Cloning and characterization of a pectin lyase gene from Colletotrichum lindemuthianum and comparative phylogenetic/structural analyses with genes from phytopathogenic and saprophytic/opportunistic microorganisms

<p>Abstract</p> <p>Background</p> <p>Microorganisms produce cell-wall-degrading enzymes as part of their strategies for plant invasion/nutrition. Among these, pectin lyases (PNLs) catalyze the depolymerization of esterified pectin by a β-elimination mechanism. PNLs are grouped together with pectate lyases (PL) in Family 1 of the polysaccharide lyases, as they share a conserved structure in a parallel β-helix. The best-characterized fungal pectin lyases are obtained from saprophytic/opportunistic fungi in the genera <it>Aspergillus </it>and <it>Penicillium </it>and from some pathogens such as <it>Colletotrichum gloeosporioides</it>.</p> <p>The organism used in the present study, <it>Colletotrichum lindemuthianum</it>, is a phytopathogenic fungus that can be subdivided into different physiological races with different capacities to infect its host, <it>Phaseolus vulgaris</it>. These include the non-pathogenic and pathogenic strains known as races 0 and 1472, respectively.</p> <p>Results</p> <p>Here we report the isolation and sequence analysis of the <it>Clpnl2 </it>gene, which encodes the pectin lyase 2 of <it>C. lindemuthianum</it>, and its expression in pathogenic and non-pathogenic races of <it>C. lindemuthianum </it>grown on different carbon sources. In addition, we performed a phylogenetic analysis of the deduced amino acid sequence of Clpnl2 based on reported sequences of PNLs from other sources and compared the three-dimensional structure of Clpnl2, as predicted by homology modeling, with those of other organisms. Both analyses revealed an early separation of bacterial pectin lyases from those found in fungi and oomycetes. Furthermore, two groups could be distinguished among the enzymes from fungi and oomycetes: one comprising enzymes from mostly saprophytic/opportunistic fungi and the other formed mainly by enzymes from pathogenic fungi and oomycetes. Clpnl2 was found in the latter group and was grouped together with the pectin lyase from <it>C. gloeosporioides</it>.</p> <p>Conclusions</p> <p>The <it>Clpnl2 </it>gene of <it>C. lindemuthianum </it>shares the characteristic elements of genes coding for pectin lyases. A time-course analysis revealed significant differences between the two fungal races in terms of the expression of <it>Clpnl2 </it>encoding for pectin lyase 2. According to the results, pectin lyases from bacteria and fungi separated early during evolution. Likewise, the enzymes from fungi and oomycetes diverged in accordance with their differing lifestyles. It is possible that the diversity and nature of the assimilatory carbon substrates processed by these organisms played a determinant role in this phenomenon.</p

Creencias y normas en México: una actualización del estudio de las premisas psico-socio-culturales = Beliefs and Norms in Mexico: An Update of the Study of Psycho-Socio-Cultural Premises

Estudiar las culturas incluye indagar en torno a los conceptos de creencias y normas para obtener el contenido de las reglas que coordinan el comportamiento, lo que se ha estudiado a través de las premisas psico-socio-culturales (PPSC; Díaz-Guerrero, 2002). La base de la etnopsicología del mexicano son las premisas originales de la familia (Díaz-Guerrero, 1994), las cuales no incluyen normas y creencias que rigen el comportamiento de parejas, la situación actual de género y aspectos de la vida postmoderna. El objetivo de la investigación fue indagar sobre las PPSC de manera que incluyera normas y creencias, en base a ítems extraídos de 4 inventarios, en una muestra no probabilística intencional de 1624 hombres y mujeres con diferentes niveles educativos, provenientes de 6 regiones diversas de México. Los resultados de los análisis de varianza factorial y de correlaciones (r) muestran la preponderancia del sexismo, machismo y marianismo, que marcan un apego mayor a la cultura conforme las personas pertenecen a ecosistemas más tradicionales, básicamente cuando la educación es más baja y cuando son hombres. ABSTRACT The study of cultures involves researching the concepts of beliefs and norms in order to reveal the content of the rules that coordinate human behavior, a task often pursued through an approach based on psycho-socio-cultural premises (PSCP; Díaz-Guerrero, 2002). The basis of the ethnopsychology of Mexicans is formed by the original premises of the family (Díaz-Guerrero, 1994), which lack norms or beliefs to regulate the behavior of couples, the current gender situation, or aspects of postmodern life. The goal of the present study was to investigate PSCPs in a manner that would include norms and beliefs. In order to do this, items were taken from 4 inventories and administered to a non probabilistic purposive sample of 1624 men and women of various educational levels, living in 6 different regions of Mexico. The analysis of variance and the correlation analysis (r) conducted reveal the predominance of sexism, machismo and marianismo, which are indicative of a stronger attachment to the culture in people belonging to more traditional ecosystems, basically people with lower education and males

Kex2 protease converts the endoplasmic reticulum α1,2-mannosidase of Candida albicans into a soluble cytosolic form

Cytosolic α-mannosidases are glycosyl hydrolases that participate in the catabolism of cytosolic free N-oligosaccharides. Two soluble α-mannosidases (E-I and E-II) belonging to glycosyl hydrolases family 47 have been described in Candida albicans. We demonstrate that addition of pepstatin A during the preparation of cell homogenates enriched α-mannosidase E-I at the expense of E-II, indicating that the latter is generated by proteolysis during cell disruption. E-I corresponded to a polypeptide of 52 kDa that was associated with mannosidase activity and was recognized by an anti-α1,2-mannosidase antibody. The N-mannan core trimming properties of the purified enzyme E-I were consistent with its classification as a family 47 α1,2-mannosidase. Differential density-gradient centrifugation of homogenates revealed that α1,2-mannosidase E-I was localized to the cytosolic fraction and Golgi-derived vesicles, and that a 65 kDa membrane-bound α1,2-mannosidase was present in endoplasmic reticulum and Golgi-derived vesicles. Distribution of α-mannosidase activity in a kex2Δ null mutant or in wild-type protoplasts treated with monensin demonstrated that the membrane-bound α1,2-mannosidase is processed by Kex2 protease into E-I, recognizing an atypical cleavage site of the precursor. Analysis of cytosolic free N-oligosaccharides revealed that cytosolic α1,2-mannosidase E-I trims free Man8GlcNAc2 isomer B into Man7GlcNAc2 isomer B. This is believed to be the first report demonstrating the presence of soluble α1,2-mannosidase from the glycosyl hydrolases family 47 in a cytosolic compartment of the cell

Tópicos selectos de las organizaciones: una visión académica

Esta obra es un compendio de trabajos realizados por diversos autores sobre el tópicos de administración en temas financieros y de tecnología de la informaciónEn una economía interdependiente y globalizada, temas como el valor del dinero, las crisis bursátiles, la optimización de portafolio de inversión, el cuestionamiento al dólar como moneda de intercambio, los cambios fiscales, la nueva revolución tecnológica y el rol de las pequeñas y microempresas, cobran vigencia en un mundo donde lo único constante es el cambio. Estas temáticas están presentes en el trabajo colectivo que el Centro Universitario UAEM Atlacomulco publica para beneplácito de la comunidad universitaria. En 2016, la empresa consultora global Price Water House Coopers (PwC), en su Reporte “Cinco megatendencias y sus posibles implicaciones”, señalaba que en 2050 el 21% de la población mundial será mayor de 60 años, y México no es ajeno a esta situación. Los datos revelan que dentro de tres décadas las personas de la tercera edad serán 32.4 millones, mientras que en 2015 eran 8.5 millones. ¿Tiene relación el dinero y el crecimiento demográfico? En nuestro país, lo que percibe más de la mitad de los pensionados es que no les alcanza para cubrir sus necesidades elementales. Es por todos conocido que el crecimiento demográfico de la población adulta tiene efectos económicos, de salud y sociales. Si bien los autores de este libro vinculan este tema con variables macroeconómicas como ahorro, inversión, inflación, tasa de interés, rendimiento, riesgo, etc., sería muy interesante analizarlo desde la perspectiva de políticas públicas, pues todos sabemos que la precariedad de las pensiones está en correspondencia con el gasto del Estado, que debe proteger y garantizar la calidad de vida de este segmento de la población. Regularmente, cuando se habla de crisis, se hace hincapié en el lado financiero-bursátil. Sin embargo, las crisis tienen múltiples facetas: alimentaria, migratoria, ecológica y cam- bio climático, gobernabilidad, etc., y existe consenso entre los expertos que estas crisis se retroalimentan entre sí y forman un círculo vicioso que se hace global. Los mercados son interdependientes y ante cualquier proceso de inestabilidad, volatilidad e incertidumbre provocan euforia en algunos y pánico en otros.Publicación financiada con recursos PFCE 201

Interacción de levaduras de Sporothrix schenckii con epitelios

Sporothirx schenckii is dimorphic fungus that usually causes a subacute and chronic infection called sporotrichosis, charactized by nodular lesions of cutaneuos and subcutaneous tissues with lymphatic involvement. The interactions of pathogen with epithelium are essential for cutaneous infections. In this work we studied the interaction between S. schenckii yeast with epithelium. The results shown that yeast of S. schenckii adhere to epithelial cells causing cell damage and alteration in the microtubules in a time dependent manner.Sporothirx schenckii es un hongo dimórfico que causa la infección subaguda y crónica llamada esporotricosis, caracterizada por lesiones nodulares cutáneas y subcutáneas con implicación linfática. Las interacciones del patógeno con el epitelio son esenciales para las infecciones cutáneas. En este trabajo, se estudio la interacción entre levaduras y epitelios. Los resultados muestran que las levaduras de S. schenckii se adhieren a los epitelios causando daño celular y alteración en los microtúbulos dependiente del tiempo

Fungal Glycosidases in <i>Sporothrix</i> Species and <i>Candida albicans</i>

Glycoside hydrolases (GHs) are enzymes that participate in many biological processes of fungi and other organisms by hydrolyzing glycosidic linkages in glycosides. They play fundamental roles in the degradation of carbohydrates and the assembly of glycoproteins and are important subjects of studies in molecular biology and biochemistry. Based on amino acid sequence similarities and 3-dimensional structures in the carbohydrate-active enzyme (CAZy), they have been classified in 171 families. Members of some of these families also exhibit the activity of trans-glycosydase or glycosyl transferase (GT), i.e., they create a new glycosidic bond in a substrate instead of breaking it. Fungal glycosidases are important for virulence by aiding tissue adhesion and colonization, nutrition, immune evasion, biofilm formation, toxin release, and antibiotic resistance. Here, we review fungal glycosidases with a particular emphasis on Sporothrix species and C. albicans, two well-recognized human pathogens. Covered issues include a brief account of Sporothrix, sporotrichosis, the different types of glycosidases, their substrates, and mechanism of action, recent advances in their identification and characterization, their potential biotechnological applications, and the limitations and challenges of their study given the rather poor available information

Heterologous expression and biochemical characterization of an 1,2-mannosidase encoded by the Candida albicans MNS1 gene

Protein glycosylation pathways, commonly found in fungal pathogens, offer an attractive new area of study for the discovery of antifungal targets. In particular, these post-translational modifications are required for virulence and proper cell wall assembly in Candida albicans , an opportunistic human pathogen. The C. albicans MNS1 gene is predicted to encode a member of the glycosyl hydrolase family 47, with 1,2-mannosidase activity. In order to characterise its activity, we first cloned the C. albicans MNS1 gene into Escherichia coli, then expressed and purified the enzyme. The recombinant Mns1 was capable of converting a Man9GlcNAc2 N-glycan core into Man8GlcNAc2 isomer B, but failed to process a Man5GlcNAc2-Asn N-oligosaccharide. These properties are similar to those displayed by Mns1 purified from C. albicansmembranes and strongly suggest that the enzyme is an α1,2-mannosidase that is localised to the endoplasmic reticulum and involved in the processing of N-linked mannans. Polyclonal antibodies specifically raised against recombinant Mns1 also immunoreacted with the soluble α1,2-mannosidases E-I and E-II, indicating that Mns1 could share structural similarities with both soluble enzymes. Due to the high degree of similarity between the members of family 47, it is conceivable that these antibodies may recognise α1,2-mannosidases in other biological systems as well

Influence of Culture Media on Biofilm Formation by Candida Species and Response of Sessile Cells to Antifungals and Oxidative Stress

The aims of the study were to evaluate the influence of culture media on biofilm formation by C. albicans, C. glabrata, C. krusei, and C. parapsilosis and to investigate the responses of sessile cells to antifungals and reactive oxygen species (ROS) as compared to planktonic cells. For biofilm formation, the Candida species were grown at different periods of time in YP or YNB media supplemented or not with 0.2 or 2% glucose. Sessile and planktonic cells were exposed to increasing concentrations of antifungals, H2O2, menadione or silver nanoparticles (AgNPs). Biofilms were observed by scanning electron microscopy (SEM) and quantified by the XTT assay. C. albicans formed biofilms preferentially in YPD containing 2% glucose (YPD/2%), C. glabrata in glucose-free YNB or supplemented with 0.2% glucose (YNB/0.2%), while C. krusei and C. parapsilosis preferred YP, YPD/0.2%, and YPD/2%. Interestingly, only C. albicans produced an exopolymeric matrix. This is the first report dealing with the in vitro effect of the culture medium and glucose on the formation of biofilms in four Candida species as well as the resistance of sessile cells to antifungals, AgNPs, and ROS. Our results suggest that candidiasis in vivo is a multifactorial and complex process where the nutritional conditions, the human immune system, and the adaptability of the pathogen should be considered altogether to provide an effective treatment of the patient

Purification and biochemical characterisation of endoplasmic reticulum α1,2-mannosidase from Sporothrix schenckii

Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous fungi and mammalian cells, α1,2-mannosidases are present in the endoplasmic reticulum (ER) and Golgi complex and are required to generate complex N-glycans. However, lower eukaryotes such Saccharomyces cerevisiae contain only one α1,2-mannosidase in the lumen of the ER and synthesise high-mannose N-glycans. Little is known about the N-glycan structure and the enzyme machinery involved in the synthesis of these oligosaccharides in the dimorphic fungus Sporothrix schenckii . Here, a membrane-bound α-mannosidase from S. schenckii was solubilised using a high-temperature procedure and purified by conventional methods of protein isolation. Analytical zymograms revealed a polypeptide of 75 kDa to be responsible for enzyme activity and this purified protein was recognised by anti-α1,2-mannosidase antibodies. The enzyme hydrolysed Man9GlcNAc2 into Man8GlcNAc2 isomer B and was inhibited preferentially by 1-deoxymannojirimycin. This α1,2-mannosidase was localised in the ER, with the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised α1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous fungi, S. schenckii lacks Golgi α1,2-mannosidases and therefore, the processing of N-glycans by α1,2-mannosidases is similar to that present in lower eukaryotes