Figure 2. Defensin-2 causes cytoplasmic leakage in R. montanensis.

R. montanensis was incubated with recombinant defensin-2 or thioredoxin (Trx) followed by centrifugation to separate the pellet and supernatant fractions. Alternatively, R. montanensis was treated with synthetic defensin-2 or diluent buffer (buffer control) followed by centrifugation to separate the pellet and supernatant fractions. Each fraction was subjected to LDS-PAGE, blotted to a PVDF membrane, and probed with anti-EF-Ts serum. (A) Lysate from E. coli expressing recombinant EF-Ts from R. montanensis was detected by EF-Ts antiserum but not rabbit preimmune serum. Recombinant R. montanensis EF-Ts migrated to 40 kDa due to the histidine tag. (B) Nonspecific band detected by EF-Ts antiserum in uninfected L929 lysate. (C) R. montanensis EF-Ts was detected in the pellet from Trx-treated, as well as in the supernatant and pellet, from defensin-2-treated R. montanensis. EF-Ts was not observed in supernatant from Trx-treated R. montanensis. The nonspecific L929 and R. montanensis EF-Ts (35–40 kDa) proteins are indicated. (D) Pellet and supernatant fractions from R. montanensis treated with increasing concentrations of synthetic defensin-2 peptide or diluent buffer (Buffer CNT). R. montanensis EF-Ts ran closer to 35 kDa due to an increased running time on the polyacrylamide gels to ensure adequate separation from the nonspecific L929 protein that cross-reacts with EF-Ts antiserum. (E) Decrease in the number of live rickettsia/mm³ with increasing concentrations of synthetic defensin-2 peptide. Live counts were performed on the same samples used to generate the blots demonstrating lysis to correlate lysis with the decrease in live counts. A T-Test was performed to test for statistical differences between the means for treatment with increasing concentrations of synthetic defensin-2 and the negative control buffer. P-values for each synthetic defensin-2 concentration are 8 μM (p=0.21), 40 μM (p=0.027), 200 μM (p=0.09), 1mM (p=0.018). Asterisks indicate significant differences from negative control buffer. M, molecular weight marker migrating to 40 kDa; R.m., R. montanensis.