Acyl Chain Specificity of the Acyltransferases LpxA and LpxD and Substrate Availability Contribute to Lipid A Fatty Acid Heterogeneity in Porphyromonas gingivalis

Abstract

Porphyromonas gingivalis lipid A is heterogeneous with regard to the number, type, and placement of fatty acids. Analysis of lipid A by matrix-assisted laser desorption ionization-time of flight mass spectrometry reveals clusters of peaks differing by 14 mass units indicative of an altered distribution of the fatty acids generating different lipid A structures. To examine whether the transfer of hydroxy fatty acids with different chain lengths could account for the clustering of lipid A structures, P. gingivalis lpxA (lpxA Pg ) and lpxD Pg were cloned and expressed in Escherichia coli strains in which the homologous gene was mutated. Lipid A from strains expressing either of the P. gingivalis transferases was found to contain 16-carbon hydroxy fatty acids in addition to the normal E. coli 14-carbon hydroxy fatty acids, demonstrating that these acyltransferases display a relaxed acyl chain length specificity. Both LpxA and LpxD, from either E. coli or P. gingivalis, were also able to incorporate odd-chain fatty acids into lipid A when grown in the presence of 1% propionic acid. This indicates that E. coli lipid A acyltransferases do not have an absolute specificity for 14-carbon hydroxy fatty acids but can transfer fatty acids differing by one carbon unit if the fatty acid substrates are available. We conclude that the relaxed specificity of the P. gingivalis lipid A acyltransferases and the substrate availability account for the lipid A structural clusters that differ by 14 mass units observed in P. gingivalis lipopolysaccharide preparations.Porphyromonas gingivalis is a gram-negative, asaccharolytic, anaerobic bacterium that is strongly associated with adult periodontitis (12,18,27). Its virulence factors include proteases (19,34,46), fimbria (2, 22, 54), hemagglutinins (17,23,35), and lipopolysaccharide (LPS) (3, 11, 13). LPS is a major component of the outer membrane of gram-negative bacteria. LPS contributes to the integrity of the bacterial cell envelope and is a strong modulator of the host innate immune system (3,5,9,10,13,14,16). Both positive and negative modulations of the host response by LPS are believed to play a role in the development of various infectious conditions (13,15,21,24,28,32,33,44,50). Most of this activity is known to be contained in the lipid portion of the LPS molecule known as lipid A (41, 51). Escherichia coli lipid A structure consists of a glucosamine disaccharide phosphorylated at the 1 and 4Ј positions and is substituted with four ␤-hydroxy-myristates in amide linkage (2 and 2Ј positions) and in ester linkage (3 and 3Ј positions). The structure also contains a laurate in the secondary ester linkage on the 2Ј ␤-hydroxy-myristate and a myristate in the secondary linkage on the 3Ј ␤-hydroxy-myristate to generate the hexa-acyl lipid A canonical structure (51).P. gingivalis LPS is significantly different from E. coli in that it consists of a heterogeneous mixture of lipid A structures that differ in the type and number of fatty acids and the number of phosphate groups (1,26,40). Com...

LPS remodeling is an evolved survival strategy for bacteria

“…Acyl chain alterations were confirmed by gas chromatography (GC) analysis ( Fig. S1A) (26). These results clearly demonstrate that Fn modifies its lipid A structure in response to temperature adaptation by altering the length of the amidelinked acyl chains: 3-OH-C16 at environmental temperature and 3-OH-C18 at mammalian temperature.…”

Structural Analysis of the Core Region of O-Lipopolysaccharide of Porphyromonas gingivalis from Mutants Defective in O-Antigen Ligase and O-Antigen Polymerase

“…While the importance of the proinflammatory properties of the LPS of P. gingivalis in periodontal disease has been recognized for some time, it is only recently that the complexity of the structural isoforms of this molecule in this organism has been acknowledged (4,6,51). The structural variation includes significant changes in the lipid A structure in terms of the degree of acylation and phosphorylation and the presence of two types of repeating units (49).…”

Assembly of Lipopolysaccharide in Escherichia coli Requires the Essential LapB Heat Shock Protein

“…It should be noted that ⌬(lapB waaC) mutant exhibits the same carbon chain polymorphism. This carbon chain polymorphism can be explained if ⌬(lapB waaC) mutant accumulates odd chain fatty acids because they can also be substrates for LpxA and LpxD (47). Also, underacylation has been reported to cause accumulation of mass peaks with similar 14-mass unit differences (48).…”