The Legionella pneumophila collagen-like protein mediates sedimentation, autoaggregation, and pathogen-phagocyte interactions

Abstract

Although only partially understood, multicellular behavior is relatively common in bacterial pathogens. Bacterial aggregates can resist various host defenses and colonize their environment more efficiently than planktonic cells. For the waterborne pathogen Legionella pneumophila, little is known about the roles of autoaggregation or the parameters which allow cell-cell interactions to occur. Here, we determined the endogenous and exogenous factors sufficient to allow autoaggregation to take place in L. pneumophila. We show that isolates from Legionella species which do not produce the Legionella collagen-like protein (Lcl) are deficient in autoaggregation. Targeted deletion of the Lcl-encoding gene (lpg2644) and the addition of Lcl ligands impair the autoaggregation of L. pneumophila. In addition, Lcl-induced autoaggregation requires divalent cations. Escherichia coli producing surface-exposed Lcl is able to autoaggregate and shows increased biofilm production. We also demonstrate that L. pneumophila infection of Acanthamoeba castellanii and Hartmanella vermiformis is potentiated under conditions which promote Lcl dependent autoaggregation. Overall, this study shows that L. pneumophila is capable of autoaggregating in a process that is mediated by Lcl in a divalent-cation-dependent manner. It also reveals that Lcl potentiates the ability of L. pneumophila to come in contact, attach, and infect amoebae.

FIG 1

L. pneumophila sediments more efficiently than strains of other Legionella species. Results of sedimentation assays with Lp02, an L. pneumophila isolate that cross-reacts with serogroups 1 to 9 (L. pneumophila sg1-9), and eight isolates from other Legionella species (A) and with L. pneumophila isolates from serogroups 1(Lp02), 1 to 9, 2, 3, 4, 5, 6, 8, 10, and 12 (B) after overnight static incubation at room temperature in deionized water with 10% BYE are shown.

FIG 2

Lcl is essential for L. pneumophila autoaggregation. (A and C) Sedimentation with the wild type (Lp02), an lpg2644 knockout (Lp02 Δlpg2644), Lp02 and Lp02 Δlpg2644 transformed with an empty vector (Lp02/pBH6119 and Lp02 Δlpg2644/pBH6119, respectively), Lp02 overexpressing Lcl (Lp02/plpg2644), and the complemented knockout (Lp02 Δlpg2644/plpg2644), measured macroscopically in test tubes (A) and with sedimentation kinetics (C). The OD₆₀₀ values in panel C have been normalized to Lp02. (B) Anti-Lcl immunoblot demonstrating different expression levels of Lcl in Lp02, Lp02 Δlpg2644, Lp02/pBH6119, Lp02 Δlpg2644/pBH6119, Lp02/plpg2644, and Lp02 Δlpg2644/plpg2644. IcdH was used as a loading control. (D) Sedimentation assays with Lp02 in the presence of 0.25 mg/ml GAGs. Mannose (0.25 mg/ml) and BSA (0.25 mg/ml) were used as negative controls.

FIG 3

Divalent cations are required for Lcl-dependent autoaggregation. (A to C) Sedimentation assays with Lp02 (A), Lp02 Δlpg2644 (B), and Lp02 Δlpg2644/plpg2644 (C) in deionized water (control) and deionized water supplemented with 500 μM CaCl₂, ZnCl₂,MgCl₂, or KCl. (D to F) Sedimentation kinetics of Lp02 with 500 μM, 100 μM, and 50 μM CaCl₂ (D), ZnCl₂ (E), or MgCl₂ (F). (G and H) Sedimentation assays with and without 50 μM DTPA in the presence of 100 μM CaCl₂ (G) and 100 μM ZnCl₂ (H) in deionized water.

FIG 4

Lcl secreted in the extracellular milieu is not sufficient to induce the aggregation of Lp02 Δlpg2644. (A) Anti-Lcl immunoblots of the supernatant (SN) and cell fraction (CF) from sedimentation assays with Lp02, Lp02 Δlpg2644, and mixed suspensions. (B and C) Fluorescence microscopy analysis of labeled autoaggregates after 5-h static incubations with Lp02/pGFP and Lp02 Δlpg2644/pRFP (B) and Lp02/pRFP and Lp02 Δlpg2644/pGFP (C) mixed suspensions in deionized water with 500 μM MgCl₂. (D and E) Relative quantifications of Lp02 and Lp02 Δlpg2644 in mixed sedimentation assays from panels B and C, respectively. Scale bars, 10 μm. *, statistically significant differences (P ≤ 0.001 by the two-tailed Student t test).

FIG 5

Lcl alone is sufficient to induce autoaggregation and biofilm production in E. coli and autoaggregation in L. feeleii. (A) Anti-Lcl immunoblot of E. coli containing the empty plasmid (E. coli/pTrc) and E. coli transformed with a plasmid containing lpg2644 under the control of a leaky IPTG-inducible promoter (E. coli/plcl). (B) Anti-Lcl immunofluorescence and FM 4-64 staining of live E. coli/pTrc and E. coli/plcl (top). The localization of Lcl (green lines) was determined by comparing its fluorescence intensity along the white lines with the inner membrane marker FM 4-64 (red lines). (C to E) Sedimentation of E. coli/pTrc and E. coli/plcl measured by test tube sedimentation assays (C), DIC microscopy of sediment (D), and sedimentation kinetics (E). (F) Biofilm production of E. coli/pTrc and E. coli/plcl as measured by crystal violet staining. *, statistically significant differences between the indicated condition and E. coli/plcl without IPTG; **, statistically significant differences with E. coli/plcl with IPTG (P ≤ 0.05 by the two-tailed Student t test). (G) Anti-Lcl immunoblot and sedimentation assays with L. feeleii transformed with the empty vector (Lf/pBH6119) and with the plpg2644 plasmid (Lf/plpg2644). IPTG was added to a final concentration of 1 mM when indicated for sedimentation experiments and immunoblotting. For biofilm assays, IPTG was added to a final concentration of 5 mM. Scale bars, 0.6 μm and 10 μm for panels B and D, respectively.

FIG 6

Lcl mediates the attachment of L. pneumophila to Acanthamoeba castellanii. (A) Infection of A. castellanii by Lp02/pGFP, Lp02 Δlpg2644/pGFP, and Lp02 Δlpg2644 clpg2644 (chromosomal insertion of gene lpg2644)/pGFP after centrifugation in deionized water in the absence or presence of 500 μM MgCl₂ as measured by flow cytometry. *, statistically significant differences with Lp02 Δlpg2644 clpg2644 without MgCl₂ by the two-tailed Student t test (P ≤ 0.01). (B and C) Infection of A. castellanii measured by flow cytometry in the presence of 2.5 × 10⁻³ to 2.5 × 10⁻⁵ mg/ml fucoidan after centrifugation in deionized water (B) and in deionized water with 500 μM MgCl₂ (C) with Lp02/pGFP (white squares), Lp02 Δlpg2644/pGFP (gray circles), and Lp02 Δlpg2644 clpg2644/pGFP (black triangles). (D) Intracellular growth of Lp02/pGFP (black diamonds) and Lp02 Δlpg2644/pGFP (gray squares) in A. castellanii.

FIG 7

Lcl-dependent autoaggregation potentiates the internalization of L. pneumophila in A. castellanii. (A) Infection of A. castellanii by Lp02/pGFP, Lp02 Δlpg2644/pGFP, and Lp02 Δlpg2644 clpg2644/pGFP in the absence or presence of 500 μM MgCl₂ as measured by flow cytometry. (B and C) Infection of A. castellanii as measured by flow cytometry in the presence of 2.5 × 10⁻³ to 2.5 × 10⁻⁵ mg/ml fucoidan in deionized water with the addition of 500 μM MgCl₂ (B) and in deionized water alone (C) with Lp02/pGFP (white squares), Lp02 Δlpg2644/pGFP (gray circles), and Lp02 Δlpg2644 clpg2644/pGFP (black triangles). (D) Fluorescence microscopy of Lp02 Δlpg2644/pGFP in the absence or presence of anti-Legionella pneumophila serogroup1 (anti-Lp1) agglutinating antibodies (1:1,000). (E) Infection of A. castellanii with isolated planktonic Lp02 Δlpg2644/pGFP and artificially aggregated Lp02 Δlpg2644/pGFP (without and with anti-Lp1 antibodies, respectively). Scale bar, 10 μm. * and **, statistically significant differences between the indicated strains (A) and compared to assays with 0 mg/ml fucoidan (B), respectively, by the two-tailed Student t test (P ≤ 0.01).

FIG 8

Lcl-dependent autoaggregation increases the number of L. pneumophila bacteria per infected A. castellanii cell. Visualization of A. castellanii infection by Lp02/pGFP, Lp02 Δlpg2644/pGFP, and Lp02 Δlpg2644 clpg2644/pGFP in the absence or presence of 500 μM MgCl₂ in deionized water by fluorescence microscopy is shown. Scale bars, 10 μm.