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J. Biol. Chem., Vol. 279, Issue 53, 55780-55791, December 31, 2004

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Periplasmic Cleavage and Modification of the 1-Phosphate Group of Helicobacter pylori Lipid A^*

An X. Tran, Mark J. Karbarz, Xiaoyuan Wang, Christian R. H. Raetz, Sara C. McGrath¶, Robert J. Cotter¶, and M. Stephen Trent||

From the Department of Microbiology, J. H. Quillen College of Medicine, Johnson City, Tennessee 37614, the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, and the ^¶Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

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.

Received for publication, June 10, 2004 , and in revised form, October 1, 2004.

^* This work was supported by National Institutes of Health Grants K22-AI53645 (to M. S. T.), R37-GM51796 (to C. R. H. R.), and RO1-GM6440 (to R. J. C). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Box 70579, Johnson City, TN 37614. Tel.: 423-439-6293; Fax: 423-439-8044; E-mail: trentms{at}mail.etsu.edu.

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