Sodium alginate potentiates antioxidant defense and PR proteins against early blight disease caused by Alternaria solani in Solanum lycopersicum Linn.

The use of biopolymers as elicitors in controlling plant diseases is gaining momentum world-wide due to their eco-friendly and non-toxic nature. In the present study, we have used an algal biopolymer (sodium alginate) and tested its applicability as an elicitor in inducing resistance factors against Alternaria solani, which causes early blight disease in Solanum lycopersicum (tomato plant). We have pre-treated tomato plants with different concentrations of sodium alginate (0.2%, 0.4%, and 0.6%) before A. solani infection. We found that sodium alginate has effectively controlled the growth of A. solani. In addition, a significant increase in the expression levels of SOD was observed in response to pathogen infection. The increased protease inhibitors activity further suggest that sodium alginate restrict the development of A. solani infection symptoms in tomato leaves. This corroborates well with the cell death analysis wherein increased sodium alginate pre-treatment results in decreased cell death. Also, the expression profile analyses reveal the induction of genes only in sodium alginate-pretreated tomato leaves, which are implicated in plant defense mechanism. Taken together, our results suggest that sodium alginate can be used as an elicitor to induce resistance against A. solani in tomato plants.

Chitosan and chitosan nanoparticles induced expression of pathogenesis-related proteins genes enhances biotic stress tolerance in tomato.

The aim of this work was to evaluate the possibility of control of wilt disease caused by Fusarium andiyazi through chitosan (CS) and chitosan nanoparticles (CNPs). In the present study, the expression pattern of pathogenesis-related (PR) proteins genes such as PR-1, PR-2 (ß-1,3-glucanase), PR-8 (chitinase), and PR-10 was analyzed using real-time RT-PCR. In vitro studies showed that among different concentrations (0.1-5.0 mg/ml), 5.0 mg/ml concentration of CS and CNPs produced maximum inhibition of radial mycelial growth, 54.8% and 73.81%, respectively. Also, upregulated expression of ß-1,3-glucanase, chitinase, PR-1 and PR-10 genes were recorded with 1.48, 1.15, 1.15, and 1.41, fold expression in 24 hpi, respectively, in plants inoculated with CNPs. The most significant up-regulation was observed in transcript profile of SOD that showed 4.5-foldexpression, at 48 hpi. Therefore, our results confirmed that CS and CNPs induced up-regulation of PR-proteins and antioxidant genes might play a significant role for successful biocontrol.

Crystal structural basis for Rv0315, an immunostimulatory antigen and inactive beta-1,3-glucanase of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) remains a leading cause of morbidity and mortality worldwide, as two billion people are latently infected with Mtb. To address Mtb drug resistance and the limitations of current vaccines, the characteristics of candidate Mtb vaccines need to be explored. Here, we report the three-dimensional structure of Rv0315 at 1.70 Å resolution, a novel immunostimulatory antigen of Mtb, and demonstrate that Rv0315 is an inactive ß-1,3-glucanase of the glycoside hydrolase 16 (GH16) family. Our study further elaborates the molecular basis for the lack of glucan recognition by Rv0315. Rv0315 has a large open groove, and this particular topology cannot bind oligosaccharide chains in solution, thus explaining the lack of detectable hydrolytic activity towards its substrate. Additionally, we identified Glu-176, a conserved catalytic residue in GH16 endo-ß-1,3-glucanases, as essential for Rv0315 to induce immunological responses. These results indicate that Rv0315 likely diverged from a broad-specificity ancestral GH16 glucanase, and this inactive member of the GH16 family offers new insights into the GH16 glucanase. Together, our findings suggest that an inactive ß-1,3-glucanase in Mtb drives T-helper 1 (Th1) immune responses, which may help develop more effective vaccines against Mtb infection.

The Immunoreactive Exo-1,3-ß-Glucanase from the Pathogenic Oomycete Pythium insidiosum Is Temperature Regulated and Exhibits Glycoside Hydrolase Activity.

The oomycete organism, Pythium insidiosum, is the etiologic agent of the life-threatening infectious disease called "pythiosis". Diagnosis and treatment of pythiosis is difficult and challenging. Novel methods for early diagnosis and effective treatment are urgently needed. Recently, we reported a 74-kDa immunodominant protein of P. insidiosum, which could be a diagnostic target, vaccine candidate, and virulence factor. The protein was identified as a putative exo-1,3-ß-glucanase (Exo1). This study reports on genetic, immunological, and biochemical characteristics of Exo1. The full-length exo1 coding sequence (2,229 bases) was cloned. Phylogenetic analysis showed that exo1 is grouped with glucanase-encoding genes of other oomycetes, and is far different from glucanase-encoding genes of fungi. exo1 was up-regulated upon exposure to body temperature, and its gene product is predicted to contain BglC and X8 domains, which are involved in carbohydrate transport, binding, and metabolism. Based on its sequence, Exo1 belongs to the Glycoside Hydrolase family 5 (GH5). Exo1, expressed in E. coli, exhibited ß-glucanase and cellulase activities. Exo1 is a major intracellular immunoreactive protein that can trigger host immune responses during infection. Since GH5 enzyme-encoding genes are not present in human genomes, Exo1 could be a useful target for drug and vaccine development against this pathogen.

An Enzymatically Active ß-1,3-Glucanase from Ash Pollen with Allergenic Properties: A Particular Member in the Oleaceae Family.

Endo-ß-1,3-glucanases are widespread enzymes with glycosyl hydrolitic activity involved in carbohydrate remodelling during the germination and pollen tube growth. Although members of this protein family with allergenic activity have been reported, their effective contribution to allergy is little known. In this work, we identified Fra e 9 as a novel allergenic ß-1,3-glucanase from ash pollen. We produced the catalytic and carbohydrate-binding domains as two independent recombinant proteins and characterized them from structural, biochemical and immunological point of view in comparison to their counterparts from olive pollen. We showed that despite having significant differences in biochemical activity Fra e 9 and Ole e 9 display similar IgE-binding capacity, suggesting that ß-1,3-glucanases represent an heterogeneous family that could display intrinsic allergenic capacity. Specific cDNA encoding Fra e 9 was cloned and sequenced. The full-length cDNA encoded a polypeptide chain of 461 amino acids containing a signal peptide of 29 residues, leading to a mature protein of 47760.2 Da and a pI of 8.66. An N-terminal catalytic domain and a C-terminal carbohydrate-binding module are the components of this enzyme. Despite the phylogenetic proximity to the olive pollen ß-1,3-glucanase, Ole e 9, there is only a 39% identity between both sequences. The N- and C-terminal domains have been produced as independent recombinant proteins in Escherichia coli and Pichia pastoris, respectively. Although a low or null enzymatic activity has been associated to long ß-1,3-glucanases, the recombinant N-terminal domain has 200-fold higher hydrolytic activity on laminarin than reported for Ole e 9. The C-terminal domain of Fra e 9, a cysteine-rich compact structure, is able to bind laminarin. Both molecules retain comparable IgE-binding capacity when assayed with allergic sera. In summary, the structural and functional comparison between these two closely phylogenetic related enzymes provides novel insights into the complexity of ß-1,3-glucanases, representing a heterogeneous protein family with intrinsic allergenic capacity.

The C-terminal domains of two homologous Oleaceae ß-1,3-glucanases recognise carbohydrates differently: Laminarin binding by NMR.

Ole e 9 and Fra e 9 are two allergenic ß-1,3-glucanases from olive and ash tree pollens, respectively. Both proteins present a modular structure with a catalytic N-terminal domain and a carbohydrate-binding module (CBM) at the C-terminus. Despite their significant sequence resemblance, they differ in some functional properties, such as their catalytic activity and the carbohydrate-binding ability. Here, we have studied the different capability of the recombinant C-terminal domain of both allergens to bind laminarin by NMR titrations, binding assays and ultracentrifugation. We show that rCtD-Ole e 9 has a higher affinity for laminarin than rCtD-Fra e 9. The complexes have different exchange regimes on the NMR time scale in agreement with the different affinity for laminarin observed in the biochemical experiments. Utilising NMR chemical shift perturbation data, we show that only one side of the protein surface is affected by the interaction and that the binding site is located in the inter-helical region between α1 and α2, which is buttressed by aromatic side chains. The binding surface is larger in rCtD-Ole e 9 which may account for its higher affinity for laminarin relative to rCtD-Fra e 9.

Molecular Interactions of ß-(1→3)-Glucans with Their Receptors.

ß-(1→3)-Glucans can be found as structural polysaccharides in cereals, in algae or as exo-polysaccharides secreted on the surfaces of mushrooms or fungi. Research has now established that ß-(1→3)-glucans can trigger different immune responses and act as efficient immunostimulating agents. They constitute prevalent sources of carbons for microorganisms after subsequent recognition by digesting enzymes. Nevertheless, mechanisms associated with both roles are not yet clearly understood. This review focuses on the variety of elucidated molecular interactions that involve these natural or synthetic polysaccharides and their receptors, i.e., Dectin-1, CR3, glycolipids, langerin and carbohydrate-binding modules.

Sublethal heavy metal stress stimulates innate immunity in tomato.

Effect of sublethal heavy metal stress as plant biotic elicitor for triggering innate immunity in tomato plant was investigated. Copper in in vivo condition induced accumulation of defense enzymes like peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), and ß-1,3 glucanase along with higher accumulation of total phenol, antioxidative enzymes (catalase and ascorbate peroxidase), and total chlorophyll content. Furthermore, the treatment also induced nitric oxide (NO) production which was confirmed by realtime visualization of NO burst using a fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2DA) and spectrophotometric analysis. The result suggested that the sublethal dose of heavy metal can induce an array of plant defense responses that lead to the improvement of innate immunity in plants.

Protocol for simultaneous isolation of three important banana allergens.

Banana fruit (Musa acuminata) has become an important food allergen source in recent years. So far, 5 IgE reactive banana proteins have been identified, and the major allergens are: Mus a 2 (a class I chitinase, 31kDa), Mus a 4 (thaumatin-like protein, 21kDa), and Mus a 5 (ß-1,3-glucanase, 33kDa). Due to variations in allergen expression levels, diagnostic reagents for food allergy can be improved by using individual allergen components instead of banana allergen extracts. The purpose of this study was to optimize the purification protocol of the three major allergens present in banana fruit: Mus a 2, Mus a 4 and Mus a 5. By employing a three-step purification protocol (a combination of anion-exchange, cation-exchange and reversed-phase chromatography) three important banana allergens were obtained in sufficient yield and high purity. Characterization of the purified proteins was performed by both biochemical (2-D PAGE, mass fingerprint and N-terminal sequencing) and immunochemical (immunoblot) methods. IgE reactivity to the purified allergens was tested by employing sera of five allergic patients. The purified allergens displayed higher sensitivity in IgE detection than the routinely used extracts. The three purified allergens are good candidates for reagents in component-based diagnosis of banana allergy.

Molecular characterization of recombinant mus a 5 allergen from banana fruit.

Allergy to banana fruit appears to have become an important cause of fruit allergy in Europe. Among five allergens that have been found, beta-1,3-glucanase denoted as Mus a 5 was identified as a candidate allergen for the component-resolved allergy diagnosis of banana allergy. Because of the variations in protein levels in banana fruit, in this study Mus a 5 was produced as a fusion protein with glutathione-S-transferase in Escherichia coli. The recombinant Mus a 5 was purified under native conditions by a combination of affinity, ion-exchange, and reversed phase chromatography. N-terminal sequence was confirmed by Edman degradation and 55 % of the primary structure was identified by mass fingerprint, while the secondary structure was assessed by circular dichroism spectroscopy. IgG reactivity of recombinant protein was shown in 2-D immunoblot with anti-Mus a 5 antibodies, while IgG and IgE binding to natural Mus a 5 was inhibited with the recombinant Mus a 5 in immunoblot inhibition test. IgE reactivity of recombinant Mus a 5 was shown in ELISA within a group of ten persons sensitized to banana fruit. Recombinant Mus a 5 is a novel reagent suitable for the component-resolved allergy diagnosis of banana allergy.

Sensitization profiles to purified plant food allergens among pediatric patients with allergy to banana.

Banana fruit allergy is well known, but neither immunoglobulin E recognition patterns to purified plant food allergens nor true prevalences of putative banana allergens have been established. This study aimed to characterize ß-1,3-glucanase and thaumatin-like protein (TLP) as banana allergens, testing them, together with other plant food allergens, in 51 children with allergic reactions after banana ingestion and both positive specific IgE and skin prick test (SPT) to banana. Banana ß-1,3-glucanase and TLP were isolated and characterized. Both banana allergens, together with kiwifruit TLP Act d 2, avocado class I chitinase Pers a 1, palm pollen profilin Pho d 2 and peach fruit lipid transfer protein (LTP) Pru p 3, were tested by in vitro and in vivo assays. Banana ß-1,3-glucanase (Mus a 5) was glycosylated, whereas banana TLP (Mus a 4) was not, in contrast with its homologous kiwi allergen Act d 2. Specific IgE to both banana allergens, as well as to peach Pru p 3, was found in over 70% of sera from banana-allergic children, and Mus a 4 and Pru p 3 provoked positive SPT responses in 6 of the 12 tested patients, whereas Mus a 5 in only one of them. Both peptidic epitopes and cross-reactive carbohydrate determinants were involved in the IgE-binding to Mus a 5, whereas cross-reactivity between Mus a 4 and Act d 2 was only based on common IgE protein epitopes. Profilin Pho d 2 elicited a relevant proportion of positive responses on in vitro (41%) and in vivo (58%) tests. Therefore, Mus a 4 and LTP behave as major banana allergens in the study population, and profilin seems to be also a relevant allergen. Mus a 5 is an equivocal allergenic protein, showing high IgE-binding to its attached complex glycan, and low in vivo potency.

Adaptive evolution in subterranean termite antifungal peptides.

We identified and analysed mRNA sequences of two immune proteins from the subterranean termites Reticulitermes flavipes and Reticulitermes virginicus. These proteins correspond to two immune proteins described in the distantly related termite genus Nasutitermes; termicin, which is a small antifungal peptide, and GNBP2, which functions both as a broad pattern recognition receptor and a direct antifungal effector. A population genetic analysis of nucleotide intraspecific polymorphism and interspecific divergence indicates that a selective sweep has reduced polymorphism in the termicins. Moreover, this selective sweep appears to have been driven by the positive selection of beneficial amino acid changes in the antifungal peptide. In contrast, the pattern of polymorphism and divergence in GNBP2 corresponds to the standard neutral model of evolution.

The 74-kilodalton immunodominant antigen of the pathogenic oomycete Pythium insidiosum is a putative exo-1,3-beta-glucanase.

The oomycetous, fungus-like, aquatic organism Pythium insidiosum is the causative agent of pythiosis, a life-threatening infectious disease of humans and animals living in tropical and subtropical areas of the world. Common sites of infection are the arteries, eyes, cutaneous/subcutaneous tissues, and gastrointestinal tract. Diagnosis of pythiosis is time-consuming and difficult. Radical excision of the infected organs is the main treatment for pythiosis because conventional antifungal drugs are ineffective. An immunotherapeutic vaccine prepared from P. insidiosum crude extract showed limited efficacy in the treatment of pythiosis patients. Many pythiosis patients suffer lifelong disabilities or die from an advanced infection. Recently, we identified a 74-kDa major immunodominant antigen of P. insidiosum which could be a target for development of a more effective serodiagnostic test and vaccines. Mass spectrometric analysis identified two peptides of the 74-kDa antigen (s74-1 and s74-2) which perfectly matched a putative exo-1,3-ss-glucanase (EXO1) of Phytophthora infestans. Using degenerate primers derived from these peptides, a 1.1-kb product was produced by PCR, and its sequence was found to be homologous to that of the P. infestans exo-1,3-ss-glucanase gene, EXO1. Enzyme-linked immunosorbent assays targeting the s74-1 and s74-2 synthetic peptides demonstrated that the 74-kDa antigen was highly immunoreactive with pythiosis sera but not with control sera. Phylogenetic analysis using part of the 74-kDa protein-coding sequence divided 22 Thai isolates of P. insidiosum into two clades. Further characterization of the putative P. insidiosum glucanase could lead to new diagnostic tests and to antimicrobial agents and vaccines for the prevention and management of the serious and life-threatening disease of pythiosis.

Mapping of IgE-binding epitopes on the major latex allergen Hev b 2 and the cross-reacting 1,3beta-glucanase fruit allergens as a molecular basis for the latex-fruit syndrome.

Nine distinct IgE-binding epitopes were identified along the entire amino acid sequence of the major latex allergen Hev b 2 (1,3beta-glucanase) using a set of synthetic 15-mer peptides frameshifted by 3 residues immobilized on cellulose membrane (Spot technique). Most of the amino acid residues building these IgE-binding epitopic regions are nicely exposed on the surface and the epitopes usually correspond to charged regions on the molecular surface of the protein. A smaller number of 5 IgE-binding epitopic areas was identified on the banana 1,3beta-glucanase, which exhibits a very similar overall conformation and charge distribution. The latter epitopes might be responsible for the IgE-binding cross-reactivity currently observed in the latex-fruit syndrome. Using rabbit polyclonal IgG anti-BanGluc as a probe instead of IgE from allergic patients the same epitopic regions were identified in both Hev b 2 and BanGluc. Additionally, surface-exposed regions with a very close conformation were predicted to occur on Ole e 9, the 1,3beta-glucanase allergen identified in olive pollen.

Hypersensitivity to black locust (Robinia pseudoacacia) pollen: "allergy mirages".

BACKGROUND: The allergenicity of the ornamental tree Robinia pseudoacacia, or black locust, is unknown. OBJECTIVE: To evaluate the prevalence of sensitization to R. pseudoacacia pollen, its possible allergenic cross-reactivity with other common pollens, and the potential implication of pollen panallergens (profilin, polcalcin, and 1,3-beta-glucanase) as a cause of sensitization to R. pseudoacacia pollen. METHODS: Skin prick testing with R. pseudoacacia pollen was performed in 149 patients with pollinosis. Nasal challenge with R. pseudoacacia pollen was performed in 10 patients. The prevalence of sensitization to the recombinant forms of profilin (rChe a 2), polcalcin (rChe a 3), and the N-terminal of the 1,3-beta-glucanase (rNtD of Ole e 9) was investigated. Immunoblotting, enzyme-linked immunosorbent assay, and competitive inhibition assays were performed with R. pseudoacacia pollen and recombinant pollen allergens. RESULTS: Sixty-four patients (43%) had positive skin prick test reactions to R. pseudoacacia pollen. Nasal challenge results were positive in 5 sensitized patients and negative in 4 controls and 1 sensitized patient. The allergenic profile of R. pseudoacacia pollen comprises at least the panallergen profilin, a calcium-binding protein, and a 1,3-beta-glucanase. The prevalence of sensitization to rChe a 2, rChe a 3, and rNtD of Ole e 9 was 60%, 33%, and 87%, respectively, among patients sensitized to R. pseudoacacia pollen. Binding of IgE to R. pseudoacacia extract was completely inhibited by Robinia, Chenopodium, Olea, Cupressus, and Lolium extracts. CONCLUSIONS: The high prevalence of R. pseudoacacia pollen sensitization in patients with pollinosis is likely to be due to cross-sensitization to panallergens (profilin, polcalcin, and 1,3-beta-glucanase) from other common pollens. This phenomenon may lead to a diagnosis of "allergy mirages."

Decoding serological response to Candida cell wall immunome into novel diagnostic, prognostic, and therapeutic candidates for systemic candidiasis by proteomic and bioinformatic analyses.

In an effort to bring novel diagnostic and prognostic biomarkers or even potential targets for vaccine design for systemic candidiasis (SC) into the open, a systematic proteomic approach coupled with bioinformatic analysis was used to decode the serological response to Candida wall immunome in SC patients. Serum levels of IgG antibodies against Candida wall-associated proteins (proteins secreted from protoplasts in active wall regeneration, separated by two-dimensional gel electrophoresis, and identified by mass spectrometry) were measured in 45 SC patients, 57 non-SC patients, and 61 healthy subjects by Western blotting. Two-way hierarchical clustering and principal component analysis of their serum anti-Candida wall antibody expression patterns discriminated SC patients from controls and highlighted the heterogeneity of their expression profiles. Multivariate logistic regression models demonstrated that high levels of antibodies against glucan 1,3-beta-glucosidase (Bgl2p) and the anti-wall phosphoglycerate kinase antibody seropositivity were the only independent predictors of SC. Receiver operating characteristic curve analysis revealed no difference between their combined evaluation and measurement of anti-Bgl2p antibodies alone. In a logistic regression model adjusted for known prognostic factors for mortality, SC patients with high anti-Bgl2p antibody levels or a positive anti-wall enolase antibody status, which correlated with each other, had a reduced 2-month risk of death. After controlling for each other, only the seropositivity for anti-wall enolase antibodies was an independent predictor of a lower risk of fatality, supporting that these mediated the protective effect. No association between serum anti-cytoplasmic enolase antibody levels and outcomes was established, suggesting a specific mechanism of enolase processing during wall biogenesis. We conclude that serum anti-Bgl2p antibodies are a novel accurate diagnostic biomarker for SC and that, at high levels, they may provide protection by modulating the anti-wall enolase antibody response. Furthermore serum anti-wall enolase antibodies are a new prognostic indicator for SC and confer protection against it. Bgl2p and wall-associated enolase could be valuable candidates for future vaccine development.

1,3-beta-glucanases as candidates in latex-pollen-vegetable food cross-reactivity.

BACKGROUND: 1,3-beta-glucanases (group 2 of pathogenesis-related proteins) are enzymes widely distributed among higher plants and have been recently proven to be significant allergens. OBJECTIVE: The aim of this work was to study the potential implication of 1,3-beta-glucanases in cross-reactivities among latex, pollen and vegetable foods. METHODS: The cDNA encoding the N-terminal domain (NtD) of Ole e 9, a major allergenic 1,3-beta-glucanase from olive pollen, was amplified by polymerase chain reaction and produced as a recombinant protein in Pichia pastoris (recombinant N-terminal domain, rNtD). Circular dichroism, ELISA, immunoblotting and immunoblotting inhibition experiments were carried out. Sera from olive pollen allergic patients and a rNtD-specific polyclonal antiserum were used. RESULTS: The NtD of Ole e 9 has been produced at high yield in the yeast P. pastoris and possesses 1,3-beta-glucanase activity. The expressed polypeptide conserves IgE and IgG immunodominant epitopes of the whole Ole e 9. A rNtD-specific polyclonal antiserum and sera from olive pollen allergic patients allowed detection of IgG and IgE reactive peptidic epitopes common to 1,3-beta-glucanase Ole e 9 in extracts from ash and birch pollen, tomato, potato, bell-pepper, banana and latex. CONCLUSION: rNtD and homologous glucanases are new molecules to be used in diagnostic protocols as they could help to identify allergic pollen patients who are at risk for developing allergic symptoms to fruits, vegetables and latex.

Characterization of cross-reactive bell pepper allergens involved in the latex-fruit syndrome.

BACKGROUND: Between 30% and 50% of individuals who are allergic to latex products are also allergic to specific plant foods, a fact that is well documented as the latex-fruit syndrome. Simultaneous sensitization to latex and bell pepper has been previously reported. Although bell pepper fruits are frequently consumed raw, cooked or as a spice, little is known about the cross-reactive allergens. OBJECTIVE: In this study we wished to identify bell pepper allergens involved in the latex-fruit syndrome. METHODS: Sera of four patients who displayed clinical symptoms to latex and bell pepper were used in immunoblot studies on protein extracts of three different cultivars of fresh bell pepper and fresh Hevea latex. Cross-reactive allergens were identified by inhibition experiments using recombinant Hev b 8 (latex profilin), and natural Hev b 2 (latex beta-1,3-glucanase) in addition to the protein extracts. A novel cross-reactive IgE-reactive 30 kDa protein was subjected to sequence analysis. RESULTS: Three patients displayed IgE to profilins from bell pepper fruits and latex. Two patients possessed IgE to Hev b 2, a latex beta-1,3-glucanase, and a homologous protein in bell pepper. One patient possessed IgE reactive with a protein of 30 kDa identified by N-terminal sequencing as an l-ascorbate peroxidase and another patient to a protein of 38 kDa. Additionally, IgE binding proteins in two higher molecular weight ranges showed cross-reactive capacities. CONCLUSION: Our findings show on the molecular level that bell pepper is part of the latex-fruit syndrome. For the first time we have identified the major latex allergen Hev b 2, a beta-1,3-glucanase, and the bell pepper l-ascorbate peroxidase as cross-reactive allergens. We were also able to show that profilins are responsible for some of the IgE cross-reactivity.