Effects of Synthetic Cecropin Analogs on in Vitro Growth of Acholeplasma laidlawii

Abstract

Four synthetic peptides (Peptidyl MIMs; Demeter Biotechnologies, Inc.) were evaluated for their in vitro activity against Acholeplasma laidlawii. Fifty percent effective concentration values ranged from 1 to 15 M. Three of these compounds are more lethal than cecropin B against A. laidlawii.Lytic polypeptides with antimicrobial activity have been isolated from insect hemolymph, amphibians, and mammals (3). Encoded by single genes, these short peptides have potent antimicrobial activity against a wide spectrum of microorganisms, including bacteria and fungi, yet have very low toxicities to higher organisms. Biologically active synthetic peptides have been produced, and gene constructs coding for lytic polypeptides have been introduced into a number of higher plants for the control of bacterial and fungal phytopathogens (1,2,7,9,10,11,12,14,17,18,19). We have evaluated the activity of four synthetic cecropin analogs for their antimicrobial activities against the mollicute Acholeplasma laidlawii. These analogs were designed for enhanced membrane lysis activity, and each possesses features characteristic of cecropin-like lytic polypeptides (6). Specifically, these peptides each are strongly basic polar molecules which are predicted to form hydrophilic amphipathic ␣-helices in their N-terminal regions and more hydophobic ␣-helices in their C-terminal regions.A. laidlawii strain PG-8 (ATCC 23206) was grown in liquid SP-4 medium (22) at 37°C. To assess the lytic activity of each peptide, early-log-phase cultures were exposed to six concentrations of four synthetic Peptidyl Membrane Interactive Molecules (Peptidyl MIMs; Demeter Biotechnologies Inc., Triangle Park, N.C.). The amino acid sequences of these peptides and cecropin B are as follows: 6M1, FKLRAKIKVRLRAKIKL; 2M5, KRKRAVKRVGRRLKKLARKIARLGVAKLAGLR AVKLF; 2M2, KRKRAVKRVGRRLKKLARKIARLGVAF; 2L2, FAKKFAKKFKKFAKKFAKFAFAF; cecropin B, KW KVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL.To each of seven 270-l aliquots of early-log-phase A. laidlawii (10 7 to 10 8 CFU/ml) was added 30 l of dilutions of each Peptidyl MIM. Solutions and cells were incubated at 37°C for 60 min, and three 20-l aliquots of each dilution were plated and allowed to dry, without spreading, onto SP-4 medium solidified with 12% Noble agar. Plates were incubated at 37°C for 2 to 3 days in a humidified chamber, and CFUs were counted after staining in Diene's stain. All treatments with each Peptidyl MIM were replicated three times. Mean viable CFU/ml values were calculated from each replicated dilution plating. Percent survival for each Peptidyl MIM at each peptide concentration was calculated based upon the CFU/ml from each dilution plating divided by the CFU/ml of the water control. EC 50 s (estimated concentrations resulting in 50% survival) were calculated for each compound by regressing the percent survival against the natural logarithm of the compound concentration. The resulting regression equation was used to calculate the concentration of compound that would result in 50% survival. Paired t tests were used...

Peptide Biocides for Engineering Bacterial Blight Tolerance and Susceptibility in Cut-flower Anthurium

“…They attributed this to a cell proliferation effect of peptides at low concentrations. In vitro, Peptidyl MIM 2L2, another synthetic cecropin analog, increased cell titers of Acholeplasma laidlawii 20% to 30% (Borth et al, 2001) at certain concentrations. These results taken together with the low level of Shiva-1 expression in 'Tropic Flame' 1-16 suggest that a sub-optimal level of Shiva-1 may actually increase disease susceptibility to anthurium blight in at least one genotype.…”