D mannose agarose beads

D-mannose agarose beads (Sigma-Aldrich Chemie GmbH, Munich, Germany) and sepharose beads (CL-4B, ø 40 μm to 165 μm, Sigma-Aldrich Chemie GmbH, Munich, Germany) were each diluted in a ratio of 1:10 in PBS and subsequently centrifuged at 1000 rpm for 5 min. The supernatant was removed and the washing procedure was repeated twice. Finally, the cleaned beads were diluted 1:10 in PBS. 3 × 10⁵A. castellanii or A. comandoni in 5 ml of PYG medium were seeded into tissue culture bottles (25 ml, Sarstedt AG & Co., Nümbrecht, Germany) and 0.5 ml of one of the bead solutions were added. The samples were observed with an inverted phase contrast microscope (Olympus CKX41) equipped with a camera (C9300, Hamamatsu, Hamamatsu, Japan) and 50 images of each bottle were taken right after the addition, after 2 h and after 4 h incubation time at room temperature. The number of acanthamoebae adhering to the beads and the total number of acanthamoebae in the resulting images were counted.

To monitor the sensitivity of bacterial strains to normal human serum (NHS), NHS was taken from healthy volunteers. We largely followed procedures described previously [10,27], using A. actinomycetemcomitans, and A. aphrophilus strains grown on agar. In brief, prior to being used in the assays, bacteria were harvested and suspensions were adjusted to 1.0 × 10⁹ cells/ml in PBS. Reaction mixtures contained 105 μl NHS, 95 μl PBS, and 10 μl bacterial suspension and were incubated at 37°C for 2 h. Cell survival was assessed by plating serial dilutions on agar. Heat-inactivated (56°C, 30 min) NHS (HI-NHS) was used as control. Survival rates were calculated as the ratio (%) of colony forming units (CFU) NHS/HI-NHS. When needed to distinguish between the classical and MBL, and the alternative pathway of complement activation, serum killing assays were performed in the presence of 5 mM MgCl₂, and 10 mM EGTA (Mg²⁺/EGTA), which selectively permits the alternative pathway only [39]. To obtain NHS depleted from the MBL pathway, serum was filtered through D-mannose-agarose beads (Sigma-Aldrich, St. Louis, USA), as described previously [27].