Divergence of a Tandem Duplication of Manganese Superoxide Dismutase in Nosema bombycis.

Manganese superoxide dismutase (MnSOD) is a key enzyme in the protection of cells from oxidative stress. A tandem duplication of the MnSOD gene (NbMnSOD1 and NbMnSOD2) in the genome of Nosema bombycis, a parasite of the silkworm Bombyx mori, was previously identified. Here, we compare the protein structures of NbMnSOD1 and NbMnSOD2 and characterize these two proteins in terms of cellular localization, timing of transcription, protein structure, and enzyme activity. Despite a similarity in the primary sequence of NbMnSOD1 and NbMnSOD2, the latter shows a remarkable degree of amino acid sequence difference on the protein's surface and in the active site, where there is a substitution of a phenylalanine for a histidine in NbMnSOD2. Immuno-electron microscopy demonstrates that NbMnSOD1 is present in the cytosol of mature spores, whereas NbMnSOD2 is localized on the polar tube and the spore wall. Immunofluorescence confirms the localization of NbMnSOD2 on the polar tube of the germinated spore. Quantitative measurement of gene expression (qRT-PCR) demonstrates production of both alleles during the first day of infection followed by a dramatic decrease during the second to fourth day of infection. From the fifth day onward, the two alleles show a complementary pattern of expression. The qRT-PCR of the host manganese superoxide dismutase (BmMnSOD) shows a notable increase in transcription upon infection, leading to a three-fold spike by the first day of infection, followed by a decrease in transcription. Measurement of overall MnSOD activity shows a similar peak at day 1 followed by a decrease to a constant rate of enzyme activity. The differences in cellular localization and pattern of gene expression of NbMnSOD2 compared to NbMnSOD1, as well as the differences in protein structure seen for NbMnSOD2 compared to other microsporidial MnSODs, strongly suggest a unique, recently evolved role for NbMnSOD2.

Heterologous expression of Paranosema (Antonospora) locustae hexokinase in lepidopteran, Sf9, cells is followed by accumulation of the microsporidian protein in insect cell nuclei.

Paranosema (Nosema, Antonospora) locustae is the only microsporidium produced as a commercial product for biological control. Molecular mechanisms of the effects of this pathogen and other invertebrate microsporidia on host cells remain uncharacterized. Previously, we immunolocalized P. locustae hexokinase in nuclei of Locusta migratoria infected adipocytes. Here, the microsporidian protein was expressed in the yeast Pichia pastoris and in lepidopteran Sf9 cells. During heterologous expression, P. locustae hexokinase was accumulated in the nuclei of insect cells but not in yeast cell nuclei. This confirms nuclear localization of hexokinase secreted by microsporidia into infected host cells and suggests convenient model for its further study.

Characterization of the First Fungal Glycosyl Hydrolase Family 19 Chitinase (NbchiA) from Nosema bombycis (Nb).

Chitinases (EC 3.2.1.14), as one kind of glycosyl hydrolase, hydrolyze the ß-(1,4) linkages of chitin. According to the sequence similarity, chitinases can be divided into glycoside hydrolase family 18 and family 19. Here, a chitinase from Nosema bombycis (NbchiA) was cloned and purified by metal affinity chromatography and molecular exclusion chromatography. Sequence analysis indicated that NbchiA belongs to glycoside hydrolase family 19 class IV chitinase. The optimal pH and temperature of NbchiA are 7.0 and 40 °C, respectively. This purified chitinase showed high activity toward soluble substrates such as ethylene glycol chitin and soluble chitosan. The degradation of chitin oligosaccharides (GlcNAc)(2-5) detected by high-performance liquid chromatography showed that NbchiA hydrolyzed mainly the second glycosidic linkage from the reducing end of (GlcNAc)(3-5). On the basis of structure-based multiple-sequence alignment, Glu51 and Glu60 are believed to be the key catalytic residues. The site-directed mutation analysis revealed that the enzymatic activity was decreased upon mutation of Glu60, whereas mutation of Glu51 totally abolished the enzymatic activity. This is the first report of a GH19 chitinase in fungi and in Microsporidia.

Characterization of a novel otubain-like protease with deubiquitination activity from Nosema bombycis (Microsporidia).

Otubains are a recently identified family of deubiquitinating enzymes (DUBs). They are involved in diverse biological processes including protein degradation, signal transduction, and cell immune response. Several microsporidian genomes have been published in the last decade; however, little is known about the otubain-like protease in these widely-spread obligate intracellular parasites. Here, we characterized a 25 kDa otubain-like protease (NbOTU1) from the microsporidian Nosema bombycis, the pathogen causing pebrine disease in the economically important insect Bombyx mori. Sequence analysis showed that this protein contained a conserved catalytic triad of otubains composed of aspartate, cysteine, and histidine residues. The expression of Nbotu1 began on day 3 postinfection as determined by the RT-PCR method. Immunofluorescence analysis indicated that NbOTU1 is localized on the spore wall of N. bombycis. The subcellular localization of the NbOTU1 was further detected with immunoelectron microscopy, which showed that NbOTU1 is localized at the regions around endospore wall and plasma membrane. Deubiquitination analysis confirmed that the recombinant NbOTU1 possessed deubiquitination activity in vitro. Taken together, a novel microsporidian otubain-like protease NbOTU1 was partially characterized in N. bombycis, demonstrating its subcellular location and deubiquitination activity. This study provided a basic reference for further dissecting the function of otubains in microsporidia.

Characterization of a subtilisin-like protease with apical localization from microsporidian Nosema bombycis.

The microsporidian Nosema bombycis is the pathogen causing pébrine leading to heavy economic loss in sericulture. Little is known of the proteases of microsporidia that are important for both parasite development and pathogenesis. Here we identified a subtilisin-like serine protease NbSLP1 which contains an inhibitor_I9 and a peptidase_S8 domain. Three dimensional modeling of the catalytic domain of the NbSLP1 exhibited a typical 3-layer sandwich structure with S1 pocket substituted by Y(359). Phylogenetic analysis confirms that subtilisin-like serine proteases of microsporidia fall into two clades: SLP1 and SLP2, suggesting the initial subtilisin gene duplication events preceded microsporidia speciation. Furthermore, transcripts of Nbslp1 were detected in the midgut of Bombyx mori infection by N. bombycis by RT-PCR. Antibodies against NbSLP1 recognized both the precursor and mature enzyme by 2D Western blotting. Besides, indirect immunofluorescence assay revealed that the NbSLP1 is mainly localized at the two poles of spore which make the spore look like "safety pins". Remarkably, the mature protease is only detected in the apical region of the spore after germination. These studies demonstrate that NbSLP1 is a conserved subtilisin protease in microsporidia and suggest that NbSLP1 play a significant role in polar tube extrusion process.

A tandem duplication of manganese superoxide dismutase in Nosema bombycis and its evolutionary origins.

Microsporidia are a group of obligate intracellular eukaryotic parasites with small genomes. They infect animals from a wide variety of phyla, including humans. Two manganese superoxide dismutase (MnSOD) genes, designated NbMnSOD1 and NbMnSOD2, were found to be organized in a tandem array within the Nosema bombycis genome. The genes, both 678 bp in length, were found to be more similar to each other than they are to homologous genes of other Microsporidia, suggesting that the tandem duplication occurred subsequent to the development of this lineage. Reverse transcript PCR shows that mRNA for both genes is present in the spores. Analysis of the primary structure, hydrophobic cluster analysis, target signal analysis, and phylogenetic analysis all indicate that NbMnSOD1 is dimeric and targeted to the cytosol. NbMnSOD2 seems to have changed more rapidly and is under less evolutionary constraint than NbMnSOD1 suggesting that NbMnSOD2 may function under different conditions or in different tissues of its host rather than simply resulting in an increase in expression. A phylogenetic analysis of MnSOD sequences from eukaryotes, Archaea, and bacteria shows the microsporidial MnSODs to be grouped with the bacteria suggesting a possible horizontal gene transfer.

Effective gene silencing in a microsporidian parasite associated with honeybee (Apis mellifera) colony declines.

Honeybee colonies are vulnerable to parasites and pathogens ranging from viruses to vertebrates. An increasingly prevalent disease of managed honeybees is caused by the microsporidian Nosema ceranae. Microsporidia are basal fungi and obligate parasites with much-reduced genomic and cellular components. A recent genome-sequencing effort for N. ceranae indicated the presence of machinery for RNA silencing in this species, suggesting that RNA interference (RNAi) might be exploited to regulate Nosema gene expression within bee hosts. Here we used controlled laboratory experiments to show that double-stranded RNA homologous to specific N. ceranae ADP/ATP transporter genes can specifically and differentially silence transcripts encoding these proteins. This inhibition also affects Nosema levels and host physiology. Gene silencing could be mediated solely by Nosema or in concert with known systemic RNAi mechanisms in their bee hosts. These results are novel for the microsporidia and provide a possible avenue for controlling a disease agent implicated in severe honeybee colony losses. Moreover, since microsporidia are pathogenic in several known veterinary and human diseases, this advance may have broader applications in the future for disease control.

Bacterial catalase in the microsporidian Nosema locustae: implications for microsporidian metabolism and genome evolution.

Microsporidia constitute a group of extremely specialized intracellular parasites that infect virtually all animals. They are highly derived, reduced fungi that lack several features typical of other eukaryotes, including canonical mitochondria, flagella, and peroxisomes. Consistent with the absence of peroxisomes in microsporidia, the recently completed genome of the microsporidian Encephalitozoon cuniculi lacks a gene for catalase, the major enzymatic marker for the organelle. We show, however, that the genome of the microsporidian Nosema locustae, in contrast to that of E. cuniculi, encodes a group II large-subunit catalase. Surprisingly, phylogenetic analyses indicate that the N. locustae catalase is not specifically related to fungal homologs, as one would expect, but is instead closely related to proteobacterial sequences. This finding indicates that the N. locustae catalase is derived by lateral gene transfer from a bacterium. The catalase gene is adjacent to a large region of the genome that appears to be far less compact than is typical of microsporidian genomes, a characteristic which may make this region more amenable to the insertion of foreign genes. The N. locustae catalase gene is expressed in spores, and the protein is detectable by Western blotting. This type of catalase is a particularly robust enzyme that has been shown to function in dormant cells, indicating that the N. locustae catalase may play some functional role in the spore. There is no evidence that the N. locustae catalase functions in a cryptic peroxisome.

[Peculiarities of metabolism of the microsporidia Nosema grylli during the intracellular development]. / Osobennosti énergeticheskogo obmena mikrosporidii Nosema grylli pri vnutrikletochnom razvitii.

A long adaptation of Microsporidia to intracellular development supposes the host-derived ATP dependence of merogony and sporogony stages. To prove this assumption the activities of ten carbohydrate and energy metabolism enzymes were compared in the microsporidia Nosema grylli intracellular stages and mature spores. This species infects the fat body of crickets Gryllus bimaculatus. We have demonstrated lower activities of glycolytic enzymes, phosphoglucomutase and glucose-6-PhDH in the metabolically active meronts and sporonts than in the dormant mature spores. Low glycolysis level indicates that carbohydrate catabolism is not a principal mechanism of ATP supply in the N. grylli intracellular stages. Furthermore, we have not revealed a preferable expenditure of glycogen in comparison with triglycerides in infected cricket fat bodies. The N. grylli infection causes an equal reduction of glycogen and lipid content approximately in 2-3 times. Microsporidia have not mitochondria, Krebs cycle and electron-transport chain. Therefore they are not able to utilise fat reserves for ATP production. It seems to be proposed that microsporidia consume exogenous ATP which is produced by host cell metabolic system. The N. grylli infection provokes an increase of ATP content and ratio of ATP/ADP concentrations in cricket fat bodies approximately in 4 times. These data indicates a rise of host cell energy metabolism rate during the infection.

Alpha and beta subunits of pyruvate dehydrogenase E1 from the microsporidian Nosema locustae: mitochondrion-derived carbon metabolism in microsporidia.

Microsporidia are highly adapted eukaryotic intracellular parasites that infect a variety of animals. Microsporidia contain no recognisable mitochondrion, but recently have been shown to have evolved from fungi and to possess heat shock protein genes derived from mitochondria. These findings make it clear that microsporidian ancestors were mitochondrial, yet it remains unknown whether they still contain the organelle, and if so what its role in microsporidian metabolism might be. Here we have characterised genes encoding the alpha and beta subunits of pyruvate dehydrogenase complex E1 (PDH, EC 1.2.4.1) from the microsporidian Nosema locustae. All other amitochondriate eukaryotes studied to date have lost the PDH complex and replaced it with pyruvate:ferredoxin oxidoreductase (PFOR). Nevertheless, molecular phylogeny shows that these Nosema enzymes are most closely related to mitochondrial PDH from other eukaryotes, demonstrating that elements of mitochondrial metabolism have been retained in microsporidia, and that PDH has not been wholly lost. However, there is still no evidence for a mitochondrion in microsporidia, and neither PDH subunit is predicted to encode an amino terminal leader sequence that could function as a mitochondrion-targeting transit peptide, raising questions as to whether these proteins function in a relic organelle or in the cytosol. Moreover, it is also unclear whether these proteins remain part of the PDH complex, or whether they have been retained for another purpose. We propose that microsporidia may utilise a unique pyruvate decarboxylation pathway involving PDH, demonstrating once again the diversity of core metabolism in amitochondriate eukaryotes.

Activities of enzymes of carbohydrate and energy metabolism of the spores of the microsporidian, Nosema grylli.

The presence of 14 enzymes was investigated using purified spores of the microsporidian Nosema grylli from fat body of the crickets Gryllus bimaculatus. Glucose 6-phosphate dehydrogenase (EC 1.1.1.49), phosphoglucomutase (EC 5.4.2.2), phosphoglucose isomerase (EC 5.3.1.9), fructose 6-phosphate kinase (EC 2.7.1.11), aldolase (EC 4.1.2.13), 3-phosophoglycerate kinase (EC 2.7.2.3), pyruvate kinase (EC 2.7.1.40) and glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) were detected with activities of 15 +/- 1, 7 +/- 1, 1,549 +/- 255, 10 +/- 1, 5 +/- 1, 16 +/- 4, 6 +/- 1 and 16 +/- 2 nmol/min mg protein, respectively. Hexokinase (EC 2.7.1.1), NAD-dependent malate dehydrogenase (EC 1.1.1.37), malic enzyme (EC 1.1.1.40), lactate dehydrogenase (EC 1.1.1.27), alcohol dehydrogenase (EC 1.1.1.1) and succinate dehydrogenase (EC 1.3.99.1) were not detectable. These results suggest the catabolism of carbohydrates in microsporidia occurs via the Embden-Meyerhof pathway. Glycerol 3-phosphate dehydrogenase may reoxidize NADH which is produced by glyceraldehyde 3-phosphate dehydrogenase in glycolysis.