Abstract
Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as a mutagen in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection.
Author Summary Cationic antimicrobial peptides (cAMPs) are small proteins naturally produced by the immune system to limit bacterial growth mainly through pore formation in the membrane. It has recently been suggested that sub-inhibitory concentrations of cAMPs promote bacterial mutagenesis, similarly to antibiotics. However, we previously reported that cAMPs do not increase mutation rate and do not activate bacterial stress responses. Here we resolve this contradiction. We report that free iron in the culture medium increases mutagenesis in the presence of cAMPs. We show that sub-inhibitory concentrations of cAMPs facilitate entry of free iron into bacterial cells, where it interacts with hydrogen peroxide, thereby resulting in production of DNA-damaging reactive oxygen species and increased mutagenesis. Moreover, these results may have clinically-relevant implications: while very little free iron is normally present in healthy individuals, this is not the case in patients suffering from cystic fibrosis, where elevated bacterial mutagenesis promotes antibiotic resistance and contributes to persistence and severity of infection. Thus, an intervention aimed at reduction of free iron in the lungs could reduce the cAMPs-facilitation of iron-mediated mutagenesis; hence antibiotic resistance and pathoadaptation.
