Boris Tefsen

Bat-derived influenza-like viruses H17N10 and H18N11

Shorebirds and waterfowls are believed to be the reservoir hosts for influenza viruses, whereas swine putatively act as mixing vessels. The recent identification of two influenza-like virus genomes (designated H17N10 and H18N11) from bats has challenged this notion. A crucial question concerns the role bats might play in influenza virus ecology. Structural and functional studies of the two major surface envelope proteins, hemagglutinin (HA) and neuraminidase (NA), demonstrate that neither has canonical HA or NA functions found in influenza viruses. However, putative functional modules and domains in other encoded proteins are conserved, and the N-terminal domain of the H17N10 polymerase subunit PA has a classical structure and function. Therefore, potential genomic reassortments of such influenza-like viruses with canonical influenza viruses cannot be excluded at this point and should be assessed.

Galactofuranose in eukaryotes: aspects of biosynthesis and functional impact

Galactofuranose (Galf) is the five-membered ring form of galactose. It is widely distributed among several branches of the eukaryotic kingdom. This review highlights recent advances in our understanding of the biosynthesis and function of Galf-containing glycoconjugates in fungal Aspergillus spp. and the protozoan trypanosomatid parasites. We give an overview of the biosynthetic pathways leading to the production of glycolipids, glycoproteins and polysaccharides containing Galf in these species and their biological relevance. Remarkably, modification of the cell surface caused by Galf absence often results in morphological abnormalities and an impaired cell wall function in these organisms. Galf-deficient mutants are generally hypersensitive to drugs, exhibit a constitutive osmotic stress phenotype and/or have an attenuated virulence. Since Galf has never been found in mammals and higher plants, Galf-biosynthetic pathways have raised much interest as targets for drug development to combat microbial infections.

Function of Neisserial Outer Membrane Phospholipase A in Autolysis and Assessment of Its Vaccine Potential

ABSTRACT Outer membrane phospholipase A (OMPLA) is an outer membrane-localized enzyme, present in many gram-negative bacterial species. It is implicated in the virulence of several pathogens. Here, we investigated the presence, function, and vaccine potential of OMPLA in the human pathogen Neisseria meningitidis. Immunoblot analysis showed the presence of OMPLA in 28 out of 33 meningococcal strains investigated. The OMPLA-negative strains all contained a pldA gene, but these alleles contained premature stop codons. All six Neisseria gonorrhoeae strains tested, but only two out of seven commensal neisserial strains investigated, expressed OMPLA, showing that OMPLA is expressed by, but not limited to, many pathogenic neisserial strains. The function of OMPLA was investigated by assessing the phenotypes of isogenic strains, expressing no OMPLA, expressing wild-type levels of OMPLA, or overexpressing OMPLA. OMPLA exhibited phospholipase activity against endogenous phospholipids. Furthermore, OMPLA was characterized as an autolysin that acted under specific conditions, such as prolonged growth of the bacteria. The vaccine potential of the protein was investigated by immunizing mice with in vitro refolded, recombinant OMPLA. High levels of antibody titers were obtained, but the murine sera were neither bactericidal nor protective. Also, convalescent patients and vaccinee sera did not contain detectable levels of anti-OMPLA antibodies, indicating that OMPLA may not be sufficiently immunogenic to be included in a meningococcal vaccine.

Galactofuranose-coated gold nanoparticles elicit a pro-inflammatory response in human monocyte-derived dendritic cells and are recognized by DC-SIGN.

Galactofuranose (Galf) is the five-membered ring form of galactose exclusively found in nonmammalian species, among which several are pathogens. To determine the putative role of this carbohydrate in host-pathogen interactions, we synthesized multivalent gold nanoparticles carrying Galf (Galf-GNPs) and show that they are recognized by the EB-A2 antibody, which is widely used to detect Galf-containing galactomannan in the serum of Aspergillosis patients. We demonstrated that human monocyte-derived dendritic cells bound Galf-GNPs via interaction with the lectin DC-SIGN. Moreover, interaction of dendritic cells with Galf-GNPs resulted in increased expression of several maturation markers on these cells and induced secretion of the pro-inflammatory cytokines IL-6 and TNF-α. These data indicate that Galf is able to modulate the innate immune response via dendritic cells. In conclusion, Galf-GNPs are a versatile tool that can be applied in multiple functional studies to gain a better understanding of the role of Galf in host-pathogen interaction.

Vaccination-induced IgG response to Galα1–3GalNAc glycan epitopes in lambs protected against Haemonchus contortus challenge infection

Lambs vaccinated with Haemonchus contortus excretory/secretory (ES) glycoproteins in combination with the adjuvant Alhydrogel are protected against H. contortus challenge infection. Using glycan micro-array analysis we showed that serum from such vaccinated lambs contains IgG antibodies that recognise the glycan antigen Galalpha1-3GalNAc-R and GalNAcbeta1-4(Fucalpha1-3)GlcNAc-R. Our studies revealed that H. contortus glycoproteins contain Galalpha1-3Gal-R as well as significant levels of Galalpha1-3GalNAc-R, which has not been previously reported. Extracts from H. contortus adult worms contain a galactosyltransferase acting on glycan substrates with a terminal GalNAc, indicating that the worms possess the enzymatic potential to synthesise terminal Gal-GalNAc moieties. These data illustrate that glycan micro-arrays constitute a promising technology for fast and specific analysis of serum anti-glycan antibodies in vaccination studies. In addition, this approach facilitates the discovery of novel, antigenic parasite glycan antigens that may have potential for developing glycoconjugate vaccines or utilization in diagnostics.

Glycan microarray profiling of parasite infection sera identifies the LDNF glycan as a potential antigen for serodiagnosis of trichinellosis

Diagnostic methods for parasite infections still highly depend on the identification of the parasites by direct methods such as microscopic examination of blood, stool and tissue biopsies. Serodiagnosis is often carried out to complement the direct methods; however, few synthetic antigens with sufficient sensitivity and specificity are available. Here we evaluated a glycan microarray approach to select for synthetic glycan antigens that could be used for serodiagnosis of parasitic infections. Using a glycan array containing over 250 different glycan antigens, we identified GalNAcβ1-4(Fucα1-3)GlcNAc-R (LDNF) as a glycan antigen that is recognized by antibodies from Trichinella-infected individuals. We synthesized a neoglycoconjugate, consisting of five LDNF molecules covalently coupled to bovine serum albumin (BSA), and used this neoglycoconjugate as an antigen to develop a highly sensitive total-Ig ELISA for serological screening of trichinellosis. The results indicate that glycan microarrays constitute a promising technology for fast and specific identification of parasite glycan antigens to improve serodiagnosis of different parasitic infections, either using an ELISA format, or parasite-specific glycan arrays.

Regulation of expression and secretion of galectin-3 in human monocyte-derived dendritic cells.

Galectin-3 (Gal-3) is a beta-galactoside binding lectin displaying both intracellular and extracellular immune functions. In Schistosoma mansoni infection, Gal-3 has been associated with the induction of a T helper 2 response. Whereas dendritic cells (DCs) play a pivotal role in the regulation of T cell differentiation, little is known about the regulation of Gal-3 expression in DCs. In this study we determined Gal-3 mRNA and protein levels during in vitro differentiation of human monocytes into immature DCs (iDCs), by culturing monocytes in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Gal-3 mRNA levels show a moderate, transient increase during iDC generation, accompanied by elevated cell-associated Gal-3 protein. Our data show that culturing monocytes with IL-4 alone strongly increases Gal-3 mRNA levels, whereas GM-CSF induces a low increase in Gal-3 mRNA. The combined data indicate that GM-CSF reduces IL-4 induced Gal-3 mRNA levels during the generation of iDC. Remarkably, stimulation of monocytes with GM-CSF results in secretion of significant amounts of Gal-3 in the medium, whereas iDCs do not release detectable amounts of Gal-3, indicating a suppressive role of IL-4 on GM-CSF induced Gal-3 secretion. Finally, our data demonstrate that all differentiated cell types tested show a significantly lower capacity to bind Gal-3 on the cell surface than monocytes. In conclusion, Gal-3 expression in iDCs is restricted, and Gal-3 protein is localized mainly intracellular, due to the opposite actions of IL-4 and GM-CSF. By these properties, the DCs may be protected against Gal-3 induced phosphatidylserine (PS) exposure and/or apoptosis.

The N-terminal domain of PA from bat-derived influenza-like virus H17N10 has endonuclease activity

ABSTRACT Influenza imposes a great burden on society, not only in its seasonal appearance that affects both humans and domesticated animals but also through the constant threat of potential pandemics. Migratory birds are considered to be the reservoir hosts for influenza viruses, but other animals must also be considered. The recently identified influenza-like virus genome, from H17N10 in bats, was shown to be markedly different from genomes of other known influenza viruses, as both its surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) do not have canonical functions. However, no studies on other individual proteins from this particular virus have been reported until now. Here, we describe the structure of the N-terminal domain of PA from H17N10 influenza-like virus at 2.7-Å resolution and show that it has a fold similar to those of homologous PA domains present in more familiar influenza A virus strains. Moreover, we demonstrate that it possesses endonuclease activity and that the histidine residue in the active site is essential for this activity. Although this particular influenza virus subtype is probably not infectious for humans (even its virus state has not been confirmed in bats, as only the genome has been sequenced), reassortment of canonical influenza viruses with certain segments from H17N10 cannot be ruled out at this stage. Therefore, further studies are urgently needed for the sake of influenza prevention and control.

Galectin-4 Reduces Migration and Metastasis Formation of Pancreatic Cancer Cells

Galectin-4 (Gal-4) is a member of the galectin family of glycan binding proteins that shows a significantly higher expression in cystic tumors of the human pancreas and in pancreatic adenocarcinomas compared to normal pancreas. However, the putative function of Gal-4 in tumor progression of pancreatic cancer is still incompletely understood. In this study the role of Gal-4 in cancer progression was investigated, using a set of defined pancreatic cancer cell lines, Pa-Tu-8988S (PaTu-S) and Pa-Tu-8988T (PaTu-T), as a model. These two cell lines are derived from the same liver metastasis of a human primary pancreatic adenocarcinoma, but differ in their growth characteristics and metastatic capacity. We demonstrated that Gal-4 expression is high in PaTu-S, which shows poor migratory properties, whereas much lower Gal-4 levels are observed in the highly metastatic cell line PaTu-T. In PaTu-S, Gal-4 is found in the cytoplasm, but it is also secreted and accumulates at the membrane at sites of contact with neighboring cells. Moreover, we show that Gal-4 inhibits metastasis formation by delaying migration of pancreatic cancer cells in vitro using a scratch assay, and in vivo using zebrafish (Danio rerio) as an experimental model. Our data suggest that Gal-4 may act at the cell-surface of PaTu-S as an adhesion molecule to prevent release of the tumor cells, but has in addition a cytosolic function by inhibiting migration via a yet unknown mechanism.

Chicken lung lectin is a functional C-type lectin and inhibits haemagglutination by influenza A virus.

Many proteins of the calcium-dependent (C-type) lectin family have been shown to play an important role in innate immunity. They can bind to a broad range of carbohydrates, which enables them to interact with ligands present on the surface of micro-organisms. We previously reported the finding of a new putative chicken lectin, which was predominantly localized to the respiratory tract, and thus termed chicken lung lectin (cLL). In order to investigate the biochemical and biophysical properties of cLL, the recombinant protein was expressed, affinity purified and characterized. Recombinant cLL was expressed as four differently sized peptides, which is most likely due to post-translational modification. Crosslinking of the protein led to the formation of two high-molecular weight products, indicating that cLL forms trimeric and possibly even multimeric subunits. cLL was shown to have lectin activity, preferentially binding to alpha-mannose in a calcium-dependent manner. Furthermore, cLL was shown to inhibit the haemagglutination-activity of human isolates of influenza A virus, subtype H3N2 and H1N1. These result show that cLL is a true C-type lectin with a very distinct sugar specificity, and that this chicken lectin could play an important role in innate immunity.