Use of Tn5-transposon mutagenesis for the identification of potential novel interactors of antimicrobial peptides in E.coli

Antimicrobial peptides (AMPs) participate in the immunity of both animals and plants. In mammals, the most important AMP families are defensins and cathelicidins. The only human cathelicidin, LL-37, is a 37-residue, ?-helical, cationic peptide with a direct membranolytic antibacterial activity and various immunomodulatory activities. The aim of our research was to establish a procedure based on an E. coli knock-out mutant library in the search for mutations conferring altered susceptibility to AMPs. The library was created by random insertions of Tn5 transposon into the bacterial genome. The selection of low-susceptibility mutants and their identification was first tested with a Bac7-derived peptide, for which at least one interactor had been found. The procedure was then applied for the identification of mutations that modulate the susceptibility of E. coli cells to LL-37 with the aim of dissecting the steps of the peptide's mode of action. The differences between the wild-type and the mutant phenotypes were characterised using assays determining the bacterial culture growth inhibition and killing, as well as cell binding and membrane permeabilisation. In the majority of the isolated Bac7-resistant mutants, Tn5 was inserted in the sbmA gene, confirming previous findings that showed an essential role for the SbmA protein in the internalisation of Bac7, allowing it to exert its antimicrobial activity. Tn5 insertion in the gene waaY, identified in 15 out of 20 resistant mutants selected with LL-37, lowered the peptide's ability to inhibit bacterial growth and to kill cells, decreased peptide binding to the bacterial surface and membrane permeabilisation. However, it did not alter the susceptibility to other AMPs. WaaY encodes a specific kinase that phosphorylates the Hep II residue in the core region of bacterial lipopolysaccharide. The inactivation of this enzyme determines a decreased negative charge of the outer membrane, which in turn causes a decrease in the peptide's binding to the cell surface and in its antibacterial activity. The results reveal a putative LPS-binding site for LL-37 and stress the importance of its initial binding to the cell surface for antimicrobial efficacy. The established procedure utilising Tn5 mutants in the search for bacterial mutations conferring resistance to AMPs, proved efficient and opened a previously unreported branch of research on the mode of action of the human cathelicidin.