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Expression and Localization of an Hsp70 Protein in the Microsporidian Encephalitozoon cuniculi
Carrie E Jolly1, Cory A Leonard, J Russell Hayman
1Department of Microbiology, James H. Quillen College of Medicine, East Tennessee State University, Box 70579, Johnson City, TN 37614, USA.
Abstract:
Microsporidia spore surface proteins are an important, under investigated aspect of spore/host cell attachment and infection. For comparison analysis of surface proteins, we required an antibody control specific for an intracellular protein. An endoplasmic reticulum-associated heat shock protein 70 family member (Hsp70; ECU02_0100; "C1") was chosen for further analysis. DNA encoding the C1 hsp70 was amplified, cloned and used to heterologously express the C1 Hsp70 protein, and specific antiserum was generated. Two-dimensional Western blotting analysis showed that the purified antibodies were monospecific. Immunoelectron microscopy of developing and mature E. cuniculi spores revealed that the protein localized to internal structures and not to the spore surface. In spore adherence inhibition assays, the anti-C1 antibodies did not inhibit spore adherence to host cell surfaces, whereas antibodies to a known surface adhesin (EnP1) did so. In future studies, the antibodies to the 'C1' Hsp70 will be used to delineate spore surface protein expression.
Insights
Researchers developed specific antibodies against an intracellular heat shock protein 70 (Hsp70) in Microsporidia. These antibodies confirmed Hsp70 is not a surface protein, aiding future studies on spore-host interactions.
Area of Science:
- Microbiology
- Cell Biology
- Parasitology
Background:
- Microsporidia spore surface proteins are crucial for host cell attachment and infection.
- Identifying specific surface proteins requires reliable controls for intracellular proteins.
Purpose of the Study:
- To generate and validate antibodies against an intracellular heat shock protein 70 (Hsp70) from Microsporidia as a control.
- To confirm the intracellular localization of Hsp70 in E. cuniculi spores.
Main Methods:
- Amplification and cloning of DNA encoding E. cuniculi Hsp70 (C1).
- Heterologous expression of C1 Hsp70 and generation of specific antiserum.
- Two-dimensional Western blotting and immunoelectron microscopy to determine antibody specificity and protein localization.
- Spore adherence inhibition assays using anti-C1 and anti-EnP1 antibodies.
Main Results:
- Monospecific antibodies against C1 Hsp70 were successfully generated.
- Immunoelectron microscopy confirmed C1 Hsp70 localizes to internal spore structures, not the surface.
- Anti-C1 antibodies did not inhibit spore adherence to host cells, unlike antibodies against surface adhesin EnP1.
Conclusions:
- The generated anti-C1 Hsp70 antibodies are specific and validate Hsp70 as an intracellular protein.
- This provides a crucial control for future investigations into Microsporidia spore surface protein expression and function.
- The findings will help delineate the roles of specific spore surface proteins in host-pathogen interactions.
