(18) Assay Card

General Information
Summary Kalata B1, circulin A and B and cyclopsychotride shown to possess some anti-bacterial activity.
Condition A sensitive and reproducible two-stage radial diffusion assay was used for testing the antimicrobial activity of cyclic peptides. A 1-4¬ó^6 colony-forming unit/ml of the organism was mixed with 10¬ml of molten underlay gel solution and was poured into 10-¬ó-cm Petri dishes (Nunc) to form a uniform layer. The gel solution contained 10¬mM sodium phosphate buffer, 0.03% TSB, and 0.02% Tween 20.¬One gel solution had high salt (100¬mM NaCl) whereas the other did not. After the gel solidified, gel wells were made by a 3-mm diameter template in an evenly spaced array. An aliquot of 5¬¬µl of a serial half-log dilution of testing peptides at seven concentrations was added to each well after removing the gel plugs. The dishes were incubated at 37¬C for 3¬h to allow the peptides to diffuse into the underlay gels. Gels were overlaid with 10¬ml of 1% agarose in 6% of TSB (wt/vol). After further incubation at 37¬C for 16-24 h, the diameter of the clear zone surrounding the wells (colony-free) was measured under the microscope. Antimicrobial activities were expressed in units (0.1¬mm¬=¬1¬unit), and the MICs were determined from the x intercepts of the dose-response curves.
Result Kalata and Circulin A were specific for the Gram-positive Staphylococcus aureus with a minimum inhibition concentration of 0.2 ¬µM. They were relatively ineffective against Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. However, Circulin B and cyclopsychotride were active against both Gram-positive and Gram-negative bacteria. In particular, Circulin B showed potent activity against E.¬coli with a minimum inhibitory concentration of 0.41¬¬µM. All four cyclic peptides were moderately active against two strains of fungi, Candida kefyr and Candida tropicalis, but were inactive against Candida albicans.
AssayType Anti-bacterial

Tam JP, Lu YA, Yang JL, Chiu KW (1999) An unusual structural motif of antimicrobial peptides containing end-to-end macrocycle and cystine-knot disulfides. Proc Natl Acad Sci U S A 96:8913-8

Proteins Assayed circulin B
kalata B1
circulin A