Icm/dot-independent entry of Legionella pneumophila into amoeba and macrophage hosts - PubMed (original) (raw)

Icm/dot-independent entry of Legionella pneumophila into amoeba and macrophage hosts

Purnima Bandyopadhyay et al. Infect Immun. 2004 Aug.

Abstract

Legionella pneumophila, the causative agent of Legionnaires' disease, expresses a type IVB secretion apparatus that translocates bacterial proteins into amoeba and macrophage hosts. When stationary-phase cultures are used to infect hosts, the type IVB apparatus encoded by the icm/dot genes is required for entry, delay of phagosome-lysosome fusion, and intracellular multiplication within host cells. Null mutants with mutations in icm/dot genes are defective in these phenotypes. Here a new model is described in which hosts are infected with stationary-phase cultures that have been incubated overnight in pH 6.5 buffer. This model is called Ers treatment because it enhances the resistance to acid, hydrogen peroxide, and antibiotic stress beyond that of stationary-phase cultures. Following Ers treatment entry into amoeba and macrophage hosts does not require dotA, which is essential for Legionella virulence phenotypes when hosts are infected with stationary-phase cultures, dotB, icmF, icmV, or icmX. Defective host entry is also suppressed for null mutants with mutations in the KatA and KatB catalase-peroxidase enzymes, which are required for proper intracellular growth in amoeba and macrophage hosts. Ers treatment-induced suppression of defective entry is not associated with increased bacterial adhesion to host cells or with morphological changes in the bacterial envelope but is dependent on protein expression during Ers treatment. By using proteomic analysis, Ers treatment was shown to induce a protein predicted to contain eight tetratricopeptide repeats, a motif previously implicated in enhanced entry of L. pneumophila. Characterization of Ers treatment-dependent changes in expression is proposed as an avenue for identifying icm/dot-independent factors that function in the entry of Legionella into amoeba and macrophage hosts.

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Figures

FIG. 1.

FIG. 1.

Effect of growth conditions on stress resistance of wild-type L. pneumophila. Exponential-phase (Exp), stationary-phase (Stat), or Ers-treated stationary-phase (Ers) cultures or bacteria recovered from A. castellanii following intracellular multiplication (Acca) were challenged with pH 3 (A) or 1 mM H2O2 (B) and titrated after different times (mean ± standard deviation). The points plotted on the x axis are the values when there were no CFU.

FIG. 2.

FIG. 2.

Effect of growth conditions on the stress resistance of the katA null mutant. Exponential-phase (Exp), stationary-phase (Stat), or Ers-treated stationary-phase (Ers) cultures of the katA::Gmr null mutant or bacteria recovered from A. castellanii following intracellular multiplication (Acca) were challenged with pH 3 or 1 mM H2O2 and titrated after different times (mean ± standard deviation). The points on the x axis are the values when there were no CFU.

FIG. 3.

FIG. 3.

Effect of growth conditions on acid resistance of icm null mutants. Acid challenge experiments were performed with icmF (A), icmP (B), and icmS (C) null mutants. Exponential-phase (Exp), stationary-phase (Stat), or Ers-treated stationary-phase (Ers) cultures were challenged with pH 3 and titrated after different times (mean ± standard deviation). Ers compl, Ers-treated stationary-phase culture of icmS mutant containing an icmS+ complementing plasmid. The points on the x axis are the values when there were no CFU.

FIG. 4.

FIG. 4.

Ers treatment reverses the defect in entry of katA, katB, and icm/dot mutants into A. castellanii. A. castellanii was infected at an MOI of 3 to 5 with wild-type L. pneumophila strain JR32 or a mutant strain grown to the stationary phase (Stat) or after Ers treatment of stationary-phase cultures (Ers). Internalized bacteria were quantified by the fluorescence microscopy method (mean ± standard deviation). (A) Strains containing a GFP-expressing plasmid. (B) Strains containing the pJN105-hygro-GFP plasmid and a second plasmid, pMMB207αB-Km14 for wild-type strain JR32 or a complementing plasmid for mutant strains. P values were determined by one-sided t tests by comparing entry of stationary-phase and Ers-treated cultures of the same strain (A) or by comparing a mutant stationary-phase or Ers-treated culture with a similar wild-type culture (B) (two asterisks, P < 0.005; one asterisk, _P_ < 0.02; no asterisk, _P_ > 0.05).

FIG. 5.

FIG. 5.

Ers treatment reverses the defect in entry of katA, katB, and icm/dot mutants into HL60-derived macrophages. The entry of wild-type strain JR32 or a mutant strain into macrophages derived from HL60 monocytes (MOI, 100) was determined as described for entry into A. castellanii in the legend of Fig. 4. P values were determined by one-sided t tests by comparing entry of stationary-phase (Stat) and Ers-treated (Ers) cultures of the same strain (one asterisk, P < 0.05; two asterisks, _P_ < 0.005; no asterisk, _P_ > 0.05).

FIG. 6.

FIG. 6.

Chloramphenicol inhibits Ers-enhanced entry into A. castellanii for icmF and katA mutants. Entry into A. castellanii was determined for wild-type strain JR32 and icmF and katA mutant strains as described in the legend to Fig. 4 with stationary-phase cultures (Stat), Ers-treated stationary-phase cultures (Ers), and Ers-treated stationary-phase cultures in the presence of chloramphenicol (ErsCm and Ers + Cm).

FIG. 7.

FIG. 7.

Two-dimensional gel electrophoresis analysis of L. pneumophila katA mutant. Isoelectric focusing (horizontal) and SDS-polyacrylamide gel electrophoresis (vertical) were performed with sonic extracts from stationary-phase (A) and Ers-treated stationary-phase (B) cultures. The regions shown are ∼20% of each gel surrounding ORF lpg2222 (arrows) (calculated molecular weight, 41,400; calculated pI 5.35). The amount of protein loaded in panel A was greater than the amount loaded in panel B.

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