Giardia Flagellar Motility Is Not Directly Required to Maintain Attachment to Surfaces

Abstract

Giardia trophozoites attach to the intestinal microvilli (or inert surfaces) using an undefined “suction-based” mechanism, and remain attached during cell division to avoid peristalsis. Flagellar motility is a key factor in Giardia's pathogenesis and colonization of the host small intestine. Specifically, the beating of the ventral flagella, one of four pairs of motile flagella, has been proposed to generate a hydrodynamic force that results in suction-based attachment via the adjacent ventral disc. We aimed to test this prevailing “hydrodynamic model” of attachment mediated by flagellar motility. We defined four distinct stages of attachment by assessing surface contacts of the trophozoite with the substrate during attachment using TIRF microscopy (TIRFM). The lateral crest of the ventral disc forms a continuous perimeter seal with the substrate, a cytological indication that trophozoites are fully attached. Using trophozoites with two types of molecularly engineered defects in flagellar beating, we determined that neither ventral flagellar beating, nor any flagellar beating, is necessary for the maintenance of attachment. Following a morpholino-based knockdown of PF16, a central pair protein, both the beating and morphology of flagella were defective, but trophozoites could still initiate proper surface contacts as seen using TIRFM and could maintain attachment in several biophysical assays. Trophozoites with impaired motility were able to attach as well as motile cells. We also generated a strain with defects in the ventral flagellar waveform by overexpressing a dominant negative form of alpha2-annexin::GFP (D122A, D275A). This dominant negative alpha2-annexin strain could initiate attachment and had only a slight decrease in the ability to withstand normal and shear forces. The time needed for attachment did increase in trophozoites with overall defective flagellar beating, however. Thus while not directly required for attachment, flagellar motility is important for positioning and orienting trophozoites prior to attachment. Drugs affecting flagellar motility may result in lower levels of attachment by indirectly limiting the number of parasites that can position the ventral disc properly against a surface and against peristaltic flow.

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).…”

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

“…Surrounding the ventral disc is another ordered structure known as the lateral crest (16). Attachment is a rapid stepwise process in which a seal with the surface is formed by the lateral crest (25) and the ventral disc adopts a dome-shaped conformation (23). This seal formed by the lateral crest may permit a suction-based mechanism of attachment.…”

“…This seal formed by the lateral crest may permit a suction-based mechanism of attachment. It remains unclear whether conformational changes of the ventral disc directly create forces for attachment or if disc conformational dynamics maintain attachment forces that are generated by some other mechanism (20,25).…”

“…To understand the function of this abundant discassociated protein, we used a morpholino-based knockdown approach to disrupt translation of MBP. We observed that introduction of an anti-MBP morpholino resulted in significant defects in attachment by both biophysical assays and microscopic examination of surface contacts using total internal reflection fluorescence microscopy (TIRFM) (25). A significant proportion of anti-MBPtreated trophozoites had open disc or horseshoe-shaped disc conformations.…”

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).…”