Figure 3

Serum albumin binds to and prevents the predator from attacking its prey. (a) The addition of human serum albumin (HSA) to HEPES buffer at a physiologically relevant concentration of 50 mg/ml significantly inhibits predation of all three prey strains. For comparison, predation tests were also attempted in heat-treated serum. The initial PPR was approximately 0.05 and the significance was determined by comparing the viabilities obtained after 24 hours with the initial values. p < 0.001 for a; p < 0.01 for b. n = 3. (b) Serum albumin prohibits predation of a bioluminescent E. coli prey strain. The bioluminescence (BL) from the prey strain was monitored over time while being predated upon in the presence of HSA. The upper panel is the control results, i.e., without B. bacteriovorus HD100 addition, and the bottom panel is with the predator added. The PPR was approximately 9.7 for these experiments. n = 9. (c ) Relative bioluminescence (RBL) results calculated using the data in (b). The RBLs were calculated by comparing the BL from the predated sample with that of its control (unpredated) every hour and the data plotted. The results show the similar levels of inhibition obtained with each of the HSA concentrations tested. n = 9. (d) Pretreatment of B. bacteriovorus HD100 with serum albumin is sufficient to block predation. The predator or its prey (E. coli) was pretreated by incubating it for one hour in a 50 mg/ml HSA solution made with HEPES buffer. Afterwards, the predator (or prey) were pelleted, washed and mixed with fresh prey (or predator) and the predatory activity determined after 24 hours. For comparison, samples containing untreated predator and prey cells were also tested. The significance was determined by comparing the viabilities obtained after 24 hours with the initial values. p < 0.001 for a. n = 3. (e) Confocal images of B. bacteriovorus HD100 after exposure to FITC-labeled BSA. B. bacteriovorus HD100 cells in HEPES buffer were incubated with the Cell Tracker live stain for 30 minutes at 30 °C to produce fluorescent predatory cells. After pelleting and washing the cells, they were exposed to FITC-labeled BSA (10 mg/ml) for one hour at 30 °C and 250 rpm. The cells were washed again to remove any free or loosely bound BSA molecules before being imaged using a laser confocal microscope (LX-700, Olympus, USA). This image shows the BSA molecules (red) bound to the predatory cells (blue). Both of the scale bars in the middle indicate 2 μm. (f) Dot blot results showing human serum albumin is biding to B. bacteriovorus HD100. Approximately 2.5 × 10⁵ B. bacteriovorus HD100 or E. coli were exposed to 50 mg/ml HSA, before being pelleted, washed and then spotted on the nylon membrane. The left-side blots were obtained as in Fig. 1e. The right-side blots were obtained using an anti-HSA rabbit IgG followed by a HRP-conjugated anti-rabbit goat IgG. Although tests with a prey (E. coli) were performed in parallel, only the predator exposed to HSA was detected.