Branching Network of Proteinaceous Filaments within the Parasitophorous Vacuole of Encephalitozoon cuniculi and Encephalitozoon hellem

Abstract

The microsporidia are a diverse phylum of obligate intracellular parasites that infect all major animal groups and have been recognized as emerging human pathogens for which few chemotherapeutic options currently exist. These organisms infect every tissue and organ system, causing significant pathology, especially in immune-compromised populations. The microsporidian spore employs a unique infection strategy in which its contents are delivered into a host cell via the polar tube, an organelle that lies coiled within the resting spore but erupts with a force sufficient to pierce the plasma membrane of its host cell. Using biochemical and molecular approaches, we have previously identified components of the polar tube and spore wall of the Encephalitozoonidae. In this study, we employed a shotgun proteomic strategy to identify novel structural components of these organelles in Encephalitozoon cuniculi. As a result, a new component of the E. cuniculi developing spore wall was identified. Surprisingly, using the same approach, a heretofore undescribed filamentous network within the lumen of the parasitophorous vacuole was discovered. This network was also present in the parasitophorous vacuole of Encephalitozoon hellem. Thus, in addition to further elucidating the molecular composition of seminal organelles and revealing novel diagnostic and therapeutic targets, proteomic analysis-driven approaches exploring the spore may also uncover unknown facets of microsporidian biology.

Microsporidia: Obligate Intracellular Pathogens Within the Fungal Kingdom

“…Many of the SWPs that have been identified in various microsporidia have posttranslational glycosylation, often involving mannosylation, which is likely important for spore wall adherence to host cells during invasion. Using various proteomic techniques additional SWPs such as SWP5 (159), SWP6 and SWP7 (L.M. Weiss manuscript in preparation) have also been identified in the Encephalitozoonidae and other putative SWPs have been identified in Nosema bombycis (160–164).…”

A new vesicular compartment in Encephalitozoon cuniculi

“…We have previously shown that proteins with biophysical properties similar to the polar tube proteins (PTPs) can copurify with PTPs during the polar tube fractionation procedure we developed and can be demonstrated to localize to distinct, unrelated compartments within the cell such as the spore wall and the branching proteinaceous filamentous network within the parasitophorous vacuole [14]. Here we have localized a novel protein in Enc.…”

“…cuniculi spores were harvested from culture supernatants of infected rabbit kidney cell line RK13 (American Type Cell Culture Collection, Manassas, VA), fractionated to enrich for polar tube proteins, and DTT-solubilized [14, 15], as previously described but omitting the urea wash. The lysate was analysed by nanoLC ESI-MS/MS and detected peptides were mapped to hypothetical Enc.…”

“…The lysate was analysed by nanoLC ESI-MS/MS and detected peptides were mapped to hypothetical Enc. cuniculi ORFs also as previously described [14]. …”

“…The ECU09_0280 ORF was cloned and expressed as a recombinant glutathione- S transferase-(GST)–tagged protein in a prokaryotic system and detected by Western blotting with a monoclonal anti-GST antibody as previously described [14]. Polyclonal antiserum to this protein was raised in murine hosts for protein localization in situ also as previously described.…”