Profiles of structural heterogeneity in native lipooligosaccharides of Neisseria and cytokine induction

Abstract

Fine differences in the phosphorylation and acylation of lipooligosaccharide (LOS) from Neisseria species are thought to profoundly influence the virulence of the organisms and the innate immune responses of the host, such as signaling through toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells (TREM). MALDI time-of-flight (TOF) mass spectrometry was used to characterize heterogeneity in the native LOS from Neisseria gonorrheae and N. meningitidis. A sample preparation methodology previously reported for Escherichia coli lipopolysaccharide (LPS) employing deposition of untreated LOS on a thin layer of a film composed of 2,4,6-trihydroxyacetophenone and nitrocellulose was used. Prominent peaks were observed corresponding to molecular ions and to fragment ions primarily formed by cleavage between the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and the lipid A (LA). Analyses of these data and comparison with spectra of the corresponding Odeacylated or hydrogen fluoride-treated LOS enabled the detection of novel species that apparently differed by the expression of up to three phosphates with one or more phosphoethanolamine (PEA) groups on the LA. We found that the heterogeneity profile of acylation and phosphorylation correlates with the induction of proinflammatory cytokines in THP-1 monocytic cells. This methodology enabled us to rapidly profile components of structural variants of native LOS that are of importance biologically.-John, C. M., M. Liu, and G. A. Jarvis. Profiles of structural heterogeneity in native lipooligosaccharides of Neisseria and cytokine induction. J. Lipid Res. 2009. 50: 424-438. Supplementary key words lipooligosaccharideThe lipooligosaccharide (LOS) of Neisseria gonorrheae and N. meningitidis is structurally related to the lipopolysaccharide (LPS) of enteric Gram-negative bacteria but does not have repeating O-antigens (1). LOS and LPS have conserved inner cores composed of L-glycero-D-manno-heptose (Hep) and 3-deoxy-D -manno-oct-2-ulosonic acid (Kdo), which are anchored in the outer membrane by lipid A (LA). The structure of the LA of gonococci and meningococci, although based on a limited number of studies, generally differs from that of Escherichia coli in the acylation and the chain length of the fatty acid residues. With regard to phosphorylation, a limited number of studies have shown that gonococci and meningococci elaborate LOS containing a LA, which is phosphorylated with variable numbers of phosphate (P) and phosphoethanolamine (PEA) residues. The recent report of a PEA transferase specific for the LA on meningococcal LOS creates the possibility of phase variation in phosphorylation (2). Thus, the LA expressed by these organisms exhibit heterogeneity in acylation and phosphorylation, both of which have been shown to influence the endotoxicity in E. coli LPS (3).The innate immune response to bacterial pathogens relies on the detection of pathogen-associated molecular patterns by pattern recognition molecules. Toll-like receptor 4 (TLR4) is one of...

Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding

“…The addition of PEA to N. flavescens lipid A resulted in an increased cytokine response from THP-1 cells (SI Appendix, Fig. S4) (25)(26)(27). These results confirmed that the recombinant DDM solubilized enzyme was enzymatically active and able to induce a cytokine response.…”

Human Lipooligosaccharide IGG That Prevents Endemic Meningococcal Disease Recognizes an Internal Lacto-N-neotetraose Structure

“…MS of intact LOS of the group C strain, 89I, did find multiple lipid phosphoforms (42,43), but this might have been an exception. What effects, if any, the different lipids might have on glycose antigen formation (48,49) were not explored.…”

“…Mass Spectrometry-Intact 7946 and 8024 LOS and O-deacylated LOS of 7946 and its ⌬lst and ⌬lgtB mutants were analyzed by negative ion matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), as described (40,42,43). MALDI-TOF MS was performed in the linear as well as the reflectron mode on a Voyager-DE STR instrument equipped with a 337-nm nitrogen laser and delayed extraction.…”

“…The spectra of the intact LOS were externally calibrated using the average fragment and molecular ion masses of the intact 1291wt LOS at m/z 1836.3, m/z 1881.6, and m/z 3718.9; a one-point internal calibration using the mass of the lipid A fragment ions at m/z 1836.3 also was used for the 8024 spectrum. The spectra of the O-deacylated 7946, 7946⌬lst, and 7946⌬lgtB LOS were acquired and externally calibrated using ions for intact 7889 LOS at m/z 1836.3 and m/z 3676.6 or 89I LOS at m/z 1738.3 and m/z 4090.1 (42,43), and then internally calibrated using either one or two calculated masses for peaks at m/z 952.0, m/z 1677.4, m/z 1839.9, and m/z 2792.6. Structures were assigned to molecular ions on the basis of composition, previously published structures (12, 15-18, 20, 42-44), L-type (19), and the genetics of LOS synthesis (45).…”

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

“…The LOS was purified, and the relative abundances of the lipid A phosphoforms were determined. We have previously shown that there can be losses of phosphoryl substituents from lipid A when the OS moiety has been cleaved by acidic hydrolysis of LOS and when the LOS is de-O-acylated to remove O-linked acyl groups (10). Thus, we analyzed the LOS intact using previously described methods on a Synapt G2 HDMS system for high mass resolution negative-ion matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) (10,11).…”