Flagellum of Legionella pneumophila positively affects the early phase of infection of eukaryotic host cells

Abstract

Legionella pneumophila, the etiologic agent of Legionnaires' disease, contains a single, monopolar flagellum which is composed of one major subunit, the FlaA protein. To evaluate the role of the flagellum in the pathogenesis and ecology of Legionella, the flaA gene of L. pneumophila Corby was mutagenized by introduction of a kanamycin resistance cassette. Immunoblots with antiflagellin-specific polyclonal antiserum, electron microscopy, and motility assays confirmed that the specific flagellar mutant L. pneumophila Corby KH3 was nonflagellated. The redelivery of the intact flaA gene into the chromosome (L. pneumophila Corby CD10) completely restored flagellation and motility. Coculture studies showed that the invasion efficiency of the flaA mutant was moderately reduced in amoebae and severely reduced in HL-60 cells. In contrast, adhesion and the intracellular rate of replication remained unaffected. Taking these results together, we have demonstrated that the flagellum of L. pneumophila positively affects the establishment of infection by facilitating the encounter of the host cell as well as by enhancing the invasion capacity.

FIG. 1

flaA region of L. pneumophila. Bars represent open reading frames; flaA is the flagellin-encoding gene, orfG is the homolog to flaG of P. aeruginosa, and accD is the homolog to accD of E. coli. Deduced promoters (P), terminators (T), binding sites for primers (arrows), and the flaA-specific probe are indicated. Restriction sites: Ec, EcoRI; Hd, HindIII; Ps, PstI; Xb, XbaI.

FIG. 2

Electron micrographs of the flagellated wild-type strain (A), the nonflagellated mutant KH3 (B), and the complemented mutant strain CD10 (C). Bacteria were grown to stationary phase, suspended in H₂O, applied to Formvar-coated copper grids, shadowed with 2% uranyl acetate, and examined with a Zeiss EM10 transmission electron microscope. Bars, 0.5 μm.

FIG. 3

Invasion efficiency of L. pneumophila Corby wild type, the flaA mutant KH3, and the complemented mutant CD10 into A. castellanii cells (A) and HL-60 cells (B). Adherent host cells were incubated with legionellae at an MOI of 10 for different time intervals and treated with gentamicin (80 μg/ml) for 1 h, and the CFU were determined by plating on ABCYE agar plates. Error bars indicate the standard deviations obtained from three independent experiments.

FIG. 4

Multiplication of L. pneumophila Corby wild type, the flaA mutant KH3, and the complemented mutant CD10 in A. castellanii and HL-60 cells. (A) A. castellanii cells (2 × 10⁵/ml) were infected with 2 × 10³ bacteria, and the CFU per well were determined daily in duplicate by plating on ABCYE plates. (B) Differentiated HL-60 cells (2 × 10⁵ cells/ml) were infected and treated as described for A. castellanii. Error bars indicate the standard deviations obtained from three independent experiments.