Astrid P. Heikema

The crucial role of Campylobacter jejuni genes in anti-ganglioside antibody induction in Guillain-Barre syndrome

Molecular mimicry of Campylobacter jejuni lipo-oligosaccharides (LOS) with gangliosides in nervous tissue is considered to induce cross-reactive antibodies that lead to Guillain-Barre 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.

The Sialylated Lipooligosaccharide Outer Core in Campylobacter jejuni Is an Important Determinant for Epithelial Cell Invasion

ABSTRACT Campylobacter jejuni is a frequent cause of bacterial gastroenteritis worldwide. Lipooligosaccharide (LOS) has been identified as an important virulence factor that may play a role in microbial adhesion and invasion. Here we specifically address the question of whether LOS sialylation affects the interaction of C. jejuni with human epithelial cells. For this purpose, 14 strains associated with Guillain-Barré syndrome (GBS), 34 enteritis-associated strains, the 81-176 reference strain, and 6 Penner serotype strains were tested for invasion of two epithelial cell lines. C. jejuni strains expressing sialylated LOS (classes A, B, and C) invaded cells significantly more frequently than strains expressing nonsialylated LOS (classes D and E) (P < 0.0001). To further explore this observation, we inactivated the LOS sialyltransferase (Cst-II) via knockout mutagenesis in three GBS-associated C. jejuni strains expressing sialylated LOS (GB2, GB11, and GB19). All knockout strains displayed significantly lower levels of invasion than the respective wild types. Complementation of a Δcst-II mutant strain restored LOS sialylation and reset the invasiveness to wild-type levels. Finally, formalin-fixed wild-type strains GB2, GB11 and GB19, but not the isogenic Δcst-II mutants that lack sialic acid, were able to inhibit epithelial invasion by viable GB2, GB11, and GB19 strains. We conclude that sialylation of the LOS outer core contributes significantly to epithelial invasion by C. jejuni and may thus play a role in subsequent postinfectious pathologies.

A novel link between Campylobacter jejuni bacteriophage defence, virulence and Guillain-Barré syndrome

Guillain–Barré syndrome (GBS) is a post-infectious disease in which the human peripheral nervous system is affected after infection by specific pathogenic bacteria, including Campylobacter jejuni. GBS is suggested to be provoked by molecular mimicry between sialylated lipooligosaccharide (LOS) structures on the cell envelope of these bacteria and ganglioside epitopes on the human peripheral nerves, resulting in autoimmune-driven nerve destruction. Earlier, the C. jejuni sialyltransferase (Cst-II) was found to be linked to GBS and demonstrated to be involved in the biosynthesis of the ganglioside-like LOS structures. Apart from a role in pathogenicity, we report here that Cst-II-generated ganglioside-like LOS structures confer efficient bacteriophage resistance in C. jejuni. By bioinformatic analysis, it is revealed that the presence of sialyltransferases in C. jejuni and other potential GBS-related pathogens correlated significantly with the apparent degeneration of an alternative anti-virus system: type II Clusters of Regularly Interspaced Short Palindromic Repeat and associated genes (CRISPR-Cas). Molecular analysis of the C. jejuni CRISPR-Cas system confirmed the bioinformatic investigation. CRISPR degeneration and mutations in the cas genes cas2, cas1 and csn1 were found to correlate with Cst-II sialyltransferase presence (p < 0.0001). Remarkably, type II CRISPR-Cas systems are mainly found in mammalian pathogens. To study the potential involvement of this system in pathogenicity, we inactivated the type II CRISPR-Cas marker gene csn1, which effectively reduced virulence in primarily cst-II-positive C. jejuni isolates. Our findings indicate a novel link between viral defence, virulence and GBS in a pathogenic bacterium.

Characterization of the Specific Interaction between Sialoadhesin and Sialylated Campylobacter jejuni Lipooligosaccharides

ABSTRACT In Campylobacter jejuni-induced Guillain-Barré syndrome (GBS), molecular mimicry between C. jejuni lipooligosaccharide (LOS) and host gangliosides leads to the production of cross-reactive antibodies directed against the peripheral nerves of the host. Currently, the presence of surface exposed sialylated LOS in C. jejuni is the single known bacterial pathogenesis factor associated with the development of GBS. Using a unique, well-characterized strain collection, we demonstrate that GBS-associated C. jejuni strains bind preferentially to sialoadhesin (Sn, Siglec-1, or CD169), a sialic acid receptor found on a subset of macrophages. In addition, using a whole-cell enzyme-linked immunosorbent assay (ELISA), C. jejuni strains with sialylated LOS bound exclusively to soluble Sn. Mass spectrometry revealed that binding was sialic acid-linkage specific with a preference for α(2,3)-linked sialic acid attached to the terminal galactose of the LOS chain as seen in the gangliosides GD1a, GM1b, and GM3. This molecular interaction was also related to functional consequences as a GBS-associated C. jejuni strain that bound Sn in a whole-cell ELISA adhered to surface-expressed Sn of Sn-transfected CHO cells but was unable to adhere to wild-type CHO cells. Moreover, a sialic acid-negative mutant of the same C. jejuni strain was unable to bind Sn-transfected CHO cells. This is the first report of the preferential binding of GBS-associated C. jejuni strains to the Sn immune receptor (P = 0.014). Moreover, because this binding is dependent on sialylated LOS, the main pathogenic factor in GBS progression, the present findings bring us closer to unraveling the mechanisms that lead to formation of cross-reactive antibodies in GBS disease.

TLR4-Mediated Sensing of Campylobacter jejuni by Dendritic Cells Is Determined by Sialylation

In Guillain-Barré syndrome (GBS), ganglioside mimicry of Campylobacter jejuni lipo-oligosaccharide (LOS) drives the production of cross-reactive Abs to peripheral nerve gangliosides. We determined whether sialic acid residues in C. jejuni LOS modulate dendritic cell (DC) activation and subsequent B cell proliferation as a possible mechanism for the aberrant humoral immune response in GBS. Highly purified sialylated LOS of C. jejuni isolates from three GBS patients induced human DC maturation and secretion of inflammatory cytokines that were inhibited by anti-TLR4 neutralizing Abs. The extent of TLR4 signaling and DC activation was greater with LOS of the wild type isolates than with nonsialylated LOS of the corresponding sialyltransferase gene knockout (cst-II mutant) strains, indicating that sialylation boosts the DC response to C. jejuni LOS. Supernatants of LOS-activated DCs induced B cell proliferation after cross-linking of surface Igs in the absence of T cells. Lower B cell proliferation indices were found with DC supernatants after DC stimulation with cst-II mutant or neuraminidase desialylated LOS. This study showed that sialylation of C. jejuni LOS enhances human DC activation and subsequent B cell proliferation, which may contribute to the development of cross-reactive anti-ganglioside Abs found in GBS patients following C. jejuni infection.

Campylobacter jejuni lipooligosaccharides modulate dendritic cell-mediated T cell polarization in a sialic acid linkage-dependent manner

ABSTRACT Carbohydrate mimicry between Campylobacter jejuni lipooligosaccharides (LOS) and host neural gangliosides plays a crucial role in the pathogenesis of Guillain-Barré syndrome (GBS). Campylobacter jejuni LOS may mimic various gangliosides, which affects the immunogenicity and the type of neurological deficits in GBS patients. Previous studies have shown the interaction of LOS with sialic acid-specific siglec receptors, although the functional consequences remain unknown. Cells that express high levels of siglecs include dendritic cells (DCs), which are crucial for initiation and differentiation of immune responses. We confirm that α2,3-sialylated GD1a/GM1a mimic and α2,8-sialylated GD1c mimic LOS structures interact with recombinant Sn and siglec-7, respectively. Although the linkage of the terminal sialic acid of LOS did not regulate expression of DC maturation markers, it displayed clear opposite expression levels of interleukin-12 (IL-12) and OX40L, molecules involved in DC-mediated Th cell differentiation. Accordingly, targeting DC-expressed siglec-7 with α2,8-linked sialylated LOS resulted in Th1 responses, whereas Th2 responses were induced by targeting with LOS containing α2,3-linked sialic acid. Thus, our data demonstrate for the first time that depending on the sialylated composition of Campylobacter jejuni LOS, specific Th differentiation programs are initiated, possibly through targeting of distinct DC-expressed siglecs.

Sialoadhesin promotes rapid proinflammatory and type I IFN responses to a sialylated pathogen, Campylobacter jejuni

Sialoadhesin (Sn) is a macrophage (Mϕ)-restricted receptor that recognizes sialylated ligands on host cells and pathogens. Although Sn is thought to be important in cellular interactions of Mϕs with cells of the immune system, the functional consequences of pathogen engagement by Sn are unclear. As a model system, we have investigated the role of Sn in Mϕ interactions with heat-killed Campylobacter jejuni expressing a GD1a-like, sialylated glycan. Compared to Sn-expressing bone marrow-derived macrophages (BMDM) from wild-type mice, BMDM from mice either deficient in Sn or expressing a non-glycan–binding form of Sn showed greatly reduced phagocytosis of sialylated C. jejuni. This was accompanied by a strong reduction in MyD88-dependent secretion of TNF-α, IL-6, IL-12, and IL-10. In vivo studies demonstrated that functional Sn was required for rapid TNF-α and IFN-β responses to i.v.-injected sialylated C. jejuni. Bacteria were captured within minutes after i.v. injection and were associated with Mϕs in both liver and spleen. In the spleen, IFN-β–reactive cells were localized to Sn+ Mϕs and other cells in the red pulp and marginal zone. Together, these studies demonstrate that Sn plays a key role in capturing sialylated pathogens and promoting rapid proinflammatory cytokine and type I IFN responses.

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

ABSTRACT 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.

Enhanced, sialoadhesin-dependent uptake of Guillain-Barre syndrome-associated Campylobacter jejuni strains by human macrophages.

ABSTRACT Molecular mimicry between Campylobacter jejuni sialylated lipooligosaccharides (LOS) and human nerve gangliosides can trigger the production of cross-reactive antibodies which induce Guillain-Barré syndrome (GBS). To better understand the immune events leading to GBS, it is essential to know how sialylated LOS are recognized by the immune system. Here, we show that GBS-associated C. jejuni strains bind to human sialoadhesin (hSn), a conserved, mainly macrophage-restricted I-type lectin. Using hSn-transduced THP-1 cells, we observed that C. jejuni strains with α(2,3)-sialylated LOS, including strains expressing GM1a- and GD1a-like epitopes, bind to hSn. This observation is of importance, as these epitopes are frequently the targets of the cross-reactive antibodies detected in GBS patients. Interestingly, the Sn binding domains were not constitutively exposed on the surface of C. jejuni. Heat inactivation and the environmental conditions which food-borne C. jejuni encounters during its passage through the intestinal tract, such as low pH and contact with bile constituents, exposed LOS and facilitated Sn binding. Sn binding enhanced bacterial uptake and increased the production of interleukin-6 (IL-6) by primary human Sn-expressing monocyte-derived macrophages compared to control conditions, where Sn was blocked using neutralizing antibodies or when nonsialylated C. jejuni was used. Sn-mediated uptake has been reported to enhance humoral immune responses. As C. jejuni strains expressing ganglioside mimics GD1a and GM1a are closely associated with GBS, Sn binding may be a determining event in the production of cross-reactive antibodies and the development of GBS.

Campylobacter jejuni capsular genotypes are related to Guillain–Barré syndrome

In about one in a thousand cases, a Campylobacter jejuni infection results in the severe polyneuropathy Guillain-Barré syndrome (GBS). It is established that sialylated lipo-oligosaccharides (LOS) of C. jejuni are a crucial virulence factor in GBS development. Frequent detection of C. jejuni with sialylated LOS in stools derived from patients with uncomplicated enteritis implies that additional bacterial factors should be involved. To assess whether the polysaccharide capsule is a marker for GBS, the capsular genotypes of two geographically distinct GBS-associated C. jejuni strain collections and an uncomplicated enteritis control collection were determined. Capsular genotyping of C. jejuni strains from the Netherlands revealed that three capsular genotypes, HS1/44c, HS2 and HS4c, were dominant in GBS-associated strains and capsular types HS1/44c and HS4c were significantly associated with GBS (p 0.05 and p 0.01, respectively) when compared with uncomplicated enteritis. In a GBS-associated strain collection from Bangladesh, capsular types HS23/36c, HS19 and HS41 were most prevalent and the capsular types HS19 and HS41 were associated with GBS (p 0.008 and p 0.02, respectively). Next, specific combinations of the LOS class and capsular genotypes were identified that were related to the occurrence of GBS. Multilocus sequence typing revealed restricted genetic diversity for strain populations with the capsular types HS2, HS19 and HS41. We conclude that capsular types HS1/44c, HS2, HS4c, HS19, HS23/36c and HS41 are markers for GBS. Besides a crucial role for sialylated LOS of C. jejuni in GBS pathogenesis, the identified capsules may contribute to GBS susceptibility.