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Microsporidia are opportunistic fungal‐like pathogens that cause microsporidiosis, which results in significant economic losses and threatens public health. Infection of domesticated silkworms by the microsporidium Nosema bombycis causes pébrine disease, for which this species of microsporidia has received much attention. Research has been conducted extensively on this microsporidium over the past few decades to better understand its infection, transmission, host–parasite interaction, and detection. Several tools exist to study this species including the complete genome sequence of N. bombycis. In addition to the understanding of N. bombycis being important for the silkworm industry, this species has become a model organism for studying microsporidia. Research on biology of N. bombycis will contribute to the development of knowledge regarding microsporidia and potential antimicrosporidia drugs. Furthermore, this will provide insight into the molecular evolution and functioning of other fungal pathogens. [ABSTRACT FROM AUTHOR]

As a common feature of eukaryotic proteins, tandem amino acid repeat has been studied extensively in both animal and plant proteins. Here, a comparative analysis focusing on the proteins having tandem repeats was conducted in eight microsporidia, including four mammal-infecting microsporidia ( Encephalitozoon cuniculi, Encephalitozoon intestinalis, Encephalitozoon hellem and Encephalitozoon bieneusi) and four insect-infecting microsporidia ( Nosema apis, Nosema ceranae, Vavraia culicis and Nosema bombycis). We found that the proteins with tandem repeats were abundant in these species. The quantity of these proteins in insect-infecting microsporidia was larger than that of mammal-infecting microsporidia. Additionally, the hydrophilic residues were overrepresented in the tandem repeats of these eight microsporidian proteins and the amino acids residues in these tandem repeat sequences tend to be encoded by GC-rich codons. The tandem repeat position within proteins of insect-infecting microsporidia was randomly distributed, whereas the tandem repeats within proteins of mammal-infecting microsporidia rarely tend to be present in the N terminal regions, when compared with those present in the C terminal and middle regions. Finally, a hypothetical protein EOB14572 possessing four tandem repeats was successfully characterized as a novel endospore wall protein, which colocalized with polar tube of N. bombycis. Our study provided useful insight for the study of the proteins with tandem repeats in N. bombycis, but also further enriched the spore wall components of this obligate unicellular eukaryotic parasite. [ABSTRACT FROM AUTHOR]

The microsporidia Nosema bombycis is the insect pathogen of pebrine disease severely destructive to sericulture production. Here, we describe the use of Escherichia coli HT115 strain ( DE3) to express double-strand RNAs targeting the gene encoding ADP/ ATP protein in N. bombycis. The results showed that ds RNAs deferentially suppressed the gene expression during N. bombycis infection in the silkworm, and the effect waned gradually. Our results, for the first time, provide a tool to utilize the ds RNA expressed by recombinant E. coli to control the pebrine disease of the domestic silkworm. [ABSTRACT FROM AUTHOR]

Chitinases ( EC 3.2.1.14), as one kind of glycosyl hydrolase, hydrolyze the β-(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 (Glc NAc)2-5 detected by high-performance liquid chromatography showed that NbchiA hydrolyzed mainly the second glycosidic linkage from the reducing end of (Glc NAc)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. [ABSTRACT FROM AUTHOR]

Microsporidia, an unusual group of unicellular parasites related to fungi, possess a highly reduced mitochondrion known as the mitosome. Since mitosomes lack an organellar genome, their proteins must be translated in the cytosol before being imported into the mitosome via translocases. We have identified a Tom40 gene ( NbTom40), the main component of the translocase of the outer mitochondrial membrane, in the genome of the microsporidian Nosema bombycis. NbTom40 is reduced in size, but it is predicted to form a β-barrel structure composed of 19 β-strands. Phylogenetic analysis confirms that NbTom40 forms a clade with Tom40 sequences from other species, distinct from a related clade of voltage-dependent anion channels ( VDACs). The NbTom40 contains a β-signal motif that the polar residue is substituted by glycine. Furthermore, we show that expression of NbTom40, as a GFP fusion protein within yeast cells, directs GFP to mitochondria of yeast. These findings suggest that NbTom40 may serve as an import channel of the microsporidian mitosome and facilitate protein translocation into this organelle. [ABSTRACT FROM AUTHOR]

Microsporidia are eukaryotic, obligate intracellular, spore-forming parasites. The resistant spores, which harbor a rigid cell wall, are critical for their host-to-host transmission and persistence in the environment. The spore wall comprises two major layers: the exospore and the endospore. In Nosema bombycis, two spore wall proteins have been characterized—an endosporal protein, SWP30, and an exosporal protein, SWP32. Here, we report the identification of the third spore wall protein of N. bombycis, SWP25, the gene of which has no known homologue. SWP25 is predicted to posses a signal peptide and a heparin-binding motif. Immunoelectron microscopy analysis showed that this protein is localized to the endospore. This characterization of a new spore wall protein of N. bombycis may facilitate our investigation of the relationship between N. bombycis and its host, Bombyx mori. [ABSTRACT FROM AUTHOR]

To understand the source of the multiple DNA sequence variants of Nosema bombi ribosomal RNA (rRNA) found in a single bumble bee host, we PCR amplified, cloned, and sequenced the partial rRNA gene from 125 clones, which were derived from four out of 46 spores individually isolated from a single host by laser microdissection. At least two rRNA variants, characterized by either (GTTT)2 or (GTTT)3 repeat units within the internal transcribed spacer (ITS) region, were found per spore in approximately equal proportions, variants which were also found in approximately equal proportions in 55 clones of the two DNA extracts of multiple spores from the same host. Firstly, we demonstrate for the first time that DNA sequences can be obtained from single-binucleate microsporidia. Secondly, it appears that concerted evolution has not homogenized the sequences of all rRNA copies within a single N. bombi spore or even within a single nucleus. We thereby demonstrate unequivocally that two or more rRNA sequence variants exist per N. bombi spore, and urge caution in the use of multicopy rRNA genes for population genetic and phylogenetic analysis of this and other Microsporidia unless homologous copies can be reliably typed. [ABSTRACT FROM AUTHOR]

The microsporidian Nosema antheraeae is a pathogen that infects the Chinese oak silkworm, Antheraea pernyi. We sequenced the complete small subunit (SSU) rRNA gene and the internal transcribed spacer (ITS) of N. antheraeae, and compared the SSU rRNA sequences in other microsporidia. The results indicated that Nosema species, including N. antheraeae, formed two distinct clades, consistent with previous observations. Furthermore, N. antheraeae is clustered with N. bombycis with high bootstrap support. The organization of the rRNA gene of N. antheraeae is LSU–ITS1–SSU–ITS2-5S, also following a pattern similar to the Nosema type species, N. bombycis. Thus, N. antheraeae is a Nosema species and has a close relationship to N. bombycis. [ABSTRACT FROM AUTHOR]

Analyzes the ribosomal DNA region in Nosema bombycis. Detection of genes coding for mitochondrial heat shock proteins in the microsporidian genomic DNA; Details on the close relationship between parasitic organisms and fungi; Isolation of the total RNA of the infected cell culture.

The microsporidian Nosema philosamiae is a pathogen that infects the eri-silkworm Philosamia cynthia ricini. The complete sequence of rRNA gene (4,314 bp) was obtained by polymerase chain reaction amplification with specific primers and sequencing. The sequence analysis showed that the organization of the rRNA of N. philosamiae was similar to the pattern of Nosema bombycis. Phylogenetic analysis of rRNA gene sequences revealed that N. philosamiae had a close relationship with other Nosema species, confirming that N. philosamiae is correctly assigned to the genus Nosema. [ABSTRACT FROM AUTHOR]

We characterized a complete Sec61 complex in Nosema bombycis, which has been shown to consist of Sec61α, Sec61β, and Sec61γ genes. Comparing the genomic regions that harbor the respective subunit genes between N. bombycis, Encephalitozoon cuniculi, and Antonospora locustae, we found that microsporidian genomes have high degree of synteny in short genomic fragment, and that this gene synteny in general might exist throughout microsporidian genomes. [ABSTRACT FROM AUTHOR]