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.

The mannose cap of mycobacterial lipoarabinomannan does not dominate the Mycobacterium-host interaction.

Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL-10 production via interaction with the mannose receptor or DC-SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M. bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose-dependent mannosyltransferase. Compared with parent strain, capless M. marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M. bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC-SIGN-transfected Raji cells, but no differences in IL-10 induction were observed. In mice, capless M. bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose-capped LAM does not dominate the Mycobacterium-host interaction.

Horizontal transmission of Mycobacterium avium subsp. paratuberculosis in cattle in an experimental setting: calves can transmit the infection to other calves.

In September 2001, two subsequent transmission experiments both lasting 3 months were carried out to study cow-calf transmission of Mycobacterium avium subsp. paratuberculosis (Map) (Period 1), followed by calf-calf transmission of the infection (Period 2). Every 2 weeks, serum, heparinised blood and faecal samples were collected from all animals. After these experiments, the 20 calves were housed individually for more than 3 years to be able to detect the infection status and excretion pattern of each animal. In autumn 2004, the animals were inseminated, to observe a possible increase in faecal excretion of Map shortly before expected calving. One month before the expected calving date in 2005, animals were slaughtered and several tissues per cow and unborn calf were sampled for culture. The results indicate that horizontal cow-calf transmission is readily achieved (Period 1). At the highest infection pressure (six shedding cows of which three high shedders in Period 1) all five calves excreted Map in their faeces during Period 1 (shortly after infection), and four of these calves during Period 2 (when the shedding cows were absent). After that, excretion became less frequently. Horizontal calf-calf transmission did take place (Period 2), as the four donor-calves infected two receiver-calves. Transmission rates during the 3 months periods were quantified as a reproduction ratio R. The R [95% CI] of cow-calf and calf-calf transmission were estimated as 2.7 [1.1, 6.6] and 0.9 [0.1, 3.2] new infections per infectious animal during 3 months.

Sensitive detection of Mycobacterium avium subsp. paratuberculosis in bovine semen by real-time PCR.

AIMS: To develop a fast and sensitive protocol for detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine semen and to make a critical evaluation of the analytical sensitivity. METHODS AND RESULTS: Processed semen was spiked with known amounts of MAP. Semen from different bulls as well as semen of different dilutions was tested. The samples were treated with lysing agents and beadbeating and the DNA was extracted with phenol and chloroform. Real-time PCR with a fluorescent probe targeting the insertion element IS900 detected as few as 10 organisms per sample of 100 mul semen. PCR-inhibition was monitored by inclusion of an internal control. Pre-treatment with immunomagnetic separation was also evaluated, but was not shown to improve the overall sensitivity. CONCLUSIONS: Real-time PCR is a sensitive method for detection of MAP in bovine semen. Lysis by mechanical disruption followed by phenol and chloroform extraction efficiently isolated DNA and removed PCR-inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: The high sensitivity of the applied method allows reliable testing of bovine semen used for artificial insemination to prevent the spread of Johne's disease, caused by MAP.

Paratuberculosis recognized as a problem at last: a review.

This article attempts to review briefly current opinions on Johne's disease, or paratuberculosis, in ruminants caused by Mycobacterium avium subsp. paratuberculosis. Paratuberculosis has been known to be prevalent in domestic livestock, such as cattle, goats, and sheep, for more than a century. Despite this knowledge only minor efforts have been made to control the disease and, with the attention being focussed on the eradication of other diseases, the problem of paratuberculosis has been neglected in most countries in the past decades. However, recent epidemiological surveys performed in Europe showed a high prevalence of paratuberculosis in cattle and sheep, indicating that the situation has become quite alarming. In addition, the possible role of M. avium subsp. paratuberculosis in the aetiology of Crohn's disease in humans is still debated, as discussed in this article. Therefore, there is suddenly a renewed interest in paratuberculosis, and the disease is recognized as a significant problem. As a consequence, there is a need for reliable diagnostic tools for large-scale use to allow the introduction of programmes to control and eventually eradicate the disease. The current status and the possibilities for such programmes are discussed.

Characterization of a periplasmic protein involved in iron utilization of Actinobacillus actinomycetemcomitans.

The periodontopathic bacterium Actinobacillus actinomycetemcomitans possesses a 35-kDa periplasmic iron-repressible protein. Its regulation is mediated by the Fur protein, as was inferred from the Fur-binding consensus sequence at the -35 position of the gene for the 35-kDa protein and from the relaxed expression of the gene in a mutant with an altered Fur-binding sequence. The 35-kDa protein, designated AfuA, has strong homology to HitA and FbpA of Haemophilus influenzae and Neisseria meningitidis, respectively, which serve as periplasmic iron transport proteins.

Identification by Tn10 transposon mutagenesis of host factors involved in the biosynthesis of K99 fimbriae of Escherichia coli: effect of LPS core mutations.

Tn10 transposon mutagenesis of Escherichia coli producing K99 fimbriae was carried out to identify host factors involved in regulation of biosynthesis of fimbriae. Two chromosomal mutants were obtained that showed a strongly reduced cell surface expression of K99 fimbriae upon colony blotting and ELISA. Analysis by inversed PCR and nucleotide sequencing showed that one mutant (EP14) contained the Tn10 transposon in rfaQ, affecting the expression of the rfaQGP gene cluster, whereas the other mutant (EP35) was affected in a, to date, unknown region of the genome. Immunoblotting analysis confirmed a Rd1 type of LPS of mutant strain EP14. These findings for the first time indicated an effect of LPS core biosynthesis on the biogenesis of fimbriae at the cell surface. Preliminary experiments indicated that K99 major subunits, in contrast to K88 subunits, strongly bind LPS molecules.

Characterization of gangliosides of epithelial cells of calf small intestine, with special reference to receptor-active sequences for enteropathogenic Escherichia coli K99.

Glycolipids were prepared from epithelial cells of the small intestine of a newborn calf and assayed for Escherichia coli K99 binding activity on thin-layer chromatograms and in microtiter wells. The bacteria did not bind to any of the non-acid glycolipids, while in the acid fraction several binding-positive glycolipids were detected. The acid glycolipids were isolated and characterized by mass spectrometry, proton NMR spectroscopy and other methods. The following gangliosides were identified, mainly from the epithelial cells from the upper part of the small intestine: NeuAc alpha 2-3Gal beta 1-4Glc beta 1-Cer (NeuAc-GM3), NeuGc alpha 2-3Gal beta 1-4Glc beta 1-Cer (NeuGc-GM3), GalNAc beta 1-4(NeuGc alpha 2-3)Gal beta 1-4Glc beta 1-Cer (NeuGc-GM2), Gal beta 1-3GalNAc beta 1-4(NeuGc alpha 2-3)Gal beta 1-4Glc beta 1-Cer (NeuGc-GM1), and NeuGc alpha 2-3Gal beta 1-3GalNAc beta 1-4(NeuGc alpha 2-3)Gal beta 1-4Glc beta 1-Cer (NeuGc-GD1a). A positive binding was demonstrated to NeuGc-GM3, NeuGc-GM2, and NeuGc-GD1a, while NeuAc-GM3 and NeuGc-GM1 were negative. The binding pattern differed somewhat for total acid glycolipids of epithelial cells from three different parts of the small intestine. Based on binding preferences of E. coli K99 to a number of glycolipids of various origins, in comparison with calculated minimum energy conformations, a binding epitope was delineated.

Multivalent binding of K99 fimbriae to the N-glycolyl-GM3 ganglioside receptor.

The ganglioside N-glycolyl-GM3 binds laterally at numerous positions to K99 fimbriae, as shown by electron microscopic detection and erythrocyte-binding activity. The data demonstrate the multivalent nature of K99 fimbriae with respect to their receptor-binding sites.

Calf small intestine receptors for K99 fimbriated enterotoxigenic Escherichia coli.

Non-acid and acid glycolipids were isolated from the small intestine of a newborn calf and tested for the ability to bind Escherichia coli carrying K99 fimbriae. The bacteria did not bind to any of the non-acid glycolipids, whereas in the acid glycolipid fraction several gangliosides were detected which bind to K99 fimbriae. Gangliosides capable of binding K99 fimbriated E. coli were characterized as NeuGc-GM3, NeuGc-GM2, NeuGc-GD1a NeuAc-SPG and NeuAc-SPG. No binding was detected to NeuAc-GM3 and NeuGc-GM1.

Identification of minor fimbrial subunits involved in biosynthesis of K88 fimbriae.

The nucleotide sequences of the genes faeF, faeH, faeI, and faeJ encoding K88 minor fimbrial subunits were determined. Analysis of the primary structure of the gene products revealed that all four proteins are synthesized with an amino-terminal signal sequence. The molecular masses of the mature FaeF, FaeH, FaeI, and FaeJ proteins were calculated to be 15,161, 25,461, 24,804, and 25,093 Da, respectively. FaeH, FaeI, and FaeJ showed significant homology with FaeG, the major fimbrial subunit of K88 fimbriae. Mutations in the respective genes were constructed. Analysis of the mutants showed that the minor fimbrial subunits FaeF and FaeH play an essential role in the biogenesis but not in the adhesive properties of the K88 fimbriae. Mutations in faeI or faeJ had no significant effect on K88 production or adhesive capacity. Specific antisera against FaeF and FaeH were raised by immunization with hybrid Cro-LacZ-FaeF and Cro-LacZ-FaeH proteins. Immunoblotting and immunoelectron microscopy revealed that FaeF and FaeH are located in or along the K88 fimbrial structure.

Age and serotype dependent binding of K88 fimbriae to porcine intestinal receptors.

The porcine small intestine contains several polypeptides that could function as receptors for K88-positive Escherichia coli. The mucus fraction contained three proteins with molecular weights of 25, 35 and 60 kDa respectively, which showed a high affinity for K88-positive E. coli cells, whereas brush borders contained a 16 kDa protein and a set of proteins ranging from 40-70 kDa. Depending on the K88 serotype tested, differences in binding to these proteins were observed. In particular, E. coli cells carrying K88ad fimbriae exhibited only a rather weak binding to mucus proteins. The influence of age of the pig on the presence of K88 receptors was also investigated. One-week-old and 35-days-old post-weaning piglets were shown to contain K88 receptors in their mucus while these receptors were hardly detectable in the mucus of 6-month-old pigs. The presence of receptors in the brush border fraction was shown to be independent of age. The binding of K88 fimbriae to mucus proteins was blocked using a lectin of Euonymus europeaus which specifically recognizes the Gal alpha(1-3)Gal sequence, indicating that this disaccharide forms a significant part of the receptor structure.

Characterization of the antigenic and adhesive properties of FaeG, the major subunit of K88 fimbriae.

The two K88 serotypes, K88ab and K88ac, differ in terms of antigenic and adhesive properties. The structural determinants of the serotype-specific epitopes and the identify of the amino acid residues involved in fimbriae-receptor interaction were studied by the construction and analysis of K88 hybrid proteins in which various parts of the K88ab and K88ac fimbrial subunit FaeG were exchanged, and by in vitro mutagenesis of non-conserved amino acid residues. Using a set of monoclonal antibodies, several regions or amino acid residues involved in the formation of serotype-specific antigenic determinants were located. The haemagglutinating activity of the hybrid and mutant proteins revealed several amino acid residues involved in the formation of the receptor binding site. A clear correlation was found between the receptor binding site and the serotype-specific antigenic determinants.

Localization and function of FanH and FanG, minor components of K99 fimbriae of enterotoxigenic Escherichia coli.

Specific antisera against FanG and against FanH were prepared by immunization with hybrid Cro-LacZ-FanG and Cro-LacZ-FanH proteins, respectively. Immunoblotting with these antisera revealed the presence of FanG and FanH as minor components in purified K99 fimbriae. Mutations were constructed in fanG and fanH and cells defective in FanG or FanH were characterized by ELISA, immunoblotting, adhesion assays and electron microscopy. A minicell experiment showed that the mutations in fanG or fanH had no effect on the expression of the other K99-specific proteins. Cells defective in FanG produced no fimbriae and did not agglutinate horse erythrocytes, but cell-free heat-shock preparations of these cells still bound the K99 glycolipid receptor. Cells defective in FanH produced 1-2% of the K99 fimbriae as compared with wild-type K99 producing cells. These mutant fimbriae appeared to be shorter but were still capable of binding the K99 glycolipid receptor. Apparently, FanG and FanH are not required for binding the K99 receptor. These results and analysis of K99 mutants by immunoblotting using a specific antiserum against another K99 minor component, FanF, indicated that the combinations FanF/FanG and FanF/FanH are required for the initiation and elongation (length determination) of K99 fimbriae formation, respectively.

Structure, localization and function of FanF, a minor component of K99 fibrillae of enterotoxigenic Escherichia coli.

The DNA sequence of the K99 fanF gene, encoding FanF, was determined. An open reading frame of 999 bp was found. The primary structure of FanF was deduced and analysis revealed the presence of a signal sequence of 22 amino acid residues. The mature protein contains 311 amino acid residues (Mr 33,905 D). The amino acid sequence of FanF showed similarity with the K88ab major subunit FaeG. A specific mouse antiserum against FanF was prepared by constructing and purifying a hybrid Cro-LacZ-FanF protein. Minicell analysis, immunoblotting and immunoelectronmicroscopy revealed a pool of FanF in the periplasm of K99-producing cells and showed, furthermore, that FanF is a minor component of K99 fibrillae, present at the top and in or along the shaft of the K99 fibrillar structures. A fanF mutant plasmid was constructed. Cells harbouring this plasmid produced all K99-specific proteins, except FanF, but produced 0.1% of the K99 fibrillae relative to 'normal' K99-producing cells. Electron microscopic observations showed that cells defective in fanF produce only a few (apparently short) K99 fibrillae. FanF, therefore, was supposed to play a role in initiation and elongation of K99 fibrillae formation. Thin-layer chromatography experiments involving purified receptor material showed that FanF is not required for binding of K99 fibrillae to the ganglioside receptor. Fibrillae produced by an adhesion-negative strain carrying a mutation in the K99 major fibrillar subunit were shown to contain a normal amount of FanF.

Protein synthesis during chloroplast development in Spirodela oligorhiza. Coordinated synthesis of chloroplast-encoded and nuclear-encoded subunits of ATPase and ribulose-1,5-bisphosphate carboxylase.

We have studied qualitative and quantitative changes of several parameters during chloroplast development in Spirodela oligorhiza (duckweed). On a dry weight basis, the amount of protein increases from 2.5% (w/w) in dark-grown to 7.8% (w/w) in light-grown fronds. At the same time the amount of starch drops from 50% to 27% (w/w). Using an immunochemical quantification method we have found that during greening of etiolated plants the amount of all subunits of the ATPase complex per frond increases 10-fold, whereas the level of the subunits of ribulose-1,5-biphosphate carboxylase increases 50-fold. Cytochrome f was found to be present in dark-grown Spirodela and the amount of this polypeptide per frond increases about 30-fold. The concentration of a polypeptide that possibly represents a cytochrome b6 subunit increases about 10-fold upon greening. The molar ratio of the CF1-beta and CF1-gamma subunits of the ATPase complex varies over 2-3, while in all stages of chloroplast development studied the molar ratio of the carboxylase subunits is about 1. As these values are in agreement with the stoichiometrical amounts in the native protein complexes, we conclude that the synthesis of CF1-beta and CF1-gamma, as well as the synthesis of the large and small carboxylase subunits, are strictly coordinated during chloroplast biogenesis in Spirodela oligorhiza.