RESUMO
Microsporidia, a large group of fungi-related intracellular parasites, are characterized by drastically reduced metabolism. They possess genes encoding glycolysis components, and the glycerol-phosphate shuttle, but lack mitochondria, Krebs cycle, respiratory chain and pyruvate-converting enzymes, except alpha and beta subunits of E(1) enzyme of pyruvate dehydrogenase (PDH) complex. Here, we have expressed PDH subunits from the microsporidum Paranosema (Antonospora) locustae in Escherichia coli. Western blot analysis with antibodies raised against recombinant proteins has revealed their specific accumulation in mature spores of P. locustae but not in the intracellular development stages. Two subunits were coprecipitated as a single heterooligomeric complex by anti-alpha or anti-beta PDH antibodies. Ultracentrifugation of spore homogenate has shown the presence of PDH in the soluble fraction. Relocalization of the mitochondrial protein in microsporidial spore cytoplasm was confirmed by immunoelectron microscopy of ultrathin cryosections with affinity-purified anti-alpha PDH antibodies. On cryosections, parasite enzyme was found partly associated with the cytoplasmic side of ER and other intraspore membranes, suggesting that electrons might be transferred to any membrane acceptor and finally to oxygen in the parasite cell.
Assuntos
Apansporoblastina/enzimologia , Subunidades Proteicas/biossíntese , Piruvato Desidrogenase (Lipoamida)/biossíntese , Apansporoblastina/química , Apansporoblastina/genética , Apansporoblastina/ultraestrutura , Microscopia Crioeletrônica , Escherichia coli/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Imunoprecipitação , Microscopia Imunoeletrônica , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Ligação Proteica , Subunidades Proteicas/genética , Piruvato Desidrogenase (Lipoamida)/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Esporos Fúngicos/química , Esporos Fúngicos/ultraestruturaRESUMO
Multiwire proportional chamber is a useful technology to build detectors that supersede the lack of interactivity of autoradiography in molecular biology experiments. Some drawbacks still limited the diffusion of existing instruments in biological laboratories. The major competitors are storage phosphor imaging systems. The simplified description of a radio-chromato-imager prototype (RCI) based on an original ultrathin multiwire proportional chamber is presented. It combines the advantage of the different existing technologies to present competitive properties in terms of efficiency, spatial resolution, robustness, manipulation easiness and production cost. Application of the RCI detector to molecular biology was performed by the analysis of karyotype and restriction display two-dimensional pulsed-field gel electrophoresis (KARD 2-D PFGE) data which are used to describe small eukaryotic genome structures. The comparative analysis with autoradiography was performed with the PDQuest software on Spraguea lophii (Microspora) genome fingerprints. The spot detection procedure applied to the different images leads to a similar conclusion considering the genome structure of S. lophii which appeared to be composed of 15 chromosomes for 13 karyotypic bands (200-880 kbp).
Assuntos
Apansporoblastina/genética , Impressões Digitais de DNA/métodos , Eletroforese em Gel de Campo Pulsado/instrumentação , Genoma de Protozoário , Genômica/métodos , Animais , Apansporoblastina/química , Impressões Digitais de DNA/instrumentação , DNA de Protozoário/análise , Genômica/instrumentação , Cariotipagem/instrumentação , Cariotipagem/métodosRESUMO
Spores of four species of microsporidia isolated from humans were analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and specific biomarkers were found for each. The microsporidia analyzed included three species, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis and the fourth organism is the recently described Brachiola algerae. Whole spores, spore shells, and soluble fractions were applied directly to the MALDI target without further purification steps. MALDI-TOF MS analysis of both whole spores and soluble fractions of the four isolates revealed a group of unique, characteristic, and reproducible spectral markers in the mass range of 2,000-8,000 Da. Statistical analysis of the averaged centroided masses uncovered two distinct sets of unique peptides or biomarkers, one originated from whole spores and the other from soluble fractions, that can differentiate the four microsporidian species studied. MALDI-TOF MS analysis of whole organisms is a rapid, sensitive, and specific option to characterize microsporidian isolates and has the potential for several applications in parasitology.