To further elucidate the antibacterial mechanism of BmKDfsin4, electron microscopy was used to visualize the potential damage to caused by the scorpion defensin. we performed an UNC569 in-depth investigation into the potential of scorpion defensins to function as neurotoxin-like potassium channel blockers on the basis of diverse neurotoxin-potassium channel interactions (8, 18,C22). As expected, BmKDfsin4, an ancient antimicrobial defensin from the scorpion Karsch (6), effectively blocked the voltage-gated potassium channels Kv1.1, Kv1.2, and Kv1.3 in the same manner as classical scorpion potassium channel toxins. These findings are the first to experimentally demonstrate that a venomous animal defensin is a potassium channel blocker, revealing that venomous animal defensins are a novel type of potassium channel blocker. Materials and Methods Peptide and Peptide Mutants Recently, BmKDfsin4 has been identified from the scorpion Karsch by our group, and BmKDfsin4 has been expressed using a prokaryotic expression technique on the basis of our previous strategy for expressing recombinant potassium channel-blocking scorpion toxins (19,C21). The KOD-Plus-Neo kit was used to produce BmKDfsin4 mutants on the basis of the wild-type pGEX-6p-1-BmKDfsin4 plasmid. All plasmids were verified by DNA sequencing before expression. The mutants were also expressed and purified using the same method. Sequence and Structure Analysis of BmKDfsin4 Multiple sequence alignments of BmKDfsin4, def, charybdotoxin (ChTX), and BmKTX were performed using the GeneDoc program. The secondary structures of peptides were measured using CD spectroscopy. The three-dimensional structure of BmKDfsin4 was modeled using micasin as a template (PDB code 2LR5) through the SWISS-MODEL server as described preciously (19). The solution structures of BmKTX and ChTX were retrieved from the PDB (BmKTX PDB code, 1BKT; ChTX PDB code, 2CRD). Bacterial Strains AB94004, AB93113, AB91021, AB92037, AB94012, and ATCC25922 were purchased from the China Center of Type Culture Collection. Methicillin-resistant P1381 and penicillin-resistant P1389 were obtained from the 302nd Military Hospital (Beijing, China). Antimicrobial Assays Antimicrobial activity was determined using the broth microdilution assay according to the procedure recommended by the Clinical and Laboratory Standards Institute, with some modifications. Briefly, bacteria were cultured in LB medium to AB94004 was cultured in LB medium to the exponential phase (AB94004 cells were cultured to the exponential phase (107 cfu/ml), and then the fluorescent dye SYTOX Green (Invitrogen) was added to a final concentration of 5 m. After incubation for 10 min, BmKDfsin4 was added to final concentrations of 1 1 MIC, 2 MIC, 5 MIC, and 10 MIC. MSI-78 and 0.9% saline were used as positive and negative controls, respectively. Fluorescence was measured using the excitation and emission wavelengths of 488 and 525 nm, respectively. Transmission electron microscopy Exponential phase genome (6). This peptide is highly homologous to the defensin def isolated from scorpion hemolymph (23) (Fig. 1and def (a UNC569 scorpion defensin from Karsch). Cysteines are shadowed in carrying pGEX-6p-1-BmKDfsin4 without isopropyl 1-thio–d-galactopyranoside induction; carrying pGEX-6p-1-BmKDfsin4 induced with 0.1 mm isopropyl 1-thio–d-galactopyranoside; UNC569 and was analyzed by Tricine SDS-PAGE. UNC569 methicillin-resistant and penicillin-resistant 3). = 3), and the determined MIC values were the same. AB94004. The bacteria were treated with BmKDfsin4 or ampicillin at 5 MIC. The experiment was repeated with similar results. AB94004 was 3.53 m. The experiment was repeated Rabbit polyclonal to MGC58753 with similar results. AB94004 in the absence or presence of BmKDfsin4. = 3). To explore the mechanism by which BmKDfsin4 inhibits bacterial growth, Abdominal94004 was selected as a standard model bacterium. The killing kinetics of BmKDfsin4 against showed that it killed this bacterium more rapidly than ampicillin, an antibiotic inhibiting bacterial cell wall biosynthesis (Fig. 2was exposed to this peptide (Fig. 2cells were exposed to BmKDfsin4 at 1 MIC, 2 MIC, 5 MIC, or 10 MIC, similar to the results for the bad control, 0.9% saline. These results showed the bacterial membrane integrity was not damaged after rBmKDfsin4 treatment, indicating.
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