dinF Elicits Nitric Oxide Signaling Induced by Periplanetasin-4 from American Cockroach in Escherichia coli.

Modern antibiotics have been developed with the aim of destroying cellular function; however, the risk of antibiotic-resistance is increasing continuously. As a result, antimicrobial peptide (AMP) is considered a novel strategy to substitute traditional drugs. This study focused on revealing the antibacterial mechanism(s) of periplanetasn-4, an AMP identified from Cockroach. To elucidate whether periplanetasin-4 generates reactive oxygen species (ROS), a crucial stress factor for cell death, intracellular ROS was measured in Escherichia coli. The degree of membrane and DNA damage was determined using the properties that ROS causes oxidative stress to cell components. Unlike normal cell death, membrane depolarization was observed but DNA fragmentation did not occur. In addition, accumulation of nitric oxide (NO), a free radical with high toxicity, was measured and the byproduct of NO also induced severe intracellular damage. Periplanetasin-4-induced NO also impacted on cytosol calcium levels and triggered lipid peroxidation and DNA oxidation. These features were weakened when NO synthesis was interrupted, and this data suggested that perplanetasin-4-induced NO participates in E. coli cell damage. Moreover, this AMP-induced NO stimulates expression of SOS repair proteins and activation of RecA, a bacterial caspase-like protein. Features of nitrosative damage did not occur especially without dinF gene which is associated with oxidative stress. Therefore, it was indicated that when there is a NO signal, dinF promotes cell death. In conclusion, the combined investigations demonstrated that the antibacterial mechanism(s) of periplanetasin-4 was a NO-induced cell death, and dinF gene is closely related to cell death pathway.