Expression and Localization of an Hsp70 Protein in the Microsporidian Encephalitozoon cuniculi

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.

Figure 1

Heterologous expression of E. cuniculi C1 Hsp70 protein as a histidine fusion protein in E. coli and Western blot analysis using purified C1 antibodies. A Coomassie stained SDS-PAGE gel ((a), right panel) of uninduced and IPTG induced expression of the recombinant protein shows a ~76 kDa protein in the induced lane. A Western blot of the SDS-PAGE gel was performed using histidine-tag-specific antibodies ((a), left panel) confirmed the recombinant protein induction. (b) A single ~76 kDa band was detected on 1D Western blot of E. cuniculi and E. intestinalis total spore protein using purified C1 Hsp70 protein antibodies. (c) The C1 (ECU02_0100) and B1 (ECU03_0520) Hsp70-related proteins were identified from a Coomassie stained 2D SDS-PAGE gel of E. cuniculi total spore protein by MALDI-MS analysis of trypsin digested gel spots. (d) Western analysis of the 2D gel using the C1 specific antibodies detected only the C1 protein (d).

Figure 2

Immuno-TEM of E. cuniculi infected rabbit kidney cells. The infected cells were prepared according to the protocol described. Ultrathin Lowicryl resin sections were cut and reacted with C1 Hsp70 specific antibodies (1 : 10) and a secondary antibody conjugated to 15-nm gold particles (1 : 200). The developmental stages include immature meronts and sporonts (the lower and upper spores in (a), resp.) and a mature spore (b). Abbreviations are as follows: PTC, polar tube coil; N, nucleus; PV, posterior vacuole; AD, anchoring disk. Each bar indicates a 500-nm scale.

Figure 3

Purified antibodies specific for the internal C1 Hsp70 protein does not inhibit E. cuniculi spore adherence to rabbit kidney host cells. Increasing concentrations of C1 antibodies (open circles) or EnP1 specific antibodies (open triangle) were incubated with spores in the presence of confluent host cells. Control samples excluded antibodies. After 4-hours, unbound spores were removed by washing and the bound spores were quantified by immunofluorescence assay. Each antibody dilution sample was performed in triplicate. Statistical significance is indicated with asterisks (P < .0001).