Periplasmic cleavage and modification of the 1-phosphate group of Helicobacter pylori lipid A.
Pathogenic bacteria modify the lipid A portion of their lipopolysaccharide to help evade the host innate immune response. Modification of the negatively charged phosphate groups of lipid A aids in resistance to cationic antimicrobial peptides targeting the bacterial cell surface. The lipid A of Helicobacter pylori contains a phosphoethanolamine (pEtN) unit directly linked to the 1-position of the disaccharide backbone. This is in contrast to the pEtN units found in other pathogenic Gram-negative bacteria, which are attached to the lipid A phosphate group to form a pyrophosphate linkage. This study describes two enzymes involved in the periplasmic modification of the 1-phosphate group of H. pylori lipid A. By using an in vitro assay system, we demonstrate the presence of lipid A 1-phosphatase activity in membranes of H. pylori. In an attempt to identify genes encoding possible lipid A phosphatases, we cloned four putative orthologs of Escherichia coli pgpB, the phosphatidylglycerol-phosphate phosphatase, from H. pylori 26695. One of these orthologs, Hp0021, is the structural gene for the lipid A 1-phosphatase and is required for removal of the 1-phosphate group from mature lipid A in an in vitro assay system. Heterologous expression of Hp0021 in E. coli resulted in the highly selective removal of the 1-phosphate group from E. coli lipid A, as demonstrated by mass spectrometry. We also identified the structural gene for the H. pylori lipid A pEtN transferase (Hp0022). Mass spectrometric analysis of the lipid A isolated from E. coli expressing Hp0021 and Hp0022 shows the addition of a single pEtN group at the 1-position, confirming that Hp0022 is responsible for the addition of a pEtN unit at the 1-position in H. pylori lipid A. In summary, we demonstrate that modification of the 1-phosphate group of H. pylori lipid A requires two enzymatic steps.
Tran
An X
AX
Department of Microbiology, J. H. Quillen College of Medicine, Johnson City, TN 37614, USA.
Karbarz
Mark J
MJ
Wang
Xiaoyuan
X
Raetz
Christian R H
CR
McGrath
Sara C
SC
Cotter
Robert J
RJ
Trent
M Stephen
MS
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D004983:Ethanolamines / Q000737:chemistry; D005822:Genetic Vectors / Q000378:metabolism; D005838:Genotype; D016480:Helicobacter pylori / Q000378:metabolism*; D008050:Lipid A / Q000737:chemistry*; D013058:Mass Spectrometry; D008954:Models, Biological; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013997:Time Factors
D018528:ATP-Binding Cassette Transporters; D023181:Antimicrobial Cationic Peptides; D001426:Bacterial Proteins; D003902:Detergents; D004983:Ethanolamines; D008050:Lipid A; C079726:MsbA protein, Bacteria; D009841:Oligonucleotides; D010710:Phosphates; C005448:phosphorylethanolamine; D004247:DNA; D010744:Phosphoric Monoester Hydrolases