Essential roles and regulation of the Legionella pneumophila collagen-like adhesin during biofilm formation

Abstract

Legionellosis is mostly caused by Legionella pneumophila (Lp) and is defined by a severe respiratory illness with a case fatality rate ranging from 5 to 80%. In a previous study, we showed that a glycosaminoglycan (GAG)-binding adhesin of Lp, named Lcl, is produced during legionellosis and is unique to the L. pneumophila species. Importantly, a mutant depleted in Lcl (Δlpg2644) is impaired in adhesion to GAGs and epithelial cells and in biofilm formation. Here, we examine the molecular function(s) of Lcl and the transcriptional regulation of its encoding gene during different stages of the biofilm development. We show that the collagen repeats and the C-terminal domains of Lcl are crucial for the production of biofilm. We present evidence that Lcl is involved in the early step of surface attachment but also in intercellular interactions. Furthermore, we address the relationship between Lcl gene regulation during biofilm formation and quorum sensing (QS). In a static biofilm assay, we show that Lcl is differentially regulated during growth phases and biofilm formation. Moreover, we show that the transcriptional regulation of lpg2644, mediated by a prototype of QS signaling homoserine lactone (3OC12-HSL), may play a role during the biofilm development. Thus, transcriptional down-regulation of lpg2644 may facilitate the dispersion of Lp to reinitiate biofilm colonization on a distal surface.

Figure 1. Lcl is expressed at the cell surface of L. pneumophila and shows a cluster distribution.

Localization of Lcl on the cell surface. (A) GFP-Lp02 (left) and GFP-Lp02 Δlpg2644 (right) cells were immuno-labelled using anti- Lcl antibodies followed by alexa 555 (red) conjugated secondary antibodies. Micrographs shown are merged images of 0.2 µm confocal z-stacks. Scale bars, 0.5 µm. (B) Surface localization of Lcl (red) as indicated by its co-localization with membrane labelling anti-Lp1 antibodies (top) and FM 4–64 dye (bottom). Images are confocal xy plane following deconvolution. Scale bars, 0.5 µm. (C) The localization of Lcl (red line) was determined by comparing its fluorescence intensity along the white lines from B, with cytoplasmic GFP (green line) and the outer and inner membrane markers anti-Lp1 (blue line, left) and FM 4–64 (blue line, right), respectively.

Figure 2. The Collagen-like tandem repeat and the C-terminal domains of Lcl are essential for biofilm formation.

(A) Anti-Lcl immunoblot analyses against Lp02 and Lp02 Δlpg2644 lysates untransformed and transformed with plpg2644, plpg2644 Δrepeat and plpg2644 ΔC-terminal. The positions of the molecular size markers are indicated on the left of the blot in kDa. (B) Static biofilm assays with Lp02 and Lp02 Δlpg2644 producing recombinant variants of Lcl (plpg2644 Δrepeat and plpg2644 ΔC-terminal) and full length recombinant Lcl (plpg2644). * Two-tailed Student’s t-test P-value <0.001 versus Lp02).

Figure 3. Role of Lcl during the initial attachment step of biofilm formation.

The attachment of Lp02, Lp02 pBH6119, Lp02 Δlpg2644, and Lp02 Δlpg2644 plpg2644 to the wells of a microtiter plate was measured by (A) qPCR and (B and C) fluorescence microscopy. (A) The binding of Lp02 Δlpg2644 was significantly reduced compared to the other strains. Values are means+/−s.d. from three individual experiments. * Two-tailed Student’s t-test P-value <0.001 Lp02 Δlpg2644 vs. all other strains. (B) Comparative analysis of the attachment of SYTO 62 stained Legionella strains attached to glass cover slips. (C) Among 10 representative microscopy fields, the average attachment of Lp02, Lp02 pBH6119 and complemented mutant were similar whereas Lp02 Δlpg2644 showed a marked decrease attachment to surface. * Two-tailed Student’s t-test P-value <0.001.

Figure 4. Adherence and persistence of L. pneumophila in a continuous-flow chamber system.

Fluorescence micrographs of GFP-expressing L.pneumophila strains Lp02, Lp02 Δlpg2644 and chromosomal complemented mutant (Lp02 Δlpg2644/clpg2644) in a continuous-flow chamber system operated at 5.5 µl/mn with BYE medium (magnification 100 X). During the initial attachment (0 h), Lp02 and Lp02 Δlpg2644/clpg2644 strains formed a monolayer on abiotic surface whereas Lp02 Δlpg2644 was unable to efficiently attach to the flow chambers and detached in a flow dependent manner (2 h to 48 h). Data are representative of three independent replicates. Scale bar, 5 µm.

Figure 5. Representative 3D composite images of mixed culture biofilms.

Total bacteria were labelled with SYTO62 and one strain of each 1∶1 mixture was labelled with GFP. Confocal scanning laser microscopy (CSLM) micrographs show the distribution of L. pneumophila within static biofilms using (A) GFP- Lp02 Δlpg2644/Lp02, (B) GFP-Lp02/Lp02, (C) GFP- Lp02 Δlpg2644/Lp02 Δlpg2644, (D) GFP- Lp02/Lp02 Δlpg2644. (Magnification 100 X).

Figure 6. Transcriptional regulation of lpg2644 and synthesis of Lcl in broth cultures, biofilm and in presence of 3OC₁₂-HSL homoserine lactone.

Amounts of lpg2644 transcripts and synthesis of Lcl were respectively measured by (A) qRT-PCR and (B) anti-Lcl immunoblot analysis of cell pellet (c) supernatant (s) and sessile cells/matrix (sm) fractions in Lp02 harvested from exponential (E), post-exponential (PE), mid-stationary (MS), late stationary (LS) broth cultures and biofilms (2 and 6 days old). Quantitative RT-PCR values are means+/−s.d. from three individual experiments. # two-tailed Student’s t-test P-value ≤0.01 versus MS phase, * two-tailed Student’s t-test P-value ≤0.001 versus MS phase. (C) Effect of 3OC12-HSL homoserine lactone on lpg2644 transcription and (D) Lcl synthesis. Lp02 was grown in absence or in presence of 3OC12-HSL (10 µM) for 24 h. Transcriptional level of lpg2644 was determined by qRT-PCR analysis. Data are representative of 3 independent experiments.* Student’s t-test P-value ≤0,001 versus untreated Lp02. (D) Synthesis of Lcl was estimated by anti-Lcl immunoblot of cell pellets (c) and supernatant fractions (s) of Lp02 and Lp02 Δlpg2644 plpg2644. The positions of the molecular size markers are indicated on the left of the blot in kDa.

Figure 7. The quorum sensing dependent regulation of Lcl plays a key-role in the control of L. pneumophila biofilm development.

Flow diagrams indicating the experimental stages when Las QS 3OC12-HSL auto-inducer (HSL) was used and the corresponding static biofilm assays (A). L. pneumophila strains Lp02, Lp02 pBH6119 (empty plasmid) and Lp02 Δlpg2644 plpg2644 were exposed to Las QS 3OC12-HSL during 48 h (B), the attachment phase (0–2 h) (C) and biofilm formation (2–48 h) (D). * P value <0.001 no HSL vs+HSL.