Natural Phosphoryl and Acyl Variants of Lipid A from Neisseria meningitidis Strain 89I Differentially Induce Tumor Necrosis Factor-α in Human Monocytes

Constance M John,Mingfeng Liu,Gary A Jarvis

doi:10.1074/jbc.m109.004887

Abstract

The native lipooligosaccharide (LOS) from Neisseria meningitidis strain 89I was analyzed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry and the spectrum compared with that of the LOS after O-deacylation and hydrogen fluoride treatment. The data are consistent with the presence of natural variations in the LOS, which include a triphosphorylated lipid A (LA) with and without a phosphoethanolamine group, and both hexa- and pentaacylated LA molecules. Thin-layer chromatography was performed on 89I LA produced by hydrolysis of the LOS, and the purified LA molecules were analyzed by MALDI-TOF and tested for their relative ability to induce the secretion of tumor necrosis factor-alpha by human monocytic THP-1 cells and primary human monocytes. The potency of tumor necrosis factor-alpha induction varied by approximately 2-10-fold, depending on the state of acylation and phosphorylation. The results highlight the significance of phosphorylation along with acylation of the LA component of LOS in stimulation of inflammatory signaling, and suggest that natural strain variation in these moieties may be a feature of meningococcal bacteria, which is of critical importance to the progression of the infection.

Highlights

The lipid A (LA)2 portion of the lipopolysaccharide (LPS) or lipooligosaccharide (LOS) of Gram-negative bacteria is an inflammatory, pathogenic component of the bacterial outer membrane (1–3)
Recent work has shown that whereas hexaacyl monophosphoryl LA was restricted to the myeloid differentiation factor 88 (MyD88)-independent pathway, which resulted in T cell activation, the diphosphoryl LA engaged the MyD88-dependent pathway, which activated NF-␬B resulting in the production of TNF-␣ and other inflammatory cytokines (15)
matrix-assisted laser desorption ionization (MALDI)-TOF mass spectrometry (MS) of Native 89I LOS—Our analysis of the native LOS from N. meningitidis strain 89I was performed using a thin layer of 2,4,6-trihydroxyacetophenone/nitrocellulose matrix as previously described for negative-ion MALDI-TOF (23)

Summary

Introduction

The lipid A (LA)2 portion of the lipopolysaccharide (LPS) or lipooligosaccharide (LOS) of Gram-negative bacteria is an inflammatory, pathogenic component of the bacterial outer membrane (1–3). 2 PEA groups, 2P, and a single Ac compared with the molecular ion peak in Fig. 1 for the native LOS at

Results

Conclusion