Cell Wall Glucanase Research Articles

The Aspartic Protease Yps3p and Cell Wall Glucanase Scw10p Are Novel Determinants That Enhance the Secretion of the Antitumor Triterpenoid GA-HLDOA in Saccharomyces cerevisiae.

Efficient bioproduction of triterpenoids is gaining increasing interest because of their significant biological applications; however, the secretion and bioproduction of triterpenoids are hindered by untapped genetic determinants. In our previous study, we observed that different engineered Saccharomyces cerevisiae strains exhibit different abilities for secreting the antitumor triterpenoid ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid (GA-HLDOA). In the present study, we performed comparative proteomics analyses of the engineered strains and identified two genes, encoding an aspartic protease, YPS3, and a cell wall glucanase, SCW10, as the most effective determinants that enhance the secretion of GA-HLDOA. Compared with this control strain, strain BJ5464-r demonstrated an overexpression of YPS3 and SCW10 resulting in 3.9-fold and 4.7-fold higher secretion of GA-HLDOA, respectively, and these increases were accompanied by an increase in cell permeability. Moreover, compared with the YPS3-overexpressing strain, the SCW10-overexpressing strain had a thinner outer mannan layer. Our findings offer valuable insights into designing microbial cell factories for the efficient secretion of triterpenoids.

A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.

Distinct and complementary roles for Aspergillus fumigatus-specific Tr1 and Foxp3+ regulatory T cells in humans and mice.

Unlike induced Foxp3+ regulatory T cells (Foxp3+ iTreg) that have been shown to play an essential role in the development of protective immunity to the ubiquitous mold Aspergillus fumigatus, type-(1)-regulatory T cells (Tr1) cells have, thus far, not been implicated in this process. Here, we evaluated the role of Tr1 cells specific for an epitope derived from the cell wall glucanase Crf-1 of A. fumigatus (Crf-1/p41) in antifungal immunity. We identified Crf-1/p41-specific latent-associated peptide+ Tr1 cells in healthy humans and mice after vaccination with Crf-1/p41+zymosan. These cells produced high amounts of interleukin (IL)-10 and suppressed the expansion of antigen-specific T cells in vitro and in vivo. In mice, in vivo differentiation of Tr1 cells was dependent on the presence of the aryl hydrocarbon receptor, c-Maf and IL-27. Moreover, in comparison to Tr1 cells, Foxp3+ iTreg that recognize the same epitope were induced in an interferon gamma-type inflammatory environment and more potently suppressed innate immune cell activities. Overall, our data show that Tr1 cells are involved in the maintenance of antifungal immune homeostasis, and most likely play a distinct, yet complementary, role compared with Foxp3+ iTreg.

Cross-protective TH1 immunity against Aspergillus fumigatus and Candida albicans

AbstractT cell–mediated heterologous immunity to different pathogens is promising for the development of immunotherapeutic strategies. Aspergillus fumigatus and Candida albicans, the 2 most common fungal pathogens causing severe infections in immunocompromised patients, are controlled by CD4+ type 1 helper T (TH1) cells in humans and mice, making induction of fungus-specific CD4+ TH1 immunity an appealing strategy for antifungal therapy. We identified an immunogenic epitope of the A fumigatus cell wall glucanase Crf1 that can be presented by 3 common major histocompatibility complex class II alleles and that induces memory CD4+ TH1 cells with a diverse T-cell receptor repertoire that is cross-reactive to C albicans. In BALB/c mice, the Crf1 protein also elicits cross-protection against lethal infection with C albicans that is mediated by the same epitope as in humans. These data illustrate the existence of T cell–based cross-protection for the 2 distantly related clinically relevant fungal pathogens that may foster the development of immunotherapeutic strategies.

Shedding of the Mucin-Like Flocculin Flo11p Reveals a New Aspect of Fungal Adhesion Regulation

Cell adhesion is a key feature in the regulation of many biological processes. In the budding yeast Saccharomyces cerevisiae, Flo11p is the major adhesion molecule that controls filamentous growth [1-3] and the expansion of interconnected cells in mats or biofilms [4]. We show here that Flo11p is shed from cells. Flo11p shedding attenuated adherence and contributed to the overall balance in adherence properties that was optimal for filamentous growth and mat formation. Shed Flo11p comprised an essential component of a fluid layer surrounding yeast mats that may be functionally analogous to the mucus secretions of higher eukaryotes. Genome-wide secretion profiling of Flo11p identified new regulatory proteins, including the furin protease Kex2p, which was required for cleavage and maturation of the Flo11p protein. Secreted mucin-like proteins may play unexpected roles in the adherence properties and virulence of microbial pathogens.

Healthy Human T-Cell Responses to Aspergillus fumigatus Antigens

Background Aspergillus fumigatus is associated with both invasive and allergic pulmonary diseases, in different hosts. The organism is inhaled as a spore, which, if not cleared from the airway, germinates into hyphal morphotypes that are responsible for tissue invasion and resultant inflammation. Hyphae secrete multiple products that function as antigens, evoking both a protective (TH1–TH17) and destructive allergic (TH2) immunity. How Aspergillus allergens (Asp f proteins) participate in the development of allergic sensitization is unknown.Methodology/Principal FindingsTo determine whether Asp f proteins are strictly associated with TH2 responses, or represent soluble hyphal products recognized by healthy hosts, human T cell responses to crude and recombinant products were characterized by ELISPOT. While responses (number of spots producing IFN-γ, IL-4 or IL-17) to crude hyphal antigen preparations were weak, responses to recombinant Asp f proteins were higher. Recombinant allergens stimulated cells to produce IFN-γ more so than IL-4 or IL-17. Volunteers exhibited a diverse CD4+ and CD8+ T cell antigen recognition profile, with prominent CD4 TH1-responses to Asp f3 (a putative peroxismal membrane protein), Asp f9/16 (cell wall glucanase), Asp f11 (cyclophilin type peptidyl-prolyl isomerase) and Asp f22 (enolase). Strong IFN-γ responses were reproduced in most subjects tested over 6 month intervals.ConclusionsProducts secreted after conidial germination into hyphae are differentially recognized by protective T cells in healthy, non-atopic individuals. Defining the specificity of the human T cell repertoire, and identifying factors that govern early responses may allow for development of novel diagnostics and therapeutics for both invasive and allergic Aspergillus diseases.

Cell wall autolytic activities and distribution of cell wall glucanases in Zea mays L. seedlings

Exo- and endo-glucanases mediate specific degradation of cell wall (1,3)(1,4)-β- d-glucans and these enzymes have been related to auxin-mediated growth and development of cereal coleoptiles. However, their distribution and functions have not been well established in other tissues. In this study the glucanase activities and cell wall autolytic activities of different maize organs were determined. Autolysis assays serve to evaluate the hydrolysis of cell wall polymers in situ by measuring the sugars released from the insoluble cell wall matrix resulting from the action of bound enzymes. Autolytic activities were observed in the cell walls of elongating young leaves, mesocotyl and roots of maize. Wall proteins extracted from all of these structures are enriched in several types of glucanases and other wall polysaccharide hydrolases. These enzymes therefore appear to have a widespread and fundamental role in wall metabolism in growing tissues

Regulation of cell wall glucanase activities by non-enzymic proteins in maize coleoptiles

Auxin stimulates cell wall glucanase activities in isolated segments of maize coleoptiles with no detectable change in the amounts of the enzymic peptides. To disclose the regulatory mechanism for this enhanced growth, we examined the possibility that protein interactions account for stimulating cell wall enzyme activities. One putative regulatory peptide, an acidic wall protein (AW), was separated from the wall protein fractionation profile, and this non enzymic protein was able to stimulate both exo- and endoglucanase activities in vitro. The results are consistent with the hypothesis that a pertinent glucanase/glucan interaction might be facilitated by AW mobilization within the wall in response to auxin-treatment.

Cell wall metabolism and autolytic activities of the yeast Saccharomyces exiguus

Autolytic activities were measured in cell walls prepared from the yeast Saccharomyces exiguus. Walls of yeast cells exhibited higher autolytic activities directed toward glucans at the exponential phase of growth when compared to cells at the stationary phase, while glucanase activities in the soluble extract fraction were higher at the stationary phase when compared to exponential phase, suggesting an important role of cell wall glucanases in growth of the yeast cells. Yeast cell walls also exhibited a substantially high autolytic activity of glycoproteins containing mannose throughout growth. These results illustrate the diverse metabolism related to functions of yeast cell walls.

Heteronuclear NMR studies of 13C-labeled yeast cell wall beta-glucan oligosaccharides.

The structures of uniformly 13C-labeled beta-glucan octa- and undeca-oligosaccharides enzymatically prepared by the yeast cell wall glucanosyl transferase of Candida albicans were characterized by using a combination of HCCH-COSY, HCCH-TOCSY, and HMBC experiments. The oligosaccharide structures indicate that the cell wall glucanosyl transferase cleaves two glucosyl units from the reducing end of the initial linear beta(1-->3) penta-oligosaccharide and subsequently transfers the remainder to another oligosaccharide at the nonreducing end via a beta(1-->6) linkage. These results indicate that the combined action of cell wall glucanase and glucanosyl transferase activities could not only introduce intrachain beta(1-->6) linkages within a single glucan strand, but also result in cross-linking of two initially separate glucan strands with concurrent introduction of intrachain beta(1-->6) linkages. Since isolated fungal membranes only synthesize linear beta(1-->3) glucan strands, wall-associated enzymes probably participate in the assembly of the final wall glucan structure during cell growth and division.

Inhibition of Auxin-Induced Cell Elongation of Maize Coleoptiles by Antibodies Specific for Cell Wall Glucanases

Polyclonal antibodies were raised in rabbits in response to the administration of purified exo- and endoglucanases extracted from cell walls of maize (Zea mays L. B37 x Mo17) coleoptiles. Since the antibodies formed specific conjugates when challenged with the glucanase antigens in immunoblot assays they were employed to evaluate the participation of glucanases in tissue growth. Indole-3-acetic acid induced cell elongation of abraded coleoptile segments was inhibited when the antibodies were supplied as a short term pretreatment (25-200 microgram/milliliter of serum protein). The extent of inhibition of IAA induced cell elongation was additive when endo- and exoglucanase antibodies were applied together. The results suggest that both enzymes have a role in mediating IAA-induced cell elongation. Pretreatment with exo- and endoglucanases antibodies also inhibited IAA induced degradation of noncellulosic beta-d-glucans and the increased level of cellulosic polymers in maize coleoptiles. Antibodies also inhibited the expression of the autohydrolytic degradation of glucans in isolated cell walls. The extent of inhibition was dependent on the antibody concentration applied. The results support the contention that enzymatic processes mediated by exo- and endoglucanases are responsible for cell wall autolytic reactions and that these reactions are linked to the mechanism for expressing auxin induced cell elongation in maize coleoptiles.

On possible connections between auxin induced growth and cell wall glucanase activities

Incubation of segments of Phaseolus aureus hypocotyls in an indoleacetic acid (IAA) solution (5 · 10 −6 M) induces an increment of the fresh weight of the segments and simultaneously a decrease of the pH of the incubation medium. This can be observed with segments from all regions of the hypocotyl although there is a gradient in growing potential along this organ. The autolysis rates as well as the glucanase activities of cell walls originated from the “upper” and “lower” parts of the hypocotyls are compared. A possible intervention of cell wall β1 → 3 endoglucanasesin the auxin-inducedgrowth is discussed.