicating that it is defective for intracellular iron acquisition and assimilation. Furthermore, this strain was unable to mediate any cytopathic effect and was impaired for infectivity of an amoebal host. Taken together, the isolation of these mutants offers genetic proof that iron acquisition and assimilation are critical for intracellular infection by L. pneumophila.

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Legionella pneumophila mutants that are defective for iron acquisition and assimilation and intracellular infection

1996

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“…The relationship between Legionella pneumophila, the agent of Legionnaires' disease, and hemin has received very little attention. The gram-negative L. pneumophila is a facultative intracellular parasite of both freshwater protozoa and human alveolar macrophages (10,20,24,34,50,60). Although ferric/ ferrous iron clearly plays a critical role in extra-and intracellular Legionella growth (7,23,26,57,58), the role of hemin is unclear.…”

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A Legionella pneumophila gene that promotes hemin binding

1996

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The ability to bind and utilize hemin is a trait common to many human pathogens. Nevertheless, the relationship between Legionella pneumophila, the agent of Legionnaires' disease, and hemin has received little attention. Thus, we explored the capacity of a virulent, serogroup 1 strain of L. pneumophila to bind hemin and use it as an iron source. Hemin, but not protoporphyrin IX, restored bacterial growth in iron-limiting media, indicating that it can serve as an iron source for L. pneumophila. In support of this idea, we observed that wildtype legionellae were able to bind 50 to 60% of added hemin, a binding capacity that was comparable to those of other pathogens. To begin to identify proteins involved in hemin acquisition, we identified a Legionella locus that conferred hemin binding upon Escherichia coli. Subcloning and nucleotide sequence analysis determined that a single open reading frame, which was designated hbp for hemin-binding promotion, was responsible for this binding activity. The hbp gene was predicted to encode a secreted, 15.5-kDa protein. To ascertain the importance of this gene in L. pneumophila biology, we used allelic exchange to construct an hbp mutant. Importantly, the mutant displayed a 42% reduction in hemin binding, confirming that hbp potentiates hemin acquisition by L. pneumophila. However, the strain was unaltered in its ability to grow within macrophage-like cells and freshwater amoebae, indicating that hbp is not required for intracellular infection. Despite this, Southern hybridization analysis and database searches demonstrated that hbp is nearly exclusive to the L. pneumophila species. A number of pathogens, including both intra-and extracellular parasites, have evolved mechanisms for interacting with hemin and heme-containing compounds (52, 66). This interaction can serve several functions. First, and perhaps most often, it serves as a means for iron acquisition. Strains of Aeromonas spp.,

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“…The mechanisms by which Legionella pneumophila, the agent of Legionnaires' disease, acquires iron from its environment have yet to be fully characterized (1,8,10,11,13,15). In particular, the suggestion of the existence of a Legionella siderophore has been controversial.…”

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Absence of siderophore-like activity in Legionella pneumophila supernatants

Liles

Cianciotto

1996

Infect Immun

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Conflicting reports have been given as to the existence of a Legionella pneumophila siderophore. Hence, we rigorously examined the reported siderophore-like activity using the chrome azurol S indicator. Although chrome azurol S reactivity was detected in supernatants, control experiments indicated that it was due to cysteine in the media. When bacteria were cultured in media without cysteine, no siderophores were detected.

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Genetic approaches to study Legionella pneumophila pathogenicity

Cited by 8 publications

References 39 publications

Legionella pneumophila mutants that are defective for iron acquisition and assimilation and intracellular infection

Legionella pneumophila mutants that are defective for iron acquisition and assimilation and intracellular infection

A Legionella pneumophila gene that promotes hemin binding

Absence of siderophore-like activity in Legionella pneumophila supernatants

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