We recently demonstrated that Campylobacter jejuni produces a capsular polysaccharide (CPS) that is the major antigenic component of the classical Penner serotyping system distinguishing Campylobacter into >60 groups. Although the wide variety of C. jejuni serotypes are suggestive of structural differences in CPS, the genetic mechanisms of such differences are unknown. In this study we sequenced biosynthetic cps regions, ranging in size from 15 to 34 kb, from selected C. jejuni strains of HS:1, HS:19, HS:23, HS:36, HS:23/36 and HS:41 serotypes. Comparison of the determined cps sequences of the HS:1, HS:19 and HS:41 strains with the sequenced strain, NCTC11168 (HS:2), provides evidence for multiple mechanisms of structural variation including exchange of capsular genes and entire clusters by horizontal transfer, gene duplication, deletion, fusion and contingency gene variation. In contrast, the HS:23, HS:36 and HS:23/36 cps sequences were highly conserved. We report the first detailed structural analysis of 81-176 (HS:23/36) and G1 (HS:1) and refine the previous structural interpretations of the HS:19, HS:23, HS:36 and HS:41 serostrains. For the first time, we demonstrate the commonality and function of a second heptose biosynthetic pathway for Campylobacter CPS independent of the pathway for lipooligosaccharide (LOS) biosynthesis and identify a novel heptosyltransferase utilized by this alternate pathway. Furthermore, we show the retention of two functional heptose isomerases in Campylobacter and the sharing of a phosphatase for both LOS and CPS heptose biosynthesis.

Molecular mimicry of Campylobacter jejuni lipo-oligosaccharides (LOS) with gangliosides in nervous tissueis considered to induce cross-reactive antibodies that lead to Guillain-Barré syndrome (GBS), an acute polyneuropathy. To determine whether specific bacterial genes are crucial for the biosynthesis of ganglioside-like structures and the induction of anti-ganglioside antibodies, we characterized the C. jejuni LOS biosynthesis gene locus in GBS-associated and control strains. We demonstrated that specific types of the LOS biosynthesis gene locus are associated with GBS and with the expression of ganglioside-mimicking structures. Campylobacter knockout mutants of 2 potential GBS marker genes, both involved in LOS sialylation, expressed truncated LOS structures without sialic acid, showed reduced reactivity with GBS patient serum, and failed to induce an anti-ganglioside antibody response in mice. We demonstrate, for the first time, to our knowledge, that specific bacterial genes are crucial for the induction of anti-ganglioside antibodies.

Molecular mimicry between lipooligosaccharides (LOS) ofCampylobacter jejuni and gangliosides in peripheral nerves plays a crucial role in the pathogenesis of C. jejuni-related Guillain-Barré syndrome (GBS). We have analyzed the LOS outer core structures of 26 C. jejuni strains associated with GBS and its variant, Miller Fisher syndrome (MFS), by capillary electrophoresis coupled with electrospray ionization mass spectrometry. Sixteen out of 22 (73%) GBS-associated and all 4 (100%) MFS-associated strains expressed LOS with ganglioside mimics. GM1a was the most prevalent ganglioside mimic in GBS-associated strains (10/22, 45%), and in eight of these strains, GM1a was found in combination with GD1a mimics. All seven strains isolated from patients with ophthalmoplegia (GBS or MFS) expressed disialylated (GD3 or GD1c) mimics. Three out of 22 GBS-associated strains (14%) did not express sialylated ganglioside mimics because their LOS locus lacked the genes necessary for sialylation. Three other strains (14%) did not express ganglioside mimics because of frameshift mutations in either the cstII sialyltransferase gene or the cgtB galactosyltransferase gene. It is not possible to determine if these mutations were already present during C. jejuni infection. This is the first report in which mass spectrometry combined with DNA sequence data were used to infer the LOS outer core structures of a large number of neuropathy-associated C. jejuni strains. We conclude that molecular mimicry between gangliosides and C. jejuni LOS is the presumable pathogenic mechanism in most cases of C. jejunirelated GBS. However, our findings suggest that in some cases, other mechanisms may play a role. Further examination of the disease etiology in these patients is mandatory.

The vast majority of proteins are posttranslationally altered, with the addition of covalently linked sugars (glycosylation) being one of the most abundant modifications. However, despite the hydrolysis of protein peptide bonds by peptidases being a process essential to all life on Earth, the fundamental details of how peptidases accommodate posttranslational modifications, including glycosylation, has not been addressed. Through biochemical analyses and X-ray crystallographic structures we show that to hydrolyze their substrates, three structurally related metallopeptidases require the specific recognition of O-linked glycan modifications via carbohydrate-specific subsites immediately adjacent to their peptidase catalytic machinery. The three peptidases showed selectivity for different glycans, revealing protein-specific adaptations to particular glycan modifications, yet always cleaved the peptide bond immediately preceding the glycosylated residue. This insight builds upon the paradigm of how peptidases recognize substrates and provides a molecular understanding of glycoprotein degradation.

Molecular mimicry of Campylobacter jejuni lipo-oligosaccharides (LOS) with gangliosides in nervous tissueis considered to induce cross-reactive antibodies that lead to Guillain-Barré syndrome (GBS), an acute polyneuropathy. To determine whether specific bacterial genes are crucial for the biosynthesis of ganglioside-like structures and the induction of anti-ganglioside antibodies, we characterized the C. jejuni LOS biosynthesis gene locus in GBS-associated and control strains. We demonstrated that specific types of the LOS biosynthesis gene locus are associated with GBS and with the expression of ganglioside-mimicking structures. Campylobacter knockout mutants of 2 potential GBS marker genes, both involved in LOS sialylation, expressed truncated LOS structures without sialic acid, showed reduced reactivity with GBS patient serum, and failed to induce an anti-ganglioside antibody response in mice. We demonstrate, for the first time, to our knowledge, that specific bacterial genes are crucial for the induction of anti-ganglioside antibodies.

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Sugar transport via the phosphoenolpyruvate (PEP) phosphotransferase system involves PEP-dependent phosphorylation of the general phosphotransferase system protein, HPr, at histidine 15. However, grampositive bacteria can also carry out ATP-dependent phosphorylation of HPr at serine 46 by means of (Ser)HPr kinase. In this study, we demonstrate that (Ser)HPr kinase in crude preparations of Streptococcus mutans Ingbritt and Streptococcus salivarius ATCC 25975 is membrane associated, with pH optima of 7.0 and 7.5, respectively. The latter organism possessed 7-to 27-fold-higher activity than S. mutans NCTC 10449, GS-5, and Ingbritt strains. The enzyme in S. salivarius was activated by fructose-1,6-bisphosphate (FBP) twofold with 0.05 mM ATP, but this intermediate was slightly inhibitory with 1.0 mM ATP at FBP concentrations up to 10 mM. Similar inhibition was observed with the enzyme from S. mutans Ingbritt. A variety of other glycolytic intermediates had no effect on kinase activity under these conditions. The activity and regulation of (Ser)HPr kinase were assessed in vivo by monitoring P- ( The principal sugar transport system in oral streptococci is the phosphoenolpyruvate (PEP):sugar phosphotransferase transport system (PTS) (33). The PTS is a group translocation process which utilizes PEP for the phosphorylation of incoming sugars via a phosphoryl transfer process involving the general, non-sugar-specific proteins enzyme I (EI) and HPr and subsequently a sugar-specific, membrane-bound enzyme II (EII) complex, which catalyzes the transport and phosphorylation of the specific carbohydrate (22). During this process, HPr is transiently phosphorylated by P-EI on histidyl residue 15 (His-15) and the phosphate group from phospho-HPr [Pϳ(His)-HPr] is then transferred to the membrane-bound EII complex. The EII complex consists of three functional domains: (i) the IIA domain (also referred to as enzyme III) possessing the first phosphorylation site, (ii) the IIB domain bearing the second phosphorylation site, and (iii) the IIC domain that forms the transmembrane channel and provides the sugar-binding site (30).In 1983, Deutscher and Saier (5) showed that the HPr of Streptococcus pyogenes could also be phosphorylated on a serine residue at the expense of ATP by a specific HPr kinase. This phosphorylation reaction was subsequently found to be widespread among gram-positive but not gram-negative bacteria (22,27) and has even been shown to occur in some species lacking a functional PTS (27). Whereas several observations over the past 10 years have suggested that P-(Ser)-HPr possesses regulatory roles, only recently have some of the physiological functions of this phosphoprotein been determined. These functions include the regulation of glucose and lactose permease activity in Lactobacillus brevis (38, 39), regulation of inducer expulsion in Lactococcus lactis (40), and involvement in catabolite repression in Bacillus subtilis (4). In vitro studies have suggested that the intracellular concentration of P-(Ser)-HPr is contro...

Campylobacter jejuni is well known for synthesizing ganglioside mimics within the glycan component of its lipooligosaccharide (LOS), which have been implicated in triggering GuillainBarré syndrome. We now confirm that this pathogen is capable of synthesizing a much broader spectrum of host glycolipid/ glycoprotein mimics within its LOS. P blood group and paragloboside (lacto-N-neotetraose) antigen mimicry is exhibited by RM1221, a strain isolated from a poultry source. RM1503, a gastroenteritis-associated strain, expresses lacto-N-biose and sialyl-Lewis c units, the latter known as the pancreatic tumorassociated antigen, DU-PAN-2 (or LSTa). C. jejuni GC149, a Guillain-Barré syndrome-associated strain, expresses an unusual sialic acid-containing hybrid oligosaccharide with similarity to both ganglio and P k antigens and can, through phase variation of its LOS biosynthesis genes, display GT1a or GD3 ganglioside mimics. We show that the sialyltransferase CstII and the galactosyltransferase CgtD are involved in the synthesis of multiple mimic types, with LOS structural diversity achieved through evolving allelic substrate specificity.