Surface Carbohydrate Epitopes Research Articles

The Beauveria bassiana Gas3 β-Glucanosyltransferase Contributes to Fungal Adaptation to Extreme Alkaline Conditions.

Fungal β-1,3-glucanosyltransferases are cell wall-remodeling enzymes implicated in stress response, cell wall integrity, and virulence, with most fungal genomes containing multiple members. The insect-pathogenic fungus Beauveria bassiana displays robust growth over a wide pH range (pH 4 to 10). A random insertion mutant library screening for increased sensitivity to alkaline (pH 10) growth conditions resulted in the identification and mapping of a mutant to a β-1,3-glucanosyltransferase gene (Bbgas3). Bbgas3 expression was pH dependent and regulated by the PacC transcription factor, which activates genes in response to neutral/alkaline growth conditions. Targeted gene knockout of Bbgas3 resulted in reduced growth under alkaline conditions, with only minor effects of increased sensitivity to cell wall stress (Congo red and calcofluor white) and no significant effects on fungal sensitivity to oxidative or osmotic stress. The cell walls of ΔBbgas3 aerial conidia were thinner than those of the wild-type and complemented strains in response to alkaline conditions, and β-1,3-glucan antibody and lectin staining revealed alterations in cell surface carbohydrate epitopes. The ΔBbgas3 mutant displayed alterations in cell wall chitin and carbohydrate content in response to alkaline pH. Insect bioassays revealed impaired virulence for the ΔBbgas3 mutant depending upon the pH of the media on which the conidia were grown and harvested. Unexpectedly, a decreased median lethal time to kill (LT50, i.e., increased virulence) was seen for the mutant using intrahemocoel injection assays using conidia grown at acidic pH (5.6). These data show that BbGas3 acts as a pH-responsive cell wall-remodeling enzyme involved in resistance to extreme pH (>9).IMPORTANCE Little is known about adaptations required for growth at high (>9) pH. Here, we show that a specific fungal membrane-remodeling β-1,3-glucanosyltransferase gene (Bbgas3) regulated by the pH-responsive PacC transcription factor forms a critical aspect of the ability of the insect-pathogenic fungus Beauveria bassiana to grow at extreme pH. The loss of Bbgas3 resulted in a unique decreased ability to grow at high pH, with little to no effects seen with respect to other stress conditions, i.e., cell wall integrity and osmotic and oxidative stress. However, pH-dependent alternations in cell wall properties and virulence were noted for the ΔBbgas3 mutant. These data provide a mechanistic insight into the importance of the specific cell wall structure required to stabilize the cell at high pH and link it to the PacC/Pal/Rim pH-sensing and regulatory system.

Extracellular vesicles from Paracoccidioides pathogenic species transport polysaccharide and expose ligands for DC-SIGN receptors

Extracellular vesicles (EVs) mediate non-conventional transport of molecules across the fungal cell wall. We aimed at describing the carbohydrate composition and surface carbohydrate epitopes of EVs isolated from the pathogenic fungi Paracoccidioides brasiliensis and P. lutzii using standard procedures. Total EV carbohydrates were ethanol-precipitated from preparations depleted of lipids and proteins, then analyzed by chemical degradation, gas chromatography-mass spectrometry, nuclear magnetic resonance and size-exclusion chromatography. EV glycosyl residues of Glc, Man, and Gal comprised most probably two major components: a high molecular mass 4,6-α-glucan and a galactofuranosylmannan, possibly an oligomer, bearing a 2-α-Manp main chain linked to β-Galf (1,3) and α-Manp (1,6) end units. The results also suggested the presence of small amounts of a (1→6)-Manp polymer, (1→3)-glucan and (1→6)-glucan. Glycan microarrays allowed identification of EV surface lectin(s), while plant lectin microarray profiling revealed terminal Man and GlcNAc residues exposed at the EVs surface. Mammalian lectin microarray profiling showed that DC-SIGN receptors recognized surface carbohydrate in Paracoccidioides EVs. Our results suggest that oligosaccharides, cytoplasmic storage, and cell wall polysaccharides can be exported in fungal EVs, which also expose surface PAMPs and lectins. The role of these newly identified components in the interaction with the host remains to be unraveled.

The MAP kinase Bbslt2 controls growth, conidiation, cell wall integrity, and virulence in the insect pathogenic fungus Beauveria bassiana

Entomopathogenic fungi, such as Beauveria bassiana, are key environmental pathogens of insects that have been exploited for biological control of insect pests. Mitogen-activated protein (MAP) kinases play crucial roles in regulating fungal development, growth, and pathogenicity, mediating responses to the environment. Bbslt2, encoding for an Slt2 family MAPK, was isolated and characterized from B. bassiana. Gene disruption of Bbslt2 affected growth, caused a significant reduction in conidial production and viability, and increased sensitivity to Congo Red and fungal cell wall degrading enzymes. ΔBbslt2 mutants were altered in cell wall structure and composition, which included temperature dependent chitin accumulation, reductions in conidial and hyphal hydrophobicity, and alterations in cell surface carbohydrate epitopes. The ΔBbslt2 strain also showed hypersensitivity to heat shock and altered trehalose accumulation, which could only be partially attributed to changes in the expression of trehalase (ntl1). Insect bioassays revealed decreased virulence in the ΔBbslt2 strain using both topical and intrahemoceol injection assays. These results indicate that Bbslt2 plays an important role in conidiation, viability, cell wall integrity and virulence in B. bassiana. Our findings are discussed within the context of the two previous MAP kinases characterized from B. bassiana.

Two hydrophobins are involved in fungal spore coat rodlet layer assembly and each play distinct roles in surface interactions, development and pathogenesis in the entomopathogenic fungus,Beauveria bassiana

The entomogenous filamentous fungus, Beauveria bassiana expresses two hydrophobin genes, hyd1 and hyd2, hypothesized to be involved in cell surface hydrophobicity, adhesion, virulence, and to constitute the protective spore coat structure known as the rodlet layer. Targeted gene inactivation of hyd1 resulted in seemingly 'bald' conidia that contained significantly altered surface fascicles or bundles. These cells displayed decreased spore hydrophobicity, loss of water mediated dispersal, changes in surface carbohydrate epitopes and β-1,3-glucan distribution, lowered virulence in insect bioassays, but no effect on adhesion. In contrast, Δhyd2 mutants retained distorted surface bundles, but truncated/incomplete rodlets could be seen within the bundles. Δhyd2 conidia displayed both decreased cell surface hydrophobicity and adhesion, but the mutant was unaffected in virulence. The double Δhyd1Δhyd2 mutant was distinct from the single mutants, lacking both bundles and rodlets, and displaying additively decreased cell surface hydrophobicity, reduced cell attachment and lowered virulence than the Δhyd1 mutant. Epitope tagged constructs of the proteins were used to examine the expression and distribution of the proteins and to demonstrate the continued presence of Hyd2 in the Δhyd1 strain and vice versa. The implications of our results with respect to fascicle and rodlet assembly on the spore surface are discussed.

Lectin mapping reveals stage-specific display of surface carbohydrates in in vitro and haemolymph-derived cells of the entomopathogenic fungus Beauveria bassiana

The entomopathogenic fungus Beauveria bassiana and its insect host target represent a model system with which to examine host-pathogen interactions. Carbohydrate epitopes on the surfaces of fungal cells play diverse roles in processes that include adhesion, non-self recognition and immune invasion with respect to invertebrate hosts. B. bassiana produces a number of distinct cell types that include aerial conidia, submerged conidia, blastospores and haemolymph-derived cells termed in vivo blastospores or hyphal bodies. In order to characterize variations in the surface carbohydrate epitopes among these cells, a series of fluorescently labelled lectins, combined with confocal microscopy and flow cytometry to quantify the response, was used. Aerial conidia displayed the most diverse lectin binding characteristics, showing reactivity against concanavalin A (ConA), Galanthus nivalis (GNL), Griffonia simplicifolia (GSII), Helix pomatia (HPA), Griffonia simplicifolia isolectin (GSI), peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEAI) and wheatgerm agglutinin (WGA), and weak reactivity against Ricinus communis I (RCA), Sambucus nigra (SNA), Limax flavus (LFA) and Sophora japonica (SJA) lectins. Lectin binding to submerged conidia was similar to that to aerial conidia, except that no reactivity against UEAI, HPA and SJA was noted, and WGA appeared to bind strongly at specific polar spots. In contrast, the majority of in vitro blastospores were not bound by ConA, GNL, GSII, GSI, SNA, UEAI, LFA or SJA, with PNA binding in large patches, and some polarity in WGA binding noted. Significant changes in lectin binding also occurred after aerial conidial germination and in cells grown on either lactose or trehalose. For germinated conidia, differential lectin binding was noted between the conidial base, the germ tube and the hyphal tip. Fungal cells isolated from the haemolymph of the infected insect hosts Manduca sexta and Heliothis virescens appeared to shed most carbohydrate epitopes, displaying binding only to the GNL, PNA and WGA lectins. Ultrastructural examination of the haemolymph-derived cells revealed the presence of a highly ordered outermost brush-like structure not present on any of the in vitro cells. Haemolymph-derived hyphal bodies placed into rich broth medium showed expression of several surface carbohydrate epitopes, most notably showing increased PNA binding and strong binding by the RCA lectin. These data indicate robust and diverse production of carbohydrate epitopes on different developmental stages of fungal cells and provide evidence that surface carbohydrates are elaborated in infection-specific patterns.

Innate immunity in the deep sea hydrothermal vent mussel Bathymodiolus azoricus

The interaction between microorganisms and host defense mechanisms is a decisive factor for the survival of marine bivalves. They rely on cell-mediated and humoral reactions to overcome the pathogens that naturally occur in the marine environment. In order to understand host defense reactions in animals inhabiting extreme environments we investigated some of the components from the immune system of the deep sea hydrothermal vent mussel Bathymodiolus azoricus. Cellular constituents in the hemolymph and extrapallial fluid were examined and led to the identification of three types of hemocytes revealing the granulocytes as the most abundant type of cell. To further characterize hemocyte types, the presence of cell surface carbohydrate epitopes was demonstrated with fluorescent WGA lectin, which was mostly ascribed to the granulocytes. Cellular reactions were then investigated by means of phagocytosis and by the activation of putative MAPKs using the microbial compounds zymosan, glucan, peptidoglycan and lipopolysaccharide. Two bacterial agents, Bacillus subtilis and Vibrio parahaemolyticus, were also used to stimulate hemocytes. The results showed that granulocytes were the main phagocytic cells in both hemolymph and extrapallial fluid of B. azoricus. Western blotting analyses using commercially available antibodies against ERK, p38 and JNK, suggested that these putative kinases are involved in signal transduction pathways during experimental stimulation of B. azoricus hemocytes. The fluorescent Ca 2+ indicator Fura-2 AM was also insightful in demonstrating hemocyte stimulation in the presence of laminarin or live V. parahaemolyticus. Finally, the expression of the antibacterial gene mytilin was analyzed in gill tissues by means of RT-PCR and whole-mount in situ hybridization. Mytilin transcripts were localized in hemocytes underlying gill epithelium. Moreover, mytilin was induced by exposure of live animals to V. parahaemolyticus. These findings support the premise of a conserved innate immune system in B. azoricus. Such system is comparable to other Bivalves and involves the participation of cellular and humoral components.

Analyzing the dynamic bacterial glycome with a lectin microarray approach

Glycosylation of bacterial cell surfaces is emerging as a critical factor in symbiosis, pathogenesis, cell-cell interactions and immune evasion. The lack of high-throughput analytical tools to examine bacterial glycans has been a major obstacle to the field and has hindered closer examination of the dynamics of carbohydrate variation. We have recently developed a lectin microarray for the analysis of glycoproteins. Herein we present a rapid analytical system based on this technology for the examination of bacterial glycans. The glycosylation pattern observed distinguishes closely related Escherichia coli strains from one another, providing a facile means of fingerprinting bacteria. In addition, dynamic alterations in the carbohydrate coat of a pathogenic E. coli strain are readily observed. The fast evaluation of real-time alterations in surface-carbohydrate epitopes allows examination of the dynamic role of bacterial sugars in response to external stimuli such as the immune system.

Thermodynamic binding studies of cell surface carbohydrate epitopes to galectins-1, -3, and -7: Evidence for differential binding specificities

Binding of a series of sialylated and non-sialylated cell surface carbohydrates to bovine heart galectin-1, recombinant murine galectin-3, and recombinant human galectin-7 was investigated by isothermal titration microcalori metry (ITC) and hemagglutination inhibition measurements. Galectin-7 shows nearly equal affinities for lactose and Galbeta(1–4)GlcNAc (LacNAc-II). Galectin-7, however, displays six- and 11-fold weaker affinity for LacNAc-II compared with galectins-1 and -3, respectively. The affinity of galectin-7 for LacNAc-II containing oligosaccharides is also weaker than the other two galectins. ITC measurements show that all three galectins bind to di- and trimeric oligomers of LacNAc-II, which are epitopes found in poly-N-acetyllactosamine chains of glycoprotein receptors, with affinity constants similar to that of LacNAc-II. The binding valencies of the di- and trimeric LacNAc-II oligomers were observed to be one from ITC measurements, indicating formation of 1:1 complexes with all three galectins. Thus, galectins-1, -3, and -7 all possess binding sites that primarily accommodate one LacNAc-II moiety per monomer of protein. Sialylated oligosaccharides show different specificities for the three galectins. While 2,3-sialyl LacNAc-II binds to all three galectins, 2,6-sialyl LacNAc-II fails to bind to any of the galectins; 2,6-sialylated diLacNAc binds well to galectin-3 and galectin-7, but only weakly to galectin-1. Similar results are obtained with 2,6-sialyl lacto-N-neo-tetraose, which has a reducing end lactose moiety. Thus, unlike galectin-1, which predominantly recognizes non-reducing terminal LacNAc-II residues in oligosaccharides, galectins-3 and -7 recognize both non-reducing terminal LacNAc-II residues as well as internal LacNAc-II and lactose residues in sialylated and non-sialylated oligosaccharides.Key words: isothermal titration microcalorimetry, galectins, binding specificities, lectins, carbohydrates.

Evolution of alpha 1,3galactosyltransferase and of the alpha-Gal epitope.

Most of the genes studied in humans have homologues in other mammals; however, theα1,3galactosyltransferase (α1,3GT) gene represents a unique exception. Whereas it is highly active in nonprimate mammals, prosimians and New World monkeys (i.e., monkeys of South America), producing one of the most abundant cell surface carbohydrate epitopes, the epitope Galα1–3Galβ1α4GlcNAc-R (termed the α-gal or α-galactosyl epitope), this gene is completely inactive in Old World primates (i.e., humans, apes and Old World monkeys). As discussed below, the inactivation of α1,3GT in ancestral primates was likely to be associated with a catastrophic evolutionary event that led primates of the Old World (i.e., primates of Asia and Africa) into almost complete extinction. Because of this event, humans, apes and Old World monkeys produce very large amounts of a natural antibody against the α-gal epitope. This antibody, termed anti-Gal, constitutes 1% of circulating immunoglobulins and it prevents transplantation of organs or tissues from nonprimate mammals (e.g., pigs) into humans because it readily binds to the α-gal epitopes on such xenografts. It is impossible to determine the actual evolutionary events which have led to inactivation of the α1,3GT gene and suppression of α-gal epitope expression in ancestral Old World primates. However, information on the expression of this epitope in various species, the structure of the α1,3GT pseudogene in primates and the fossil record of primates, enable us to speculate on both the evolutionary period in which α1,3GT gene inactivation occurred, as well as possible causes for this event.

Induction of Band 3 Aggregation in Erythrocytes Results in Anti-band 3 Autoantibody Binding to the Carbohydrate Epitopes of Band 3

Involvement of band 3 aggregation in the mechanism of anti-band 3 autoantibody binding to the cell surface carbohydrate epitopes of band 3 was investigated. When erythrocytes were treated nonoxidatively with a known protein-aggregating agent acridine orange, protein aggregates of the cell membrane which are insoluble in a nonionic detergent C 12E 8solution were remarkably increased. Analysis of the protein aggregates by SDS–PAGE indicated that they were composed of several species of noncovalently associated membrane proteins including band 3. 125I-labeled anti-band 3 bound to the acridine orange-treated cells, and the binding increased depending on the concentrations of acridine orange used. The binding was inhibited by band 3 and its oligosaccharides but not by the oligosaccharides pretreated with endo-β-galactosidase, an enzyme specifically cleaves poly- N-acetyllactosaminyl saccharide chains of band 3. When erythrocytes were pretreated with endo-β-galactosidase to remove poly- N-acetyllactosaminyl saccharide chains from cell surface prior to acridine orange treatment, the cells did not become susceptible to anti-band 3 binding. The results indicate that induction of band 3 aggregation in erythrocyte membrane leads to anti-band 3 binding to the poly- N-acetyllactosaminyl saccharide chains of band 3. Consistently, membrane proteins including band 3 were found to be aggregated when erythrocytes were oxidized with ADP-chelated Fe 3+under the conditions that induce anti-band 3 binding to the cells. Similar band 3 aggregation was observed on senescent erythrocytes whose carbohydrate epitopes of band 3 had been occupied with anti band 3. These results indicate that anti-band 3 binds to the carbohydrate epitopes of band 3 on erythrocytes when band 3 is aggregated by oxidative and nonoxidative mechanisms.

Carbohydrates on the surface of Echinococcus granulosus protoscoleces are immunodominant in mice.

A carbohydrate enriched soluble fraction (CHP) was prepared by mild treatment of viable Echinococcus granulosus protoscoleces (PSC) with the enzyme endoglycosidase-F (endo-F) and characterized by SDS-PAGE, glycoside- inhibition ELISA, and immunoblotting. Three groups of four BALB/c mice were immunized with the CHP, with the remaining deglycosylated PSC (DGP) and with dead PSC (DPSC) to analyse the relative immunogenicity of carbohydrates on the surface of PSC. A fourth sentinel group was not immunized. The antibody response was analyzed during primary and hyperimmune responses. Specific antibody titres (IgG and IgM) against somatic PSC antigens (PSA) were evaluated by ELISA and their antigen recognition pattern by immunoblotting, discriminating carbohydrate and peptide specific antibody responses by periodate treatment of PSA. The avidity index of those antibodies and the titer of non-specific immunoglobulins during the whole protocol were evaluated by ELISA in vitro mitogenic activity of CHP was also evaluated. The results indicated: 1) immunodominance of surface carbohydrate epitopes from PSC, 2) predominant IgM and low avidity response against these epitopes, 3) a dramatic increase of non-specific antibody titres and 4) in vitro mitogenic activity of CHP.

Quantitation of the density of cell surface carbohydrate antigens on cancer cells with a sensitive cell-suspension ELISA

The density of carbohydrate epitopes on the surface of tumor cells is a governing factor for immune recognition and antibody-mediated targeting of tumor-associated carbohydrate antigens in cancer immunotherapy. A sensitive cell-suspension ELISA (cs-ELISA) is developed for quantitation of the functionally exposed carbohydrate epitopes on the cell surface. The factors affecting the measurement of tumor-cell surface glycoconjugates are evaluated using three human melanoma cell lines before and after exposure to various cell preservation treatments. The results of cs-ELISA are compared with the quantitative profile obtained by biochemical and flow cytometry assays. Cs-ELISA measures the density of the functionally exposed specific sugar epitopes on the surface of tumor cells, even in the presence of other similar carbohydrate antigens, provided that the monoclonal antibodies to carbohydrate epitopes are monospecific and sensitive, and that the cells are viable and present in optimal density. Of the three melanoma cell lines, M10-v and M101 expressed disialolactosyl residues of G D3 at concentrations of 5–6 pmol/10 6 cells and 2–3 pmol/10 6 cells, respectively. In both cell lines, the cell-surface G D2 was less than 1.0 pmol/10 6 cells. M24 melanoma cells expressed trace quantities (<0.1 pmol/10 6 cells) of G D3 and G D2. Trypsinization of M10-v and M101 cells significantly reduced the cell-surface expression of G D3, suggesting G D3 loss, but increased the expression of G D2, suggesting crypticity of membrane-bound G D2. Cs-ELISA results showed that cryopreservation with 10% DMSO and irradiation at 15 krad decreased melanoma cell viability and ganglioside expression for M10-v but not M101 and M24. Formalinization did not affect cs-ELISA measurement of cell-surface carbohydrates. Cs-ELISA was used to monitor the quantity of incorporation of exogenous G D3 onto the surface of G D3-deficient M24 cells. Cs-ELISA for assessment of density of cell surface carbohydrate epitopes may be useful to characterize different types of tumors, to develop carbohydrate-based whole cell vaccines from tumor biopsies, to monitor the effects of cell preservation treatments commonly used in a whole cell vaccine preparation, and to evaluate the incorporation of a particular glycolipid (antigen or adjuvant) into glycolipid-deficient cells that are useful for carbohydrate-based active specific immunotherapy.

Chapter 9 Monoclonal Antibodies to Cell-Specific Cell Surface Carbohydrates in Plant Cell Biology and Development

Publisher Summary This chapter illustrates the variety of ways in which monoclonal antibody (MAb) technology can be used for the analysis of cell- and tissue-specific carbohydrate antigens present at the surfaces of plant cells. MAbs to cell-specific cell surface carbohydrate epitopes are versatile molecular probes with multiple applications for plant cell biology and development, for plant molecular biology, and for plant cell and tissue culture. MAbs have been essential probes for identifying and characterizing developmentally regulated cell surface epitopes in plants, and now can be used for the manipulation of the cells in which they are expressed. The chapter focuses on the use of MAbs for the purification and characterization of cell- and tissue-specific carbohydrate antigens and for the manipulation of defined cellular targets. Indirect immunolabeling techniques are used in this chapter. The MAb is first bound to the antigen then, after washing to remove surplus MAb, is tagged with an enzyme, radioisotope, fluorochrome, electron-opaque particle, or paramagnetic bead. The chapter illustrates the variety of ways in which MAb technology can be used for the analysis of cell- and tissue-specific carbohydrate antigens present at the surfaces of plant cells.

Antibody to schistosomulum surface carbohydrate epitopes in subjects infected with Schistosoma haematobium

The binding of immunoglobulin to carbohydrate epitopes on the surface of Schistosoma mansoni schistpsomula was measured with 125I-labelled protein A in sera from 67 Gambian subjects living in a focus of intense S. haematobium transmission. The levels of such antibody differed considerably between subjects and there was significant variation between age groups. The highest mean level, in subjects aged between 8 and 14 years, was significantly greater than that in older subjects. Previous studies have shown that resistance to post-treatment reinfection with S. haematobium is related to age in this focus, and is greatest in those aged 15 or more years. These differing relationships with age suggest that antibodies directed against carbohydrate epitopes on the surface of the schistosomulum do not have a major protective role in man.

Surface carbohydrate epitopes of Thy-1.1 and Thy-1.2 thymocytes are distinguished by a monoclonal antibody with a specificity common to peanut agglutinin.

NIMF4-31.7 is a monoclonal IgM antibody with affinity for terminal galactose residues of membrane glycoproteins, Sepharose and agarose. It is cytotoxic for a subpopulation of murine thymocytes and peripheral lymphocytes, corresponding to the peanut agglutinin-positive population, but reacts with a majority of cells after neuraminidase treatment. It has revealed a genetic linkage between the Thy-1 locus and quantitative expression of carbohydrate determinants on the thymocyte membrane.