Quantitative Proteomic Analysis of Germination of Nosema bombycis Spores under Extremely Alkaline Conditions

Abstract

The microsporidian Nosema bombycis is an obligate intracellular pathogen of the silkworm Bombyx mori, causing the epidemic disease Pebrine and extensive economic losses in sericulture. Although N. bombycis forms 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 an in vitro spore germination model, how the proteome of N. bombycis changes during germination, analyse specific metabolic pathways employed in detail, and validate key functional proteins in vivo in 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 analyses in vitro and in 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 of N. bombycis spores, 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.

Invasion of Host Cells by Microsporidia

“…05M halogen anion Br − , Cl − , or I − in combination with Na + or K + pH 9.5; or 0.05M F − in combination with Na + or K + pH 5.5 Undeen and Avery, 1988 Nosema algerae 0.1M NaCl buffered at pH 9.5 with 20 mM glycine-NaOH or borate-NaOH Avery, 1988, Undeen andFrixione, 1991 Nosema algerae 0.1M NaCl buffered at pH 9.5 with 20 mM Tris-borate Frixione et al, 1992 Nosema algerae Alkali metal cations in 0.1M NaCl or KCl, pH 9.5 or 0.1M NaNO 2 , pH 9.5 or Na + ionophore monesin in 0.04M NaCl pH 9.5 Frixione et al, 1994 Nosema apis Dehydration in air, followed by rehydration with neutral distilled H 2 O Kramer, 1960 Nosema apis Dehydration in air, followed by rehydration in phosphate buffered saline, pH 7.1 Olsen et al, 1986 Nosema apis 0.5M NaCl with 0.5M NaHCO 3 , pH 6 de Graaf et al, 1993 Nosema bombycis 30% H 2 O 2 or 30% H 2 O 2 with 1% NaHCO 3 Kudo, 1918 Nosema bombycis Boiled digestive fluid of silkworm or 3% H 2 O 2 Ohshima, 1927 Nosema bombycis Digestive fluid of silkworm or liver extract medium pH > 8. 0 Trager, 1937 Nosema bombycis NaOH (N/10 to N/160) pH 11-13 neutralized with HCl to pH 6.0-9.0 Ohshima, 1937 Nosema bombycis KOH (N/7 to N/640) neutralized with HCl to pH 6.5-8.0 Ohshima, 1964a Nosema bombycis 0.375M KCl, 0.05M Glycine, 0.05M KOH pH 9.4-10.0 Ohshima, 1964b Nosema bombycis 1.5 to 3% H 2 O 2 Ohshima, 1966 Nosema bombycis 0.1N KOH followed by preheated silkworm hemolymph Ishihara, 1968 Nosema bombycis 0.05M Glycine, 0.05M KOH, and 0.375M KCl, pH 10.5 Liu et al, 2016 Nosema costelytrae Pretreatment with 0.2M KCl pH 12 followed by 0.2M KCl pH 7 Malone, 1990 Nosema heliothidis Pretreatment with 0.15M cation (K, Na, Li, Rb, or Cs) at pH 11, followed by 0.15M cation (K), pH 7 Undeen, 1978 Nosema helminthorum Mechanical pressure Dissanaike, 1955 Nosema locustae Dehydration with 2.5M sucrose or 5% polyethylene glycol followed by 0.1M Tris-HCl, 0.1M NaCl or 0.1M glycine-NaOH, 0.1M NaCl pH 9-10…”

3-Dimensional Organization and Dynamics of the Microsporidian Polar Tube Invasion Machinery

“…To better understand the kinetics and mechanics of PT germination, we performed high-speed, live-cell imaging to capture in vitro PT germination events in three microsporidian species that infect humans: A. algerae, E. hellem, and Encephalitozoon intestinalis. Although the in vivo triggers for PT firing are not well understood, in vitro triggers have been reported [46][47][48][49] . We used small variations of these conditions to optimize PT firing in vitro for our light microscopy assay (see Methods), and captured the entire germination process, including release of the cargo after transport through the PT ( Fig.…”

Morphology and Transcriptome Analysis of Nosema bombycis Sporoplasm and Insights into the Initial Infection of Microsporidia

“…Germination of microsporidia is the means of releasing sporoplasms, which involves a series of biologically complex changes (24). To elucidate the molecular basis of the germination process, transcriptome sequencing and quantitative proteomic analysis of ungerminated and germinated spores of N. bombycis have been investigated (25,26). Several genes associated with germination were identified, especially genes involved in protein dephosphorylation (25), and a number of important changes in metabolic pathways were detected during the germination process, such as glycolysis, the pentose phosphate pathway, and purine and pyrimidine metabolism (26).…”

“…To elucidate the molecular basis of the germination process, transcriptome sequencing and quantitative proteomic analysis of ungerminated and germinated spores of N. bombycis have been investigated (25,26). Several genes associated with germination were identified, especially genes involved in protein dephosphorylation (25), and a number of important changes in metabolic pathways were detected during the germination process, such as glycolysis, the pentose phosphate pathway, and purine and pyrimidine metabolism (26). After the infective sporoplasm enters the host cytoplasm, it develops into meronts and undergoes proliferation, which demands much energy (7,27).…”