Sialylation of campylobacter jejuni lipo-oligosaccharides: impact on phagocytosis and cytokine production in mice

<p>Background: Guillain-Barré syndrome (GBS) is a post-infectious polyradiculoneuropathy, frequently associated with antecedent Campylobacter jejuni (C. jejuni) infection. The presence of sialic acid on C. jejuni lipo-oligosaccharide (LOS) is considered a risk factor for development of GBS as it crucially determines the structural homology between LOS and gangliosides, explaining the induction of cross-reactive neurotoxic antibodies. Sialylated C. jejuni are recognised by TLR4 and sialoadhesin; however, the functional implications of these interactions in vivo are unknown.</p> <p>Methodology/Principal Findings: In this study we investigated the effects of bacterial sialylation on phagocytosis and cytokine secretion by mouse myeloid cells in vitro and in vivo. Using fluorescently labelled GM1a/GD1a ganglioside-mimicking C. jejuni strains and corresponding (Cst-II-mutant) control strains lacking sialic acid, we show that sialylated C. jejuni was more efficiently phagocytosed in vitro by BM-MΦ, but not by BM-DC. In addition, LOS sialylation increased the production of IL-10, IL-6 and IFN-β by both BM-MΦ and BM-DC. Subsequent in vivo experiments revealed that sialylation augmented the deposition of fluorescent bacteria in splenic DC, but not macrophages. In addition, sialylation significantly amplified the production of type I interferons, which was independent of pDC.</p> <p>Conclusions/Significance: These results identify novel immune stimulatory effects of C. jejuni sialylation, which may be important in inducing cross-reactive humoral responses that cause GBS</p&gt

Campylobacter jejuni Translocation across Intestinal Epithelial Cells Is Facilitated by Ganglioside-Like Lipooligosaccharide Structures

Translocation across intestinal epithelial cells is an established pathogenic feature of the zoonotic bacterial species Campylobacter jejuni. The number of C. jejuni virulence factors known to be involved in translocation is limited. In the present study, we investigated whether sialylation of C. jejuni lipooligosaccharide (LOS) structures, generating human nerve ganglioside mimics, is important for intestinal epithelial translocation. We here show that C. jejuni isolates expressing ganglioside-like LOS bound in larger numbers to the Caco-2 intestinal epithelial cells than C. jejuni isolates lacking such structures. Next, we found that ganglioside-like LOS facilitated endocytosis of bacteria into Caco-2 cells, as visualized by quantitative microscopy using the early and late endosomal markers early endosome-associated protein 1 (EEA1), Rab5, and lysosome-associated membrane protein 1 (LAMP-1). This increased endocytosis was associated with larger numbers of surviving and translocating bacteria. Next, we found that two different intestinal epithelial cell lines (Caco-2 and T84) responded with an elevated secretion of the T-cell attractant CXCL10 to infection by ganglioside-like LOS-expressing C. jejuni isolates. We conclude that C. jejuni translocation across Caco-2 cells is facilitated by ganglioside-like LOS, which is of clinical relevance since C. jejuni ganglioside-like LOS-expressing isolates are linked with severe gastroenteritis and bloody stools in C. jejuni-infected patients

Generation of heat shock protein-based vaccines by intracellular loading of gp96 with antigenic peptides

Several studies have shown that immunization with heal shock proteins (HSPs) purified from tumors of virus-infected cells induces specific cytotoxic T-cell (CTL) activity. This immune response is directed against peptides bound to the HSPs rather than against the HSPs themselves. The peptides are derived from tumor- or virus-specific proteins which are degraded in the course of normal protein turnover and processing for presentation by MHC class I molecules. The HSPs appear to function as carriers for the antigenic peptides. Upon immunization they ensure their uptake by specialized macrophages and their introduction into the MHC class I presentation route which is otherwise accessible only for intracellular proteins. Using influenza virus nucleoprotein (NP) as a model antigen, we have tested whether an HSP-based vaccine can be produced by overexpressing an antigen in cultured cells prior to purification of the HSP's. The transfection system based on the Semliki Forest virus (SFV) replicon was employed to achieve high expression of NP. Since SFV-mediated transfection of murine cells was inefficient we used the hamster-derived cell line BHK21, which can be transfected with 100% efficiency, as a source for NP peptide-loaded gp96. The protein was purified from transfected cells and used for first vaccination studies. The hamster gp96 preparation was well tolerated in mice, an antibody response against the foreign protein was not observed. Preliminary results suggest that a cellular immune response against NP was indeed induced. SFV transfection is applicable for any known antigen and is therefore considered to be an elegant means for the production of HSP-based vaccines capable of inducing a cellular immune response. (C) 1997 Elsevier Science B.V

Morphology of the mitochondria in heat shock protein 60 deficient fibroblasts from mitochondrial myopathy patients:Effects of stress conditions

We have described two mitochondrial (mt) myopathy patients with reduced activities of various mt enzymes associated with significantly decreased amounts of heat shock protein 60 (hsp60). Experimental evidence suggested that the lack of hsp60 was the primary defect. Since hsp60 is essential for the proper folding of enzyme subunits in the mt matrix a partial deficiency of this protein can explain the observed defects of the mitochondria. Here we report on morphological studies aimed at obtaining more insight into the relation between lack of hsp60 and pathological changes of the mitochondria. Under standard culture conditions mitochondria in the partially hsp60 deficient fibroblasts showed profound morphological aberrations. In contrast, the mitochondria in fibroblasts from a MELAS patient and a cytochrome c oxidase-deficient patient appeared normal. Under stress conditions the integrity of the hsp60 deficient mitochondria declined even further: heat shock induced a temporary collapse of the electrochemical potential across the inner mt membrane, but did not affect the ultrastructure of the mitochondria; prolonged growth in confluent cultures resulted in decrease in mt number. The altered mt morphology in the hsp60 deficient cells is probably indicative of the severely impaired mt metabolism whereas the decreased stress tolerance is likely to be a direct result of paucity of the heat shock protein. Both variables are potentially useful in the diagnosis and molecular characterization of mt disorders with systemic manifestation and multiple enzyme deficiency

Mass Spectrometric Analysis of Intact Lipooligosaccharide: Direct Evidence for O-Acetylated Sialic Acids and Discovery of O-Linked Glycine Expressed by Campylobacter jejuni: Biochemistry

The lipooligosaccharides (LOS) of Campylobacter jejuni is an important virulence factor. Its core oligosaccharide component is frequently sialylated and bears a close resemblance with host gangliosides. The display of ganglioside mimics by this bacterium is believed to trigger the onset of the autoimmune condition Guillain-Barre syndrome (GBS) in some individuals. Considerable effort has been directed toward the structural characterization of the glycan component of the LOS of C. jejuni strains isolated from GBS patients. Capillary electrophoresis-mass spectrometry (CE-MS) has been a particularly useful analytical technique applied toward this task. Conventional analysis of bacterial LOS by CE-MS has generally involved the prior removal of O-acyl lipid chains, which is necessary for the effective solubilization and separation of the heterogeneous ensemble of LOS species. Unfortunately, O-deacylation causes the undesired removal of important glycan-associated O-linked modifications, such as O-acetate and O-linked amino acids. In this report, we describe a CE-MS technique developed for the rapid analysis of fully intact LOS from C. jejuni. Using this method, we report the structural characterization of the glycan from 10 GBS-associated strains and two enteritis strains, using material isolated from as little as one colony. The application of this technique has enabled us to unambiguously identify LOS-bound O-acetylated sialic acid in a number of strains and has revealed for the first time that C. jejuni frequently modifies its core with O-linked glycine. Our studies demonstrate that MS-based structural analysis of bacterial LOS can be optimized to the level where only a single-colony quantity of material is required and time-consuming chemical treatments can be avoidedNRC publication: Ye

The sialylated lipooligosaccharide outer core in Campylobacter jejuni is an important determinant for epithelial cell invasion.: Infect.Immun.

NRC publication: Ye

Siglec-7 specifically recognizes Campylobacter jenuni strains associated with oculomotor weakness in Guillain-Barre syndrome and Miller Fisher syndrome

AbstractDue to molecular mimicry, Campylobacter jejuni lipo-oligosaccharides can induce a cross-reactive antibody response to nerve gangliosides, which leads to Guillain–Barré syndrome (GBS). Cross-reactive antibodies to ganglioside GQ1b are strongly associated with oculomotor weakness in GBS and its variant, Miller Fisher syndrome (MFS). Antigen recognition is a crucial first step in the induction of a cross-reactive antibody response, and it has been shown that GQ1b-like epitopes expressed on the surface of C. jejuni are recognized by sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7). We aimed to determine the epitope specificity of C. jejuni binding to Siglec-7, and correlate the outcome to disease symptoms in GBS and MFS patients. Using a well-defined GBS/MFS-associated C. jejuni strain collection, which included three sialic acid knockout strains, we found that Siglec-7 exclusively binds to C. jejuni strains that express terminal disialylated ganglioside mimics. When serological and diagnostic patient records were correlated with the Siglec-7-binding properties, we observed an association between Siglec-7 binding and the presence of anti-GQ1b antibodies in patient serum. In addition, Siglec-7 binding was associated with oculomotor weakness in GBS and MFS patients. Lipo-oligosaccharide-specific binding of C. jejuni to Siglec-7 may be an initiating event in immune recognition and presentation, and lead to anti-GQ1b antibody production and the development of ocular weakness in GBS or MFS

Rapid method for sensitive screening of oligosaccharide epitopes in the lipooligosaccharide from Campylobacter jejuni strains isolated from Guillain-Barre syndrome and Miller Fisher syndrome patients.: J.Clin.Microbiol.

NRC publication: Ye