Showing papers on "Chitinase published in 2004"

Acidic Mammalian Chitinase in Asthmatic Th2 Inflammation and IL-13 Pathway Activation

Abstract: Chitin is a surface component of parasites and insects, and chitinases are induced in lower life forms during infections with these agents. Although chitin itself does not exist in humans, chitinases are present in the human genome. We show here that acidic mammalian chitinase (AMCase) is induced via a T helper-2 (Th2)-specific, interleukin-13 (IL-13)-mediated pathway in epithelial cells and macrophages in an aeroallergen asthma model and expressed in exaggerated quantities in human asthma. AMCase neutralization ameliorated Th2 inflammation and airway hyperresponsiveness, in part by inhibiting IL-13 pathway activation and chemokine induction. AMCase may thus be an important mediator of IL-13-induced responses in Th2-dominated disorders such as asthma.

Fungal cell wall chitinases and glucanases.

Abstract: The fungal cell wall is a complex structure composed of chitin, glucans and other polymers, and there is evidence of extensive cross-linking between these components. The wall structure is highly dynamic, changing constantly during cell division, growth and morphogenesis. Hydrolytic enzymes, closely associated with the cell wall, have been implicated in the maintenance of wall plasticity and may have roles during branching and cross-linking of polymers. Most fungal cell wall hydrolases identified to date have chitinase or glucanase activity and this short article reviews the apparent functions of these enzymes in unicellular and filamentous fungi, and the mechanisms that regulate enzyme activity in yeasts.

The chitinolytic cascade in vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor/kinase

Abstract: Chitin, a highly insoluble polymer of GlcNAc, is produced in massive quantities in the marine environment. Fortunately for survival of aquatic ecosystems, chitin is rapidly catabolized by marine bacteria. Here we describe a bacterial two-component hybrid sensor/kinase (of the ArcB type) that rigorously controls expression of ≈50 genes, many involved in chitin degradation. The sensor gene, chiS, was identified in Vibrio furnissii and Vibrio cholerae (predicted amino acid sequences, full-length: 84% identical, 93% similar). Mutants of chiS grew normally on GlcNAc but did not express extracellular chitinase, a specific chitoporin, or β-hexosaminidases, nor did they exhibit chemotaxis, transport, or growth on chitin oligosaccharides such as (GlcNAc)2. Expression of these systems requires three components: wild-type chiS; a periplasmic high-affinity chitin oligosaccharide, (GlcNAc)n (n > 1), binding protein (CBP); and the environmental signal, (GlcNAc)n. Our data are consistent with the following model. In the uninduced state, CBP binds to the periplasmic domain of ChiS and “locks” it into the minus conformation. The environmental signal, (GlcNAc)n, dissociates the complex by binding to CBP, releasing ChiS, yielding the plus phenotype (expression of chitinolytic genes). In V. cholerae, a cluster of 10 contiguous genes (VC0620–VC0611) apparently comprise a (GlcNAc)2 catabolic operon. CBP is encoded by the first, VC0620, whereas VC0619–VC0616 encode a (GlcNAc)2 ABC-type permease. Regulation of chiS requires expression of CBP but not (GlcNAc)2 transport. (GlcNAc)n is suggested to be essential for signaling these cells that chitin is in the microenvironment.

Structure-based exploration of cyclic dipeptide chitinase inhibitors

Abstract: Family 18 chitinases play an essential role in a range of pathogens and pests. Several inhibitors are known, including the potent inhibitors argadin and allosamidin, and the structures of these in complex with chitinases have been elucidated. Recent structural analysis has revealed that CI-4 [cyclo-(L-Arg-D-Pro)] inhibits family 18 chitinases by mimicking the structure of the proposed reaction intermediate. Here we report the high-resolution structures of four new CI-4 derivatives, cyclo-(L-Arg-L-Pro), cyclo-(Gly-L-Pro), cyclo-(L-His-L-Pro), and cyclo-(L-Tyr-L-Pro), in complex with a family 18 chitinase. In addition, details of enzyme inhibition and in vivo activity against Saccharomyces cerevisiae are presented. The structures reveal that the common cyclo-(Gly-Pro) substructure is sufficient for binding, allowing modification of the side chain of the nonproline residue. This suggests that design of cyclic dipeptides with a view to increasing inhibition of family 18 chitinases should be possible through relatively accessible chemistry. The derivatives presented here in complex with chitinase B from Serratia marcescens provide further insight into the mechanism of inhibition of chitinases by cyclic dipeptides as well as providing a new scaffold for chitinase inhibitor design.

Digestive chitinolytic activity in marine fishes of Monterey Bay, California.

Abstract: Chitinolytic activities, both chitinase (EC 3.2.1.14) and minimum chitobiase (β-N-acetyl-d-glucosaminidase; EC 3.2.1.30), were measured in stomach and intestinal tissues and their contents, from 13 fish species. Higher activities were found in the tissues than in the gut contents, and higher activities were seen in the stomachs than in the intestines. Demersal species exhibited chitobiase activities very close to their chitinase activities, suggesting that these fishes can degrade chitin completely to its soluble, absorbable monomer, N-acetyl-glucosamine. This suggests that these species may catabolize chitin not just to penetrate prey exoskeletons but also to derive nutrients from the chitin itself. In contrast, three mesopelagic species exhibited low chitobiase but high chitinase activities. This chitobiase limitation correlated strongly with gastrointestinal tract morphology, with the myctophids having the greatest chitobiase limitation and the shortest alimentary tracts. The high chitinase activities measured in the myctophids reflect their ability to rapidly disrupt prey exoskeletons ingested during their nightly feeding in surface waters. Their chitobiase activities are greatly reduced because with rapid meal evacuation through a short gut there is little time for processing and limited energetic advantage in the complete degradation of chitin. These results suggest multiple roles for chitinolytic enzymes in marine fishes and that feeding habits and frequency may have a bearing on the evolution of their digestive enzymes systems.

Characterization of extracellular lytic enzymes produced by the yeast biocontrol agent Candida oleophila

Abstract: The yeast Candida oleophila, the base of the commercial product Aspire, is recommended for the control of postharvest decay of citrus and pome fruit. Competition for nutrients and space is believed to be the major mode of action. Involvement of fungal cell wall-degrading enzymes is also suggested to play a role in the mechanism of action of yeast antagonists. The present study showed that the yeast C. oleophila is capable of producing and secreting various cell wall-degrading enzymes, including exo-beta-1,3-glucanase, chitinase and protease. Exo-beta-1,3-glucanase and chitinase were produced and maximized in the early stages of growth, whereas protease reached a maximum level only after 6-8 days. Production of exo-beta-1,3-glucanase, chitinase and protease was stimulated by the presence of cell wall fragments of Penicillium digitatum in the growth medium, in addition to glucose. This study also provided evidence that C. oleophila is capable of secreting exo-beta-1,3-glucanase into the wounded surface of grapefruit. The role of exo-beta-1,3-glucanase ( CoEXG1) in the biocontrol activity of C. oleophila was tested using CoEXG1-knockouts and double- CoEXG1 over-producing transformants. In vitro bioassays showed that wild-type C. oleophila and exo-beta-1,3-glucanase over-expressing transformants had similar inhibitory effects on spore germination and germ-tube elongation; and both were more inhibitory to the fungus than the knockout transformant. In experiments conducted on fruit to test the biocontrol activity against infection by P. digitatum, no significant difference in inhibition was observed between transformants and untransformed C. oleophila cells at the high concentrations of cells used, whereas at a lower concentration of yeast cells the knockout transformants appeared to be less effective.

Lytic enzymes induced by Pseudomonas fluorescens and other biocontrol organisms mediate defence against the anthracnose pathogen in mango

Abstract: Talc-based bioformulations containing cells of Pseudomonas fluorescens, Bacillus subtilis and Saccharomyces cerevisiae were evaluated for their potential to attack the mango (Mangifera indica L.) anthracnose pathogen Colletotrichum gloeosporioides Penz. under endemic conditions. The preharvest aerial spray was given at fortnightly and monthly intervals. The plant growth-promoting rhizobacteria Pseudomonas fluorescens (FP7) amended with chitin sprayed at fortnightly intervals gave the maximum induction of flowering, a yield attribute in the preharvest stage, consequently reduced latent symptoms were recorded at the postharvest stage. An enormous induction of the defence-mediating lytic enzymes chitinase and β-1,3-glucanase was recorded in colorimetric assay and the expression of discrete bands in native PAGE analysis after FP7 + chitin treatment. The enhanced expression of defence-mediating enzymes may collectively contribute to suppress the anthracnose pathogen, leading to improved yield attributes.

Constitutive and induced resistance to pathogens in Arabidopsis thaliana depends on nitrogen supply

Abstract: Knowledge about the induced pathogen resistance of plants is rapidly increasing, but little information exists on its dependence on abiotic growing conditions. Arabidopsis thaliana plants that had been cultivated under different nitrogen regimes were treated with BION ® , a chemical resistance elicitor. The activities of three enzyme classes functionally involved in resistance (chitinase, chitosanase and peroxidase) were quantified over 8 d following treatment as resistance markers. Constitutive levels of three markers and the induced level of peroxidase and chitinase activity were significantly lower under limiting nitrogen supply. Under such conditions the increase of chitosanase activity after resistance induction was severely delayed, although the induced maximum activity of chitosanase was not significantly affected. Total soluble protein content decreased during the first 12 h after resistance elicitation. Thereafter, the induced plants cultivated under high N conditions reached higher protein contents than controls, whereas N-limited induced plants continuously had reduced protein contents. A plant’s investment in resistance-related compounds can be severely constrained under limiting nitrogen supply.

Increased antifungal and chitinase specific activities of Trichoderma harzianum CECT 2413 by addition of a cellulose binding domain.

Abstract: Trichoderma harzianum is a widely distributed soil fungus that antagonizes numerous fungal phytopathogens. The antagonism of T. harzianum usually correlates with the production of antifungal activities including the secretion of fungal cell walls that degrade enzymes such as chitinases. Chitinases Chit42 and Chit33 from T. harzianum CECT 2413, which lack a chitin-binding domain, are considered to play an important role in the biocontrol activity of this strain against plant pathogens. By adding a cellulose-binding domain (CBD) from cellobiohydrolase II of Trichoderma reesei to these enzymes, hybrid chitinases Chit33-CBD and Chit42-CBD with stronger chitin-binding capacity than the native chitinases have been engineered. Transformants that overexpressed the native chitinases displayed higher levels of chitinase specific activity and were more effective at inhibiting the growth of Rhizoctonia solani, Botrytis cinerea and Phytophthora citrophthora than the wild type. Transformants that overexpressed the chimeric chitinases possessed the highest specific chitinase and antifungal activities. The results confirm the importance of these endochitinases in the antagonistic activity of T. harzianum strains, and demonstrate the effectiveness of adding a CBD to increase hydrolytic activity towards insoluble substrates such as chitin-rich fungal cell walls.

Chitinase Gene Sequences Retrieved from Diverse Aquatic Habitats Reveal Environment-Specific Distributions

Abstract: Chitin is an abundant biopolymer whose degradation is mediated primarily by bacterial chitinases. We developed a degenerate PCR primer set to amplify a ∼900-bp fragment of family 18, group I chitinase genes and used it to retrieve these gene fragments from environmental samples. Clone libraries of presumptive chitinase genes were created for nine water and six sediment samples from 10 aquatic environments including freshwater and saline lakes, estuarine water and sediments, and the central Arctic Ocean. Putative chitinase sequences were also retrieved from the Sargasso Sea metagenome sequence database. We were unable to obtain PCR product with these primers from an alkaline, hypersaline lake (Mono Lake, California). In total, 108 partial chitinase gene sequences were analyzed, with a minimum of 5 and a maximum of 13 chitinase sequences obtained from each library. All chitinase sequences were novel compared to previously identified sequences. Intralibrary sequence diversity was low, while we found significant differences between libraries from different water column samples and between water column and sediment samples. However, identical sequences were retrieved from samples collected at widely distributed locations that did not necessarily represent similar environments, suggesting homogeneity of chitinoclastic communities between some environments.

Extracellular chitinase production by Trichoderma harzianum in submerged fermentation.

Abstract: Extra-cellular chitinase production by a chitinolytic fungus Trichoderma harzianum TUBF 966 using submerged fermentation was studied. Colloidal chitin (1.5% w/v) was used as sole carbon source. Maximum chitinase production (14.7 U/ml) was obtained when fermentation was carried out at 30 degrees C for 96 h using 72 h old mycelium in a medium containing colloidal chitin 1.5% (w/v) as carbon source and 0.42 (% w/v) peptone as nitrogen source (pH 5.5). Supplementation of additional carbon sources (0.75% w/v) showed no further enhancement in chitinase production while supplementation of nitrogen sources (0.42% w/v) such as peptone and tryptone in the fermentation medium showed a marked increase in production. The process parameters that controlled chitinase production by the fungus were studied and presented here.

Distribution and phylogenetic analysis of family 19 chitinases in Actinobacteria

Abstract: In organisms other than higher plants, family 19 chitinase was first discovered in Streptomyces griseus HUT6037, and later, the general occurrence of this enzyme in Streptomyces species was demonstrated. In the present study, the distribution of family 19 chitinases in the class Actinobacteria and the phylogenetic relationship of Actinobacteria family 19 chitinases with family 19 chitinases of other organisms were investigated. Forty-nine strains were chosen to cover almost all the suborders of the class Actinobacteria, and chitinase production was examined. Of the 49 strains, 22 formed cleared zones on agar plates containing colloidal chitin and thus appeared to produce chitinases. These 22 chitinase-positive strains were subjected to Southern hybridization analysis by using a labeled DNA fragment corresponding to the catalytic domain of ChiC, and the presence of genes similar to chiC of S. griseus HUT6037 in at least 13 strains was suggested by the results. PCR amplification and sequencing of the DNA fragments corresponding to the major part of the catalytic domains of the family 19 chitinase genes confirmed the presence of family 19 chitinase genes in these 13 strains. The strains possessing family 19 chitinase genes belong to 6 of the 10 suborders in the order Actinomycetales, which account for the greatest part of the Actinobacteria. Phylogenetic analysis suggested that there is a close evolutionary relationship between family 19 chitinases found in Actinobacteria and plant class IV chitinases. The general occurrence of family 19 chitinase genes in Streptomycineae and the high sequence similarity among the genes found in Actinobacteria suggest that the family 19 chitinase gene was first acquired by an ancestor of the Streptomycineae and spread among the Actinobacteria through horizontal gene transfer.

Members of a new group of chitinase-like genes are expressed preferentially in cotton cells with secondary walls.

Abstract: Two homologous cotton (Gossypium hirsutum L.) genes, GhCTL1 and GhCTL2, encode members of a new group of chitinase-like proteins (called the GhCTL group) that includes other proteins from two cotton species, Arabidopsis, rice, and pea. Members of the GhCTL group are assigned to family GH19 glycoside hydrolases along with numerous authentic chitinases (http://afmb.cnrs-mrs.fr/CAZY/index.html), but the proteins have novel consensus sequences in two regions that are essential for chitinase activity and that were previously thought to be conserved. Maximum parsimony phylogenetic analyses, as well as Neighbor-Joining distance analyses, of numerous chitinases confirmed that the GhCTL group is distinct. A molecular model of GhCTL2 (based on the three-dimensional structure of a barley chitinase) had changes in the catalytic site that are likely to abolish catalytic activity while retaining potential to bind chitin oligosaccharides. RNA blot analysis showed that members of the GhCTL group had preferential expression during secondary wall deposition in cotton lint fiber. Cotton transformed with a fusion of the GhCTL2 promoter to the β-d-glucuronidase gene showed preferential reporter gene activity in numerous cells during secondary wall deposition. Together with evidence from other researchers that mutants in an Arabidopsis gene within the GhCTL group are cellulose-deficient with phenotypes indicative of altered primary cell walls, these data suggest that members of the GhCTL group of chitinase-like proteins are essential for cellulose synthesis in primary and secondary cell walls. However, the mechanism by which they act is more likely to involve binding of chitin oligosaccharides than catalysis.

Independent regulation of chitin synthase and chitinase activity in Candida albicans and Saccharomyces cerevisiae.

Abstract: Chitin is an essential structural polysaccharide in fungi that is required for cell shape and morphogenesis. One model for wall synthesis at the growing cell surface suggests that the compliance that is necessary for turgor-driven expansion of the cell wall involves a delicate balance of wall synthesis and lysis. Accordingly, de novo chitin synthesis may involve coordinated regulation of members of the CHS chitin synthase and CHT chitinase gene families. To test this hypothesis, the chitin synthase and chitinase activities of cell-free extracts were measured, as well as the chitin content of cell walls isolated from isogenic mutant strains that contained single or multiple knock-outs in members of these two gene families, in both Candida albicans and Saccharomyces cerevisiae. However, deletion of chitinase genes did not markedly affect specific chitin synthase activity, and deletion of single CHS genes had little effect on in vitro specific chitinase activity in either fungus. Chitin synthesis and chitinase production was, however, regulated in C. albicans during yeast-hypha morphogenesis. In C. albicans, the total specific activities of both chitin synthase and chitinase were higher in the hyphal form, which was attributable mainly to the activities of Chs2 and Cht3, respectively. It appeared, therefore, that chitin synthesis and hydrolysis were not coupled, but that both were regulated during yeast-hypha morphogenesis in C. albicans.

A Chitinase from Tex6 Maize Kernels Inhibits Growth of Aspergillus flavus.

Abstract: The maize inbred Tex6 has resistance to colonization and aflatoxin accumulation by Aspergillus flavus. A protein inhibitory to growth of A. flavus has been identified from aqueous extracts of mature Tex6 seeds. This study reports the purification of a chitinase associated with this inhibitory activity to electrophoretic homogeneity and the further characterization of its properties. The inhibitory protein, which has an M(r) of 29,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is an endochitinase that is also capable of exochitinase activity. The enzyme has an optimal pH of 5.5 and a temperature optimum of 45 degrees C. Chitinase activity in maize kernels peaked approximately 36 days after pollination. The Tex6 chitinase purified in this study is capable of inhibiting the growth of A. flavus by 50% at a concentration of 20 mug/ml. Our data indicate that chitinase activity in Tex6 kernels makes a major contribution to the antifungal activity in this maize genotype. Partial peptide sequence of the chitinase showed it to differ from previously reported chitinases.

Field performance of chitinase transgenic silver birches (Betula pendula): resistance to fungal diseases.

Abstract: A field trial of 15 transgenic birch lines expressing a sugar beet chitinase IV gene and the corresponding controls was established in southern Finland to study the effects of the level of sugar beet chitinase IV expression on birch resistance to fungal diseases. The symptoms caused by natural infections of two fungal pathogens, Pyrenopeziza betulicola (leaf spot disease) and Melampsoridium betulinum (birch rust), were analysed in the field during a period of 3 years. The lines that had shown a high level of sugar beet chitinase IV mRNA accumulation in the greenhouse also showed high sugar beet chitinase IV expression after 3 years in the field. The level of sugar beet chitinase IV expression did not significantly improve the resistance of transgenic birches to leaf spot disease. Instead, some transgenic lines were significantly more susceptible to leaf spot than the controls. The level of sugar beet chitinase IV expression did have an improving effect on most parameters of birch rust; the groups of lines showing high or intermediate transgene expression were more resistant to birch rust than those showing low expression. This result indicates that the tested transformation may provide a tool for increasing the resistance of silver birch to birch rust.

Association of the Hydrolytic Enzyme Chitinase against Rhizoctonia solani in Rhizobacteria‐treated Rice Plants

Abstract: Twenty-two strains of Pseudomonas fluorescens isolated from the rhizosphere soil of nine plant species were screened in vitro for their inhibitory effect on the mycelial growth of the rice sheath blight fungus, Rhizoctonia solani. Of the 22 strains, two promising strains (Pf1 and FP7) were assessed for their effect on seedling vigour and their ability to promote growth in vitro of four cultivars of rice. Both bacterial strains induced systemic resistance in rice cv. IR 50, which is susceptible to sheath blight. After inoculation of the sheaths with the pathogen, Pseudomonas-treated plants showed an increase in chitinase activity significantly higher than that of untreated control plants. A twofold increase in chitinase activity occurred 2 days after inoculation of plants with the pathogen. Western blot analysis of chitinase indicated the expression of 28 and 38 kDa proteins in rice sheaths against R. solani. Increased induction of the pathogenesis-related chitinase isoform in Pseudomonas-treated rice in response to R. solani infection indicates that the induced chitinase has a definite role in suppressing disease development.