Gerardo Gutierrez-Sanchez

Microbial tannases: advances and perspectives

In the last years, tannase has been the subject of a lot of studies due to its commercial importance and complexity as catalytic molecule. Tannases are capable of hydrolyzing complex tannins, which represent the main chemical group of natural anti-microbials occurring in the plants. The general outline of this work includes information of the substrates, the enzyme, and the applications. This review considers in its introduction the concepts and history of tannase and explores scientific and technological aspects. The “advances” trace the route from the general, molecular, catalytic, and functional information obtained under close to optimal conditions for microbial production through purification, description of the enzyme properties, and the commercial applications to the “perspectives” including expression studies, regulation, and potential uses; aspects related to the progress in our understanding of tannin biodegradation are also included.

The Botrytis cinerea early secretome.

The extracellular proteome, or secretome, of phytopathogenic fungi is presumed to be a key element of their infection strategy. Especially interesting constituents of this set are those proteins secreted at the beginning of the infection, during the germination of conidia on the plant surfaces or wounds, since they may play essential roles in the establishment of a successful infection. We have germinated Botrytis cinerea conidia in conditions that resemble the plant environment, a synthetic medium enriched with low molecular weight plant compounds, and we have collected the proteins secreted during the first 16 h by a double precipitation protocol. 2‐D electrophoresis of the precipitated secretome showed a spot pattern similar for all conditions evaluated and for the control medium without plant extract. The proteins in 16 of these spots were identified by PMF and corresponded to 11 different polypeptides. Alternative determination of secretome composition by LC‐MS/MS of tryptic fragments rendered a much larger number, 105 proteins, which included all previously identified by PMF. All proteins were functionally classified according to their putative function in the infection process. Key features of the early secretome include a large number of proteases, the abundance of proteins involved in the degradation of plant defensive barriers, and plenty of proteins with unknown function.

Site Mapping and Characterization of O-Glycan Structures on α-Dystroglycan Isolated from Rabbit Skeletal Muscle

The main extracellular matrix binding component of the dystrophin-glycoprotein complex, α-dystroglycan (α-DG), which was originally isolated from rabbit skeletal muscle, is an extensively O-glycosylated protein. Previous studies have shown α-DG to be modified by both O-GalNAc- and O-mannose-initiated glycan structures. O-Mannosylation, which accounts for up to 30% of the reported O-linked structures in certain tissues, has been rarely observed on mammalian proteins. Mutations in multiple genes encoding defined or putative glycosyltransferases involved in O-mannosylation are causal for various forms of congenital muscular dystrophy. Here, we explore the glycosylation of purified rabbit skeletal muscle α-DG in detail. Using tandem mass spectrometry approaches, we identify 4 O-mannose-initiated and 17 O-GalNAc-initiated structures on α-DG isolated from rabbit skeletal muscle. Additionally, we demonstrate the use of tandem mass spectrometry-based workflows to directly analyze glycopeptides generated from the purified protein. By combining glycomics and tandem mass spectrometry analysis of 91 glycopeptides from α-DG, we were able to assign 21 different residues as being modified by O-glycosylation with differing degrees of microheterogeneity; 9 sites of O-mannosylation and 14 sites of O-GalNAcylation were observed with only two sites definitively exhibiting occupancy by either type of glycan. The distribution of identified sites of O-mannosylation suggests a limited role for local primary sequence in dictating sites of attachment.

A proteomic study of pectin‐degrading enzymes secreted by Botrytis cinerea grown in liquid culture

Botrytis cinerea is a pathogenic filamentous fungus, which infects more than 200 plant species. The enzymes secreted by B. cinerea play an important role in the successful colonization of a host plant. Some of the secreted enzymes are involved in the degradation of pectin, a major component of the plant cell wall. A total of 126 proteins secreted by B. cinerea were identified by growing the fungus on highly or partially esterified pectin, or on sucrose in liquid culture. Sixty‐seven common proteins were identified in each of the growth conditions, of which 50 proteins exhibited a SignalP motif. Thirteen B. cinerea proteins with functions related to pectin degradation were identified in both pectin growth conditions, while only four were identified in sucrose. Our results indicate it is unlikely that the activation of B. cinerea from the dormant state to active infection is solely dependent on changes in the degree of esterification of the pectin component of the plant cell wall. Further, these results suggest that future studies of the B. cinerea secretome in infections of ripe and unripe fruits will provide important information that will describe the mechanisms that the fungus employs to access nutrients and decompose tissues.

Proteomic Analysis of Ripening Tomato Fruit Infected by Botrytis cinerea

Botrytis cinerea, a model necrotrophic fungal pathogen that causes gray mold as it infects different organs on more than 200 plant species, is a significant contributor to postharvest rot in fresh fruit and vegetables, including tomatoes. By describing host and pathogen proteomes simultaneously in infected tissues, the plant proteins that provide resistance and allow susceptibility and the pathogen proteins that promote colonization and facilitate quiescence can be identified. This study characterizes fruit and fungal proteins solubilized in the B. cinerea-tomato interaction using shotgun proteomics. Mature green, red ripe wild type and ripening inhibited (rin) mutant tomato fruit were infected with B. cinerea B05.10, and the fruit and fungal proteomes were identified concurrently 3 days postinfection. One hundred eighty-six tomato proteins were identified in common among red ripe and red ripe-equivalent ripening inhibited (rin) mutant tomato fruit infected by B. cinerea. However, the limited infections by B. cinerea of mature green wild type fruit resulted in 25 and 33% fewer defense-related tomato proteins than in red and rin fruit, respectively. In contrast, the ripening stage of genotype of the fruit infected did not affect the secreted proteomes of B. cinerea. The composition of the collected proteins populations and the putative functions of the identified proteins argue for their role in plant-pathogen interactions.

Microbial Enzymes Involved in Polyurethane Biodegradation: A Review

Plastics are present in a lot of aspects of everyday life. They are very versatile and resistant to microbial attack. Polyurethanes are used in several industries and are divided in polyester and polyether polyurethanes and there are different types among them. Despite their microbial resistance, they are susceptible to the attack of fungi and bacteria but the mechanism to elucidate its biodegradation are unknown. There are reports from bacteria and fungi that are capable of degrading polyurethane but the studies about the enzymes that attack the plastic are focused on bacterial enzymes only. The enzymes reported are of type esterase and protease mainly since these enzymes are very unspecific and can recognize some regions in the polyurethane molecule and hydrolyze it. Fungal enzymes have been studied prior the 1990s decade but recently, some authors report the use of filamentous fungi to degrade polyurethane and also report some characteristics of the enzymes involved in it. This review approaches polyurethane biodegradation by focusing on the enzymes reported to date.

Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is a common birth malformation with a heterogeneous etiology. In this study, we report that ablation of the heparan sulfate biosynthetic enzyme NDST1 in murine endothelium (Ndst1ECKO mice) disrupted vascular development in the diaphragm, which led to hypoxia as well as subsequent diaphragm hypoplasia and CDH. Intriguingly, the phenotypes displayed in Ndst1ECKO mice resembled the developmental defects observed in slit homolog 3 (Slit3) knockout mice. Furthermore, introduction of a heterozygous mutation in roundabout homolog 4 (Robo4), the gene encoding the cognate receptor of SLIT3, aggravated the defect in vascular development in the diaphragm and CDH. NDST1 deficiency diminished SLIT3, but not ROBO4, binding to endothelial heparan sulfate and attenuated EC migration and in vivo neovascularization normally elicited by SLIT3-ROBO4 signaling. Together, these data suggest that heparan sulfate presentation of SLIT3 to ROBO4 facilitates initiation of this signaling cascade. Thus, our results demonstrate that loss of NDST1 causes defective diaphragm vascular development and CDH and that heparan sulfate facilitates angiogenic SLIT3-ROBO4 signaling during vascular development.

Heparin-induced Leukocytosis Requires 6-O-Sulfation and Is Caused by Blockade of Selectin- and CXCL12 Protein-mediated Leukocyte Trafficking in Mice

Background: Heparin-induced leukocytosis (HIL) is commonly seen in patients. Results: In mice, heparin requires 6-O-sulfation to induce leukocytosis. Heparin alters selectin- and/or CXCL12-mediated lymphocyte homing and release, neutrophil release, and distribution in the vasculature. Conclusion: HIL requires 6-O-sulfation and is caused by blocking selectin- and CXCL12-mediated leukocyte trafficking. Significance: This study elucidated the structural determinants and the underlying mechanisms of heparin in HIL. Leukocytosis refers to an increase in leukocyte count above the normal range in the blood and is a common laboratory finding in patients. In many cases, the mechanisms underlying leukocytosis are not known. In this study, we examined the effects, the structural determinants, and the underlying mechanisms of heparin-induced leukocytosis, a side effect occurring in 0.44% of patients receiving heparin. We observed that heparin induced both lymphocytosis and neutrophilia, and the effects required heparin to be 6-O-sulfated but did not require its anticoagulant activity. Cell mobilization studies revealed that the lymphocytosis was attributable to a combination of blockage of lymphocyte homing and the release of thymocytes from the thymus, whereas the neutrophilia was caused primarily by neutrophil release from the bone marrow and demargination in the vasculature. Mechanistic studies revealed that heparin inhibits L- and P-selectin, as well as the chemokine CXCL12, leading to leukocytosis. Heparin is known to require 6-O-sulfate to inhibit L- and P-selectin function, and in this study we observed that 6-O-sulfate is required for its interaction with CXCL12. We conclude that heparin-induced leukocytosis requires glucosamine 6-O-sulfation and is caused by blockade of L-selectin-, P-selectin-, and CXCL12-mediated leukocyte trafficking.

Fungal cultures of tar bush and creosote bush for production of two phenolic antioxidants (Pyrocatechol and Gallic acid)

Abstract‘Tar bush’ and ‘creosote bush’ were substrates of fungal cultivation for tannase production and gallic acid and pyrocatechol accumulation. Aspergillus niger GH1 grew similarly on both plant materials under solid state culture conditions, reaching maximal levels after 4 d. Fungal strain degraded all tannin content of creosote bush after 4 d of fermentation and >75 % of tar bush after 5 d. Higher level of tannase activity was detected in tar bush fermentation. Biotransformation of tannins to gallic acid was high (93 % in creosote bush and 89 % in tar bush). Pyrocatechol was released poorly. Kinetic parameters of tannin conversion were calculated.

The secondary cell wall polysaccharide of Bacillus anthracis provides the specific binding ligand for the C-terminal cell wall-binding domain of two phage endolysins, PlyL and PlyG

Endolysins are bacteriophage enzymes that lyse their bacterial host for phage progeny release. They commonly contain an N-terminal catalytic domain that hydrolyzes bacterial peptidoglycan (PG) and a C-terminal cell wall-binding domain (CBD) that confers enzyme localization to the PG substrate. Two endolysins, phage lysin L (PlyL) and phage lysin G (PlyG), are specific for Bacillus anthracis. To date, the cell wall ligands for their C-terminal CBD have not been identified. We recently described structures for a number of secondary cell wall polysaccharides (SCWPs) from B. anthracis and B. cereus strains. They are covalently bound to the PG and are comprised of a -ManNAc-GlcNAc-HexNAc- backbone with various galactosyl or glucosyl substitutions. Surface plasmon resonance (SPR) showed that the endolysins PlyL and PlyG bind to the SCWP from B. anthracis (SCWPBa) with high affinity (i.e. in the μM range with dissociation constants ranging from 0.81 × 10(-6) to 7.51 × 10(-6) M). In addition, the PlyL and PlyG SCWPBa binding sites reside with their C-terminal domains. The dissociation constants for the interactions of these endolysins and their derived C-terminal domains with the SCWPBa were in the range reported for other protein-carbohydrate interactions. Our findings show that the SCWPBa is the ligand that confers PlyL and PlyG lysin binding and localization to the PG. PlyL and PlyG also bound the SCWP from B. cereus G9241 with comparable affinities to SCWPBa. No detectable binding was found to the SCWPs from B. cereus ATCC (American Type Culture Collection) 10987 and ATCC 14579, thus demonstrating specificity of lysin binding to SCWPs.