Factors influencing glycosylation of Trichoderma reesei cellulases. II: N-glycosylation of Cel7A core protein isolated from different strains

A systematic analysis of the N-glycosylation of the catalytic domain of cellobiohydrolase I (Cel7A or CBH I) isolated from several Trichoderma reesei strains grown in minimal media was performed. Using a combination of chromatographic, electrophoretic, and mass spectrometric methods, the presence of glucosylated and phosphorylated oligosaccharides on the three N-glycosylation sites of Cel7A core protein (from T. reesei strains Rut-C30 and RL-P37) confirms previous findings. With N-glycans isolated from other strains, no end-capping glucose could be detected. Phosphodiester linkages were however found in proteins from each strain and these probably occur on both the alpha1-3 and the alpha1-6 branch of the high-mannose oligosaccharide tree. Evidence is also presented for the occurrence of mannobiosyl units on the phosphodiester linkage. Therefore the predominant N-glycans on Cel7A can be represented as (ManP)(0-1)GlcMan(7-8)GlcNAc2 for the hyperproducing Rut-C30 and RL-P37 mutants and as (Man(1-2)P)(0-1-2)Man(5-6-7)GlcNAc2 for the wild-type strain and the other mutants. As shown by ESI-MS, random substitution of these structures on the N-glycosylation sites explains the heterogeneous glycoform population of the isolated core domains. PAG-IEF separates up to five isoforms, resulting from posttranslational modification of Cel7A with mannosyl phosphodiester residues at the three distinct sites. This study clearly shows that posttranslational phosphorylation of glycoproteins is not atypical for Trichoderma sp. and that, in the case of the Rut-C30 and RL-P37 strains, the presence of an end-capped glucose residue at the alpha1-3 branch apparently hinders a second mannophoshoryl transfer.     

Controlling chronic Pseudomonas aeruginosa infections by strategically interfering with the sensory function of SagS

The hybrid sensor SagS plays a central role in the formation of Pseudomonas aeruginosa biofilms, by enabling the switch from the planktonic to the biofilm mode of growth and by facilitating the transition of biofilm cells to a highly tolerant state. In this study, we examined the importance of the SagS key amino acid residues associated with biofilm formation (L154) and antibiotic tolerance (D105) in P. aeruginosa virulence. Recombinant P. aeruginosa ΔsagS and ΔsagS chromosomally expressing wild‐type sagS, or its two variants D105A and L154A, were tested for their potential to form biofilms and cause virulence in plants and mouse models of acute and chronic pneumonia. Although mutation of sagS did not alter P. aeruginosa virulence during acute infections, a significant difference in pathogenicity of sagS mutants was observed during chronic infections, with the L154A variant showing reduced bacterial loads in the chronic pneumonia model, while interference with the D105 residue enhanced the susceptibility of P. aeruginosa biofilms during tobramycin treatment. Our findings suggest that interference with the biofilm or tolerance regulatory circuits of SagS affects P. aeruginosa pathogenicity in chronic but not acute infections, and reveal SagS to be a promising new target to treat P. aeruginosa biofilm infections.     

Development of a triclosan scaffold which allows for adaptations on both the A- and B-ring for transport peptides

The enoyl acyl-carrier protein reductase (ENR) enzyme is harbored within the apicoplast of apicomplexan parasites providing a significant challenge for drug delivery, which may be overcome through the addition of transductive peptides, which facilitates crossing the apicoplast membranes. The binding site of triclosan, a potent ENR inhibitor, is occluded from the solvent making the attachment of these linkers challenging. Herein, we have produced 3 new triclosan analogs with bulky A- and B-ring motifs, which protrude into the solvent allowing for the future attachment of molecular transporters for delivery.     

Calreticulin mRNA and protein are localized to protein bodies in storage maize callus cells

Maize callus cells possess numerous protein bodies which develop as sub-compartments of the endoplasmic reticulum. We localized maize calreticulin mRNAs and protein in maize callus cells using in situ hybridization and immunocytochemistry. Calreticulin mRNAs were selectively targeted to the endoplasmic reticulum (ER) subdomains surrounding protein bodies. Profilin mRNAs, used as a positive control for in situ hybridization experiments, showed distinct and rather diffuse localization pattern. Using both, immunofluorescence and immunogold electron microscopy localization techniques, calreticulin was found to be enriched around and within protein bodies in maize callus storage cells. As a positive control for reticuloplasmins, HDEL antibody revealed labelling of protein bodies and of the nuclear envelope. The identity of protein bodies was confirmed by specific binding of an α zein antibody. These data suggest that calreticulin mRNA is targeted towards protein body forming subdomains of the ER, and that calreticulin is localized and enriched in these protein bodies. The possibility that calreticulin plays an important role in zein retention within the ER and/or its assembly and packaging into protein bodies during protein body biogenesis in maize callus is discussed.     

Statistical distribution of amino acid sequences: a proof of Darwinian evolution

MOTIVATION: The article presents results of the listing of the quantity of amino acids, dipeptides and tripeptides for all proteins available in the UNIPROT-TREMBL database and the listing for selected species and enzymes. UNIPROT-TREMBL contains protein sequences associated with computationally generated annotations and large-scale functional characterization. Due to the distinct metabolic pathways of amino acid syntheses and their physicochemical properties, the quantities of subpeptides in proteins vary. We have proved that the distribution of amino acids, dipeptides and tripeptides is statistical which confirms that the evolutionary biodiversity development model is subject to the theory of independent events. It seems interesting that certain short peptide combinations occur relatively rarely or even not at all. First, it confirms the Darwinian theory of evolution and second, it opens up opportunities for designing pharmaceuticals among rarely represented short peptide combinations. Furthermore, an innovative approach to the mass analysis of bioinformatic data is presented. CONTACT: eitner@amu.edu.pl SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Recombinant production of <Emphasis Type="Italic">Streptococcus equisimilis</Emphasis> streptokinase by <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Background  

Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. It is naturally secreted by several strains of beta-haemolytic streptococci. The low yields obtained in SK production, lack of developed gene transfer methodology and the pathogenesis of its natural host have been the principal reasons to search for a recombinant source for this important therapeutic protein. We report here the expression and secretion of SK by the Gram-positive bacterium Streptomyces lividans. The structural gene encoding SK was fused to the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) signal sequence or to the Streptomyces lividans xylanase C (xlnC) signal sequence. The native Vsi protein is translocated via the Sec pathway while the native XlnC protein uses the twin-arginine translocation (Tat) pathway.     

Toxoplasma gondii: chimeric Dr fimbriae as a recombinant vaccine against toxoplasmosis

Toxoplasma gondii is responsible for fetopathy in farm animals and humans and severe disease in immunocompromised individuals (i.e. AIDS patients). Effective vaccines, inducing protective and long-lasting immunity to this global parasite, are still desired. In the work, we evaluated the immunogenic and immunoprotective activity of Escherichia coli chimeric Dr fimbriae bearing selected antigenic epitopes of three T. gondii antigens (SAG1, GRA1 and MAG1), in comparison with conventional recombinant antigens obtained in E. coli expression system. Our data demonstrate a very high protective efficacy of recombinant antigens supplemented with Freund's adjuvants, whereas chimeric Dr fimbriae as a vaccine proved non-protective. The recombinant antigen vaccine induced a strong specific antibody response and prevented the brain cysts formation by 89%. The results are promising and should be confirmed in further study on farm animals by use of less aggressive than Freund's adjuvant preparations.