(62i) Elucidation of the Mechanism of Action of Antifungal ß-Peptides | AIChE

(62i) Elucidation of the Mechanism of Action of Antifungal ß-Peptides

Authors 

Acevedo, S. - Presenter, University of Puerto Rico
Palecek, S. P. - Presenter, University of Wisconsin-Madison


Antimicrobial ß-peptides are oligomers made from artificial amino acids, which have been designed to mimic natural host defense peptides (HDPs). These ß-peptides have shown potent antibacterial and antifungal activities and key structural features have been identified that determine these activities. Despite the amount of literary information found on mechanisms of action for other antifungal agents, the mechanisms of action of the ß-peptides used in this research are not completely understood. It is believed they kill target cells by interacting with and disrupting the integrity of cellular membranes. In this study we have devised a biomolecular strategy to elucidate the genes involved in the sensitivity of yeast to well-characterized antifungal ß-peptides. In order to do this we chose two strains of Saccharomyces cerevisiae with the same background where one of them (SNYDER 760) was mutagenized with a mini-Tn3::LEU2 transposon that contained a lacZ gene lacking a promoter and initiator codon. We hypothesize that an increased resistance of this strain to the ß-peptides might indicate a disruption of the genes involved in the sensitivity to these ß-peptides. Therefore, as a control an unmutagenized strain (SKY 760) was submitted to the same treatment. Two different ß-peptides were used in this study, (ACHC-V-K)3 and Y-(ACHC-V-K)3. First the Minimum Inhibitory Concentration (MIC) of the two S. cerevisiae strains when exposed to the ß-peptides was determined. To gain further insight into the mechanism of action of these ß-peptides, Molecular Biology techniques were employed in order to determine the genes that were active upon exposure of the yeast to the ß-peptides. In the MIC Assays for the Y-(ACHC-V-K)3 ß-peptide, both strains of S. cerevisiae grew to the same concentration indicating equal resistance to the ß-peptide. In the MIC Assays for the (ACHC-V-K)3 ß-peptide, the strain SNYDER 760 proved more resistant to the ß-peptide by a two-fold dilution. Therefore, a genetic analysis was performed on that strain in order to identify if indeed the cause for resistance was the transposon inserted into this strain's genome. After obtaining these results, we will determine by means of a PCR Vectorette Assay, the genes that were interrupted by this transposon and knowing these interrupted genes might give us some insight into the mechanism of action of these ß-peptides.

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