The antibacterial activity of PAD-1.17 and its effects on the Bacillus subtilis ribosome

PAD-1.17 is a synthetic cationic antimicrobial peptide (amino acid sequence TRKKLFHIFHATIRSR) that is a derivative of a larger peptide originally identified in a screen for peptide activators of the diphtheria toxin repressor, DtxR. Following the observation that PAD-1.17 inhibits the growth of several Gram-negative and Gram-positive species, research described in this thesis was designed to gain an understanding of the underlying mechanism of this antibacterial activity. Using Bacillus subtilis and Bacillus anthracis Sterne as model organisms, time-kill assays, germination assays, and determination of minimal inhibitory concentrations demonstrated that PAD-1.17 inhibits vegetative growth as well as outgrowth from spores. While the majority of cationic antimicrobial peptides are known to function by disrupting bacterial membranes, PAD-1.17 did not affect membrane permeability as assessed by red blood cell lysis and bacterial membrane assays. Addition of the antibiotic efflux pump inhibitor reserpine to bacterial cultures was found to increase the sensitivity to PAD-1.17, providing additional evidence that it acts intracellularly. Subsequent analysis demonstrated that exposure of B. subtilis to PAD-1.17 resulted in major changes in ribosomal RNA and ribosomes.;Examination of total RNA harvested from B. subtilis treated with PAD-1.17 using gel electrophoresis, Agilent Bioanalyzer analysis, and Northern blotting revealed that PAD-1.17 causes degradation of 16S and 23S rRNA. Ribosomes harvested from bacteria treated with PAD-1.17 were analyzed by sucrose gradient ultracentrifugation, SDS-PAGE, and 2D gel electrophoresis. Following exposure to PAD-1.17, ribosomes displayed altered sedimentation profiles and protein compositions. Uridine pulse and chase labeling assays performed to assess ribosomal subunit formation determined that PAD-1.17 inhibits the assembly of both 50S and 30S subunits Further analysis of subunit stability demonstrated that PAD-1.17 facilitates degradation of 50S and 30S subunits, whereas 70S ribosomes are resistant. Results presented here demonstrate that PAD-1.17 inhibits the assembly of 30S and 50S subunits, as well as inducing the degradation of free subunit particles. In contrast, PAD-1.17 does not affect the structure or function of 70S ribosomes. As such, the findings reported here describe a possible mechanism of action for PAD-1.17 as a cationic antimicrobial peptide.