Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria

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 mutagens 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. Colistin had the same effect. 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.

Fig 1. The joint action of LL-37 and ferrous iron induces an increase in the mutation rate of P. aeruginosa.

No changes occur when LL-37 or iron are added to the culture separately. Error bars represent 95% confidence intervals for mutation rates.

Fig 2. The mutagenesis of LL-37+Fe²⁺ combination is suppressed by the addition of an iron chelator.

This supports the notion that iron is causal in increasing mutagenesis (A). In the same line, over-expression of Dps, a natural iron chelator in bacteria, also decreases the mutant frequency to rifampicin (B). Error bars represent 95% confidence intervals for mutant frequencies.

Fig 3. Mutagenesis induced by colistin and Fe²⁺ combination.

The mutation rate is only increased if both, iron and colistin, are present. Error bars represent 95% confidence intervals for mutation rates.

Fig 4. Mutation hotspots in glpT.

Distribution of the mutations in the 1160 bp-long fragment of glpT in P. aeruginosa PA14 fosfomycin-resistant clones treated with iron, LL-37, or a combination of both. The number of mutations (nucleotide substitutions, indels) is plotted against respective nucleotide positions within the gene fragment. Note the overlapping mutations at positions 220–225, 409 and 524 bp in iron and LL-37 treatments and absence of common mutations in LL-37+Fe²⁺ treatment. The fig was generated using the mutational spectrum analysis software iMARS [39].

Fig 5. A model for LL-37-mediated, iron–induced mutagenesis in Pseudomonas aeruginosa.

The interactions of LL-37 with the membrane at sub-inhibitory concentrations lead to transient permeability changes in the membranes that promote iron movement in favour of the electrochemical gradient. Uncontrolled uptake of ferrous ions stimulates Fenton reaction that leads to hydroxyl radical formation and results in DNA damage and mutagenesis.