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Microsporidia are ubiquitous in the environment, infecting almost all invertebrates, vertebrates, and some protists. The microsporidian Nosema bombycis causes silkworms pébrine disease and leads to huge economic losses. Parasite secreted proteins play vital roles in pathogen–host interactions. Serine protease inhibitors (serpins), belonging to the largest and most broadly distributed protease inhibitor superfamily, are also found in Microsporidia. In this study, we characterized 19 serpins (NbSPNs) in N. bombycis; eight of them were predicted with signal peptides. All NbSPN proteins contain a typical conserved serpin (PF00079) domain. The comparative genomic analysis revealed that microsporidia serpins were only found in the genus Nosema. In addition to N. bombycis, a total of 34 serpins were identified in another six species of Nosema including N. antheraeae (11), N. granulosis (8), Nosema sp. YNPr (3), Nosema sp. PM-1 (3), N. apis (4), and N. ceranae (5). Serpin gene duplications in tandem obviously occurred in Nosema antheranae. Notably, the NbSPNs were phylogenetically clustered with serpins from the Chordopoxvirinae, the subfamily of Poxvirus. All 19 NbSPN transcripts were detected in the infected midgut and fat body, while 19 NbSPN genes except for NbSPN12 were found in the transcriptome of the infected silkworm embryonic cell line BmE-SWU1. Our work paves the way for further study of serpin function in microsporidia. [ABSTRACT FROM AUTHOR]

Nosema bombycis is a destructive and specific intracellular parasite of silkworm, which is extremely harmful to the silkworm industry. N. bombycis is considered as a quarantine pathogen of sericulture because of its long incubation period and horizontal and vertical transmission. Herein, two single-chain antibodies targeting N. bombycis hexokinase (NbHK) were cloned and expressed in fusion with the N-terminal of Slmb (a Drosophila melanogaster FBP), which contains the F-box domain. Western blotting demonstrated that Sf9-III cells expressed NSlmb–scFv-7A and NSlmb–scFv-6H, which recognized native NbHK. Subsequently, the NbHK was degraded by host ubiquitination system. When challenged with N. bombycis, the transfected Sf9-III cells exhibited better resistance relative to the controls, demonstrating that NbHK is a prospective target for parasite controls and this approach represents a potential solution for constructing N. bombycis-resistant Bombyx mori. [ABSTRACT FROM AUTHOR]

Nosema bombycis is a destructive, obligate intracellular parasite of the Bombyx mori. In this study, a single-chain variable fragment (scFv) dependent technology is developed for the purpose of inhibiting parasite proliferation in insect cells. The scFv-G4, which we prepared from a mouse G4 monoclonal antibody, can target the N. bombycis spore wall protein 12 (NbSWP12). Indirect immunofluorescence assays showed that NbSWP12 located mainly on the outside of the N. bombycis cytoskeleton, although some of it co-localized with β-tubulin in the meront-stage of parasites. When meront division began, NbSWP12 became concentrated at both ends of each meront. Western blotting showed that scFv-G4 could express in Sf9-III cells and recognized native NbSWP12. The transgenic Sf9-III cell line showed better resistance than the controls when challenged with N. bombycis, indicating that NbSWP12 is a promising target in this parasite and this scFv dependent strategy could be a solution for construction of N. bombycis-resistant Bombyx mori. [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]

• Nosema bombycis detection by NALFS is 10–100× more sensitive than agarose gel electrophoresis. • The presence of ≥2% infected eggs can be detected by NALFS. • Primer LSU323 targeting N. bombycis LSU rDNA is specific for some insect microsporidia. Nosema bombycis, the pathogen of silkworm pébrine, causes enormous economic losses to sericulture. As such, quarantine of commercial silkworm eggs represents an important safeguard to the silkworm industry. Here, we established a user-friendly detection system based on a nucleic acid lateral flow strip (NAFLS) that combines polymerase chain reaction (PCR) and a colloidal gold strip. PCR primers were designed based on the sequence of LSU rDNA of N. bombycis and has favourable specificity for common microsporidian isolates in silkworms. The forward and reverse primers were labeled on the 5′ end with biotin and carboxyfluorescein (FAM), respectively. Genomic DNA was extracted from egg samples and was used as a template for PCR, followed by subsequent detection by NALFS. The detection limit of purified N. bombycis genomic DNA was 1 pg, 100× more sensitive than that of agarose gel electrophoresis (AGE). Furthermore, the sensitivity of detection of simulated "infected" silkworm eggs was 10–100× higher than that of AGE. NALFS detected infection in 27 of 29 samples of silkworm eggs oviposited by female moths infected in lab; ≥2% infected eggs per batch are detected as positive, while ≥40% infected eggs per batch are required for detection by AGE. Collectively, NALFS is easy to use and has great potential for widespread use in the detection of N. bombycis in silkworm egg production. [ABSTRACT FROM AUTHOR]

Nosema bombycis is an obligate intracellular parasite, which can cause pébrine disease. To investigate the effects of N. bombycis infection, 5th-instar silkworms were challenged with N. bombycis isolate CQ1, and two-dimensional gel electrophoresis analysis was performed to analyze the differentially expressed proteins in infected and uninfected silkworm fat bodies 1, 2, 4, 6 and 8 days post-infection (dpi). 46 differentially expressed proteins were identified at the 5 time points using MALDI-TOF/TOF MS. The changed proteins mainly involved in immune response, energy metabolism, and molecular synthesis. Overall, the identified proteins may provide important insights into the mechanisms of the silkworm response to N. bombycis infection. [ABSTRACT FROM AUTHOR]

Highlights: [•] We isolated the deproteinated chitin spore coats (DCSCs) employing boiling alkali. [•] The DCSC was a loose, curled ring and devoid of organelles or nuclei. [•] SWPs fraction could bind to DCSCs, though SWP25 was unable to interact. [•] Some SWPs lost the ability to bind the DCSC after 0.1M NaOH treatment. [ABSTRACT FROM AUTHOR]

• H20, a monoclonal antibody, was generated to label mature spores of Nosema bombycis. • The target protein of H20 was highly stable. • The target protein was little affected by alkali and SDS treatments. • Spore wall protein 26 on the mature spore surface, was the likely target of H20. • H20 is a more sensitive labeling method for mature spores than Calcofluor White M2R. Microsporidia are a fascinating phylum of obligate intracellular pathogens with unique infection processes and complicated life cycles. Microsporidian life cycles can be divided roughly into intracellular and extracellular stages. Currently, research on their life cycles were mainly explored by morphology because there are few molecular markers available with which to distinguish the different life stages. In this study, we generated H20, a monoclonal antibody (MAb) to label mature spores of Nosema bombycis. Immunofluorescence assays showed that the target protein of H20, which is highly stable and was barely affected by alkali and sodium dodecyl sulfate (SDS) treatments, was located on the mature spore surface. Western blot analysis showed that spore wall protein 26 (SWP26) was the likely target of H20. This MAb can specifically identify mature spores in a complex biological sample based on immunological detection of the parasite. [ABSTRACT FROM AUTHOR]