Abstract
Lipid A molecules of certain Gram-negative bacteria, including Salmonella typhimurium and Pseudomonas aeruginosa, may contain secondary S-2-hydroxyacyl chains. S. typhimurium has recently been shown to synthesize its S-2-hydroxymyristate-modified lipid A in a PhoP/PhoQ-dependent manner, suggesting a possible role for the 2-OH group in pathogenesis. We postulated that 2-hydroxylation might be catalyzed by a novel dioxygenase. Lipid A was extracted from a PhoP-constitutive mutant of S. typhimurium grown in the presence or absence of O(2). Under anaerobic conditions, no 2-hydroxymyristate-containing lipid A was formed. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipid A from cells grown in the presence of (18)O(2) confirmed the direct incorporation of molecular oxygen into 2-hydroxyacyl-modified lipid A. Using several well characterized dioxygenase protein sequences as probes, tBLASTn searches revealed unassigned open reading frame(s) with similarity to mammalian aspartyl/asparaginyl beta-hydroxylases in bacteria known to make 2-hydroxyacylated lipid A molecules. The S. typhimurium aspartyl/asparaginyl beta-hydroxylase homologue (designated lpxO) was cloned into pBluescriptSK and expressed in Escherichia coli K-12, which does not contain lpxO. Analysis of the resulting construct revealed that lpxO expression is sufficient to induce O(2)-dependent formation of 2-hydroxymyristate-modified lipid A in E. coli. LpxO very likely is a novel Fe(2+)/alpha-ketoglutarate-dependent dioxygenase that catalyzes the hydroxylation of lipid A (or of a key precursor). The S. typhimurium lpxO gene encodes a polypeptide of 302 amino acids with predicted membrane-anchoring sequences at both ends. We hypothesize that 2-hydroxymyristate chains released from lipopolysaccharide inside infected macrophages might be converted to 2-hydroxymyristoyl coenzyme A, a well characterized, potent inhibitor of protein N-myristoyl transferase.
Highlights
Lipid A molecules of certain Gram-negative bacteria, including Salmonella typhimurium and Pseudomonas aeruginosa, may contain secondary S-2-hydroxyacyl chains
The ability of S. typhimurium to adapt to the acidic pH and the low divalent cation concentrations found inside macrophage vacuoles is critical to the infection process [6]
Previously thought to be a relatively static structure, recent studies of S. typhimurium and Pseudomonas aeruginosa have demonstrated that lipid A may be modified in a PhoP/PhoQ-dependent manner under conditions that mimic the phagolysosomal environment [22, 23]
Summary
Be attached to the 4Ј- and/or 1-phosphates (Fig. 1B) once the PmrA/PmrB system is activated [22]. In Escherichia coli K-12, modification of lipid A with L-Ara4N, pEtN, and/or palmitate is seen in polymyxin-resistant mutants [26] or in wild type cells treated with metavanadate [27], but 2-hydroxymyristate is not made [28]. We report the discovery, cloning, and heterologous expression of a novel gene from S. typhimurium, designated lpxO, encoding a 302-amino acid polypeptide with significant sequence similarity to mammalian aspartyl/asparaginyl -hydroxylase, an Fe2ϩ/␣-ketoglutarate-dependent dioxygenase [33, 34]. The lpxO gene is highly homologous to a family of bacterial genes that may be responsible for the biosynthesis of 2-hydroxy fatty acids found in lipid A molecules of certain other Gram-negative pathogens, including Klebsiella pneumonia, P. aeruginosa, Bordetella pertussis, Legionella pneumophila, and all types of Salmonella. The release of 2-hydroxyacyl chains from lipid A within phagolysosomes, known to be catalyzed by mammalian acyloxyacyl hydrolase [36], might allow animal cells to synthesize 2-hydroxyacyl-coenzyme A species, some of which are very potent inhibitors of protein N-myristoylation [37]
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