Capsular genotype and lipooligosaccharide locus class distribution in Campylobacter jejuni from young children with diarrhea and asymptomatic carriers in Bangladesh.

Campylobacter jejuni-related diarrheal diseases is one of the major health issues among young children (0-59 months old) in low-income countries. Monitoring of the capsular (capsule polysaccharide, CPS) types of virulent C. jejuni strains in regions where the disease is endemic is of great importance for the development of a customized capsule-based multivalent vaccine. Therefore, we aimed to determine the prevalence of CPS genotypes among C. jejuni strains isolated from young children with enteritis (n = 152) and asymptomatic carriers matched by age, sex, and residence defined as the control group (n = 215) in Bangladesh. CPS genotyping was performed using a newly established multiplex polymerase chain reaction (PCR) method and lipooligosaccharide (LOS) locus classes (A-E) were characterized using PCR as well. We identified 24 different CPS genotypes among the 367 isolates. Four prevalent capsular types, HS5/31 complex (n = 27, 18%), HS3 (n = 26, 17%), HS4A (n = 10, 7%), and HS8/17 (n = 10, 7%) covered almost 50% of the strains from enteritis patients and 43% of the isolates from controls. In combination, the CPS genotype and LOS class was not discriminative between cases and controls. Dominant capsular types previously identified in C. jejuni strains isolated from patients with Guillain-Barré syndrome in Bangladesh were rarely detected in strains isolated from the young children. A similar distribution was evident among enteritis- and control-related strains when comparison was done between CPS types and LOS classes. This is the first systematic study presenting the distribution of CPS genotypes of C. jejuni strains isolated in Bangladesh from children with diarrhea and controls, with capsular genotypes HS5/31 complex, HS3, HS4A, and HS8/17 being prevalent in both. In conclusion, systematic studies are required to develop a multivalent capsule-based vaccine for children in low-income countries.

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.

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.

Siglec-7 specifically recognizes Campylobacter jejuni strains associated with oculomotor weakness in Guillain-Barré syndrome and Miller Fisher syndrome.

Due 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.

Hydrophobic ligand binding by Zn-alpha 2-glycoprotein, a soluble fat-depleting factor related to major histocompatibility complex proteins.

Zn-alpha(2)-glycoprotein (ZAG) is a member of the major histocompatibility complex (MHC) class I family of proteins and is identical in amino acid sequence to a tumor-derived lipid-mobilizing factor associated with cachexia in cancer patients. ZAG is present in plasma and other body fluids, and its natural function, like leptin's, probably lies in lipid store homeostasis. X-ray crystallography has revealed an open groove between the helices of ZAG's alpha(1) and alpha(2) domains, containing an unidentified small ligand in a position similar to that of peptides in MHC proteins (Sanchez, L. M., Chirino, A. J., and Bjorkman, P. J. (1999) Science 283, 1914-1919). Here we show, using serum-derived and bacterial recombinant protein, that ZAG binds the fluorophore-tagged fatty acid 11-(dansylamino)undecanoic acid (DAUDA) and, by competition, natural fatty acids such as arachidonic, linolenic, eicosapentaenoic, and docosahexaenoic acids. Other MHC class I-related proteins (FcRn, HFE, HLA-Cw*0702) showed no such evidence of binding. Fluorescence and isothermal calorimetry analysis showed that ZAG binds DAUDA with K(d) in the micromolar range, and differential scanning calorimetry showed that ligand binding increases the thermal stability of the protein. Addition of fatty acids to ZAG alters its intrinsic (tryptophan) fluorescence emission spectrum, providing a strong indication that ligand binds in the expected position close to a cluster of exposed tryptophan side chains in the groove. This study therefore shows that ZAG binds small hydrophobic ligands, that the natural ligand may be a polyunsaturated fatty acid, and provides a fluorescence-based method for investigating ZAG-ligand interactions.

Crystal structure and ligand binding properties of the D1D2 region of the inhibitory receptor LIR-1 (ILT2).

LIR-1 is an inhibitory receptor that recognizes class I MHC molecules and the human cytomegalovirus class I homolog UL18. Here, we report the 2.1 A resolution crystal structure of the ligand binding portion of LIR-1 (domains 1 and 2 [D1D2]) and localize the binding region for UL18. LIR-1 D1D2 is composed of two immunoglobulin-like domains arranged at an acute angle to form a bent structure resembling the structures of natural killer inhibitory receptors (KIRs). The LIR-1 binding site comprises a portion of D1 distant from the interdomain hinge region that constitutes the KIR binding site, consistent with differences in LIR-1 and KIR recognition properties and functions.

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

Several studies have shown that immunization with heat 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 preduced 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.

Transient expression of a mitochondrial precursor protein. A new approach to study mitochondrial protein import in cells of higher eukaryotes.

In order to study mitochondrial protein import in the context of whole cell metabolism, we have used the transfection technique based on Semliki Forest virus (SFV) to express a mitochondrial precursor protein within BHK21 cells and human fibroblasts. Recombinant SFV particles mediate a highly efficient, transient transfection of higher eukaryotic cells. The mitochondrial precursor protein used is a fusion protein consisting of the mitochondrial targeting sequence of Neurospora crassa ATPase subunit 9 and mouse dihydrofolate (H2folate) reductase. Transfected BHK21 cells synthesized substantial amounts of subunit-9-H2folate-reductase. Immunofluorescence staining revealed that the protein colocalized with the mitochondria. The precursor protein was processed to the intermediate and mature form, implying that is was successfully imported into the mitochondrial matrix. Import was dependent on a proton gradient across the mitochondrial membranes since uncoupling of oxidative phosphorylation inhibited the process. The mature-sized protein was folded into a protease-resistant conformation. These results indicate that, in mammalian cells, transport of the precursor subunit-9-H2folate-reductase into mitochondria and its subsequent maturation occurs in a similar way as in lower eukaryotes. Import and processing of the fusion protein proceeded very rapidly in BHK21 cells but were substantially slower in human fibroblasts. SFV-mediated transfection proved to be excellently suited to study protein import into mitochondria of living cells and is probably applicable to transport studies with other organelles as well. The approach could also be helpful in the diagnosis of hereditary disorders or organelle protein import.

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.