Tuesday, December 13, 2011

Investigation of the polar tube proteins of the Microsporidia parasite: Encephalitozoon cuniculi

The microsporidia parasite Encephalitozoon cuniculi is an intracellular parasite that is unique in that its complete genome is just under 2000 genes and 2.9 Mb in size (1). Microsporidia parasites infect all major mammal species, and are now one of the most common infections among immunocompromised humans. Infections of this parasite are transmitted by internalization of spores via the respiratory system or gastrointestinal tracks. This organism can survive outside of a host for several years as dormant microscopic spores that range in size from 1 to 40 μm. The spores are covered in a thick double wall composed of a chitin-rich endospore and a protein based exospores layer. Within the double wall is the sporoplasm that contains the nucleus and cytoplasm of the microsporidia along with the polar tube organelle required for the infection process, and transfer of the sporoplasm into the host cells (2).
This defining trait of the microsporidia phylum is the polar tube that is wrapped tightly around the periphery of the sporoplasm until time of spore germination, yet its polar tube protein structure is still not well understood. E. cuniculi became the model microsporidia organism when the organism’s full genome was successfully sequenced. The polar tube of E. cuniculi is a proteinaceous structure composed of three separate proteins: polar tube protein 1 (PTP1), PTP2, and PTP3 (2). These three proteins share little sequence homology with one another or with the BLAST or PFAM databases. This suggests that these proteins are specific to microsporidia and evolved during the transition from fungal like cells to intracellular parasites (2).  Following an external signal for spore germination, the contents within the endospore begin to swell and pressure begins to build. This swelling occurs due to a rapid influx of water across aquaporin channels in the plasma membrane (2). After the spore wall reaches its maximum threshold for pressure build up, the anterior end of the spore wall ruptures and the polar tube extends as the sporoplasm is forced out of the spore and down the tube. It is hypothesized that the the central core PTP1 of the polar tube binds to the host cell membrane and then through endocytosis of the polar tube, the sporoplasm enters the host cell (2).
Recently research was conducted to investigate the interaction of the polar tube proteins of E. cuniculi to better understand the formation of the polar tube. An understanding of the protein structure of the polar tube is crucial in developing methods to prevent microsporidia infection and subsequent disease caused by the microbe. The three distinct polar tube proteins were the focus of the study: PTP1 that is rich in proline protein, PTP2 that is rich in lysine protein, and PTP3 that is uniquely larger than the other two polar tube proteins. The preservation of cysteine residues in PTP1 suggest that it may be involved in intraprotein or interprotein linking leading to the formation of the polar tube. The study was focused on the interactions between the major protein PTP1 with itself and the other polar tube proteins (3).
In order to investigate where the PTP associated on the polar tube, E. cuniculi PTP1, PTP2 and PTP3 were expressed as fusion proteins and then from these recombinant proteins were used to prepare polyclonal antibodies. The accuracy of the antisera prepared for each polar tube protein was confirmed on an immunoblot analysis. Each corresponding antisera was able to detect its corresponding polar tube protein from the E. cuniculi lysate. In addition, all of the antisera, anti-EcPTP2, anti-EcPTP2, and anti-EcPTP3, reacted with the E. cuniculi polar tube by indirect immunofluoresence assay and viewed under fluorescence microscopy (3).  The staining of the polar tube with anti-EcPTP2 covered the entire structure with green staining and the staining of the polar tube with the anti-EcPTP3 also covered the entire structure of the polar tube with red staining. When these two images were overlaid the image was yellow which suggested that the polar tube proteins overlap within the polar tube and are not isolated to specific locations (Figure 1). This immunofluoresence assay was repeated with anti-EcPTP1 and anti-EcPTP2, and resulted in the same type of overlap of the polar tube proteins on the polar tube (3).  Also, it’s important to note that none of the antisera reacted with surfaces of nongerminated spores.
Figure 1. Immunofluorescent analysis of PTP antibodies.
Images of extruded polar tubes of
E. cuniculi
that were incubated
with one of the three polar tube antibodies: anti-EcPTP1,
anti-EcPTP2, and anti-EcPTP3. The polar tubes were then
labeled with a second polar tube antibody and a fluorescent
label. (A)The polar tube was incubated with anti-EcPTP1 and
a green fluorescent marker. (B) Polar tube incubated with
anti-EcPTP3 with fluorescent red. (C) Merged image of image
A and B. (D). Polar tube incubated with anti-EcPTP2 and a
green fluorescent marker. (E). Polar tube with anti-EcPTP3 and
red fluorescent marker. (F) Merged image of image D and E.
(G) anti-EcPTP1 with green fluorescent marker. (H) anti-EcPTP2
with red fluorescent marker. (I). Merged image of G and H.
In order to investigate the possibility of an interaction among the three different PTPs a yeast two hybrid analysis was conducted. In these experiments, the E. cuniculi PTPs were analyzed in all possible pair-wise combinations; fused either to the bait or prey vector (3). A two hybrid yeast analysis consists of amplifying the PTP gene by PCR amplification, integrating it into a plasmid vector, and then transforming yeast cells with both the bait and prey vectors. The growth of the transformed yeast colonies was observed as evidence of the in vivo interaction of the two polar proteins. The researchers show that growth of all the yeast cells containing the different PTP combination suggest that all three of the E. cuniculi PTP can interact with themselves and each other, although the domain responsible for this interaction remains to be determined. Another two-hybrid yeast analysis focused on the compatibility of the N- and C- terminal regions of PTP1 provided a similar results that suggested that both the N- and C- terminal regions of PTP1 can interact with each other and themselves (3). 
Overall, this study makes a first attempt to better understand how the proteins of the polar tube of the microsporidia E. cuniculi are associated to gain insight to how the polar tube functions. This provides the first steps into more research of understanding how the proteins interact to produce the invasive polar tube that is used by microsporidia for infection, and thereby gaining a perspective how to prevent this pathogen from infection and prolonged disease.

Submitted by KB

Works Cited
1. Sibley, L. D. Invasion and intracellular survival by protozoan parasites. Immunological Reviews, 2011. (240) p.72-91.
2. Williams, Bryony. Unique physiology of host-parasite interactions in microsporidia infections. Cellular Microbiology, 2009. (11):11. p. 1551-1560.
3.  Bouzahzah, Boumediene, et. al., Interactions of Encephalitozoon cuniculi polar tube proteins, 2010. (78):6 p. 2745-2753. 

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