title: Quantitative Proteomic Analysis of Germination ofNosema bombycisSpores under Extremely Alkaline Conditions.
abstract: The microsporidianNosema bombycisis an obligate intracellular pathogen of the silkwormBombyx mori, causing the epidemic disease Pebrine and extensive economic losses in sericulture. AlthoughN. bombycisforms spores with rigid spore walls that protect against various environmental pressures, ingested spores germinate immediately under the extremely alkaline host gut condition (Lepidoptera gut pH > 10.5), which is a key developmental turning point from dormant state to infected state. However, to date this process remains poorly understood due to the complexity of the animal digestive tract and the lack of genetic tools for microsporidia. Here we show, using anin vitrospore germination model, how the proteome ofN. bombycischanges during germination, analyse specific metabolic pathways employed in detail, and validate key functional proteinsin vivoin silkworms. By a label-free quantitative proteomics approach that is directly based on high-resolution mass spectrometry (MS) data, a total of 1136 proteins were identified with high confidence, with 127 proteins being significantly changed in comparison to non-germinated spores. Among them, structural proteins including polar tube protein 1 and 3 and spore wall protein (SWP) 4 and 30 were found to be significantly down-regulated, but SWP9 significantly up-regulated. Some nucleases like polynucleotide kinase/phosphatase and flap endonucleases 1, together with a panel of hydrolases involved in protein degradation and RNA cleavage were overrepresented too upon germination, which implied that they might play important roles during spore germination. The differentially regulated trends of these genes were validated, respectively, by quantitative RT-PCR and 3 proteins of interest were confirmed by Western blotting analysesin vitroandin vivo. Furthermore, the pathway analysis showed that abundant up- and down-regulations appear involved in the glycolysis, pentose phosphate pathway, purine, and pyrimidine metabolism, suggesting preparations of energy generation and substance synthesis for the following invasion and proliferation inside the host. This report, to our knowledge, provides the first proteomic landscape ofN. bombycisspores, and also a stepping stone on the way to further study of the unique infection mode of this economically important pathogen and other microsporidia in general.
authors.affiliation: Laboratory of Invertebrate Pathology, College of Animal Sciences, Zhejiang University Hangzhou, China.
authors.forename: Han
authors.initials: H
authors.lastname: Liu
authors.affiliation: Laboratory of Invertebrate Pathology, College of Animal Sciences, Zhejiang University Hangzhou, China.
authors.forename: Bosheng
authors.initials: B
authors.lastname: Chen
authors.affiliation: Laboratory of Invertebrate Pathology, College of Animal Sciences, Zhejiang University Hangzhou, China.
authors.forename: Sirui
authors.initials: S
authors.lastname: Hu
authors.affiliation: Laboratory of Invertebrate Pathology, College of Animal Sciences, Zhejiang University Hangzhou, China.
authors.forename: Xili
authors.initials: X
authors.lastname: Liang
authors.affiliation: Laboratory of Invertebrate Pathology, College of Animal Sciences, Zhejiang University Hangzhou, China.
authors.forename: Xingmeng
authors.initials: X
authors.lastname: Lu
authors.affiliation: Laboratory of Invertebrate Pathology, College of Animal Sciences, Zhejiang University Hangzhou, China.
authors.forename: Yongqi
authors.initials: Y
authors.lastname: Shao
keywords: Nosema bombycis; germination; metabolic pathway; microsporidia; proteome