Identification of Legionella pneumophila Genes Important for Infection of Amoebas by Signature-Tagged Mutagenesis

Abstract

Legionella pneumophila is a facultative intracellular gram-negative rod that causes pneumonia in humans. Free-living amoebas are thought to serve as a reservoir for Legionella infections. Signature-tagged mutagenesis was employed to identify Legionella pneumophila genes necessary for survival in the amoeba Acanthamoeba castellanii. Six mutant strains were defective in assays of invasion and intracellular growth. Four mutants also exhibited invasion and replication defects in Hartmannella vermiformis, an amoeba linked to hospital outbreaks of Legionella pneumonia. The six mutants also were tested in macrophages derived from peripheral blood mononuclear cells. Two mutants had intracellular replication defects, and two different strains entered cells less efficiently. Two transposon insertions were in known L. pneumophila genes, lspK and aroB. The other four were in novel genes. One gene has similarity to a cytochrome c-type biogenesis protein of Pseudomonas fluorescens. Another has similarity to a transcriptional activator regulating flagellar biosynthesis in Vibrio cholera. The third is similar to traA of Rhizobium sp. strain NGR234, which is involved in conjugal transfer of DNA. The fourth has no homology. By using survival in amoeba as a selection, we have isolated mutant strains with a range of phenotypes; and we have potentially identified new L. pneumophila virulence genes.

Genetic and Phenotypic Differences between Legionella pneumophila Strains

“…However, an E. coli mutant with a mutation in traJ, an F ϩ plasmid regulatory gene, is less invasive and less able to cause meningitis in the neonatal rat than is the wild type (2). In addition, an L. pneumophila mutant with a mutation in traA, involved in DNA processing during conjugation, does not replicate in amoebae and macrophages (44). Since this and other loci involved in type IV secretion affect L. pneumophila virulence (45,52,61), examination of a larger number of Legionella strains for the presence of this locus as well as evaluation of the effects of a specific tra1 mutant are mandated to clarify its role, if any, in pathogenesis.…”

The Type II Secretion System of Legionella pneumophila Dampens the MyD88 and Toll-Like Receptor 2 Signaling Pathway in Infected Human Macrophages

“…After washing of the pellet in this manner two more times, the mononuclear fraction was suspended in RPMI medium containing 15% human serum (catalog number H6914; Sigma), and the cell suspension was then added to tissue culture-treated, 24-well polystyrene plates (Falcon, Corning, NY) and incubated overnight at 37°C. To enrich for the adherent mononuclear cell population, as described previously (28,(96)(97)(98), the newly formed monolayers were washed three times to exclude nonadherent cells from the plate, and RPMI medium containing 15% human serum was then added back. The adherent mononuclear cells were then allowed to differentiate in human serum-containing medium for an additional 6 days, as previously described (96,98).…”

“…To enrich for the adherent mononuclear cell population, as described previously (28,(96)(97)(98), the newly formed monolayers were washed three times to exclude nonadherent cells from the plate, and RPMI medium containing 15% human serum was then added back. The adherent mononuclear cells were then allowed to differentiate in human serum-containing medium for an additional 6 days, as previously described (96,98).…”

Co-ordination of iron acquisition, iron porphyrin chelation and iron–protoporphyrin export via the cytochrome c biogenesis protein CcmC in Pseudomonas fluorescens

“…One obvious phenotype of mutants affected in the biogenesis of c-type cytochromes is their reduced capacity to grow under conditions of iron limitation (Gaballa et al, 1996(Gaballa et al, , 1998Yeoman et al, 1997;Pearce et al, 1998;Polesky et al, 2001;Baysse et al, 2002, Viswanathan et al, 2002. The decrease in haem production can explain some of the observed phenotypes of the ccmC mutant, such as reduced production of siderophores PVD and QB.…”