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Identification of a microsporidian polar tube protein reactive monoclonal antibody
E M Keohane1, P M Takvorian, A Cali
1Department of Biological Sciences, Rutgers University, Newark, New Jersey 07102, USA.
Abstract:
The microsporidia are characterized by spores containing a single polar tube that coils around the sporoplasm. When triggered by appropriate stimuli, the polar tube rapidly discharges out of the spore forming a hollow tube. The sporoplasm passes out of the spore through this tube serving as a unique vehicle of infection. Due to the unusual functional and solubility properties of the polar tube, the proteins comprising it are likely to be members of a protein family with a highly conserved amino acid composition among the various microsporidia. Polar tube proteins were separated from the majority of other proteins in glass bead disrupted spores of Glugea americanus using sequential 1% sodium dodecyl sulfate (SDS) and 9M urea extractions. The resultant spore pellet demonstrated broken, empty spore coats and numerous polar tubes in straight and twisted formations by negative stain transmission electron microscopy. After subsequent incubation of the pellet with 2% dithiothreitol (DTT), empty spore coats were still observed but the polar tubes were no longer present in the pellet. The DTT supernatant demonstrated four major protein bands by SDS-PAGE: 23, 27, 34 and 43 kDa. Monoclonal antibodies were produced to these proteins using Hunter's Titermax adjuvant. Mab 3C8.23.1 which cross-reacted with a 43-kDa antigen by immunoblot analysis, demonstrated strong reactivity with the polar tube of G. americanus spores by immunogold electron microscopy. This antibody will be useful in further characterization of polar tube proteins and may lead to novel diagnostic and therapeutic reagents.
Insights
Microsporidia spores infect hosts using a unique polar tube. Researchers isolated and identified key proteins within this polar tube, paving the way for new diagnostic tools.
Area of Science:
- Parasitology
- Microbiology
- Molecular Biology
Background:
- Microsporidia are obligate intracellular parasites.
- Their spores possess a unique polar tube for host cell invasion.
- The proteins forming the polar tube are poorly characterized.
Purpose of the Study:
- To isolate and identify proteins of the Glugea americanus microsporidian polar tube.
- To develop tools for further characterization of these proteins.
Main Methods:
- Spore disruption using glass beads.
- Sequential extraction with sodium dodecyl sulfate (SDS) and urea.
- Dithiothreitol (DTT) treatment to solubilize polar tube proteins.
- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis.
- Production of monoclonal antibodies.
- Immunogold electron microscopy.
Main Results:
- Polar tube proteins were successfully separated from spore coats.
- SDS-PAGE revealed four major protein bands (23, 27, 34, and 43 kDa).
- A monoclonal antibody (Mab 3C8.23.1) specifically recognized the 43-kDa polar tube antigen.
Conclusions:
- The study successfully isolated and identified major components of the microsporidian polar tube.
- A specific monoclonal antibody was generated, useful for further research.
- These findings may lead to novel diagnostic and therapeutic strategies against microsporidian infections.