Lipooligosaccharide Structures of Invasive and Carrier Isolates of Neisseria meningitidis Are Correlated with Pathogenicity and Carriage

Abstract

The degree of phosphorylation and phosphoethanolaminylation of lipid A on neisserial lipooligosaccharide (LOS), a major cell-surface antigen, can be correlated with inflammatory potential and the ability to induce immune tolerance in vitro. On the oligosaccharide of the LOS, the presence of phosphoethanolamine and sialic acid substituents can be correlated with in vitro serum resistance. In this study, we analyzed the structure of the LOS from 40 invasive isolates and 25 isolates from carriers of Neisseria meningitidis without disease. Invasive strains were classified as groups 1-3 that caused meningitis, septicemia without meningitis, and septicemia with meningitis, respectively. Intact LOS was analyzed by high resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Prominent peaks for lipid A fragment ions with three phosphates and one phosphoethanolamine were detected in all LOS analyzed. LOS from groups 2 and 3 had less abundant ions for highly phosphorylated lipid A forms and induced less TNF-␣ in THP-1 monocytic cells compared with LOS from group 1. Lipid A from all invasive strains was hexaacylated, whereas lipid A of 6/25 carrier strains was pentaacylated. There were fewer O-acetyl groups and more phosphoethanolamine and sialic acid substitutions on the oligosaccharide from invasive compared with carrier isolates. Bioinformatic and genomic analysis of LOS biosynthetic genes indicated significant skewing to specific alleles, dependent on the disease outcome. Our results suggest that variable LOS structures have multifaceted effects on homeostatic innate immune responses that have critical impact on the pathophysiology of meningococcal infections.Our previous studies of neisserial lipooligosaccharide (LOS) 2 and the human innate immune system have shown that the degree of phosphorylation of the lipid A component is correlated with the potential of the LOS to induce inflammation stimulated by innate immunity as revealed by cytokine induction in human monocytes in vitro and, in general, with the severity of infections (1-3). In the LOS of Neisseria meningitidis, the modification of lipid A with phosphoethanolamine (PEA) also has been shown to inhibit the bactericidal activity of cathepsin G within neutrophil extracellular traps (4) and to increase adhesion of the bacteria to human cells (5). Expression of PEA on the oligosaccharide (OS) of meningococcal LOS (Fig. 1), particularly in the O-3 position on heptose (Hep) II, or expression of sialic acid (Neu5Ac) have been shown to inhibit activation of complement via the classical and alternative pathways (6 -9). In this study, we postulated that analysis of the structures of the LOS and the genomic diversity of genes for LOS biosynthesis would reveal differences associated with the severity of meningococcal infection and with carrier versus invasive strains of N. meningitidis.To ascertain whether such differences exist, we characterized the structures of LOS from isolates from infected patients or from carriers of N. meningitidi...

Analysis of Bacterial Lipooligosaccharides by MALDI-TOF MS with Traveling Wave Ion Mobility

“…Lacto-N-neotetraose (Galβ1→4GlcNAcβ1→3Galβ1→4Glc) expression is associated with greater ability of N. gonorrhoeae to infect men [19], and induces bactericidal IgG for L2, L3, L4, and L7 N. meningitidis immunotypes [20,21]. Our data show that addition of Neu5Ac, acetate, and PEA to the oligosaccharide moieties of LOS impacts the pathophysiology of infection with N. meningitidis [13]. The expression of PEA, particularly on O-3 of HepII, or of Neu5Ac on the oligosaccharide of meningococcal LOS, inhibits complement activation [20][21][22][23].…”

“…N. meningitidis serogroup B strain 701 is an isolate from a throat culture of a carrier of N. meningitidis without disease obtained during a serogroup B vaccination trial in Norway. We recently characterized the LOS from a large sampling of invasive and carrier strains of N. meningitidis from this study in order to correlate clinical outcomes with LOS structural features [13]. N. flavescens strain 4322 was available in the Center for Immunochemistry from the collection of the late Herman Schneider, formerly of the Walter Reed Army Institute of Research, and has been described previously [12].…”

“…Modification of lipid A with PEA increases resistance of N. gonorrhoeae to complement killing and increases survival of both gonococci and meningococci in vivo [8]. We have shown that the degree of phosphorylation of lipid A of Neisserial LOS is correlated with its inflammatory potential [9][10][11][12][13] and appears to play a role in the immune privilege enjoyed by commensal Neisseria [12,14].…”

“…As we have previously reported [9,10,12,13], intact LOS was deposited on top of a prespotted matrix layer for MALDI-TOF MS analysis. The matrix solution was prepared by mixing a solution of THAP (200 mg/mL in methanol; Sigma-Aldrich) with a solution of nitrocellulose transblot membrane (15 mg/ mL in acetone/isopropanol, 1:1; Bio-Rad) in a 3:1 (v/v) ratio.…”

“…Prompt fragments representing both domains of the molecule corroborate proposed compositions of the LOS molecular ions detected. Although previously only conducted in linear mode, analysis of intact LOS by high-resolution, reflectron mode MALDI-TOF MS has now been achieved [12,13,32,35]. The ability to measure monoisotopic molecular ions with good mass accuracy (<20 ppm) has dramatically improved the quality of the information that can be obtained from MALDI-TOF MS analysis of intact LOS.…”