The Lipid A 1-Phosphatase ofHelicobacter pyloriIs Required for Resistance to the Antimicrobial Peptide Polymyxin

Abstract

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 g/ml). However, disruption of lpxE HP in H. pylori results in a dramatic decrease in polymyxin resistance (MIC, 10 g/ml). In conclusion, we have characterized the first gram-negative LpxEdeficient mutant and have shown the importance of modifying the 1 position of H. pylori lipid A for resistance to polymyxin.

Selective Activation of the p38 MAPK Pathway by Synthetic Monophosphoryl Lipid A

“…Alteration of lipid A structures such as hypophosphorylation is known to be used by pathogens as an immune evasion mechanism (37,38). It is possible that active dephosphorylation of lipid A structures allows pathogens to escape MyD88-dependent initial inflammatory response and thus rapid clearance as well as to induce strong anti-inflammatory cytokine expression through p38 activation and TRIF-dependent endotoxin tolerance (27,28).…”

A link between the assembly of flagella and lipooligosaccharide of the Gram-negative bacterium Campylobacter jejuni

“…The amplicon was digested with KpnI and SacI, and inserted into vector pBluescript II SK(+) creating pBflgGHis. The vector pBflgGHis was then used as template for an inverse PCR (primers: 13,14) engineered to add an in-frame His 6 coding sequence before the stop codon of flgG and insert the restriction site NdeI to both ends of the amplicon. A aph3 cassette, obtained by PCR (primers: 15, 16) from cloning vector pRY107 (32), was inserted into the NdeI sites on the inverse PCR amplicon creating pFlgGHISKO:Kan R .…”

“…In C. jejuni the glucosamine disaccharide backbone can be replaced with the analogue 2,3-diamino-2,3-dideoxy-D-glucopyranose, resulting in two additional amide linked (brown) acyl chains. polymyxin B (PMB), a positively charged lipopeptide that binds to the phosphate groups of lipid A, killing Gram-negative bacteria in a manner similar to CAMPs of the innate immune system (14). Strain 81-176A1 (Δcj0256) showed a striking decrease in resistance to PMB (MIC of 0.8 ± 0.2 μg/mL) compared with WT (MIC of 17.3 ± 3.3 μg/mL) and cj0256 complemented strain (17.6 ± 3.6 μg/mL; Table S1).…”

Colonize, evade, flourish

“…H. pylori possesses only eight homologs to the nine Raetz pathway enzymes, with the exception in the last step of the pathway. 19 In E. coli, the last two acylation events are catalyzed by LpxL and LpxM ( Fig. 2 and 3), which each add an acyl chain to the existent 2' and 3' hydroxyacyl chains, respectively.…”

“…21 These successive modifications reduce the net negative charge of the LPS, thereby promoting resistance to cationic antimicrobial peptides (CAMPs) like polymyxin B. 19 Next, the terminal Kdo sugar is removed by the two-protein Kdo-hydrolase complex, Kdo H1/H2 (Hp0579 and Hp0580). 22 Kdo hydrolysis is required for expression of Lewis antigens (addressed in next section) and fully extended O-antigen.…”