Decreased Cell Wall Galactosaminogalactan in Aspergillus nidulans Mediates Dysregulated Inflammation in the Chronic Granulomatous Disease Host

Author Disclosure Statement No competing financial interests exist.

Lead (pb) toxicity in saccharomyces cerevisiae: the role of cell wall

O chumbo é utilizado em muitos produtos, tais como baterias, gasolina, tintas e corantes, resultando na sua libertação no meio ambiente. Neste trabalho, foi examinado o papel da parede celular da levedura Saccharomyces cerevisiae como uma barreira ou como alvo da toxicidade do chumbo. A biodisponibilidade do Pb é muito reduzida pelos componentes do meio de cultura YEPD, o que dificulta a avaliação da toxicidade deste elemento em concentrações ambientalmente realistas. Para avaliar a toxicidade de Pb em S. cerevisiae, em condições de crescimento, foram efetuadas diferentes diluições (10-100 vezes) do meio YEPD, as quais foram misturadas com várias concentrações de Pb (0,1-1,0 mmol/l). Observou-se que o YEPD diluído 25 vezes constituía a melhor condição de compromisso entre o crescimento celular e a precipitação de Pb. Os genes CWP1 e CWP2 codificam para duas grandes manoproteínas da parede celular da levedura S. cerevisiae; a deleção destes genes CWP aumenta a permeabilidade da parede celular. A suscetibilidade de células de levedura interrompidas no gene CWP1 (estirpe cwp1Δ) ou CWP2 (estirpe cwp2Δ) foi comparada com a da estirpe, isogénica, selvagem (WT). Verificou-se que o crescimento das estirpes cwp1Δ e cwp2Δ, no meio de cultura YEPD 25 vezes diluído, na presença de Pb, não diferiu do crescimento da estirpe WT. Este resultado sugere que a alteração da permeabilidade da parede celular não altera a sensibilidade de células de levedura ao Pb. Foi investigada o impacto do Pb na parede celular de levedura. Para este efeito, comparou-se a suscetibilidade ao dodecil sulfato de sódio (SDS), ao calcofluor (CFW) e a uma enzima que degrada a parede da célula (liticase), em células da estirpe WT não expostas ou expostas a Pb durante 4, 8 ou 24 h. Além disso, o conteúdo de quitina da parede celular de levedura foi investigada por coloração das células com CFW. Os resultados não mostraram uma alteração da suscetibilidade ao SDS e ao CFW, nas células tratadas com Pb; contudo, nas células tratadas durante 24 h com Pb, observou-se um aumento da sensibilidade à liticase e um aumento da coloração com CFW. Estes resultados sugerem que o chumbo interage com a parede celular da levedura e influencia a sua composição. Deve ser levado a cabo trabalho adicional a fim de confirmar estes resultados.

Alteration of cell-wall porosity is involved in osmotic stress-induced enhancement of aluminium resistance in common bean (Phaseolus vulgaris L.)

Aluminium (Al) toxicity and drought are the two major abiotic stress factors limiting common bean production in the tropics. Using hydroponics, the short-term effects of combined Al toxicity and drought stress on root growth and Al uptake into the root apex were investigated. In the presence of Al stress, PEG 6000 (polyethylene glycol)-induced osmotic (drought) stress led to the amelioration of Al-induced inhibition of root elongation in the Al-sensitive genotype VAX 1. PEG 6000 (>> PEG 1000) treatment greatly decreased Al accumulation in the 1 cm root apices even when the roots were physically separated from the PEG solution using dialysis membrane tubes. Upon removal of PEG from the treatment solution, the root tips recovered from osmotic stress and the Al accumulation capacity was quickly restored. The PEG-induced reduction of Al accumulation was not due to a lower phytotoxic Al concentration in the treatment solution, reduced negativity of the root apoplast, or to enhanced citrate exudation. Also cell-wall (CW) material isolated from PEG-treated roots showed a low Al-binding capacity which, however, was restored after destroying the physical structure of the CW. The comparison of the Al(3+), La(3+), Sr(2+), and Rb(+) binding capacity of the intact root tips and the isolated CW revealed the specificity of the PEG 6000 effect for Al. This could be due to the higher hydrated ionic radius of Al(3+) compared with other cations (Al(3+) >> La(3+) > Sr(2+) > Rb(+)). In conclusion, the results provide circumstantial evidence that the osmotic stress-inhibited Al accumulation in root apices and thus reduced Al-induced inhibition of root elongation in the Al-sensitive genotype VAX 1 is related to the alteration of CW porosity resulting from PEG 6000-induced dehydration of the root apoplast.

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins

International audience

Fruit cell culture as a model system to study cell wall changes during strawberry fruit ripening

Strawberry (Fragaria x ananassa, Duch.) fruit is characterized by its fast ripening and soft texture at the ripen stage, resulting in a short postharvest shelf life and high economic losses. It is generally believed that the disassembly of cell walls, the dissolution of the middle lamella and the reduction of cell turgor are the main factors determining the softening of fleshy fruits. In strawberry, several studies indicate that the solubilisation and depolymerisation of pectins, as well as the depolymerisation of xyloglucans, are the main processes occurring during ripening. Functional analyses of genes encoding pectinases such as polygalacturonase and pectate lyase also point out to the pectin fraction as a key factor involved in textural changes. All these studies have been performed with whole fruits, a complex organ containing different tissues that differ in their cell wall composition and undergo ripening at different rates. Cell cultures derived from fruits have been proposed as model systems for the study of several processes occurring during fruit ripening, such as the production of anthocyanin and its regulation by plant hormones. The main objective of this research was to obtain and characterize strawberry cell cultures to evaluate their potential use as a model for the study of the cell wall disassembly process associate with fruit ripening. Cell cultures were obtained from cortical tissue of strawberry fruits, cv. Chandler, at the stages of unripe-green, white and mature-red. Additionally, a cell culture line derived from strawberry leaves was obtained. All cultures were maintained in solid medium supplemented with 2.5 mg.l-1 2,4-D and incubated in the dark. Cell walls from the different callus lines were extracted and fractionated to obtain CDTA and sodium carbonate soluble pectin fractions, which represent polyuronides located in the middle lamella or the primary cell wall, respectively. The amounts of homogalacturonan in both fractions were estimated by ELISA using LM19 and LM20 antibodies, specific against demethylated and methyl-esterified homogalacturonan, respectively. In the CDTA fraction, the cell line from ripe fruit showed a significant lower amount of demethylated pectins than the rest of lines. By contrast, the content of methylated pectins was similar in green- and red-fruit lines, and lower than in white-fruit and leaf lines. In the sodium carbonate pectin fraction, the line from red fruit also showed the lowest amount of pectins. These preliminary results indicate that cell cultures obtained from fruits at different developmental stages differ in their cell wall composition and these differences resemble to some extent the changes that occur during strawberry softening. Experiments are in progress to further characterize cell wall extracts with monoclonal antibodies against other cell wall epitopes.

Mineral stress response of Vitis cell wall transcriptome: identification, characterization and expression of genes from key families involved in wall biosynthesis and modification

Doutoramento em Engenharia Agronómica - Instituto Superior de Agronomia - UL

Drug development:the cell wall as a drug target

The complex and essential cell wall of Mycobacterium tuberculosis represents a plethora of new and old drug targets that collectively form an apparent mycobacterial “Achilles’ heel”. The mycolic acids are long-chain α-alkyl-β-hydroxy fatty acids (C70–90), which are unique to mycobacterial species, forming an integral component of the mycolyl–arabinogalactan–peptidoglycan complex. Their apparent uniqueness to the M. tuberculosis complex has rendered components of mycolic acid biosynthesis as powerful drug targets for specific tuberculosis (TB) chemotherapy. Here, I will discuss a contribution to TB drug discovery by deconvolution of the inhibitory mechanisms of a number of antitubercular compounds targeting mycolic acid biosynthesis. I will begin with the early days, elucidating the mode of action of ethionamide [1] and thiolactomycin [2], each targeting two separate components of the fatty acid synthase II (FAS-II) pathway. I will further discuss the recently discovered tetrahydropyrazo[1,5-a]pyrimidine-3-carboxamide compounds [3] which selectively target the essential, catalytically silent M. tuberculosis EchA6, providing a crucial lipid shunt between β-oxidation and FAS-II and supplying lipid precursors for essential mycolate biosynthesis. Finally, I will discuss the recent discovery of the mode of action of the indazole sulfonamides [4], inhibiting M. tuberculosis KasA by, a completely novel inhibitory mechanism.

Secretome analysis of the mycoparasitic fungus Trichoderma harzianum ALL 42 cultivated in different media supplemented with Fusarium solani cell wall or glucose.

2016

Étude comparative des propagules extraracinaires et intraracinaires du champignon mycorhizien Glomus irregulare

La germination des spores est une étape essentielle dans le cycle de vie de la majorité des champignons filamenteux. Les champignons mycorhiziens à arbuscules (CMA) forment un certain nombre de propagules infectieuses différentes qui augmentent leur potentiel à coloniser les racines. Parmi elles se trouvent les spores extraracinaires et intraracinaires. La paroi cellulaire des spores joue un rôle majeur dans la survie de ces propagules en étant une barrière physique et osmotique. Puisque une cellule peut faire des ajustements considérables dans la composition et la structure de sa paroi, en réponse aux conditions environnementales, il est possible que les parois des spores intraracinaires et extraracinaires montrent des propriétés mécaniques et osmotiques différentes affectant leur germination et leur survie. Pourtant, contrairement à la connaissance de la génétique moléculaire et de la formation de la paroi cellulaire des CMA, peu d’information est disponible au sujet de ces propriétés mécaniques. Les informations sur la germination des CMA dans des conditions hypertoniques sont aussi rares, et les modèles expérimentaux ne séparent généralement pas les effets directs de la forte pression osmotique externe sur la germination des champignons et les effets attribuables aux plantes. Cette étude avait pour but de répondre à deux importantes séries de questions concernant le comportement des spores mycorhiziennes. Nous avons d'abord déterminé la relation entre la composition de la paroi cellulaire, la structure et les propriétés mécaniques du champignon modèle Glomus irregulare (isolat DAOM 197198). La micro-indentation a été utilisée pour mesurer quantitativement les propriétés mécaniques de la paroi cellulaire. La composition (contenu de chitine et de glomaline) de la paroi cellulaire a été quantifiée par immunofluorescence tandis que la microscopie optique a été utilisée pour mesurer l'épaisseur de la paroi cellulaire. La densité locale en glomaline et l’épaisseur de la paroi étaient significativement plus élevées pour les parois des spores extraracinaires alors que la densité locale en chitine et la rigidité n’ont pas montré de variations entre les spores extraracinaires et intraracinaires. La grande variabilité dans les paramètres étudiés nous a empêchés de cibler un facteur principal responsable de la force totale de la paroi lors de la compression. La diminution des concentrations de chitine et de glomaline a été corrélée à l'évolution de la paroi du champignon au cours de son cycle de vie. On a aussi observé une composition différentielle des couches de la paroi: les polymères de chitine et de glomaline furent localisés principalement dans les couches externes et internes de la paroi, respectivement. Dans la deuxième partie de notre travail, nous avons exploré les effets directs d'engrais, par rapport à leur activité de l'eau (aw), sur la germination des spores et la pression de turgescence cellulaire. Les spores ont été soumises à trois engrais avec des valeurs de aw différentes et la germination ainsi que la cytorrhyse (effondrement de la paroi cellulaire) des spores ont été évaluées après différents temps d'incubation. Les valeurs de aw des engrais ont été utilisées comme indicateurs de leurs pressions osmotiques. L'exposition des spores de Glomus irregulare au choc osmotique causé par les engrais dont les valeurs de aw se situent entre 0,982 et 0,882 a provoqué des changements graduels au niveau de leur cytorrhyse et de leur germination. Avec l'augmentation de la pression de turgescence externe, la cytorrhyse a augmenté, tandis que le taux de germination a diminué. Ces effets ont été plus prononcés à des concentrations élevées en éléments nutritifs. La présente étude, bien qu’elle constitue une étape importante dans la compréhension des propriétés mécaniques et osmotiques des spores de CMA, confirme également que ces propriétés dépendent probablement de plusieurs facteurs, dont certains qui ne sont pas encore identifiés.

RNA interference suppression of genes in glycosyl transferase families 43 and 47 in wheat starchy endosperm causes large decreases in arabinoxylan content

The cell walls of wheat (Triticum aestivum) starchy endosperm are dominated by arabinoxylan (AX), accounting for 65% to 70% of the polysaccharide content. Genes within two glycosyl transferase (GT) families, GT43 (IRREGULAR XYLEM9 [IRX9] and IRX14) and GT47 (IRX10), have previously been shown to be involved in the synthesis of the xylan backbone in Arabidopsis, and close homologs of these have been implicated in the synthesis of xylan in other species. Here, homologs of IRX10 TaGT47_2 and IRX9 TaGT43_2, which are highly expressed in wheat starchy endosperm cells, were suppressed by RNA interference (RNAi) constructs driven by a starchy endosperm-specific promoter. The total amount of AX was decreased by 40% to 50% and the degree of arabinosylation was increased by 25% to 30% in transgenic lines carrying either of the transgenes. The cell walls of starchy endosperm in sections of grain from TaGT43_2 and TaGT47_2 RNAi transgenics showed decreased immunolabeling for xylan and arabinoxylan epitopes and approximately 50% decreased cell wall thickness compared with controls. The proportion of AX that was water soluble was not significantly affected, but average AX polymer chain length was decreased in both TaGT43_2 and TaGT47_2 RNAi transgenics. However, the long AX chains seen in controls were absent in TaGT43_2 RNAi transgenics but still present in TaGT47_2 RNAi transgenics. The results support an emerging picture of IRX9-like and IRX10-like proteins acting as key components in the xylan synthesis machinery in both dicots and grasses. Since AX is the main component of dietary fiber in wheat foods, the TaGT43_2 and TaGT47_2 genes are of major importance to human nutrition.

Degradação in vitro de tecidos da lâmina foliar e do colmo de gramíneas forrageiras tropicais, em função do estádio de desenvolvimento

O experimento foi realizado com o objetivo de avaliar o efeito da idade sobre o potencial de degradação dos diferentes tecidos da lâmina foliar e do colmo de capim-braquiária (Brachiaria decumbens), capim-gordura (Melinis minutiflora) e capim-tifton 85 (Cynodon sp). Foram amostradas a 7ª (capim-braquiária e capim-gordura) e a 11ª (capim-tifton 85) lâminas foliares, no dia da exposição da lígula e 20 dias após. Por meio de observações ao microscópio foram estimadas a extensão da digestão in vitro dos tecidos da lâmina e do colmo e a redução na espessura da parede de células do esclerênquima do colmo. Lâminas foliares e segmentos de colmos jovens apresentaram maiores áreas digeridas. Permaneceram intactos os tecidos com células de parede espessada e lignificada, a bainha parenquimática dos feixes, o esclerênquima, o xilema e a epiderme do colmo. Tecidos com células de parede delgada, normalmente não-lignificada, o mesofilo, o floema e o parênquima, desapareceram completamente. O avanço na idade reduziu a digestão do mesofilo, em lâminas de capim-braquiária e capim-gordura, e do parênquima em colmos, principalmente de capim-gordura. A epiderme na lâmina foliar foi parcialmente digerida, independentemente da idade e da espécie. Embora aparentemente intactas, células esclerenquimáticas do colmo sofreram redução da espessura da parede com a incubação em líquido ruminal. A porcentagem de redução variou de 7 a 37% e a taxa de redução da espessura de 0,007 a 0,018 µm/h.

Características anatômicas da lâmina foliar e do colmo de gramíneas forrageiras tropicais, em função do nível de inserção no perfilho, da idade e da estação de crescimento

Foram avaliados os efeitos da idade, do nível de inserção da folha no perfilho e da estação de crescimento (verão ou outono), sobre a proporção de tecidos e a espessura da parede celular em lâminas foliares e segmentos de colmo de capim-braquiária (Brachiaria decumbens), capim-gordura (Melinis minutiflora) e capim-tifton 85 (Cynodon sp). Lâminas foliares das posições inferior e superior do perfilho foram colhidas no dia da exposição da lígula e 20 dias após. Foram determinadas as proporções relativas de epiderme, xilema, floema, bainha parenquimática dos feixes, esclerênquima, parênquima no colmo e mesofilo na lâmina foliar. Foram medidas as espessuras das paredes dos vasos de metaxilema e do esclerênquima da lâmina e do colmo. A proporção de tecidos em lâminas foliares não foi alterada pela idade nem pela estação de crescimento, sendo a espessura da parede de células do esclerênquima da lâmina a única característica modificada pela idade. Lâminas do nível de inserção superior apresentaram mais elevadas proporções de esclerênquima, bainha parenquimática dos feixes e xilema e células do esclerênquima e do metaxilema com paredes mais espessas, enquanto as lâminas do nível de inserção inferior se destacaram por apresentar mais elevada proporção de mesofilo e paredes celulares mais delgadas. Enquanto a proporção de parênquima decresceu, a área relativa de esclerênquima e as espessuras das paredes celulares variaram diretamente com a idade do colmo e foram maiores, em geral, no verão.

Anatomia e ultra-estrutura do pulvino primário de Pterodon pubescens Benth. (Fabaceae - Faboideae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

First report of vancomycin-resistant staphylococci isolated from healthy carriers in Brazil

Reduced susceptibility or resistance to vancomycin has been reported among clinical isolates of staphylococci in previous studies. In the present study we report on the isolation of four vancomycin-resistant staphylococcal strains from healthy carriers inside and outside the hospital environment. These carriers did not receive treatment with any antibiotic. All coagulase-negative staphylococcal strains showed variable levels of resistance to several antimicrobial agents, including oxacillin, and unstable resistance to vancomycin, with decreased vancomycin MICs (<4 mg/liter) after 10 days of passage in a nonselective medium. However, exposure of these revertants to vancomycin selected staphylococcal strains resistant to vancomycin at very high frequencies (10(-2) and 10(-3)). The vancomycin resistance in these staphylococcal strains was not mediated by the van gene. The cell wall of the staphylococcal strains studied became thickest after culture in medium containing vancomycin, and the differences in cell wall thickness were statistically significant (P < 0.001). Thus, the thickening of the cell wall in these staphylococcal strains may be an important contributor to vancomycin resistance.

Processamento, caracterização microestrutural e propriedades elásticas de metais celulares do sistema Ti-6Al-4V para fins de regeneração óssea

Pós-graduação em Ciência e Tecnologia de Materiais - FC