Добірка наукової літератури з теми "Fungal Lectin"

Fungal lectins are a large family of carbohydrate-binding proteins with no enzymatic activity. They play fundamental biological roles in the interactions of fungi with their environment and are found in many different species across the fungal kingdom. In particular, their contribution to defense against feeders has been emphasized, and when secreted, lectins may be involved in the recognition of bacteria, fungal competitors and specific host plants. Carbohydrate specificities and quaternary structures vary widely, but evidence for an evolutionary relationship within the different classes of fungal lectins is supported by a high degree of amino acid sequence identity. The UniLectin3D database contains 194 fungal lectin 3D structures, of which 129 are characterized with a carbohydrate ligand. Using the UniLectin3D lectin classification system, 109 lectin sequence motifs were defined to screen 1223 species deposited in the genomic portal MycoCosm of the Joint Genome Institute. The resulting 33,485 putative lectin sequences are organized in MycoLec, a publicly available and searchable database. These results shed light on the evolution of the lectin gene families in fungi.

Clitocybe nebularis lectin (CNL) is present in fruiting bodies of clouded agaric along with several similar isolectins that are all small and stable proteins. It is a beta-trefoil type lectin forming homodimers that are essential for its functionality. It binds specifically N,N’-diacetyllactosediamine (GalNAcβ1-4GlcNAc, LacDiNac) and human blood group A determinant-containing glycan epitopes. Its most probable function is to defend fruiting bodies against predators and parasites. In addition, an endogenous regulatory function is possible for CNL, as indicated by its interaction with fungal protease inhibitors sharing the beta-trefoil fold. CNL is toxic to insects, nematodes and amoebae, as well as to leukemic T-cell lines. Bivalent carbohydrate binding is essential for the toxicity of CNL, against both invertebrates and cancer-derived cell lines. In addition, CNL exhibits potent immunostimulation of human dendritic cells, resulting in a strong T helper cell type 1 response. Based on its unique characteristics, CNL is a promising candidate for applications in human and veterinary medicine as well as in agriculture, for plant protection.

Actinoporins are potent eukaryotic pore-forming toxins specific for sphingomyelin-containing membranes. They are structurally similar to members of the fungal fruit-body lectin family that bind cell-surface exposed Thomsen–Friedenreich antigen. In the present study we found a number of sequences in public databases with similarity to actinoporins. They originate from three animal and two plant phyla and can be classified in three families according to phylogenetic analysis. The sequence similarity is confined to a region from the C-terminal half of the actinoporin molecule and comprises the membrane binding site with a highly conserved P-[WYF]-D pattern. A member of this novel actinoporin-like protein family from zebrafish was cloned and expressed in Escherichia coli. It displays membrane-binding behaviour but does not have permeabilizing activity or sphingomyelin specificity, two properties typical of actinoporins. We propose that the three families of actinoporin-like proteins and the fungal fruit-body lectin family comprise a novel superfamily of membrane binding proteins, tentatively called AF domains (abbreviated from actinoporin-like proteins and fungal fruit-body lectins).

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.

Abstract Plant lectins are carbohydrate-binding proteins with nonimmune origin, which can reversibly bind with carbohydrates, agglutinate cells, and precipitate polysaccharides and glycoconjugates. Plant lectins have attracted much attention for their anti-virus, anti-proliferation, and pro-apoptosis properties. Thus the exploration of new lectins has received special attention. Here we purified a mannose-binding lectin from the rhizomes of Liparis nervosa by ion exchange chromatography on DEAE-Sepharose, affinity chromatography on Mannose-Sepharose 4B, and gel filtration chromatography on Sephacryl S-100. The purified L. nervosa lectin (LNL) was identified to be a monomeric protein with a molecular mass of 13 kDa. LNL exhibited hemagglutinating activity towards rabbit erythrocytes, and its activity could be strongly inhibited by D-mannose, N-acetyl glucosamine and thyroglobulin. In vitro experiments showed that LNL exhibited a comparable anti-fungal activity against Piricularia oryzae (Cavara), Bipolaris maydis, Fusarium graminearum, and Sclerotium rolfsii, and anti-proliferation activity against tumor cells by inducing apoptosis. The full-length cDNA sequence of LNL is 715 bp in length and contains a 525 bp open reading frame (ORF) encoding a 110-residue mature protein. It was predicted to have three mannose-binding conserved motifs ‘QXDXNXVXY’. The binding pattern of LNL was further revealed by homology modeling and molecular docking. We demonstrated that LNL is not only a potential therapeutic candidate against tumor but also a new anti-fungal agent.

We report the development of a cytochemical affinity technique for detection of galacturonic acids at the ultrastructural level. The highly purified gonad lectin from Aplysia depilans (AGL) was tagged with colloidal gold particles and used for labeling carbohydrates in resin-embedded sections of various plant and fungal tissues. Patterns of AGL binding sites were compared to those obtained with a D-galactose-specific lectin, Ricinus communis agglutinin I. Differences in labeling patterns were noted, indicating that the lectins exhibited differential carbohydrate binding. In addition, the considerable loss of labeling over isolated wheat coleoptile walls treated for removal of pectin, after incubation with the AGL-gold complex, strongly suggested an affinity of AGL for pectic substances. A series of cytochemical controls, including sugar inhibition tests, has proven the specificity of the technique and the high affinity of AGL towards galacturonic acids. The potential value of this new lectin for ultrastructural studies on cell wall pectic substances in plant biology and pathology is demonstrated.

Lectins are involved in recognition phenomena and their ability to bind particular Carbohydrate structures are the key to their biological functions. Bacteria typically attaches to prospective host cell membranes in receptors with lectin like sugar specificity. This is of great importance as the adherence of bacteria to host tissue surfaces is the initial event in bacterial infection. Lectins are also known to play important roles in immune system by recognizing carbohydrates that are found exclusively on pathogens, or that are inaccessible on host cells. This ability of lectins to selectively bind or agglutinate specific sugars have made them useful tools for the characterization of certain cell types or fragments, to detect cells in different states of development, to distinguish normal from tumour cells and to separate different cell types by affinity chromatography. A total of 120 samples of local Achatina achatina snail specie were collected, authenticated at the Zoology Department of the University of Nigeria, Nsukka and 80mls of pooled crude Lectin extract was obtained. Purifications were performed on 20mls of the crude extract in three steps viz, Ammonium sulphate precipitation and Dialysis (Partial purifications), Con A Sepharose 4B affinity chromatography column (Complete purification). The affinity purified lectin was used in all the tests conducted in this research. The crude, partially and complete/affinity purified lectin extracts were subjected to Haemagglutination and Protein Assay tests. The Molecular weight was deduced by Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. The microbial agglutination potentials of the lectin was assessed by testing typed bacterial organisms viz, Salmonella typhimurium, Escherichia coli, Lactobacilli acidophilus, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella aeruginosa and four typed fungal organisms: Aspergillus niger, Trichophyton mentagrophytes, Candida albicans and A. flavus. The lectin’s Lymphocyte blastogenesis activities was determined by its incubation with human lymphocytes for mitogenic stimulation assay. The results of the research showed as follows: On complete/affinity purification, 15mls of pure sample containing only the high molecular weight lectin was obtained. On standardization, the respective haemagglutination tests on the crude, partially and affinity purified lectin showed preferential agglutinations with Blood group A type. Only S. typhimurium (+++), E. coli (+) and L. acidophilus (+) reacted with the lectin but in different strengths. Incubation of the lectin with lymphocytes from human serum showed that it has the ability to stimulate lymphocytes to undergo mitosis. This research has therefore succeeded in assessing the Microbial agglutination and Lymphocyte blastogenesis potentials of the isolated and characterised A. achatina snail lectin.

Lectins have been increasingly utilized as carriers for targeted drug delivery based on their specific binding to glycans located on mammalian cells. This study employed two lectins, B subunit of bacterial Shiga holotoxin (Stx1B) and fungal Clitocybe nebularis lectin (CNL), for surface display on the lactic acid bacterium Lactococcus lactis. The specific adhesion of these engineered, lectin-displaying L. lactis to cancer cells was evaluated. The expression and surface display of both lectins on L. lactis were demonstrated by western blotting and flow cytometry, respectively. MTS assays revealed that recombinant Stx1B had no effect on Caco-2 cell viability at concentrations of ≤25 µg/mL, whereas CNL was non-toxic even at relatively high concentrations of ≤250 µg/mL. Stx1B bound to Caco-2, HT-29 and HeLa cells after 1 h of incubation. CNL bound to Caco-2 cells and recognized several glycoproteins in HT-29 and Caco-2 cell homogenates of which a 70 kDa protein predominated. Confocal microscopy revealed adhesion of Stx1B-displaying L. lactis to HeLa, Caco-2, and, to a lesser extent, HT-29 cells; CNL-displaying L. lactis showed a relatively similar level of adherence to HT-29 and Caco-2 cells. Thus, lectin-displaying L. lactis might serve as a carrier in targeted drug delivery when coupled to a therapeutic moiety.

Innate signals are fundamental to determine the class of adaptive response against infection. The translation of microbial signatures into adaptive immunity is mediated by pattern recognition receptors (PRRs), which are expressed in specialized leukocytes called dendritic cells (DCs) and results in responses matched to the nature of the offending microbe. Here, I provide evidence that two Syk-coupled C-type lectin receptors (CLRs), Dectin-1 and Dectin-2, act in the coordination of adaptive immune responses to fungal stimuli and fungal pathogens. DC activated via Dectin-1 are strong elicitors of IL-17 production by CD4+ T cells. Results presented in this thesis demonstrate that Dectin-1 signalling in DCs results in the generation and Foxp3+-IL-17+ T cells, a cell type that defies either regulatory or Th17 classification. This process is dependent on IL-23, which is produced by DCs upon Dectin-1 ligation. In addition, this work identified an additional CLR responsible for the induction of Th17 responses during the course of fungal infections. This thesis demonstrates that Dectin-2 is a second Syk-coupled PRR involved in DC activation by fungi. In a model of Candida albicans systemic infection, Dectin-2 is essential for the induction of Th17 responses to the organism. Finally, I have generated tools to study responses of T cells specific for a fungal-associated antigen. Furthermore, I provide preliminary evidence regarding the activation, proliferation and trafficking of antigen-specific CD4+ and CD8+ T cells during systemic fungal infections.

Strawberry (Fragaria × ananassa) is an important soft fruit but easily to be infected by pathogens. Anthracnose and gray mold are two of the most destructive diseases of strawberry which lead to serious fruit rot. The first chapter introduced strawberry anthracnose caused by Colletotrichum acutatum. The infection strategy, disease cycle and management of C. acutatum on strawberry were reported. Likewise, the second chapter summarized the infection strategy of Botrytis cinerea and the defense responses of strawberry. As we already know white unripe strawberry fruits are more resistant to C. acutatum than red ripe fruits. During the interaction between strawberry white/red fruit and C. acutaum, a mannose binding lectin gene, FaMBL1, was found to be the most up-regulated gene and induced exclusively in white fruit. FaMBL1 belongs to the G-type lectin family which has important roles in plant development and defense process. To get insight into the role of FaMBL1, genome-wide identification was carried out on G-type lectin gene family in Fragaria vesca and the results were showed in chapter 3. G-type lectin genes make up a large family in F. vesca. Active expression upon biotic/abiotic stresses suggested a potential role of G-lectin genes in strawberry defenses. Hence, stable transgenic strawberry plants with FaMBL1 gene overexpressed were generated. Transformed strawberry plants were screened and identified. The results were showed in chapter 4, content of disease-related phytohormone, jasmonic acid, was found decreased in overexpressing lines compared with wild type (WT). Petioles inoculated by C. fioriniae of overexpressing lines had lower disease incidence than WT. Leaves of overexpressing lines challenged by B. cinerea showed remarkably smaller lesion diameters compared with WT. The chitinase 2-1 (FaChi2-1) showed higher expression in overexpressing lines than in WT during the interaction with B. cinerea, which could be related with the lower susceptibility of overexpressing lines.

Cryptococcus sp é um fungo saprófita, cosmopolita, que causa micose sistêmica, geralmente, subaguda ou crônica, conhecida, sobretudo, por sua localização meníngea, após aquisição da infecção por via respiratória Embora seja ubíquo, a criptococose ocorre predominantemente em indivíduos imunodeficientes e podendo ocorrer, também, em indivíduos imunocompetentes. Os estudos experimentais e em humanos avaliando a ativação do sistema complemento e a produção de anticorpos específicos mostram que a resposta inata e de anticorpos são importantes para a delimitação do processo infeccioso por Cryptococcus sp, como também, a administração de anticorpos monoclonais podem induzir uma resposta eficaz na disseminação da doença. O sistema complemento contribui para a defesa do organismo contra o Cryptococcus sp de diferentes maneiras: secretando opsoninas e fatores quimiotáticos e colaborando com a ação dos anticorpos específicos, aumentando a interação entre a imunidade inata e adquirida. Os anticorpos antiglicuroxilomanana (GXM) possuem numerosas atividades biológicas: a) opsonização para fagocitose, b) ativação da via clássica do complemento resultando na deposição precoce de fragmentos de C3 no fungo, c) supressão do excesso de acúmulo de C3 pela via alternativa; d) facilitação do clareamento do GXM do soro in vivo, resultando no maior acúmulo de GXM nos tecidos ricos em células do sistema fagocítico mononuclear; e) proteção em modelos murinos da criptococose e f) facilitação de vários aspectos da imunidade celular ao Cryptococcus sp. O objetivo desse estudo foi avaliar a resposta humoral ao GXM e às proteínas da parede celular (Ag S) avaliando a atividade do sistema complemento como também a produção de anticorpos específicos em amostras séricas de adultos com e sem neurocriptococose. Foram coletadas 106 amostras de soro e divididas em 3 grupos: grupo 1- 21 indivíduos com neurocriptococose e baixa exposição a levedura, grupo 2- foi composto por 23 indivíduos saudáveis com alta exposição ao fungo e HIV negativos, granjeiros da cidade de Jumirim localizada a 164 km de São Paulo, na região de Sorocaba e, o grupo 3- 60 indivíduos saudáveis, HIV negativos e com baixa exposição ao Cryptococcus sp. Dois pacientes foram excluídos do estudo por apresentarem tumores (timona e câncer de pulmão). O sistema complemento foi avaliado por ensaio hemolítico (CH 50 e AP 50) e, a dosagem da proteína ligadora de manose (MBL) foi feita por ELISA. Os valores de CH 50 estiveram dentro da normalidade em 17/21, 13/23, 59/60 indivíduos dos grupos 1, 2 e 3 respectivamente. A média dos valores de CH 50 foi diferente significativamente entre o três grupos (P < 0,0001). O grupo 2 mostrou níveis reduzidos significantes em comparação aos dois outros grupos. Os valores de AP 50 estiveram dentro da normalidade em 11/21; 21/23 e 60/60 indivíduos dos grupos 1, 2 e 3 respectivamente. Houve diferença nos valores de AP 50 (P = 0,0005) e apenas um paciente do grupo 1 apresentou valores indetectáveis desta via. Houve diferença significante na dosagem de MBL entre os três grupos (P = 0,0277). Anticorpos IgG anti-GXM foram quantificados por ELISA e expressos por densidade óptica (DO). IgG anti GXM foi detectado em todos os grupos com diferença significante entre eles (P= 0,0127). As médias de IgG anti- GXM (DO) foram: 1.191 (0,49 a 1.217) no grupo 1, 1.572 (0,815 a 2.479) no grupo 2 e 0,965 (0,321 a 1.295) no grupo 3. Dois indivíduos assintomáticos do grupo 2 tiveram títulos de GXM detectáveis (1/256 e 1/32). Quatro pacientes com neurocriptococose faleceram (19%) e seus resultados mostravam: CH 50 normal, 2/4 tinham valores de AP 50 baixo (12 UI/mL) e indetectável; 3/4 tinham altos níveis de MBL e apenas um tinha baixa DO de IgG anti-GXM. Baseado em nosso estudo, podemos concluir que a resposta humoral (sistema complemento e anticorpos) não é suficiente para explicar a susceptibilidade a neurocriptococose, porém a alta e constante exposição ao Cryptococcus sp pode prevenir o desenvolvimento de doença, ou seja, a constante e intensa exposição ao fungo induz a produção de anticorpos que previnem a doença clínica mas não a infecção. Por outro lado fatores genéticos que determinam as concentrações de MBL podem influenciar na susceptibilidade a neurocriptococose. Os anticorpos contribuem para o clearence de GXM, entretanto as concentrações séricas não se correlacionam com resistência à doença
Cryptococcus sp is a fungal pathogen with a worldwide distribution. Although it is ubiquitous in the environment, cryptococcal disease occurs predominantly in immunocompromised hosts and can also occur in apparently immunocompetent individuals. The innate immunity is of special relevance for the antifungal reaction, as it allows an immediate reaction and recognizes a broad variety of fungal pathogens. The host immune response is a major determinant of the outcome of cryptococcal infection; however, the antibodies response is poorly understood. In addition, most of the studies are experimental and there is restricted knowledge concerning the human immune response. Complement system has soluble factors, restrictive regulator proteins and cellular receptors involved in defense mechanism. Glucuroxylomannan (GXM) monoclonal antibodies (MAbs) have numerous biological activities: a) opsonization for phagocytosis, b) activation of the classical complement pathway leading to early deposition of C3 fragments on the yeast, c) suppression overall accumulation of C3 via the alternative pathway; d) clearance facilitation of GXM from serum in vivo, leading to increased accumulation of GXM in tissues rich in mononuclear phagocyte system; e) protection in murine models of cryptococcosis and f) facilitation of various aspects of cellular immunity to Cryptococcus sp. The goal of our study was to evaluate if the antibody response to GXM and cell wall proteins regarding specific antibodies as well as complement system in sera of immunocompetent adults with and without neurocryptococcosis. The aim of our research was to evaluate classical and alternative complement system pathway, to quantify mannose-binding lectin (MBL) as well antibody response to GXM and cell wall proteins (AgS) regarding specific antibodies in sera of immunocompetent adults with and without neurocryptococcosis. One hundred and six samples were collected and classified in 3 groups: group 1- 21 individuals with neurocryptococcosis and low exposure to the yeast; group 2- was composed by 23 healthy individuals, chicken farmings from Jurumirim, a town 164 km to São Paulo, and with high exposure to Cryptoccocus spp and HIV negative. The third group included 60 healthy HIV negative individuals with presumed low exposure to Cryptococcus. Two patients were excluded by report of previous malignancies (timoma and pulmonary cancer). The complement system was evaluated by hemolytic assay and ELISA to MBL. CH 50 and AP 50 values were within the normal range in 17/21; 13/23; 59/60 patients in groups 1, 2 and 3 respectivelly. Mean CH 50 values were significantly different among the three groups (P < 0,0001). Group 2 showed significantly reduced levels in comparison with groups 1 and 3. AP 50 values were within the normal range in 11/21; 21/23; 60/60 patients in groups 1, 2 and 3 respectivelly. There was difference in the AP 50 values (P=0,0005) and one no activation of this pathway in group 1. There was significant difference in MBL among the groups (P = 0,0277). GXM antibodies IgG was measured by ELISA and expressed as optical density (OD). GXM- IgG was detected in all the groups with significant difference among them (P = 0,0127). The means of IgG anti-GXM (OD) were: 1.191 (range 0,49 to 1.217) in group 1, 1.572 (range 0,815 to 2.479) in group 2 and 0,965 (range 0,321 to 1.295) in the group 3. Two of the group 2 individuals had low GXM titers (1/256 and 1/32) and no symptoms. Four patients (4/21; 19%) with neurocryptococcosis died and the results showed: normal classical pathway activation, 2/4 had low (12 UI/mL) or undetectable alternative pathway values ; 3/4 had high MBL concentrations and only one had low OD for IgG anti-GXM. In conclusion, our results suggest that constant and high exposure to Cryptococcus sp can prevent the development of cryptococcosis, i.e. constant and intensive fungal exposition induces protective antibodies to clinical disease but not to the infection. In the other side, genetic factors which determine MBL concentrations could influence the susceptibility to neurocryptococcosis. The antibodies contribute to GXM clearance, however, the concentrations did not correlate with the resistance to the disease

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O Rio Grande do Sul é considerado o maior produtor de arroz irrigado do Brasil. Contudo, devido às condições climáticas da região, o potencial produtivo da lavoura de arroz ainda é limitado pela incidência de doenças fúngicas. A utilização de cultivares resistentes ou tolerantes é uma forma alternativa sustentável para reduzir as perdas na produção orizícola. A transformação genética, aliada a cultura de tecidos, vem auxiliando os programas de melhoramento vegetal na geração de plantas resistentes e tolerantes a estresses bióticos e abióticos, contribuindo para a sustentabilidade da orizicultura. Lectinas são proteínas que se ligam a carboidrados e estão envolvidas em diferentes processos biológicos, inclusive na defesa de plantas contra diversos tipos de patógenos. Desta forma, o objetivo do presente trabalho foi introduzir o gene da lectina bvl de Bauhinia veriegata em diferentes explantes da cultivar IRGA 426 a fim de se obter plantas transformadas geneticamente com o gene bvl. Para a obtenção das plantas transformadas foram usados calos organogênicos, mesocótilos e a técnica de transformação de botões florais. No experimento para a obtenção de calos via organogênese indireta, o 2,4-D na concentração de 2,0 mg L-1 foi efetiva, induzindo uma média de 47,3 calos, sendo 89% calos organogênicos aptos para à regeneração. Na regeneração dos calos, observou-se a formação média de 12,73 brotações no meio com 0,5 mg L-1 de ANA e 2,5 mg L-1 de BAP. Já na regeneração do mesocótilo, o incremento de BAP no meio de cultivo reduziu diretamente o comprimento das brotações primárias, no entanto esse efeito foi compensado pelo aumento da multiplicação dos explantes, principalmente com 5 mg L-1 de BAP, onde observou-se uma média de 21,16 brotações. Após o processo de infecção com o vetor pH7WGD2::bvl e seleção com antibiótico, os calos da cv. IRGA 426 apresentaram hiperhidricidade e não foram regenerados. Dos mesocótilos que passaram pela transformação, 20,66% sobreviveram ao meio de seleção. A análise de PCR revelou uma eficiência de transformação de 6,35% em relação ao total de brotos e, a partir do Western Blot, confirmou-se que 3 plantas estavam expressando a lectina BVL. No método de transformação imersão floral, em todas as sementes putativamente transformadas o gene gfp estava ativo, sendo observada a expressão transiente. De acordo com os resultados obtidos, os protocolos de regeneração dos explantes foram eficientes. Análises moleculares das plantas transformadas deverão ser realizadas para determinar o número de cópias do transgene no gDNA e o papel da lectina BVL na fisiologia vegetal e defesa da planta de arroz.
Rio Grande do Sul is the largest producer of irrigated rice in Brazil. However, due to the region's climate, the productive potential of crop farms is still limited by fungal diseases. The use of tolerant or resistant cultivars is a sustainable alternative to reduce loss in rice production. Genetic transformation, coupled with tissue culture, has been assisting vegetable breeding in the production of crops resistant and tolerant to abiotic/biotic stress, contributing to the sustainability of rice culture. Lectins are proteins that bind to carbohydrates and are involved in several biological processes, including defense against diseases in plants. The main goal of this study was the insertion of the Bauhinia variegata BVL Lectin gene in different explants of cultivar IRGA 426 to obtain transformed plants with the BVL gene. To obtain the transformed plants, organogenic callus, mesocotyls and flower buds transformation technique were used. In the experiment for obtaining callus through indirect organogenisis, 2,4-D at 2,0 mg L-1 was effective, inducing the formation of an average of 47,3 calluses, of which 89% of embriogenic callus were apt for regeneration. In the regeneration step, an average of 12,73 sprouts in medium with 0,5 mg L-1 of ANA and 2,5 mg L-1 of BAP was observed. In the mesocotyls regeneration, the BAP increment in the growing medium directly reduced the length of primary shoots; however, this effect was compensated by the increse of explant multiplication, mainly with 5 mg L-1 of BAP, where an average of 21,16 sprouts were observed. After infection with the pH7WGD2::bvl vector and antibiotic selection, the callus of cv. IRGA 426 showed hyperhydricity and were not regenerated. Of the mesocotyls that went through transformation, 20,66% survived the selection medium. PCR analysis revealed a transformation efficiency of 6,35% in all sprouts and, through Western Blot, 3 plants were confirmed to express the BVL lectin. In the floral immersion transformation method, in all transformed seeds the GFP gene was active, with transient expression being observed. Based on the results obtained, the explant regeneration protocols were efficient. Molecular analysis of the transformed plants should be made to determine the number of copies of the transgene in gDNA and the role of BVL lectin in the plant physiology and its role in the plant's defense.

L'objectif de cette thèse a été de contribuer à la compréhension des stratégies d'infection du pathogène opportuniste Aspergillus fumigatus. Ce pathogène est une cause émergente de morbidité et de mortalité chez les patients dont l'immunité est compromise et dans les milieux hospitaliers. Une infection avec Aspergillus est en général appelée une Aspergillose et ellepeut se développer dans un certain nombre d'organes, le plus fréquemment dansl'appareil respiratoire (poumons et sinus). Outre les infections (Aspergillosis invasive), la colonisation par ce champignon peut causer des réactions allergiques (Aspergilloses broncho pulmonaire allergique) et de l'asthme. Le nombre de patients immunodéprimés augmente régulièrement à cause des avancées des traitements du SIDA, du cancer, de la mucoviscidose ainsi que par le nombre grandissant de transplantation d'organes. De nouveaux médicaments antifongiques et des médicaments préventifs sont nécessaires pour venir en soutienaux soins médicaux des patients. Bien que plusieurs fongicides existent déjà sur le marché, l'Aspergillosisinvasive reste souvent fatale. Ceci est lié d'une part à la difficulté d'établir le diagnostic, et d'autre part au fait que des résistances émergent rapidement. La motivation de cette thèse est de comprendre les mécanismes impliqués dans le premier contact entre les conidies et le tissu de l'hôte. Ces mécanismes d'adhésion initiaux sont souvent réalisés par des liaisons entre les lectines et les carbohydrates. Le tissu épithélial et la surface muqueuse du système respiratoire sont couverts de structures possédants des carbohydratestels que les glycoprotéines, les glycolipides et les glycosaminoglycanes. L'identification des lectines d'A. fumigatus et leurs caractérisations devraient dorénavant contribuer à la compréhension de la glycostratégie de ce pathogène opportuniste ainsi que des mécanismes impliqués dans l'adhésion et l'infection. L'analyse détaillée de la structure des lectines permettra d'établir le rôle de cesprotéinesdans la virulence et de guider la conception de glycomimétiques, afin d'inhiber le phénomène d'adhésion. Cettenouvelle approche consistant à bloquer l'adhésion de l'agent pathogène plutôt que sa prolifération, vise à diminuer les résistances par une réduction de la pression évolutive. Deuxstratégies différentes ont été utilisées pouridentifier de nouvelleslectines. Tout d'abord une purification des lectinesà partir d'extraits fongiques brutsa été tentée et d'autre part un criblage pour trouver des séquences similaires avec les protéines codées par A. fumigatusa été réalisé parmi une banque de lectines fongiques connues
The aim of this thesis was to contribute to the understanding of infection strategies of the opportunistic pathogen Aspergillusfumigatus. This pathogenic mould is an emerging cause of morbidity and mortality in immuno-compromised patients and hospital environments. An infection with Aspergillus is generally referred to as Aspergillosis; it can develop in a variety of organs but the most common sites are the respiratory apparatus i.e. lungs and sinuses. Besides infections (invasive aspergillosis), colonization with the fungus can cause allergic reactions (allergic broncho pulmonary aspergillosis) and asthma. The number of immuno-suppressed patients is steadily increasing due to advancement in the HIV, cancer and cystic fibrosis medical care, as well as an increasing number of organ transplantations. Needless to say that new antifungal drugs and preventive medication is desperately needed to support medical care for those patients. Even though several fungicides already exist on the market, invasive aspergillosis remains to be often fatal. On one hand, this is due to difficulties in diagnosis and on the other hand, resistances are emerging rapidly. The motivation behind this thesis is to understand the underlying mechanisms that are involved in the first contact between conidial spores and host tissues. Initial adhesion steps often involve carbohydrate binding proteins, called lectins. They recognize glycoconjugates such as glycoproteins, glycolipids and glycosaminoglycans which cover the epithelial tissue and mucosal surface of the respiratory tract.. Identification and characterization of the lectins from A. fumigatus will therefore contribute to the understanding of the glycostrategy of this opportunistic pathogen and of the mechanisms involved in adhesion and infection. Detailed structural analysis of the carbohydrate-protein interactions will allow ascertaining the lectins role in virulence and guide the design of glycomimetics, as adhesion inhibitors. With this novel approach of targeting the pathogen adhesion rather than its proliferation, resistances are believed to be less frequent due to the lack of evolutionary pressure. In this work, two different strategies were employed to obtain novel lectins. Firstly, lectins were purified from crude fungal extracts and secondly the A. fumigatus genome was screened for encoded proteins showing sequence similarity with known fungal lectins. While lectin purification from the crude extracts was inconclusive due to low lectin activity in the starting material, genome screening showed that several putative lectins were present. One of these lectins, named AFL6, belonged to the cyanovirin-N homolog (CVNH) family and it was recombinantly expressed and purified. Glycan array and micro calorimetry techniques were carried out to investigate its carbohydrate binding specificityand the three dimensional structure was determined using X-ray crystallography. The structure showed an overall similarity with other CVNHs with slight differences in the presumed carbohydrate binding sites. Unlike other family members, it shows a low affinity for mannosides and an apparent affinity for lactosamine containing glycan structures

Orientador: Maria Ligia Rodrigues Macedo
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Este estudo descreve a purificação e a caracterização parcial de uma nova proteína de sementes de Pouteria torta, pertencente à família Sapotaceae. A proteína foi purificada pela combinação de filtração em gel, cromatografias de troca iônica e fase reversa. PAGE-SDS da proteína purificada resultou em uma única banda de 14 kDa e aglutinou eritrócitos humanos e animais. A atividade de lectin-like foi melhor inibida pelas glicoproteínas fetuína, asialofetuina, heparina, orosomucoide e ovoalbumina. A proteína mostrou um conteúdo de carboidrato de 22 %, estabilidade entre 37- 60 ºC e pH 5.0-10.0. Pouterin teve um conteúdo relativamente extenso de aminoácidos como Asx, Glx e Leu, e também um número alto de resíduos de Cys. Pouterin inibiu o crescimento dos fungos Fusarium oxysporum, Colletotrichum musae e da levedura Saccharomyces cerevisiae. A proteína lectina-like foi avaliada em relação ao seu papel inseticida sobre C. maculatus (Coleoptera) e A. kuehniella (Lepidoptera). A incorporação de pouterin em dieta artificial (0,12 %) causou 50 % de mortalidade das larvas de Callosobruchus maculatus, enquanto que a 0,08 % pouterin produziu uma ED50. Pouterin não produziu efeitos significativos na sobrevivência larval de A. kuehnuella; entretanto, a uma ca 1 %, produziu uma redução de 71, 4 % no peso médio larval. Os resultados de utilização da dieta realizados com larvas de A. kuehniella apresentaram uma redução em ECI, ECD e AD, e um aumento no CM. Pouterin aumentou os níveis de atividade triptica no intestino médio e nas fezes das larvas de A. kuehniella. A citotoxicidade de pouterin em linhagens celulares tumorigênicas e não tumorigênicas também foram investigadas. Nós verificamos que as células tumorais HeLa, Hep-2 e HT-29 foram altamente sensíveis a citotoxicidade de pouterin de maneira dose-dependente, enquanto células Vero não tumorigênica foram relativamente resistentes a proteína. Dentre as linhagens de células tumorais, células HeLa mostraram uma maior susceptibilidade a citotoxicidade de pouterin, exibindo um aumento tempo-dependente na dosagem de LDH e um valor de IC50 de 5 µg/mL. Alterações morfológicas como arredondamento, retração citoplasmática e condensação da cromatina, consistente com morte celular apoptótica foram observados. A indução de apoptose foi demonstrada pela fragmentação de DNA como detectado pelo TUNEL. Além disso, células HeLa incubadas com pouterin mostraram rompimento do citoesqueleto de actina. Análise em western blot revelou que pouterin provocou um aumento na expressão da p21, indicando parada do ciclo celular. Estudos subseqüentes forneceram evidencias de que a apoptose pode ser parcialmente explicada pela ativação da sinalização do receptor 1 TNF (TNFR1). Interessantemente, uma diminuição tempo dependente da expressão da subunidade do fator nuclear kappa B p65 (NF?B), concomitante com a dowregulação do inibidor da proteína 1 da apoptose (IAP1) foram observados, sugerindo que a apoptose mediado pelo TNF é a via predominante induzida por pouterin em células HeLa. Nossos resultados sugerem que pouterin é uma proteína lectina-like com ampla aplicabilidade biológica, e pode ser uma ferramenta para produzir plantas transgênicas mais resistentes a patogênos e insetos utilizando técnicas de biologia molecular. Finalmente, desde que as células Hela, Hep-2 e HT-29 foram altamente sensíveis a citotoxicidade induzida pela lectina-like, futuras investigações são necessárias, pois suas propriedades podem ser uma ferramenta útil, importante nos estudos da terapia contra o câncer humano
Abstract: This study describes the purification and characterization of a novel protein from the seeds of Pouteria torta, belong to the Sapotaceae family. The protein was purified by a combination of gel filtration, ion-exchange and reverse phase chromatographies. SDS-PAGE of the purified protein resulted in a single protein band of 14 kDa and agglutinated human and animal erythrocytes. The lectin-like activity of pouterin was best inhibited by glycoproteins such as fetuin, asialofetuin, heparin, orosomucoid and ovoalbumin. The protein showed a carbohydrate content of 22 %; stability between 37 and 60 ºC and at pH 5.0-10.0. Pouterin had a relatively large content of amino acids such as Asx, Glx and Leu, and there were also a high number of Cys residues. Pouterin inhibited the growth of the fungi Fusarium oxysporum and Colletotrichum musae and of the yeast Saccharomyces cerevisiae. The lectin-like protein was tested for anti-insect activity against C. maculatus (Coleoptera) and A. kuehniella (Lepidoptera). The incorporation of pouterin into an artificial diet (0.12%) caused 50% mortality in larvae of the insect Callosobruchus maculatus, whereas 0.08 % Pouterin produced an ED50. Pouterin did not produce significant effects on survival of A. kuehnuella larvae; however, at a ca 1 %, it produced a 71.4 % reduction in the average weight of the larvae. The results from dietary utilization experiments realized with A. kuehniella larvae presented a reduction in ECI, ECD and AD, and an increase in CM. Pouterin increased the level of trypsin activity in larval midgut and faeces of A. kuehniella. The cytotoxicity of pouterin in tumourigenic and non-tumourigenic mammalian cell lines also was investigated. We found that HeLa, Hep-2 and HT-29 tumor cells were highly sensitive to pouterin cytotoxicity in a dose-dependent manner, whereas non-tumourigenic Vero cells were relatively resistant to the protein. Among the tumor cell lines, HeLa cells showed the highest susceptibility to pouterin cytotoxicity, exhibiting a time-dependent increase in LDH leakage and an IC50 value of 5 µg/mL. Morphological alterations such as rounding, cell shrinkage and chromatin condensation, consistent with apoptotic cell death were observed. Apoptosis induction was demonstrated by DNA fragmentation as detected by terminal dUTP nick-end-labeling (TUNEL). Furthermore, HeLa cells incubated with pouterin showed disruption of the actin cytoskeleton. Western blot analysis revealed that pouterin caused increased expression of p21, thus indicating cell cycle arrest. Subsequent studies provided evidence that apoptosis may be partially explained in the activation of the TNF receptor 1 (TNFR1) signalling. Interestingly, a time-dependent decrease of the expression of p65 nuclear factor kappa B (NF?B) subunit, concomitant with a downregulation of the inhibitor of apoptosis protein 1 (IAP1) was observed, suggesting that TNFR-mediated apoptosis is the predominant pathway induced by pouterin in HeLa cells. Our results suggest that pouterin is a lectin-like protein with wide biological application and could be a tool for the molecular biology. The transformation of the genes coding for this lectin-like could be useful in the development of insect resistance in important agricultural crops. Finally, since Hela, Hep-2 and Ht-29 cells were highly sensitive to lectin-like-induced cytotoxicity, pouterin merits further investigation due to its properties can be a useful tool in therapy against human cancer
Doutorado
Bioquimica
Doutor em Biologia Funcional e Molecular

La Lección Cajal es una conferencia anual dictada desde 2019 en la Universidad de Zaragoza por una figura académica relevante en su campo del saber, impulsada por el Vicerrectorado de Cultura y Proyección Social para conmemorar el 150 aniversario de la entrada de Santiago Ramón y Cajal en esta universidad, su «venerada alma mater». MARIANO BARBACID estudió Ciencias Químicas en la Universidad Complutense y se doctoró en 1974. Entre 1974 y 1977 completó su formación postdoctoral en el Instituto del Cáncer (NCI) de Estados Unidos. En 1978 formó su propio grupo de investigación en el NCI, donde trabajó hasta 1988. Durante la siguiente década (1988-1998) fue vicepresidente de Oncología Preclínica de la multinacional Bristol-Myers Squibb. En 1998 regresó a España para fundar y diri- gir el Centro Nacional de Investigaciones Oncológicas (CNIO). El Dr. Barbacid es miembro extranjero de la Academia de Ciencias de EE. UU., un honor que tan solo ostentan otros siete investigado- res españoles. En 2014 fue nombrado Fellow de la Academia de la Asociación Americana de Investigación en Cáncer (AACR), el primer español en recibir esta distinción. Es doctor honoris causa por la Universidad Internacional Menéndez Pelayo (1995), la Universidad de Cantabria (2011) y la Universidad de Barcelona (2014). En 2011 reci- bió la Gran Cruz del Dos de Mayo, la más alta distinción que otorga la Comunidad de Madrid. Entre los premios internacionales destacan la Medalla Burkitt (Irlanda, 2017), la Medalla de Honor de la Agencia Internacional del Cáncer de la Organización Mundial de la Salud (Francia, 2007) y el Premio Charles Rodolphe Brupbacher (Suiza, 2005). En la actualidad, el «Índice h» (Hirsch Index) del Dr. Barbacid es de 121, el más alto de España en las áreas de Bioquímica y Biología Molecular y el segundo más alto en Oncología.

Mushrooms belonging to basiodmycetes with their high nutritional value and biologically active compounds of medicinal importance can be developed into potential food products. They have been used as a traditional food, and their medicinal property is also appreciable all over the world. Naturally occurring active compounds such as polysaccharides, proteins, lipids, and glucans, etc. are obtained from various sources including plants, animals, bacteria, algae, and fungi. The efficiency of naturally derived compounds in food industry, as well as factors influencing its effectiveness, has been reported by researchers. Mushrooms produce a diversity of biologically active compounds such as proteoglucans, polysaccharides, phenolic compounds, lectins, steroids β-glucan, chitosan, and terpenoids, etc. The bioactive compounds and their concentration differ from species to species. Thus, these bioactives can be effectively used in the fabrication of fungal (mushroom)-derived nanoemulsions applicable for the food industry.

In normal human microbiome, the polymorphic fungus Candida albicans is a crucial member. C. albicans resides mostly in individual as harmless commensal life. In specific situations, however, C. albicans can cause diseases that cause contaminations of the skin to life-threatening fundamental contaminations. Pathogenesis of Candida species is contributed by multiple factors. Some of the major contributors are enlisted here. These include host pathogen interaction, receptors molecule like TLR recognition, TLR signaling, C type lectin receptors, Dectin 1,2 and 3, mannose receptor, mincle, DC sign, Nod-Like Receptors (NLRs) and inflammasomes, soluble molecules in candida recognition, cellular responses to candida such as neutrophils, macrophages. This chapter enlightens all the components of candida pathogenicity by the assessment of Candida species pathogenic determinants. All together these will explain the current knowledge about how these determinant factors and receptors modulate virulence as well as consequent infection. Better understanding of candida pathogenicity mechanism can be the resultant of better treatment guidelines along with development of novel antifungal agents. Overall, in this review we present an update in the current understanding of the insight of pathogenicity mechanisms in this important human pathogen.