The lipid A 1-phosphatase of Helicobacter pylori is required for resistance to the antimicrobial peptide polymyxin

Modification of the phosphate groups of lipid A with amine-containing substituents, such as phosphoethanolamine, reduces the overall net negative charge of gram-negative bacterial lipopolysaccharide, thereby lowering its affinity to cationic antimicrobial peptides. Modification of the 1 position of Helicobacter pylori lipid A is a two-step process involving the removal of the 1-phosphate group by a lipid A phosphatase, LpxE(HP) (Hp0021), followed by the addition of a phosphoethanolamine residue catalyzed by EptA(HP) (Hp0022). To demonstrate the importance of modifying the 1 position of H. pylori lipid A, we generated LpxE(HP)-deficient mutants in various H. pylori strains by insertion of a chloramphenicol resistance cassette into lpxE(HP) and examined the significance of LpxE with respect to cationic antimicrobial peptide resistance. Using both mass spectrometry analysis and an in vitro assay system, we showed that the loss of LpxE(HP), activity in various H. pylori strains resulted in the loss of modification of the 1 position of H. pylori lipid A, thus confirming the function of LpxE(HP). Due to its unique lipid A structure, H. pylori is highly resistant to the antimicrobial peptide polymyxin (MIC > 250 mu g/ml). However, disruption of lpxE(HP) in H. pylori results in a dramatic decrease in polymyxin resistance (MIC, 10 mu g/ml). In conclusion, we have characterized the first gram-negative LpxE-deficient mutant and have shown the importance of modifying the 1 position of H. pylori lipid A for resistance to polymyxin.