Research ArticleMICROBIOLOGY

Social amoebae establish a protective interface with their bacterial associates by lectin agglutination

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Science Advances  24 Jul 2019:
Vol. 5, no. 7, eaav4367
DOI: 10.1126/sciadv.aav4367
  • Fig. 1 CadA promotes amoebal plaque formation on dense bacterial lawns.

    The viability of D. discoideum amoebae was assessed by plating them clonally on growing lawns of K. pneumoniae (K. p.). (A) Plaques resulting from equal numbers of AX4 (top) and cadA mutant (bottom) amoebae plated on K. pneumoniae on SM agar plates (left) or SM agar plates with the nutrient components diluted fivefold (SM/5) (right). (B) Amoebal viability as assessed by plating efficiency of AX4 and cadA (y axis) (left) on K. pneumoniae lawns growing on SM agar plates in which the nutrient components were diluted 1.5- to 5-fold, as indicted. Number of K. pneumoniae cells in representative samples of these platings when grown without amoeba (red line; y axis) (right). Lower and upper bounds of the box plots represent the first and third quartile, respectively, while the whiskers represent maximum and minimum values; the centerline is the median, and the “x” represents mean of triplicate samples for three biological replicates [Kruskal-Wallis one-way analysis of variance (ANOVA) and ad hoc pairwise Wilcoxon rank sum test]. ns, not significant; CFU, colony-forming units. (C) Plating efficiency of AX4 and cadA amoebae on SM agar in the presence of exogenous CadA protein. Statistical significance was observed between cadA and cadA amoebae plated with exogenous CadA protein produced in E. coli [recombinant CadA protein (rCadA)], where ***P < 0.001. Box plots represent data from triplicate samples of three biological replicates where statistical significance was estimated by the Wilcoxon rank sum test.

  • Fig. 2 CadA prevents mixing of bacteria and amoebae across plaque borders.

    (A) Growing edge localization of CadA protein. AX4 and cadA plaques growing on K. pneumoniae on SM/5 agar were extracted and electroblotted onto nitrocellulose membranes. CadA protein was visualized using anti-CadA monoclonal antibody MLJ11. Scale bars, 1 cm. The inset shows the result of 2 μg of purified CadA spotted on top of a cadA plaque before blotting. (B) AX4 and cadA amoebae were plated with K. pneumoniae onto SM agar plates and incubated overnight. Plates were harvested and amoebal (AP) and bacterial (BP) pellets were separated from the supernatant (S) by successive centrifugation and probed for the presence of CadA by Western blot, with pure CadA as a control. AX4 cells were also recovered from growth media (HL5) after overnight incubation and processed such as the plated cells into amoebal pellet and supernatant fractions. Anti-actin monoclonal antibodies (mAb) were used to detect an amoebae-associated protein in the same samples. (C) Representative images of AX4 (top) and cadA (bottom) plaques grown on K. pneumoniae on SM/2.5 agar. AX4 plaques form roughly circular plaques with defined edges (top left), while cadA plaques appear erose with satellite plaques (bottom left). No satellite colonies are observed when cadA amoebae are grown with exogenously added recombinant CadA protein. Representative images of H2B-mCherry–expressing AX4 amoebae, showing a defined plaque edge with few cells outside of the plaque, and H2B-mCherry–expressing cadA amoebae migrating away from the plaque edge into the bacterial lawn. The table shows the numbers of cells observed outside of plaques. (D) Representative images of the edge of AX4 (top) and cadA (bottom) plaques on K. pneumoniae on SM/2.5 agar, stained with LIVE/DEAD BacLight stain and visualized by differential interference contrast (DIC) or fluorescence microscopy. SYTO9 (green) labels live bacteria and propidium iodide (PI; red) labels dead bacteria. In the composite images of the plaque edges (far right), live bacteria are seen within the growing edge of the cadA plaque, while the AX4 has predominantly dead bacteria within the border of the plaque. Scale bars, 100 μm.

  • Fig. 3 CadA binds and agglutinates bacteria.

    (A) Increasing concentrations of recombinant CadA (1.25 to 74 μg) were incubated with 109 K. pneumoniae in 600 μl for 60 min. After bacteria were removed by centrifugation, unbound CadA was used to determine the amount of CadA bound to bacteria. Saturation binding of CadA suggests an association constant (Ka) of 1.0 × 107 M−1 and 7.5 × 105 binding sites per bacterium. Line shown is best fit for specific binding with Hill coefficient with the Bmax and Hill coefficient calculated to be 15.58 and 1.16, respectively. (B) K. pneumoniae (109) were incubated with recombinant CadA (8 μg) for 60 min, with increasing concentrations (10 to 300 mM) of d-glucose (white), d-glucosamine (black), and d-galactose (gray). Unbound CadA was quantified and used to calculate the amount of CadA bound to the bacteria. Data represent the means ± SD values of three technical replicates of three independent biological replicates, and the presence of glucosamine and galactose shows significantly reduced CadA binding relative to glucose (Wilcoxon rank sum test, ***P < 0.001). (C) K. pneumoniae (109) bacteria were incubated with buffer (top) or recombinant CadA protein (0.66 mg/ml) (bottom), with glucose (middle) or galactose (right) for 1 hour and visualized by fluorescence microscopy. Scale bars, 100 μm. GFP, green fluorescent protein.

  • Fig. 4 Species-specific restriction of bacterial colony expansion by CadA correlates with CadA bacterial agglutination.

    (A) A fraction from a mock protein purification from E. coli, recombinant CadA (10 μg), or BSA (10 μg) was spotted onto SM agar and allowed to dry. Overnight cultures of K. pneumoniae (top left) and other species of bacteria, as indicated, were spotted adjacent but not touching the CadA spot. Dashed circles represent the original deposition of the protein solutions (or mock fraction), and the bars indicate the diameter of the original bacterial spot. (B) The bacterial species in (A) were incubated with recombinant CadA protein (0.66 mg/ml) for 1 hour and visualized by fluorescence microscopy after labeling the bacteria with the cell-permeable dye SYTO9. Scale bars, 100 μm.

  • Fig. 5 Altered amoebal predation on CadA-agglutinated bacteria.

    H2B-mCherry–expressing AX4 amoebae (A) or cadA mutant amoebae (B) were mixed with K. pneumoniae clumped by CadA (A and B, top) or by mechanically by centrifugation (A and B, bottom) under agar on glass bottom plates. White arrows highlight amoebae traveling through a clump, while orange arrows highlight amoebae traveling away from a clump. Individual cells were imaged and tracked for 3 hours using Nikon Eclipse Ti. t, minutes. Scale bars, 100 μm. (C) Violin plots of the chemotactic index of individual AX4 or cadA amoebae for clumps of K. pneumoniae that were mechanically produced, CadA-induced, or CadA-induced with 100 μM folic acid added to the buffer before plating.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaav4367/DC1

    Fig. S1. Growth of cadA mutant amoebae on dense lawns of bacteria.

    Fig. S2. Elevated ROS in cadA mutant amoebae feeding on bacteria.

    Fig. S3. CadA agglutinates K. pneumoniae and protects them from killing.

    Fig. S4. Differential amoebal movement toward CadA clumped bacteria.

    Movie S1. Amoebal predation on CadA-agglutinated bacteria.

    Movie S2. Amoebal predation on mechanically generated bacterial clumps.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Growth of cadA mutant amoebae on dense lawns of bacteria.
    • Fig. S2. Elevated ROS in cadA mutant amoebae feeding on bacteria.
    • Fig. S3. CadA agglutinates K. pneumoniae and protects them from killing.
    • Fig. S4. Differential amoebal movement toward CadA clumped bacteria.
    • Legends for movies S1 and S2

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Amoebal predation on CadA-agglutinated bacteria.
    • Movie S2 (.mp4 format). Amoebal predation on mechanically generated bacterial clumps.

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