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This study explored a novel systemcombining plant-based production and the elastin-like peptide (ELP) fusion strategy to produce vaccinal antigens against tuberculosis. Transgenic tobacco plants expressing the mycobacterial antigens Ag85B and ESAT-6 fused to ELP (TBAg-ELP) were generated. Purified TBAg-ELP was obtained by the highly efficient, cost-effective, inverse transition cycling (ICT)method and tested in mice. Furthermore, safety and immunogenicity of the crude tobacco leaf extracts were assessed in piglets. Antibodies recognizing mycobacterial antigens were produced in mice and piglets. A T-cell immune response able to recognize the native mycobacterial antigens was detected in mice. These findings showed that the native Ag85B and ESAT- 6 mycobacterial B- and T-cell epitopes were conserved in the plant-expressed TBAg-ELP. This study presents the first results of an efficient plant-expression system, relying on the elastin-like peptide fusion strategy, to produce a safe and immunogenic mycobacterial Ag85B-ESAT-6 fusion protein as a potential vaccine candidate against tuberculosis. [ABSTRACT FROM AUTHOR]

Abstract: Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick cell wall composed of glycoproteins and chitin. Despite an extensive description of the fibrillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the ECU11_0510 open reading frame and presents two isoforms of 33 and 55kDa. Sequence analysis supports an assignment to the polysaccharide deacetylase family with a suspected chitin deacetylase activity (EcCDA). As demonstrated by TEM studies, EcCDA is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identification of an EcCDA homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore wall formation and spore resistance. [Copyright &y& Elsevier]

The microsporidian Encephalitozoon cuniculi is an intracellular eukaryotic parasite considered to be an emerging opportunistic human pathogen. The infectious stage of this parasite is a unicellular spore that is surrounded by a chitin containing endospore layer and an external proteinaceous exospore. A putative chitin deacetylase (ECU11_0510) localizes to the interface between the plasma membrane and the endospore. Chitin deacetylases are family 4 carbohydrate esterases in the CAZY classification, and several bacterial members of this family are involved in evading lysis by host glycosidases, through partial de-N-acetylation of cell wall peptidoglycan. Similarly, ECU11_0510 could be important for E. cuniculi survival in the host, by protecting the chitin layer from hydrolysis by human chitinases. Here, we describe the biochemical, structural, and glycan binding properties of the protein. Enzymatic analyses showed that the putative deacetylase is unable to deacetylate chitooligosaccharides or crystalline beta-chitin. Furthermore, carbohydrate microarray analysis revealed that the protein bound neither chitooligosaccharides nor any of a wide range of other glycans or chitin. The high resolution crystal structure revealed dramatic rearrangements in the positions of catalytic and substrate binding residues, which explain the loss of deacetylase activity, adding to the unusual structural plasticity observed in other members of this esterase family. Thus, it appears that the ECU11_0510 protein is not a carbohydrate deacetylase and may fulfill an as yet undiscovered role in the E. cuniculi parasite.

The microsporidian Encephalitozoon cuniculi is a unicellular obligate intracellular parasite considered as an emerging opportunistic human pathogen. The differentiation phase of its life cycle leads to the formation of stress-resistant spores. The E. cuniculi genome (2.9 Mbp) having been sequenced, we undertook a descriptive proteomic study of a spore-rich cell population isolated from culture supernatants. A combination of 2-DE and 2-DE-free techniques was applied to whole-cell protein extracts. Protein identification was performed using an automated MALDI-TOF-MS platform and a nanoLC-MS/MS instrument. A reference 2-DE map of about 350 major spots with multiple isoforms was obtained, and for the first time in microsporidia, a large set of unique proteins (177) including proteins with unknown function in a proportion of 25.6% was identified. The data are mainly discussed with reference to secretion and spore structural features, energy and carbohydrate metabolism, cell cycle control and parasite survival in the environment.