Francisella (F.) tularensis is a gram-negative, zoonotic, facultative intracellular pathogen that causes tularemia. The disease of tularemia in animals and humans is characterized by infection of the macrophages, followed by infection of other cell types and organs including lung, liver and spleen. While Francisella is not normally a respiratory pathogen, the most severe infections by Francisella species occur via inhalation or direct inoculation of the lungs leading to pneumonic tularemia. Due to its potential use as a biological weapon or bio-terrorist threat (via aerosol) and the probable development of antibiotic resistant strains of Francisella, new approaches to the treatment of pneumonic tularemia are a priority. Antimicrobial peptides are small (3-6 kDa), cationic peptides that exert a direct antimicrobial effect on microbes. Interest in antimicrobial peptides has grown due to increasing resistance of microorganisms to commonly used antibiotics and the potential therapeutic applications of these peptides. In humans, only one cathelicidin LL-37 has been characterized, which is derived by proteolysis from the C-terminal end of the human CAP18 protein. Recently, helical cathelicidins have been discovered in various species of snakes including the Chinese cobra, Naja atra. Two 11-residue peptides (NA-CATH-1 and NA-CATH-2) containing one of the two repeated motifs were designed from NA-CATH. We hypothesize that smaller synthetic peptides modeled after the reptile cathelicidin NA-CATH can be designed with increased antimicrobial effectiveness and decreased hemolysis making them better candidates for development into useful and broad spectrum antimicrobial compounds. We tested the susceptibility of Francisella and E. coli to NA-CATH cathelicidin and four truncated peptides compared to the effectiveness of the human cathelicidin, LL-37. A killing time curve was also performed to know the activity time of the peptides. Finally, we have also shown that infection of Human alveolar type II epithelial cells (A549) with F. novicida induces an increase in LL-37 gene expression. Data from the antimicrobial studies shows that full length snake cathelicidin peptide (NA-CATH) is considerably more potent than LL-37 in vitro against E.coli, but less potent against Francisella. The antimicrobial activity of the four truncated peptides is presented, one of which is found to be extremely potent in the nM range. The peptides also show no hemolytic activity even at high peptide concentration, indicating low cytotoxicity to host cells. These peptides represent a new approach to antimicrobial drug development. We also determined that LL-37 mRNA levels were elevated 3 fold relative to the levels of the corresponding LL-37 mRNAs in uninfected control cells. This is the first report of the induction of LL-37 expression in A549 cells as a consequence of bacterial stimulus.
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