Chemorepulsion from the Quorum Signal Autoinducer-2 Promotes Helicobacter pylori Biofilm Dispersal
- Jeneva K. Andersona,
- Julie Y. Huangb,
- Christopher Wredena,
- Emily Goers Sweeneya,
- John Goersc,
- S. James Remingtond,
- Karen Guillemina
- aInstitute of Molecular Biology, University of Oregon, Eugene, Oregon, USA
- bDepartment of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA
- cDepartment of Chemistry, California Polytechnic State University, San Luis Obispo, California, USA
- dDepartment of Physics, University of Oregon, Eugene, Oregon, USA
- Vanessa Sperandio, Editor
- Address correspondence to Karen Guillemin, guillemin{at}molbio.uoregon.edu.
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FIG 1Endogenous AI-2 production and chemotaxis alter biofilm organization. (A to E) Epifluorescence images of wild-type and mutant G27 H. pylori biofilms grown on glass slides for 5 days. Cells were fixed and stained with DAPI (white). Bar, 100 µm. (F) Lacunarity scores for biofilm images. Each dot represents the lacunarity score for a single epifluorescence biofilm image postthresholding, with corresponding means and standard deviations. (G) Schematic of glass frit biofilm assay with crystal violet analysis and examples of percentages of cells in the biofilm calculation. (H) Effect of AI-2 production and chemotaxis on adherence of cells in biofilms on glass frit after 2 days, as quantified by the crystal violet assay. Error bars represent standard errors of the means of the results of a minimum of three experiments for each strain. Daggers indicate a significant (P < 0.05) difference from the wild-type results determined using Student’s t test (F) or one-way analysis of variance (H).
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FIG 2AI-2 chemotaxis requires two periplasmic binding proteins, AibA and AibB, that bind AI-2 independently. (A) Chemotaxis response of wild-type and ΔHPG27_277 (ΔaibA) and ΔHPG27_431 (ΔaibB) mutant H. pylori to brucella broth (BB10), 100 mM HCl, and 100 µM synthetic DPD (AI-2). Representative wet-mount images of bacterial cells (white dots) are shown. Formation of a barrier (marked by white arrows) indicates a chemotactic response. Bar, 200 µm. (B) In vitro AI-2 binding assay performed with purified proteins using a V. harveyi bioluminescence readout. Percentages of relative luminescence units (RLU) were normalized to an AI-2 positive control in each independent experiment. Error bars represent standard errors of the means of the results of comparisons of experimental data. Daggers indicate a significant (P < 0.05) difference from the buffer negative-control results determined using Student’s t test.
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FIG 3Chemorepulsion from AI-2 is necessary for biofilm organization. (A to G) Epifluorescence images of AI-2 chemotaxis-defective mutants and complemented-strain biofilms grown on glass slides for 5 days. Cells were fixed and stained with DAPI (white). (H) Lacunarity scores for biofilm images. Each point represents the lacunarity score for a single epifluorescence biofilm image postthresholding, with corresponding means and standard deviations. (I) Effect of AI-2 chemotaxis disruption on adherence of cells in biofilms on glass frit after 2 days, as quantified by the crystal violet assay. Error bars represent standard errors of the means of the results of a minimum of 3 experiments for each strain. Daggers indicate a significant (P < 0.05) difference from the wild-type results determined using Student’s t test (H) or one-way analysis of variance (I).
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FIG 4Chemorepulsion from AI-2 is sufficient to decrease adherence in biofilms. (A) Change in adherence of strains in biofilm with addition of exogenous AI-2 (37 nM) at the time of inoculation. Daggers indicate a significant (P < 0.05) difference from 0 (no change) determined using Student’s t test. (B) Fraction changes in biofilm size (grey bars) and numbers of planktonic cells (white bars) measured 72 h after the initiation of the biofilms and 24 h after rinsing and exchange with fresh media containing 37 nM exogenous AI-2. Daggers indicate a significant (P < 0.05) difference from a value of 1 (no change) determined using Student’s t test. Box plots show means and standard deviations of the results of a minimum of three experiments for each strain.
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FIG 5Disruption of either AI-2 production or AI-2 chemotaxis results in larger microcolonies on cultured epithelial cell monolayers. (A to C) Representative images of WT, ΔluxS, and ΔtlpB H. pylori microcolonies on polarized MDCK monolayers at 3 days postinfection. H. pylori cells are visualized in red, nuclei in blue, and cell junctions in green. Bar, 10 µm. (D) Estimated numbers of attached bacteria on the MDCK epithelial cell layer at 5 min and 3 days postinfection for all strains. Each point represents the estimated number of bacteria averaged over multiple images per transwell. Error bars represent standard errors of the means. Daggers represent significant increases in numbers of attached bacteria from 5 min to 3 days of attachment for each strain. (E) Plot of the surface area covered by contiguous clusters of bacterial cells (microcolonies) for each strain at 5 min (predominantly isolated single cells) and 3 days postinoculation. Each dot represents the average area (in pixels) of all the microcolonies in an image. Three images were taken per transwell, with three replicate transwells per experiment. Error bars represent standard errors of the means. Daggers represent significant (P < 0.05) increases of pixel sizes compared to the WT results through the use of one-way analysis of variance.
- Copyright © 2015 Anderson et al.
