SDS–polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 12% gels with 30% acrylamide/bis-acrylamide (29:1) solution (Bio-Rad). Western blot transfer from SDS-PAGE gels to nitrocellulose membranes were performed with methanol/tris transfer buffer [25 mM tris (pH 7.6) and 20% (v/v) methanol] at 100 V for 1 hour. Membranes were blocked with tris-buffered saline with Tween 20 (TBST; pH 7.6, 0.5% Tween 20) and 5% nonfat milk for 1 hour at room temperature. Monoclonal anti-CadA antibodies (MLJ11, obtained from D. Fuller; 1:10,000 dilution) were incubated for 1 hour at room temperature. Filters were washed with TBST three times for 10 min each. Secondary horseradish peroxidase–conjugated goat anti-mouse antibodies (Thermo Fisher Scientific) were incubated for 1 hour at 1:10,000 before repeating the wash. Blots were then visualized with a chemiluminescent substrate kit (SuperSignal WestPico, Thermo Fisher Scientific).

To determine CadA localization during growth on bacterial lawns, we constructed layered 10-cm agar plates by laying the agar of one SM agar plate (10-ml volume) on top of a 25-ml SM agar plate. An overnight culture of K. pneumoniae was mixed with ~100 amoebae and plated onto the layered agar plates. After the amoebal plaques appeared, the top 10-ml agar layer was separated from the petri dish and gently washed to remove bacteria and amoebae. The 10-ml agar disk was then placed on a nitrocellulose membrane with the side previously containing bacteria and amoeba facing away from the membrane. The contents of the agar were then electroblotted onto the membrane with a standard Western blot apparatus and buffer system, as described above, and CadA protein localization was determined as described above. As a control, 2 μg of recombinant CadA protein was spotted on top of a cadA mutant plaque to demonstrate that CadA could migrate through the SM agar.

CadA secretion during vegetative growth was determined by plating AX4 or cadA amoebae with K. pneumoniae on SM agar and incubating them overnight at room temperature. Before any clearing of the bacteria by the amoebae had occurred, the bacteria and amoebae were collected and suspended in Sor buffer. Differential low-speed centrifugation was used to separate amoebae (400g for 5 min), bacteria (3000g for 10 min), and supernatant. The presence of CadA was probed by Western blot as described above, and anti-actin monoclonal antibodies were used to detect cell-associated actin (224-236-1, Developmental Studies Hybridoma Bank, University of Iowa).

We determined the apparent binding affinity of CadA for K. pneumoniae by measuring the amount of protein remaining unbound. K. pneumoniae was grown to an OD600 of 1.0 in SM. One milliliter of bacteria was spun down and washed with Sor buffer. The bacteria were then resuspended in 60 μl of Sor buffer with 0 to 70 μg of CadA and incubated for 1 hour. The bacteria were pelleted, 30 μl of supernatant was collected, and unbound protein was measured by Bio-Rad protein assay. By subtracting the unbound protein from the known input concentration, we calculated the amount of CadA bound to the bacteria. Estimated binding affinity of CadA for K. pneumoniae was calculated using the method of Steck and Wallach (41).

To determine the possible lectin-binding activity of CadA, we repeated the above experiment in the presence of 0 to 300 mM d-galactose, d-trehalose, or d-glucosamine (EMD Millipore). K. pneumoniae was grown as previously described. Next, bacteria, saccharide, and 10 μg of CadA were mixed in 60 μl of Sor buffer for 1 hour. Supernatants were collected, and unbound protein was measured by the Bio-Rad protein assay.

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