| The Microsporidia are ubiquitous organisms that are emerging pathogens in humans. The defining characteristic of these organisms is an extrusion apparatus consisting of a polar tube that is attached to the anterior end of the spore by an anchoring disc. During germination the polar tube rapidly everts forming a hollow tube that brings the sporoplasm into intimate contact with the host cell. The polar tube provides a bridge to deliver the sporoplasm to the host cell. We have previously developed and reported an HPLC technique for the purification of major polar tube protein (PTP1). The lectin ConA binds to PTP1, bud doesn't bind to other polar tube proteins. In fungi, ConA has been demonstrated to bind to O-mannosylated proteins. Analysis suggests this binding is due to O-linked glycosylation. Examination of the Enc. cuniculi genome demonstrates a limited number of glycosylation enzymes, but there the presence of a complete O-mannosylation pathway. We evaluated the ability of Enc. hellem to infect RK13 cells in media containing mannose, glucose and sucrose as well as the ability of this microsporidia to infect various CHO cell lines that have glycosylation defects resulting in changes in ConA binding to these cells. Our hypothesis is that mannosylation of PTP1, which is conserved among the microsporidia, may provide a "sticky" surface to the polar tube that facilitates invasion. In addition, our data suggests that PTP1 can provide immunoprotection against microsporidia infection. Both anti PTP1 antibody in cell culture media and immunization with PTP1-GST fusion protein in mice inhibit microsporidia infection.;To further study the composition, formation and function of the polar tube, HPLC, Mass Spectrometry, 2-Dimentional Electrophoresis, Monoclonal Antibody, Mass Mapping and Electron Microscopy were used to look for other polar tube proteins or polar tube related proteins. Among the proteins identified, we characterized a 30-kDa protein. This protein appears to be a spore wall protein and may play a role in spore development. |
