The caudal complex of Giardia lamblia and its relation to motility

Abstract

This paper presents a detailed study of the caudal complex of Giardia lamblia and its relation to movements observed in this region. The caudal complex of Giardia, composed of axonemes from the caudal flagella plus associated microtubular sheets, was investigated by light, electron microscopy, and 3D reconstruction tools. By the use of video-microscopy and digital image processing techniques, we were able to visualize in detail the caudal movements. A non-ionic detergent, Triton X-100, was used to isolate the complex that was afterwards analyzed by video-microscopy and transmission electron microscopy (TEM). We showed for the first time, using video-microscopy, that the intracellular portion of the caudal flagella axonemes presented motility, even after the disrupture of the cell membrane, contrasting with the caudal flagella themselves, that do not show active beating pattern. To check if actin filaments play a role in the above described movements, as previously supposed, we incubated the cells with jasplakinolide, a drug that induces the disruption of actin filaments in living cells. The experiments demonstrated that the drug did not affect the caudal motility. The analysis of the caudal complex by transmission electron microscopy (TEM) revealed that, even after the exposure to higher detergent concentrations, the connections between their components remained intact. The information obtained by TEM and 3D reconstruction tools showed that the region between both nuclei marks the intracellular end of the caudal complex, which proceeds toward the caudal portion of the cell following its longitudinal axis, where the axonemes emerge as the caudal flagella. The results obtained from video-microscopy assays of the isolated beating complex together with the 3D reconstruction data indicated that the internal portion of the caudal flagella is the force-generator of the movements in this region.

Introduction

The study of cell motility in protozoans presents several peculiarities. These peculiarities are mainly associated with the fact that these unicellular organisms can display very specific structures in their cytoskeleton, being a trait of a group and being not found, at least in the great majority of cases, in more complex metazoan. Examples of such structures are: the axostyle, found in protozoa such as Trichomonadidae, which was studied by Heuser (1986), McIntosh et al. (1973), Woodrum and Linck (1980), and McIntosh (1973), the myoneme from Acantaria revealed by Febvre (1981), and the costa of Trichomonas analyzed by Amos et al. (1979) and Monteiro-Leal et al. (1993), just to list a few.

Giardia lamblia is a multiflagellated protozoan that parasitizes the small intestine epithelium of mammals, causing diarrhea, malabsorption, and growth failure in children (Adam, 2001, Campanati et al., 2001, Farthing et al., 1986). The trophozoites of Giardia have a pear-shaped body, are ventrally flattened and bilaterally symmetric. Besides, they have an adhesive disk on the ventral surface, a microtubular median body, two nuclei and four pairs of flagella: anterior, ventral, latero-posterior, and caudal (Owen et al., 1979). As a parasite of the intestinal tract, Giardia needs to swim through a turbulent environment in the lumen and to adhere to the intestinal cells. To accomplish that, the protozoan uses its four pairs of flagella and specialized cytoskeleton structures (Campanati et al., 2002, Elmendorf et al., 2003). In relation to the motility of G. lamblia, it is possible to describe two kinds of movements: those arising from flagellar beating and those that are totally independent of flagella. In a previous paper, our group presented a detailed analysis of Giardia movement (Campanati et al., 2002). We demonstrated that only the anterior and the ventral pairs present active beating patterns, while the latero-posterior and caudal pairs are not active. Movements produced within the caudal portion of the cell body were also described. These include movements looking like flexions of the caudal cell portion leading to bending during attachment to surfaces, as well as, during swimming (Campanati et al., 2002). Feely and co-workers (1982) showed by immunoassaying and SDS–PAGE the presence of contractile proteins, like actin, α-actinin, tropomyosin, and myosin, in the cell and speculated that they may participate in the caudal movements. Contrasting with these findings, our group suggested that a microtubular cytoskeleton complex, found in the caudal region, is the actual responsible for these non-flagellar movements (Campanati et al., 2002).

The present work illustrates a detailed analysis of the caudal complex, using video-microscopy, digital image processing, and computer modelling, following the protocol used in our previous studies of the multiflagellated protozoan, Tritrichomonas foetus (Monteiro-Leal et al., 1995, Monteiro-Leal et al., 1996) and of the Giardia flagellar beatings (Campanati et al., 2002). To analyze the hypothesis suggested by Campanati and co-workers (2002), that the caudal complex is responsible for flexions of this region, we performed 3D reconstruction analysis. We also isolated the complex and studied it by video-microscopy and by transmission electron microscopy. Besides that, we incubated the cells with jasplakinolide, which acts by disrupting actin filaments in living cells (Ou et al., 2002), to evaluate their participation in the above-described movements. Finally, our results showed, for the first time, that the isolated microtubular complex presented active motion and was the responsible for the movements observed in this region of the cell.