Acquisition of adaptive mutations is essential for microbial persistence during chronic infections. This is particularly evident during chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) patients. Thus far, mutagenesis has been attributed to the generation of reactive species by polymorphonucleocytes (PMN) and antibiotic treatment. However, our current studies of mutagenesis leading to P. aeruginosa mucoid conversion have revealed a potential new mutagen. Our findings confirmed the current view that reactive oxygen species can promote mucoidy in vitro, but revealed PMNs are proficient at inducing mucoid conversion in the absence of an oxidative burst. This led to the discovery that cationic antimicrobial peptides can be mutagenic and promote mucoidy. Of specific interest was the human cathelicidin LL-37, canonically known to disrupt bacterial membranes leading to cell death. An alternative role was revealed at sub-inhibitory concentrations, where LL-37 was found to induce mutations within the mucA gene encoding a negative regulator of mucoidy and to promote rifampin resistance in both P. aeruginosa and Escherichia coli. The mechanism of mutagenesis was found to be dependent upon sub-inhibitory concentrations of LL-37 entering the bacterial cytosol and binding to DNA. LL-37/DNA interactions then promote translesion DNA synthesis by the polymerase DinB, whose error-prone replication potentiates the mutations. A model of LL-37 bound to DNA was generated, which reveals amino termini α-helices of dimerized LL-37 bind the major groove of DNA, with numerous DNA contacts made by LL-37 basic residues. This demonstrates a mutagenic role for antimicrobials previously thought to be insusceptible to resistance by mutation, highlighting a need to further investigate their role in evolution and pathoadaptation in chronic infections.
Antimicrobial peptides (AMPs) are produced by the mammalian immune system to fight invading pathogens. The best understood function of AMPs is to interact with the membranes of microbes, thereby disrupting and killing cells. However, the amount of AMP available during chronic bacterial infections may not be sufficient to kill pathogens (sub-inhibitory). In this study, we found that at sub-inhibitory levels, AMPs promote mutations in bacterial DNA, a function not previously attributed to them. In particular, we found that in the bacteria Pseudomonas aeruginosa, one AMP called LL-37 can promote mutations, which enable the bacteria to overproduce a protective sugar coating, a process called mucoid conversion. P. aeruginosa mucoid conversion is a major risk factor for those suffering from cystic fibrosis (CF), the most common lethal, heritable disease in the US. We found that LL-37 is able to produce these mutations by penetrating the bacterial cell and binding to the bacterial DNA. DNA binding disrupts normal DNA replication and allows mutations to occur. Furthermore, we observed LL-37 induced mutagenesis in processes apart from mucoid conversion, in both P. aeruginosa and E. coli. This suggests that AMP-induced mutagenesis may be important for a broad range of chronic diseases and pathogens.