Fatty acids of Helicobacter pylori lipoproteins CagT and Lpp20

Abstract

Bacterial lipoproteins are post-translationally modified by the addition of acyl chains that anchor the protein to bacterial membranes. This modification includes two ester-linked and one amide-linked acyl chain on lipoproteins from Gram-negative bacteria. Helicobacter pylori lipoproteins have important functions in pathogenesis (including delivering the CagA oncoprotein to mammalian cells) and are recognized by host innate and adaptive immune systems. The number and variety of acyl chains on lipoproteins impact the innate immune response through Toll-like receptor 2. The acyl chains added to lipoproteins are derived from membrane phospholipids. H. pylori membrane phospholipids have previously been shown to consist primarily of C14:0 and C19:0 cyclopropane-containing acyl chains. However, the acyl composition of H. pylori lipoproteins has not been determined. In this study, we characterized the acyl composition of two representative H. pylori lipoproteins, Lpp20 and CagT. Fatty acid methyl esters were prepared from both purified lipoproteins and analyzed by gas chromatography-mass spectrometry. For comparison, we also analyzed H. pylori phospholipids. Consistent with previous studies, we observed that the H. pylori phospholipids contain primarily C14:0 and C19:0 cyclopropane-containing fatty acids. In contrast, both the ester-linked and amide-linked fatty acids found in H. pylori lipoproteins were observed to be almost exclusively C16:0 and C18:0. A discrepancy between the acyl composition of membrane phospholipids and lipoproteins as reported here for H. pylori has been previously reported in other bacteria including Borrelia and Brucella. We discuss possible mechanisms.IMPORTANCEColonization of the stomach by Helicobacter pylori is an important risk factor in the development of gastric cancer, the third leading cause of cancer-related death worldwide. H. pylori persists in the stomach despite an immune response against the bacteria. Recognition of lipoproteins by TLR2 contributes to the innate immune response to H. pylori. However, the role of H. pylori lipoproteins in bacterial persistence is poorly understood. As the host response to lipoproteins depends on the acyl chain content, defining the acyl composition of H. pylori lipoproteins is an important step in characterizing how lipoproteins contribute to persistence.

Keywords: Helicobacter pylori; Toll-like receptor 2; acyltransferases; lipoproteins; phospholipids; posttranslational protein modification.

Fig 1

Lipoprotein synthesis. The post-translational modification of lipoproteins is carried out by three membrane-embedded enzymes. First, a short cysteine-containing amino acid sequence known as a lipobox is recognized by prolipoprotein diacylglyceryl transferase (Lgt) which transfers a diacylglyceride with two ester-linked acyl chains from a membrane lipid to the cysteine sulfhydryl of the preprolipoprotein. Second, prolipoprotein signal peptidase (LspA, signal peptidase II) cleaves the amino acids preceding the cysteine, resulting in a diacylated apolipoprotein. Finally, apolipoprotein N-acyltransferase (Lnt) transfers an acyl chain from a membrane lipid to the amino-terminal cysteine via an amide bond to produce the mature triacylated lipoprotein. For simplicity, the lipoprotein is illustrated as uniformly acylated with C16:0.

Fig 2

Gas chromatograms of fatty-acid methyl esters (FAMEs). (A and B) Commercially available FAMEs were combined to form two standard preparations containing palmitic, stearic, arachidonic, behenic, and lignoceric acid methyl esters (A), or myristic, palmitic, and cis-9,10-methyleneoctadecanoic acid methyl esters (B). (C) H. pylori phospholipids were extracted and converted to FAMEs as described in Materials and Methods. The resulting FAMEs were analyzed by GC-MS. The results are representative of four or more independent samples. The chemical identities were determined based on elution relative to standards and confirmed based on the fragmentation patterns (data not shown).

Fig 3

GC-MS analysis of fatty-acid methyl esters derived from H. pylori lipoproteins. (A and B) Coomassie-stained gels of purified Lpp20-HA (A) and CagT-DDK (B). (C and D) The acyl chains were hydrolyzed from purified Lpp20-HA and CagT-DDK and converted to FAMEs as described in Materials and Methods. The resulting FAMEs were analyzed by GC-MS (C, Lpp20-HA; D, CagT-DDK). The gas chromatograms are representative of three or more independent samples of each lipoprotein. The elution times for the 6.9- and 7.9-minute peaks are consistent with the elution times of palmitic acid methyl ester and stearic acid methyl ester standards, respectively (Fig. 2). Analysis of the MS fragmentation patterns of molecules eluting at 9.4 and 9.7 minutes suggests that these are contaminants (e.g., from labware) rather than CagT-DDK-derived fatty acid methyl esters. (E and F) MS fragmentation patterns of the 6.9-minute peak (E) and 7.9-minute peak (F) are consistent with the expected fragmentation patterns of palmitic acid methyl ester (E) and stearic acid methyl ester (F). Insets illustrate characteristic fragment ions seen in FAMES. The series of ions uniformly 14 amu apart (e.g., at m/z = 87, 101, 115, 129, 143, 157, 199, and so forth) is evidence that there are unlikely to be other functional groups in the acyl chain.

Fig 4

Gas chromatograms of ester- and amide-linked fatty-acid methyl esters. (A) The ester-linked acyl chains of CagT-DDK were hydrolyzed under alkaline conditions and converted to FAMES for analysis by GC-MS. The chemical identities were determined based on elution relative to standards and confirmed based on the fragmentation patterns (data not shown). Results reveal a mixture of palmitic and stearic acids. Similar results were obtained when analyzing acyl chains released from Lpp20-HA by lipoprotein lipase and when analyzing CagT-DDK and Lpp20-HA isolated from lnt mutant H. pylori (which contain the ester-linked acyl chains but not the amide-linked acyl chain, data not shown). (B) CagT-DDK remaining following alkaline hydrolysis (panel A) was treated under acidic conditions to hydrolyze the amide-linked acyl chain which was subsequently converted to FAMES and analyzed by GC-MS. Results reveal a mixture of palmitic and stearic acids. Similar results were obtained when analyzing CagT-DDK which had first been treated with lipoprotein lipase to remove ester-linked acyl chains and then treated under acidic conditions to hydrolyze the amide-linked acyl chain. Similar results also were obtained by treating Lpp20-HA with lipoprotein lipase to remove the ester-linked fatty acids, followed by acid hydrolysis of the remaining amide-linked fatty acid (data not shown).