Campylobacter jejuni lipooligosaccharide sialylation, phosphorylation, and amide/ester linkage modifications fine-tune human Toll-like receptor 4 activation

Abstract

Campylobacter jejuni is a leading cause of acute gastroenteritis. C. jejuni lipooligosaccharide (LOS) is a potent activator of Toll-like receptor (TLR) 4-mediated innate immunity. Structural variations of the LOS have been previously reported in the oligosaccharide (OS) moiety, the disaccharide lipid A (LA) backbone, and the phosphorylation of the LA. Here, we studied LOS structural variation between C. jejuni strains associated with different ecological sources and analyzed their ability to activate TLR4 function. MALDI-TOF MS was performed to characterize structural variation in both the OS and LA among 15 different C. jejuni isolates. Cytokine induction in THP-1 cells and primary monocytes was correlated with LOS structural variation in each strain. Additionally, structural variation was correlated with the source of each strain. OS sialylation, increasing abundance of LA d-glucosamine versus 2,3-diamino-2,3-dideoxy-d-glucose, and phosphorylation status all correlated with TLR4 activation as measured in THP-1 cells and monocytes. Importantly, LOS-induced inflammatory responses were similar to those elicited by live bacteria, highlighting the prominent contribution of the LOS component in driving host immunity. OS sialylation status but not LA structure showed significant association with strains clustering with livestock sources. Our study highlights how variations in three structural components of C. jejuni LOS alter TLR4 activation and consequent monocyte activation.

Keywords: Campylobacter; Innate Immunity; Lipopolysaccharide (LPS); Monocytes; Toll-like Receptors (TLR).

FIGURE 1.

Negative ion MALDI-TOF spectra for O-deacylated and intact C. jejuni LOS. Representative MALDI-TOF MS spectra for O-deacylated LOS of strain 40917 (A) and intact LOS of strain 31481 (B). Observed peaks are postulated to be due to Y-type reducing terminal lipid A fragment ions, non-reducing terminal B-type OS fragment ions, and ions for the entire intact or O-deacylated LOS. In the inset in the high resolution spectrum (B), the labeled masses represent the monoisotopic ions. The asterisks in B indicate peaks for sodiated (M + Na − 2H)⁻ ions.

FIGURE 2.

Variation in the number of amide linkages and phosphorylation in C. jejuni LA. A, hexaacylated C. jejuni lipid A with GlcN (R = O) and/or GlcN3N (R = N) disaccharide backbone originally described in Ref. and confirmed in the present study. O-Deacylated LA composed of a GlcN3N-GlcN3N disaccharide backbone with four amide-linked acyl chains (B), a GlcN-GlcN3N disaccharide backbone with three amide-linked acyl chains (C), or a GlcN-GlcN disaccharide backbone with two amide-linked acyl chains (D) is shown. The LA backbone is depicted with two phosphate and two phosphoethanolamine groups, although the abundance of these modifications can vary. Y-type reducing terminal LA fragment ions were observed as shown in the portion of the negative ion MALDI-TOF MS spectra of the O-deacylated LOS of strain 40917 (E) and 31485 (F). G, the relative abundance of amide linkages was assessed by summing the areas of peaks corresponding to four amide linkages and expressing this as a percentage of the areas of all LA peaks observed in the MS analysis. Portions of negative ion MALDI-TOF spectra of the intact lipid of strains 33106 (H) and KJCattle8 (I) in which postulated Y-type LA fragment ions were observed are shown. J, the relative abundance of LA with three or four phosphoryl groups was calculated by summing the area of peaks corresponding to LA with three or four phosphoryl moieties and expressing this as a percentage of the total area of all LA peaks observed.

FIGURE 3.

Impact of C. jejuni LOS sialylation, amide linkages, and phosphorylation on TNFα induction in THP-1 cells. THP-1 cells were stimulated with 10 ng/ml LOS for 20 h, and TNFα levels were assessed by ELISA. TNFα levels were compared with the level of sialylation of the LOS from individual strains (A), levels from neuraminidase-treated LOS (N-treated; B), the relative abundance of four amide linkages (C), and the relative abundances of three or four phosphoryl moieties based on the areas of the respective LA peaks for each molecule (D). Data points represent mean values for an individual strain from a minimum of four independent experiments. A, one-way analysis of variance (*, p < 0.05). B, unpaired Student's t test analysis (ns, not significant). C and D, linear regression analyses were performed.

FIGURE 4.

Combinatorial effect of C. jejuni lipooligosaccharide sialylation, amide linkages, and phosphorylation on TNF-α induction in THP-1 cells. Supernatants from THP-1 cells stimulated with 10 ng/ml isolated LOS (A), 100 ng/ml isolated LOS (B), or live bacteria at a multiplicity of infection (MOI) 10 for 20 h (C) were assessed for TNFα levels by ELISA. To show the combined influence of all three modifications (sialylation, amide linkage, and phosphorylation) on TNFα levels, the relative abundance of two amide and three amide linkages plus 3 or 4 phosphoryl residues were added to either 100 (for strains containing 2 SA residues) or 50 (for strains containing 1 SA residue). TNFα levels were plotted against this relative score out of 300 for these modifications. Data points represent mean values for an individual strain from a minimum of four independent experiments. Linear regression analyses were performed.

FIGURE 5.

C. jejuni LOS modifications modulate TLR4 signaling. THP-1 cells were stimulated with 10 ng/ml LOS for 20 h in the presence of either a 50 or 500 ng/ml concentration of the TLR4 antagonist lipid IVa, and TNFα levels were assessed by ELISA. Data points represent mean values for an individual strain from a minimum of four independent experiments. One-way analysis of variance was performed (**, p < 0.01; *** p < 0.001; ns, not significant).

FIGURE 6.

Combinatorial effect of C. jejuni LOS sialylation, amide linkages, and phosphorylation on induction of TNFα in primary human monocytes. Primary monocytes were stimulated with 10 ng/ml LOS for 20 h, and TNFα was assessed by ELISA. TNFα levels were compared with the level of LOS sialylation (A), the relative abundance of four amide linkages (B), the relative abundance of three or four phosphoryl moieties in each strain (C), and a combination of relative sialylation, abundance of amide linkages, and phosphorylation (D). Data points represent mean values for an individual strain from five donors. A, one-way analysis of variance (*, p < 0.05). B, C, and D, linear regression analyses were performed.