Easy labeling of proliferative phase and sporogonic phase of microsporidia Nosema bombycis in host cells

Abstract

Microsporidia are eukaryotic, unicellular parasites that have been studied for more than 150 years. These organisms are extraordinary in their ability to invade a wide range of hosts including vertebrates and invertebrates, such as human and commercially important animals. A lack of appropriate labeling methods has limited the research of the cell cycle and protein locations in intracellular stages. In this report, an easy fluorescent labeling method has been developed to mark the proliferative and sporogonic phases of microsporidia Nosema bombycis in host cells. Based on the presence of chitin, Calcofluor White M2R was used to label the sporogonic phase, while β-tubulin antibody coupled with fluorescence secondary antibody were used to label the proliferative phase by immunofluorescence. This method is simple, efficient and can be used on both infected cells and tissue slices, providing a great potential application in microsporidia research.

Engineered resistance to Nosema bombycis by in vitro expression of a single-chain antibody in Sf9-III cells

“…Therefore, N . bombycis β-tubulin antibody and chitin dye (FWA)(Sigma, Saint Louis, Missouri, USA) were used as markers to distinguish mature spores and the meront [ 29 ]. Infected Sf9-III cells were used for IFAs three days after infection.…”

“…In our recent study, β-tubulin, the N . bombycis cytoskeletal protein on the inner side of the spore wall was proved to be a reliable meront stage marker protein, facilitating our further research on SWP12 [ 29 ]. In the IFAs, we failed to detected the SWP12 signal in the mature spore, which may cause by insufficient exposure of mAb G4 recognition epitope of SWP12.…”

A secretory hexokinase plays an active role in the proliferation of Nosema bombycis

“…Nbβ-tubulin was used as a marker protein to distinguish the meront stage from mature spores. Meronts can be labeled using the Nbβ-tubulin antibody, while the mature spores can be observed visibly in differential-interference microscopy (DIC) ( Chen et al, 2017 ; Huang et al, 2018 ). The IFA demonstrated the NbHK can be secreted into host cells ( Fig.…”

“…Proteins were separated by sodium dodecyl SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (Roche, Basel, Switzerland). After blocking in blocking buffer (5% (w/v) skim milk, 20 mM Tris–HCl, 150 mM NaCl and 0.05% Tween-20), membrane incubated with an HK-antiserum or Nbβ-tubulin-antiserum (diluted 1:3,000) ( Chen et al, 2017 ), washed, and incubated with HRP-labeled goat anti-mouse IgG (diluted 1:6,000; Sigma, Saint Louis, MI, USA). The bound antibodies were detected by ECL Plus Western Blotting Detection Reagents (Bio-Rad, Richmond, CA, USA).…”

Peer Review #4 of "A secretory hexokinase plays an active role in the proliferation of Nosema bombycis (v0.1)"

“…Nbβ-tubulin was used as a marker protein to distinguish the meront stage from mature spores. Meronts can be labeled using the Nbβ-tubulin antibody, while the mature spores can be observed visibly in differentialinterference microscopy (DIC) (Chen et al 2017;Huang et al 2018). The IFA demonstrated the NbHK can be secreted into host cells (Fig 2).…”