Caenorhabditis elegans Bacterial Pathogen Resistant bus-4 Mutants Produce Altered Mucins

Caenorabditis elegans bus-4 glycosyltransferase mutants are resistant to infection by Microbacterium nematophilum, Yersinia pestis and Yersinia pseudotuberculosis and have altered susceptibility to two Leucobacter species Verde1 and Verde2. Our objective in this study was to define the glycosylation changes leading to this phenotype to better understand how these changes lead to pathogen resistance. We performed MALDI-TOF MS, tandem MS and GC/MS experiments to reveal fine structural detail for the bus-4 N- and O-glycan pools. We observed dramatic changes in O-glycans and moderate ones in N-glycan pools compared to the parent strain. Ce core-I glycans, the nematode''s mucin glycan equivalent, were doubled in abundance, halved in charge and bore shifts in terminal substitutions. The fucosyl O-glycans, Ce core-II and neutral fucosyl forms, were also increased in abundance as were fucosyl N-glycans. Quantitative expression analysis revealed that two mucins, let-653 and osm-8, were upregulated nearly 40 fold and also revealed was a dramatic increase in GDP-Man 4,6 dehydratease expression. We performed detailed lectin binding studies that showed changes in glycoconjugates in the surface coat, cuticle surface and intestine. The combined changes in cell surface glycoconjugate distribution, increased abundance and altered properties of mucin provide an environment where likely the above pathogens are not exposed to normal glycoconjugate dependent cues leading to barriers to these bacterial infections.     

The C. elegans glycosyltransferase BUS-8 has two distinct and essential roles in epidermal morphogenesis

Ventral enclosure in Caenorhabditis elegans involves migration of epidermal cells over a neuroblast substrate and subsequent adhesion at the ventral midline. Organisation of the neuroblast layer by ephrins and their receptors is essential for this migration. We show that bus-8, which encodes a predicted glycosyltransferase, is essential for embryonic enclosure and acts in or with ephrin signalling to mediate neuroblast organisation and to permit epidermal migration. BUS-8 acts non-cell-autonomously in this process, and likely modifies an extracellular regulator of ephrin signalling and cell organisation. Weak and cold-sensitive alleles of bus-8 show that the gene has a separate and distinct post-embryonic role, being essential for epidermal integrity and production of the cuticle surface. This disorganisation of the epidermis and cuticle layers causes increased drug sensitivity, which could aid the growing use of C. elegans in drug screening and chemical genomics. The viable mutants are also resistant to infection by the pathogen Microbacterium nematophilum, due to failure of the bacterium to bind to the host surface. The two separate essential roles of BUS-8 in epidermal morphogenesis add to our growing understanding of the widespread importance of glycobiology in development.     

Site-specific glycan analysis of human chorionic gonadotropin beta-subunit from malignancies and pregnancy by liquid chromatography--electrospray mass spectrometry

Glycosylation is an important posttranslational modificationin proteins, and aberrant glycosylation occurs in malignancies.Human chorionic gonadotropin (hCG) is a glycoprotein hormoneproduced in high concentrations during pregnancy. It is alsoexpressed as particular glycoforms by certain malignancies.These glycoforms, which are called "hyperglycosylated" hCG (hCGh),have been reported to contain more complex glycan moieties.We have analyzed tryptic glycopeptides of the ß-subunitof hCG of various origins by liquid chromatography (LC) connectedto an electrospray mass spectrometer. Site-specific glycan structureswere visualized by the use of differential expression analysissoftware. hCGß was purified from urine of two patientswith testicular cancer, one with choriocarcinoma, one with aninvasive mole, two pregnant women at early and late gestation,from a pharmaceutical preparation and culture medium of a choriocarcinomacell line. N-glycans at Asn-13 and Asn-30 as well as O-glycansat Ser-121, Ser-127, Ser-132, and Ser-138 were characterized.In all samples, the major type of N-glycan was a biantennarycomplex-type structure, but triantennary structures linked toAsn-30 as well as fucosylation of the Asn-13-bound glycan areincreased in cancer-derived hCGß. There were significantsite-specific differences in the O-glycans, with constant core-2glycans at Ser-121, core-1 glycans at Ser-138, and putativesites unoccupied by any glycan. Core-2 glycans at either Ser-127or Ser-132 were enriched in cancer. The glycans of free hCGßwere larger and had a higher fucose content of Asn-13-linkedoligosaccharides than intact hCG. This may facilitate the detectionof this malignancy-associated variant by a lectin assay. Analysisof hCGh affinity purified with antibody B152 confirmed thatthis antibody recognizes a core-2 glycan on Ser-132.     

Survival Signals of Hepatic Stellate Cells in Liver Regeneration Are Regulated by Glycosylation Changes in Rat Vitronectin,Especially Decreased Sialylation

The extracellular matrix (ECM) molecules play important roles in many biological and pathological processes. During tissue remodeling, the ECM molecules that are glycosylated are different from those of normal tissue owing to changes in the expression of many proteins that are responsible for glycan synthesis. Vitronectin (VN) is a major ECM molecule that recognizes integrin on hepatic stellate cells (HSCs). The present study attempted to elucidate how changes in VN glycans modulate the survival of HSCs, which play a critical role in liver regeneration. Plasma VN was purified from partially hepatectomized (PH) and sham-operated (SH) rats at 24 h after operation and non-operated (NO) rats. Adhesion of rat HSCs (rHSCs), together with phosphorylation of focal adhesion kinase, in PH-VN was decreased to one-half of that in NO- or SH-VN. Spreading of rHSCs on desialylated NO-VN was decreased to one-half of that of control VN, indicating the importance of sialylation of VN for activation of HSCs. Liquid chromatography/multiple-stage mass spectrometry analysis of Glu-C glycopeptides of each VN determined the site-specific glycosylation. In addition to the major biantennary complex-type N-glycans, hybrid-type N-glycans were site-specifically present at Asn¹⁶⁷. Highly sialylated O-glycans were found to be present in the Thr¹¹⁰–Thr¹²⁴ region. In PH-VN, the disialyl O-glycans and complex-type N-glycans were decreased while core-fucosylated N-glycans were increased. In addition, immunodetection after two-dimensional PAGE indicated the presence of hyper- and hyposialylated molecules in each VN and showed that hypersialylation was markedly attenuated in PH-VN. This study proposes that the alteration of VN glycosylation modulates the substrate adhesion to rat HSCs, which is responsible for matrix restructuring.     

Glycomics Profiling of Chinese Hamster Ovary Cell Glycosylation Mutants Reveals N-Glycans of a Novel Size and Complexity

Identifying biological roles for mammalian glycans and the pathways by which they are synthesized has been greatly facilitated by investigations of glycosylation mutants of cultured cell lines and model organisms. Chinese hamster ovary (CHO) glycosylation mutants isolated on the basis of their lectin resistance have been particularly useful for glycosylation engineering of recombinant glycoproteins. To further enhance the application of these mutants, and to obtain insights into the effects of altering one specific glycosyltransferase or glycosylation activity on the overall expression of cellular glycans, an analysis of the N-glycans and major O-glycans of a panel of CHO mutants was performed using glycomic analyses anchored by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. We report here the complement of the major N-glycans and O-glycans present in nine distinct CHO glycosylation mutants. Parent CHO cells grown in monolayer versus suspension culture had similar profiles of N- and O-GalNAc glycans, although the profiles of glycosylation mutants Lec1, Lec2, Lec3.2.8.1, Lec4, LEC10, LEC11, LEC12, Lec13, and LEC30 were consistent with available genetic and biochemical data. However, the complexity of the range of N-glycans observed was unexpected. Several of the complex N-glycan profiles contained structures of m/z ∼13,000 representing complex N-glycans with a total of 26 N-acetyllactosamine (Galβ1–4GlcNAc)_n units. Importantly, the LEC11, LEC12, and LEC30 CHO mutants exhibited unique complements of fucosylated complex N-glycans terminating in Lewis^x and sialyl-Lewis^x determinants. This analysis reveals the larger-than-expected complexity of N-glycans in CHO cell mutants that may be used in a broad variety of functional glycomics studies and for making recombinant glycoproteins.     

A C-type lectin of Caenorhabditis elegans: its sugar-binding property revealed by glycoconjugate microarray analysis

C-type lectins are a family of proteins with an affinity to carbohydrates in the presence of Ca²⁺. In the genome of Caenorhabditis elegans, almost 300 genes encoding proteins containing C-type lectin-like domains (CTLDs) have been assigned. However, none of their products has ever been shown to have carbohydrate-binding activity. In the present study, we selected 6 potential C-type lectin genes and prepared corresponding recombinant proteins. One of them encoded by clec-79 was found to have sugar-binding activity by using a newly developed glycoconjugate microarray based on evanescent-field excited fluorescence. CLEC-79 exhibited affinity to sugars containing galactose at the non-reducing terminal, especially to the Galβ1-3GalNAc structure, in the presence of Ca²⁺. Combined with structural information of the glycans of C. elegans, these results suggest that CLEC-79 preferentially binds to O-glycans in vivo.     

Engineering of Sialylated Mucin-type O-Glycosylation in Plants

Proper N- and O-glycosylation of recombinant proteins is important for their biological function. Although the N-glycan processing pathway of different expression hosts has been successfully modified in the past, comparatively little attention has been paid to the generation of customized O-linked glycans. Plants are attractive hosts for engineering of O-glycosylation steps, as they contain no endogenous glycosyltransferases that perform mammalian-type Ser/Thr glycosylation and could interfere with the production of defined O-glycans. Here, we produced mucin-type O-GalNAc and core 1 O-linked glycan structures on recombinant human erythropoietin fused to an IgG heavy chain fragment (EPO-Fc) by transient expression in Nicotiana benthamiana plants. Furthermore, for the generation of sialylated core 1 structures constructs encoding human polypeptide:N-acetylgalactosaminyltransferase 2, Drosophila melanogaster core 1 β1,3-galactosyltransferase, human α2,3-sialyltransferase, and Mus musculus α2,6-sialyltransferase were transiently co-expressed in N. benthamiana together with EPO-Fc and the machinery for sialylation of N-glycans. The formation of significant amounts of mono- and disialylated O-linked glycans was confirmed by liquid chromatography-electrospray ionization-mass spectrometry. Analysis of the three EPO glycopeptides carrying N-glycans revealed the presence of biantennary structures with terminal sialic acid residues. Our data demonstrate that N. benthamiana plants are amenable to engineering of the O-glycosylation pathway and can produce well defined human-type O- and N-linked glycans on recombinant therapeutics.     

A novel bacterial pathogen, Microbacterium nematophilum, induces morphological change in the nematode C. elegans

The Dar (deformed anal region) phenotype, characterized by a distinctive swollen tail, was first detected in a variant strain of Caenorhabditis elegans which appeared spontaneously in 1986 during routine genetic crosses [1] and [2]. Dar isolates were initially analysed as morphological mutants, but we report here that two independent isolates carry an unusual bacterial infection different from those previously described [3], which is the cause of the Dar phenotype. The infectious agent is a new species of coryneform bacterium, named Microbacterium nematophilum n. sp., which fortuitously contaminated cultures of C. elegans. The bacteria adhere to the rectal and post-anal cuticle of susceptible nematodes, and induce substantial local swelling of the underlying hypodermal tissue. The swelling leads to constipation and slowed growth in the infected worms, but the infection is otherwise non-lethal. Certain mutants of C. elegans with altered surface antigenicity are resistant to infection. The induced deformation appears to be part of a survival strategy for the bacteria, as C. elegans are potentially their predators.     

ABH blood group antigens in N-glycan of human glycophorin A

We previously showed that a small proportion of the O-linked oligosaccharide chains of human glycophorin A (GPA) contains blood group A, B or H antigens, relevant to the ABO phenotype of the donor. The structures of these minor O-glycans have been established (Podbielska et al. (2004) [20]). By the use of immunochemical methods we obtained results indicating that ABH blood group epitopes are also present in N-glycan of human GPA (Podbielska and Krotkiewski (2000) [22]). In the present paper we report a detailed analysis of GPA N-glycans using nanoflow electrospray ionization tandem mass spectrometry. N-glycans containing A-, B- and H-related sequences were identified in GPA preparations obtained from erythrocytes of blood group A, B and O donors, respectively. The ABH blood group epitopes are present on one antenna of the N-glycan, whereas a known sialylated sequence NeuAcα2-6Galβ1-4GlcNAc- occurs on the other antenna and other details are in agreement with the known major structure of the GPA N-glycan. In the bulk of the biantennary sialylated N-glycans released from GPA preparations, the blood group ABH epitopes-containing N-glycans, similarly O-glycans, constituted only a minor part. The amount relative to other N-glycans was estimated to 2-6% of blood group H epitope-containing glycans released from GPA-O preparations and 1-2% of blood group A and B epitope-containing glycans, released from GPA-A and GPA-B, respectively.     

Comparison of two approaches for quantitative O-linked glycan analysis used in characterization of recombinant proteins

The principal aim of this study was to demonstrate the optimization and fine-tuning of quantitative and nonselective analysis of O-linked glycans released from therapeutic glycoproteins. Two approaches for quantitative release of O-linked glycans were examined: ammonia-based β-elimination and hydrazinolysis deglycosylation strategies. A significant discrepancy in deglycosylation activity was observed between the ammonia-based and hydrazinolysis procedures. Specifically, the release of O-glycans from glycoproteins was approximately 20 to 30 times more efficient with hydrazine compared with ammonia-based β-elimination reagent. In addition, the ammonia-based reagent demonstrated bias in the release of particular glycan species. A robust quantitative hydrazinolysis procedure was developed for characterization of O-glycans. The method performance parameters were evaluated. It was shown that this procedure is superior for quantitative nonselective release of O-glycans. Identity confirmation and structure elucidation of O-glycans from hydrophilic interaction chromatography (HILIC) fractions was also demonstrated using linear ion trap Fourier transform mass spectrometry (LTQ FT MS) with mass accuracy below 1 ppm.     

Monosialylated biantennary N-glycoforms containing GalNAc-GlcNAc antennae predominate when human EPO is expressed in goat milk

Recently, our group reported the expression of recombinant human erythropoietin in goat milk (rhEPO-milk) as well as in the mammary epithelial cell line GMGE (EPO-GMGE) by cell culture using the adenoviral transduction system. N-Glycosylation characterization of rhEPO-milk by Normal-Phase HPLC profiling of the fluorophore, 4-aminobenzoic acid-labeled enzymatically released N-glycan pool from rhEPO-goat milk, combined with MALDI, ESI-MS and LC/MS, revealed that low branched, core-fucosylated, N-glycans predominate. The labeled N-glycans were separated into neutral and charged fractions by anion exchange chromatography and the charged N-glycans were found to be mostly α2,6-monosialylated with Neu5Ac or Neu5Gc in a ratio of 1:1. Unlike the N-glycans from rhEPO produced in CHO cells, where the glycans are multiantennary highly sialylated, core-fucosylated oligosaccahrides, or even in the goat mammary gland epithelial cell line cultured in vitro in which multiantennary, core- and outer-arm fucosylated, monosialylated N-glycans are the most abundant species, a large proportion of the N-glycans from rhEPO-milk were monosialylated, biantennary, antennae mostly terminating with the more unusual GalNAc-GlcNAc motive and without outer-arm fucosylation. These findings, emphasizing the difference in the N-glycan repertoire between the rhEPO-milk and EPO-GMGE, are consistent with the principle that glycosylation is cell-type dependent and that the cell environment is crucial as well.     

Sulfation of Colonic Mucins by N-Acetylglucosamine 6-O-Sulfotransferase-2 and Its Protective Function in Experimental Colitis in Mice

N-Acetylglucosamine 6-O-sulfotransferase-2 (GlcNAc6ST-2) catalyzes the sulfation of mucin-like glycoproteins, which function as ligands for a lymphocyte homing receptor, L-selectin, in the lymph node high endothelial venules (HEVs). We previously showed that GlcNAc6ST-2 is expressed not only in lymph node HEVs but also in the colonic epithelial cells in mice. Here we investigated the regulatory mechanism and physiological significance of colonic expression of GlcNAc6ST-2 in mice. Treatment of a mouse colonic epithelial cell line with butyrate, a short-chain fatty acid produced by anaerobic bacteria, induced GlcNAc6ST-2 expression in the presence of epidermal growth factor. Administration of butyrate in the drinking water stimulated GlcNAc6ST-2 expression in the mouse intestine, indicating that butyrate could serve as a regulatory molecule for the GlcNAc6ST-2 expression in vivo. Immunohistochemical analysis indicated that the sulfation of colonic mucins was greatly diminished in GlcNAc6ST-2-deficient mice. Liquid chromatography coupled to electrospray ionization tandem mass spectrometry of the colonic-mucin O-glycans from wild-type and GlcNAc6ST-2-deficient mice showed that GlcNAc-6-O-sulfation was the predominant sulfate modification of these mucins, and it was exclusively mediated by GlcNAc6ST-2. After colitis induction by dextran sulfate sodium, significantly more leukocyte infiltration was observed in the colon of GlcNAc6ST-2-deficient mice than in that of wild-type mice, indicating that the sulfation of colonic mucins by GlcNAc6ST-2 has a protective function in experimental colitis. These findings indicate that GlcNAc6ST-2, whose expression is regulated by butyrate, is a major sulfotransferase in the biosynthesis of sulfomucins in the mouse colon, where they serve as a mucosal barrier against colonic inflammation.     

Improved nonreductive O-glycan release by hydrazinolysis with ethylenediaminetetraacetic acid addition

The study of protein O-glycosylation is receiving increasing attention in biological, medical, and biopharmaceutical research. Improved techniques are required to allow reproducible and quantitative analysis of O-glycans. An established approach for O-glycan analysis relies on their chemical release in high yield by hydrazinolysis, followed by fluorescent labeling at the reducing terminus and high-performance liquid chromatography (HPLC) profiling. However, an unwanted degradation known as “peeling” often compromises hydrazinolysis for O-glycan analysis. Here we addressed this problem using low-molarity solutions of ethylenediaminetetraacetic acid (EDTA) in hydrazine for O-glycan release. O-linked glycans from a range of different glycoproteins were analyzed, including bovine fetuin, bovine submaxillary gland mucin, and serum immunoglobulin A (IgA). The data for the O-glycans released by hydrazine with anhydrous EDTA or disodium salt dihydrate EDTA show high yields of the native O-glycans compared with the peeled product, resulting in a markedly increased robustness of the O-glycan profiling method. The presented method for O-glycan release demonstrates significant reduction in peeling and reduces the number of sample handling steps prior to release.     

Exploring the Unique N-Glycome of the Opportunistic Human Pathogen Acanthamoeba

Glycans play key roles in host-pathogen interactions; thus, knowing the N-glycomic repertoire of a pathogen can be helpful in deciphering its methods of establishing and sustaining a disease. Therefore, we sought to elucidate the glycomic potential of the facultative amoebal parasite Acanthamoeba. This is the first study of its asparagine-linked glycans, for which we applied biochemical tools and various approaches of mass spectrometry. An initial glycomic screen of eight strains from five genotypes of this human pathogen suggested, in addition to the common eukaryotic oligomannose structures, the presence of pentose and deoxyhexose residues on their N-glycans. A more detailed analysis was performed on the N-glycans of a genotype T11 strain (4RE); fractionation by HPLC and tandem mass spectrometric analyses indicated the presence of a novel mannosylfucosyl modification of the reducing terminal core as well as phosphorylation of mannose residues, methylation of hexose and various forms of pentosylation. The largest N-glycan in the 4RE strain contained two N-acetylhexosamine, thirteen hexose, one fucose, one methyl, and two pentose residues; however, in this and most other strains analyzed, glycans with compositions of Hex_8–9HexNAc₂Pnt_0–1 tended to dominate in terms of abundance. Although no correlation between pathogenicity and N-glycan structure can be proposed, highly unusual structures in this facultative parasite can be found which are potential virulence factors or therapeutic targets.     

Identification of Glycosyltransferase 8 Family Members as Xylosyltransferases Acting on O-Glucosylated Notch Epidermal Growth Factor Repeats

The epidermal growth factor repeats of the Notch receptor are extensively glycosylated with three different O-glycans. O-Fucosylation and elongation by the glycosyltransferase Fringe have been well studied and shown to be essential for proper Notch signaling. In contrast, biosynthesis of O-glucose and O-N-acetylglucosamine is less well understood. Recently, the isolation of the Drosophila mutant rumi has shown that absence of O-glucose impairs Notch function. O-Glucose is further extended by two contiguous α1,3-linked xylose residues. We have identified two enzymes of the human glycosyltransferase 8 family, now named GXYLT1 and GXYLT2 (glucoside xylosyltransferase), as UDP-d-xylose:β-d-glucoside α1,3-d-xylosyltransferases adding the first xylose. The enzymes are specific for β-glucose-terminating acceptors and UDP-xylose as donor substrate. Generation of the α1,3-linkage was confirmed by nuclear magnetic resonance. Activity on a natural acceptor could be shown by in vitro xylosylation of a Notch fragment expressed in a UDP-xylose-deficient cell line and in vivo by co-expression of the enzymes and the Notch fragment in insect cells followed by mass spectrometric analysis of peptide fragments.     

Improved Method for Drawing of a Glycan Map,and the First Page of Glycan Atlas,Which Is a Compilation of Glycan Maps for a Whole Organism

Glycan Atlas is a set of glycan maps over the whole body of an organism. The glycan map that includes data of glycan structure and quantity displays micro-heterogeneity of the glycans in a tissue, an organ, or cells. The two-dimensional glycan mapping is widely used for structure analysis of N-linked oligosaccharides on glycoproteins. In this study we developed a comprehensive method for the mapping of both N- and O-glycans with and without sialic acid. The mapping data of 150 standard pyridylaminated glycans were collected. The empirical additivity rule which was proposed in former reports was able to adapt for this extended glycan map. The adapted rule is that the elution time of pyridylamino glycans on high performance liquid chromatography (HPLC) is expected to be the simple sum of the partial elution times assigned to each monosaccharide residue. The comprehensive mapping method developed in this study is a powerful tool for describing the micro-heterogeneity of the glycans. Furthermore, we prepared 42 pyridylamino (PA-) glycans from human serum and were able to draw the map of human serum N- and O-glycans as an initial step of Glycan Atlas editing.     

Dual-gradient high-performance liquid chromatography for identification of cytosolic high-mannose-type free glycans

It has been shown that free oligosaccharides derived from N-linked glycans accumulate in the cytosol of animal cells. Most of the glycans have only a single GlcNAc at their reducing termini (Gn1 glycans), whereas the original N-glycans retain N,N′-diacetylchitobiose at their reducing termini (Gn2 glycans). Under the conditions of high-performance liquid chromatography (HPLC) mapping established for pyridylamine (PA)-labeled Gn2 N-glycans, Gn1 glycans are not well retained on reversed-phase HPLC, making simultaneous analysis of Gn1 and Gn2 glycans problematic. We introduced a dual gradient (i.e., pH and butanol gradient) for the separation of Gn1 and Gn2 glycans in a single reversed-phase HPLC. Determination of elution time for various standard Gn2 high-mannose-type glycans, as well as Gn1 glycans found in the cytosol of animal cells, showed that elution of Gn1 and Gn2 glycans could be separated. Sufficient separation for most of the structural isomers could be achieved for Gn1 and Gn2 glycans. This HPLC, therefore, is a powerful method for identification of the structures of PA-labeled glycans, especially Gn1-type glycans, isolated from the cytosol of animal cells.     

Several N-Glycans on the HIV Envelope Glycoprotein gp120 Preferentially Locate Near Disulphide Bridges and Are Required for Efficient Infectivity and Virus Transmission

The HIV envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 N-linked glycans. Using a probability calculation, we here demonstrate that there is a co-localization of disulphide bridges and N-linked glycans in HIV-1 gp120, with a predominance of N-linked glycans in close proximity to disulphide bridges, at the C-terminal side of the involved cysteines. Also, N-glycans are frequently found immediately adjacent to disulphide bridges in gp120 at the N-terminal side of the involved cysteines. In contrast, N-glycans at positions close to, but not immediately neighboring disulphide bridges seem to be disfavored at the N-terminal side of the involved cysteines. Such a pronounced co-localization of disulphide bridges and N-glycans was also found for the N-glycans on glycoprotein E1 of the hepatitis C virus (HCV) but not for other heavily glycosylated proteins such as E2 from HCV and the surface GP from Ebola virus. The potential functional role of the presence of N-glycans near disulphide bridges in HIV-1 gp120 was studied using site-directed mutagenesis, either by deleting conserved N-glycans or by inserting new N-glycosylation sites near disulphide bridges. The generated HIV-1_NL4.3 mutants were subjected to an array of assays, determining the envelope glycoprotein levels in mutant viral particles, their infectivity and the capture and transmission efficiencies of mutant virus particles by DC-SIGN. Three N-glycans located nearby disulphide bridges were found to be crucial for the preservation of several of these functions of gp120. In addition, introduction of new N-glycans upstream of several disulphide bridges, at locations where there was a significant absence of N-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity. It was shown that the N-glycan environment around well-defined disulphide bridges of gp120 is highly critical to allow efficient viral infection and transmission.     

A new model system for the study of the animal innate immune response to fungal infections

The association of the model organism Caenorhabditis elegans and the fungus Pleurotus ostreatus gives the possibility to study the molecular and genetic mechanisms of the early stages of the spatial and temporal interactions of animals with fungal pathogens. We identified the stages of the infection process of P. ostreatus on the nematode C. elegans. We found that prior to penetration inside a worm a fungal toxin paralyzed and immobilized, but did not kill C. elegans. This finding opens the possibility for the further study of the effect of paralyzing toxins on host organisms. The membrane permeability of paralyzed worms increased dramatically and leakage products initiated the growth of directional hyphae towards the nematodes. The hyphae penetrated into live C. elegans animals either through natural openings or directly by piercing the cuticle. Upon contact with the nematode cuticle, P. ostreatus attached to it, formed appressoria-like structures and infection pegs, piercing the cuticle and penetrating inside the nematode body. The small zones around the penetration loci are of special interest for the evaluation of initial contacts between two organisms and for the study of the C. elegans local defense response against fungal infection.