Campylobacter jejuni Lipooligosaccharide Sialylation, Phosphorylation, and Amide/Ester Linkage Modifications Fine-tune Human Toll-like Receptor 4 Activation

Stephenson, Holly; John, Constance M.; Naz, Neveda; Gundogdu, Ozan; Dorrell, Nick; Wren, Brendan W.; Jarvis, Gary A.; Bajaj-Elliott, Mona

doi:10.1074/jbc.m113.468298

Cited by 41 publications

(46 citation statements)

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“…Using a thin-layer matrix composed of THAP and nitrocellulose [31], intact LOS can be analyzed by MALDI MS, generally in the negative-ion mode [9,10,12,13,[30][31][32]35]. This method produces an envelope of (M-H) -ions, as well as informative prompt fragments arising from the lipid A and oligosaccharide domains of the molecule.…”

Section: Molecular Weight Profiling Of Intact Losmentioning

confidence: 99%

“…Recently, an improved method was introduced for the analysis of intact LOS by negative-ion, matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that utilizes a thin layer matrix composed of 2,4,6-trihydroxyacetophenone (THAP) and nitrocellulose [31]. We and others have exploited this methodology to profile complex LOS mixtures without any chemical modifications [9,10,30,32]. In the MS analysis, LOS molecular ions readily undergo Bprompt^fragmentation, a type of insource decay occurring at the sample surface in a few picoseconds to nanoseconds before or during desorption [33], to give fragments arising from the oligosaccharide and lipid A domains of the molecule through cleavage at the labile ketosidic linkage [31,34].…”

mentioning

confidence: 99%

“…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.…”

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

Phillips

John

Jarvis

2016

J. Am. Soc. Mass Spectrom.

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LOS MALDI IMS-MSAbstract. Lipooligosaccharides (LOS) are major microbial virulence factors displayed on the outer membrane of rough-type Gram-negative bacteria. These amphipathic glycolipids are comprised of two domains, a core oligosaccharide linked to a lipid A moiety. Isolated LOS samples are generally heterogeneous mixtures of glycoforms, with structural variability in both domains. Traditionally, the oligosaccharide and lipid A components of LOS have been analyzed separately following mild acid hydrolysis, although important acid-labile moieties can be cleaved. Recently, an improved method was introduced for analysis of intact LOS by MALDI-TOF MS using a thin layer matrix composed of 2,4,6-trihydroxyacetophenone (THAP) and nitrocellulose. In addition to molecular ions, the spectra show in-source Bprompt^fragments arising from regiospecific cleavage between the lipid A and oligosaccharide domains. Here, we demonstrate the use of traveling wave ion mobility spectrometry (TWIMS) for IMS-MS and IMS-MS/MS analyses of intact LOS from Neisseria spp. ionized by MALDI. Using IMS, the singly charged prompt fragments for the oligosaccharide and lipid A domains of LOS were readily separated into resolved ion plumes, permitting the extraction of specific subspectra, which led to increased confidence in assigning compositions and improved detection of less abundant ions. Moreover, IMS separation of precursor ions prior to collision-induced dissociation (CID) generated time-aligned, clean MS/MS spectra devoid of fragments from interfering species. Incorporating IMS into the profiling of intact LOS by MALDI-TOF MS exploits the unique domain structure of the molecule and offers a new means of extracting more detailed information from the analysis.

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Section: Molecular Weight Profiling Of Intact Losmentioning

confidence: 99%

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confidence: 99%

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

Phillips

John

Jarvis

2016

J. Am. Soc. Mass Spectrom.

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“…Current knowledge on LOS diversity has been based primarily on work on C. jejuni and its role in promoting severe clinical symptoms (9)(10)(11)(12). C. jejuni LOS is a potent Toll-like receptor 4 (TLR4) agonist, and the subsequent immune response is affected by changes in LOS structure and composition (10)(11)(12)(13)(14). Additionally, due to molecular mimicry between human gangliosides and certain LOS structures, C. jejuni has been identified as one of the causative agents of Guillain-Barré syndrome (GBS) (15).…”

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Large Sequence Diversity within the Biosynthesis Locus and Common Biochemical Features of Campylobacter coli Lipooligosaccharides

et al. 2016

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Despite the importance of lipooligosaccharides (LOSs) in the pathogenicity of campylobacteriosis, little is known about the genetic and phenotypic diversity of LOS in Campylobacter coli. In this study, we investigated the distribution of LOS locus classes among a large collection of unrelated C. coli isolates sampled from several different host species. Furthermore, we paired C. coli genomic information and LOS chemical composition for the first time to investigate possible associations between LOS locus class sequence diversity and biochemical heterogeneity. After identifying three new LOS locus classes, only 85% of the 144 isolates tested were assigned to a class, suggesting higher genetic diversity than previously thought. This genetic diversity is at the basis of a completely unexplored LOS structural heterogeneity. Mass spectrometry analysis of the LOSs of nine isolates, representing four different LOS classes, identified two features distinguishing C. coli LOS from that of Campylobacter jejuni. 2-Amino-2-deoxy-D-glucose (GlcN)-GlcN disaccharides were present in the lipid A backbone, in contrast to the ␤-1=-6-linked 3-diamino-2,3-dideoxy-D-glucopyranose (GlcN3N)-GlcN backbone observed in C. jejuni. Moreover, despite the fact that many of the genes putatively involved in 3-acylamino-3,6-dideoxy-D-glucose (Quip3NAcyl) were apparently absent from the genomes of various isolates, this rare sugar was found in the outer core of all C. coli isolates. Therefore, regardless of the high genetic diversity of the LOS biosynthesis locus in C. coli, we identified species-specific phenotypic features of C. coli LOS that might explain differences between C. jejuni and C. coli in terms of population dynamics and host adaptation. IMPORTANCEDespite the importance of C. coli to human health and its controversial role as a causative agent of Guillain-Barré syndrome, little is known about the genetic and phenotypic diversity of C. coli LOSs. Therefore, we paired C. coli genomic information and LOS chemical composition for the first time to address this paucity of information. We identified two species-specific phenotypic features of C. coli LOS, which might contribute to elucidating the reasons behind the differences between C. jejuni and C. coli in terms of population dynamics and host adaptation. C ampylobacteriosis is the most common bacterial foodborne disease in developed countries, with over 200,000 human cases being reported annually in the European Union alone (1). The true burden of the disease in the population is likely underestimated, as many infections result in mild gastroenteritis (1). Approximately 80% of reported infections are caused by Campylobacter jejuni, and 7 to 18% of cases are attributed to C. coli. Therefore, C. coli is among the five most important bacterial etiological agents of human gastroenteritis (2, 3).As in other Gram-negative bacteria, Campylobacter species cell surface glycoconjugates, including lipooligosaccharides (LOSs), play an important role in serum and bile resistance; resistanc...

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“…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). Some prompt fragmenta-tion of the LOS occurs during the MS analysis, enabling assignment of proposed compositions for the OS moieties as well as a comparison of the relative ion abundance of the lipid A phosphoforms.…”

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

John

Phillips²,

Din

et al. 2016

Journal of Biological Chemistry

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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...

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Campylobacter jejuni Lipooligosaccharide Sialylation, Phosphorylation, and Amide/Ester Linkage Modifications Fine-tune Human Toll-like Receptor 4 Activation

Cited by 41 publications

References 43 publications

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

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

Large Sequence Diversity within the Biosynthesis Locus and Common Biochemical Features of Campylobacter coli Lipooligosaccharides

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

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