Identification of Proteus mirabilis mutants with increased sensitivity to antimicrobial peptides

Antimicrobial peptides (APs) are important components of the innate defense s of animals, plants, and microorganisms. However, some bacterial pathogens are resistant to the action of APs, For example, Proteus mirabilis is high ly resistant to the action of APs, such as polymyxin B (PM), protegrin, and the synthetic protegrin analog IB-367. To better understand this resistanc e, a transposon mutagenesis approach was used to generate P. mirabilis muta nts sensitive to APs. Four unique PM-sensitive mutants of P, mirabilis were identified (these mutants mere >2 to >128 times more sensitive than the wi ld type). Two of these mutants were also sensitive to IB-367 (16 and 128 ti mes more sensitive than the wild type). Lipopolysaccharide (LPS) profiles o f the PM- and protegrin-sensitive mutants demonstrated marked differences i n both the lipid A and O-antigen regions, while the PM-sensitive mutants ap peared to have alterations of either lipid A or O antigen. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of th e wild type and PM-sensitive mutant lipid A showed species with one or two aminoarabinose groups, while lipid A from the PM- and protegrin-sensitive m utants was devoid of aminoarabinose. When the mutants were streaked on an a gar-containing medium, the swarming motility of the PM- and protegrin-sensi tive mutants was completely inhibited and the swarming motility of the muta nts sensitive to only PM was markedly decreased. DNA sequence analysis of t he mutagenized loci revealed similarities to an O-acetyltransferase (PM and protegrin sensitive) and ATP synthase and sap loci (PM sensitive). These d ata further support the role of LPS modifications as an elaborate mechanism in the resistance of certain bacterial species to APs and suggest that LPS surface charge alterations may play a role in P. mirabilis swarming motili ty.