The Giardia Median Body Protein Is a Ventral Disc Protein That Is Critical for Maintaining a Domed Disc Conformation during Attachment

Abstract

Giardia has unique microtubule structures, including the ventral disc, the primary organelle of attachment to the host, and the median body, a structure of undefined function. During attachment, the ventral disc has a domed conformation and enables Giardia to attach to the host intestinal epithelia within seconds. The mechanism of attachment via the ventral disc and the overall structure, function, and assembly of the ventral disc are not well understood. Our recent proteomic analysis of the ventral disc indicated that the median body protein (MBP), previously reported to localize exclusively to the median body, was primarily localized to the ventral disc. Using high-resolution light and electron microscopy, we confirm that the median body protein localizes primarily to the overlap zone of the ventral disc. The MBP also occasionally localized to the median body during prophase. To define the contribution of MBP to the ventral disc structure, we depleted MBP using an anti-MBP morpholino. We found that the ventral disc was no longer able to form properly and that the disc structure often had an aberrant nondomed or flattened horseshoe conformation. The ability of attached anti-MBP morpholino-treated trophozoites to withstand shear forces and normal forces was significantly decreased. Most notably, the plasma membrane contacts with the surface, including those of the bare area, were defective after the anti-MBP knockdown. To our knowledge, this is the first ventral disc protein whose depletion directly alters ventral disc structure, confirming that the domed ventral disc conformation is important for robust attachment.

A detailed look at the cytoskeletal architecture of the Giardia lamblia ventral disc

“…Here we employed ns-ET to gain a large-field overview of a complete Giardia ventral disc by montaging 4 × 4 tomograms into one continuous volume of which a ~180 nm thick xy-slice is shown in figure 2A. The overall orientation of the disc in figure 2A is the same as in 2B, a 3View® reconstruction (Gatan Inc. Pleasanton CA; Denk & Horstmann, 2004; see also Schwartz et al, 2012) where the ventral disc is shown in pink, with the two nuclei in cyan, the median body in brown (Woessner & Dawson, 2012), and the four pairs of flagella in green (anterior), blue (caudal), yellow (ventral), and red (post-lateral; Dawson & House, 2010B). The flagellar basal bodies are clearly visible in the tomographic reconstructions of the disc preparations (Figs.…”

“…B: 3-D reconstructions of Giardia lamblia from Schwartz et al (2012) by a procedure called 3View (Denk & Horstmann, 2004). This SEM and microtomy 3-D reconstruction approach reveals the overall architecture of the Giardia cell with the ventral disc (pink) the nuclei (cyan), the median body (brown; Woessner & Dawson, 2012), and the four flagellar pairs in green (anterior), blue (caudal), yellow (ventral) and red (post-lateral; Dawson & House, 2010B). …”

Stable Transfection of the Diplomonad Parasite Spironucleus salmonicida

“…The unregulated maintenance of episomes might lead to elevated and uneven expression levels, as noticed previously in G. intestinalis transfectants (32). We utilized singlecrossover homologous recombination, previously shown to be effective in G. intestinalis (10,14,34), for the construction of a chromosomally integrated BiP-2ϫOLLAS strain (see Fig. S3 in the supplemental material).…”

Microvesicles released from Giardia intestinalis disturb host-pathogen response in vitro

“…Although still poorly understood, it is clear that the attachment of the parasite to the mucosal surface is the critical point for its persistence in the host. The parasite contains a ventral disk that seems to be important for attachment (Woessner and Dawson, 2012), while the flagella contributes to correct positioning and orientation of the trophozoites before the attachment (House et al, 2011). The parasite actively engages mucosal immunity and the infection progresses with a low or absent inflammation in most cases (Oberhuber and Stolte, 1997).…”