Abstract
Lipopolysaccharide is one of the major constituents of the Gram-negative bacterial outer membrane and is a potent stimulator of the host innate immune response. The biosynthesis of the lipid A moiety of lipopolysaccharide is a complex process in which multiple gene products are involved. Two late lipid A acyl transferases, LpxL and LpxM, were first identified in Escherichia coli and shown to be responsible for the addition of secondary acyl chains to the 2' and 3' positions of lipid A, respectively. Here, we describe the identification of two lpxL homologues in the genome of Bordetella pertussis. We show that one of them, LpxL2, is responsible for the addition of the secondary myristate group that is normally present at the 2' position of B. pertussis lipid A, whereas the other one, LpxL1, mediates the addition of a previously unrecognized secondary 2-hydroxy laurate at the 2 position. Increased expression of lpxL1 results in the appearance of a hexa-acylated lipopolysaccharide form with strongly increased endotoxic activity. In addition, we show that an lpxL1-deficient mutant of B. pertussis displays a defect in the infection of human macrophages.
Highlights
Current knowledge about lipid A biosynthesis is mainly derived from studies in Escherichia coli and Salmonella enterica serovar Typhimurium (Salmonella typhimurium), where the biosynthetic pathway has been completely elucidated
B. pertussis lipid A has been reported to consist of pentaacylated lipid A species containing four primary hydroxylated acyl chains and one secondary acyl chain, i.e. a C14 acyl chain at the 2Ј position [22]
We found that LpxL2, but not LpxL1, was capable of fully restoring the temperature-sensitive phenotype of an E. coli lpxL mutant, indicating that the function of B. pertussis LpxL2 resembles that of E. coli LpxL, and, that LpxL2 mediates the addition of the secondary C14 acyl chain to the 2Ј position of B. pertussis lipid A
Summary
Current knowledge about lipid A biosynthesis is mainly derived from studies in Escherichia coli and Salmonella enterica serovar Typhimurium (Salmonella typhimurium), where the biosynthetic pathway has been completely elucidated. After 4Ј-phosphorylation by LpxK, creating a molecule known as lipid IVA [10], two 2-keto-3-deoxyoctulosonic acid residues are added by KdtA [11], and the secondary acyl chains are added by the late acyltransferases LpxL and LpxM [12, 13]. The late acyltransferase LpxL of E. coli was found to be responsible for the addition of a secondary laurate (C12) moiety to the 2Ј position of lipid A [12, 14]. The structure of B. pertussis lipid A (Fig. 1) resembles that of E. coli It typically consists of a GlcN disaccharide substituted with 3OH C14 residues at positions 2, 2Ј, and 3Ј via ester or OH. Limited information on the genetics of Bordetella lipid A biosynthesis is currently available, and detailed analyses have only been performed for the acyl transferase LpxA and the 2-keto-3-deoxyoctulosonic acid transferase KdtA [23, 24]
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