Nanoarchitecture of the ventral disc of Giardia intestinalis as revealed by high-resolution scanning electron microscopy and helium ion microscopy.

The parasitic protozoan Giardia intestinalis, the causative agent of giardiasis, presents a stable and elaborated cytoskeleton, which shapes and supports several intracellular structures, including the ventral disc, the median body, the funis, and four pairs of flagella. Giardia trophozoite is the motile form that inhabits the host small intestine and attaches to epithelial cells, leading to infection. The ventral disc is considered one important element of adhesion to the intestinal cells. It is adjacent to the plasma membrane in the ventral region of the cell and consists of a spiral layer of microtubules and microribbons. In this work, we studied the organization of the cytoskeleton in the ventral disc of G. intestinalis trophozoites using high-resolution scanning electron microscopy or helium ion microscopy in plasma membrane-extracted cells. Here, we show novel morphological details about the arrangement of cross-bridges in different regions of the ventral disc. Results showed that the disc is a non-uniformly organized structure that presents specific domains, such as the margin and the ventral groove region. High-resolution scanning electron microscopy allowed observation of the labeling pattern for several anti-tubulin antibodies using secondary gold particle-labeled antibodies. Labeling in the region of the emergence of the microtubules and supernumerary microtubules using an anti-acetylated tubulin antibody was observed. Ultrastructural analysis and immunogold labeling for gamma-tubulin suggest that disc microtubules originate from a region bounded by the bands of the banded collar and merge with microtubules formed at the perinuclear region. Actin-like filaments and microtubules of the disc are associated, showing an interconnection between elements of the cytoskeleton of the trophozoite.

The structural organization of Giardia intestinalis cytoskeleton.

Giardia intestinalis, the causative agent of giardiasis, has complex cytoskeleton organization with structures involved in motility, adhesion, cell division, and cell differentiation. Microtubules are key components of the cytoskeleton and are the main elements of the ventral disc, median body, funis, in addition to four pairs of flagella. These cytoskeletal elements are basically stable microtubule arrangements. Although tubulins are the main proteins of these elements, molecular and biochemical analyses of Giardia trophozoites have revealed the presence of several new and not yet characterized proteins in these structures, which may contribute to their nanoarchitecture (mainly in the ventral disc). Despite these findings, morphological data are still required for understanding the organization and biogenesis of the cytoskeletal structures. In the study of this complex and specialized network of filaments in Giardia, two distinct and complementary approaches have been used in recent years: (a) transmission electron microscopy tomography of conventionally processed as well as cryo-fixed samples and (b) high-resolution scanning electron microscopy and helium ion microscopy in combination with new plasma membrane extraction protocols. In this review we include the most recent studies that have allowed better understanding of new Giardia components and their association with other filamentous structures of this parasite, thus providing new insights in the role of the cytoskeletal structures and their function in Giardia trophozoites.

Helium ion microscopy and ultra-high-resolution scanning electron microscopy analysis of membrane-extracted cells reveals novel characteristics of the cytoskeleton of Giardia intestinalis.

Giardia intestinalis presents a complex microtubular cytoskeleton formed by specialized structures, such as the adhesive disk, four pairs of flagella, the funis and the median body. The ultrastructural organization of the Giardia cytoskeleton has been analyzed using different microscopic techniques, including high-resolution scanning electron microscopy. Recent advances in scanning microscopy technology have opened a new venue for the characterization of cellular structures and include scanning probe microscopy techniques such as ultra-high-resolution scanning electron microscopy (UHRSEM) and helium ion microscopy (HIM). Here, we studied the organization of the cytoskeleton of G. intestinalis trophozoites using UHRSEM and HIM in membrane-extracted cells. The results revealed a number of new cytoskeletal elements associated with the lateral crest and the dorsal surface of the parasite. The fine structure of the banded collar was also observed. The marginal plates were seen linked to a network of filaments, which were continuous with filaments parallel to the main cell axis. Cytoplasmic filaments that supported the internal structures were seen by the first time. Using anti-actin antibody, we observed a labeling in these filamentous structures. Taken together, these data revealed new surface characteristics of the cytoskeleton of G. intestinalis and may contribute to an improved understanding of the structural organization of trophozoites.

Assembly of the Leishmania amazonensis flagellum during cell differentiation.

The flagellar cytoskeleton of Leishmania promastigotes contains the canonical 9+2 microtubular axoneme and a filamentous structure, the paraflagellar rod (PFR), which is present alongside the axoneme. In contrast to promastigotes, which contain a long and motile flagellum, the amastigote form of Leishmania displays a short flagellum without a PFR that is limited to the flagellar pocket domain. Here, we investigated the biogenesis of the Leishmania flagellum at 0, 4, 6 and 24h of differentiation. Light and electron microscopy observations of the early stages of L. amazonensis differentiation showed that the intermediate forms presented a short and wider flagellum that did not contain a PFR and presented reduced motion. 3D-reconstruction analysis of electron tomograms revealed the presence of vesicles and electron-dense aggregates at the tip of the short flagellum. In the course of differentiation, cells were able to adhere and proliferate with a doubling time of about 6h. The new flagellum emerged from the flagellar pocket around 4h after initiation of cell cycle. Close contact between the flagellar membrane and the flagellar pocket membrane was evident in the intermediate forms. At a later stage of differentiation, intermediate cells exhibited a longer flagellum (shorter than in promastigotes) that contained a PFR and electron dense aggregates in the flagellar matrix. In some cells, PFR profiles were observed inside the flagellar pocket. Taken together, these data contribute to the understanding of flagellum biogenesis and organisation during L. amazonensis differentiation.

New associated structures of the anterior flagella of Giardia duodenalis.

Giardia duodenalis is a protozoan parasite that causes intestinal disorders. The trophozoites present four pairs of flagella. Here we further analyze the structural organization of the anterior flagella associated structures of G. duodenalis. High resolution scanning electron microscopy of detergent-extracted trophozoites revealed novel aspects of the interaction of the anterior flagella axonemes with the marginal plates. Images of the marginal plates showed that it was located in the anterior region of the parasite, above the crossing point of the anterior flagella axonemes toward the periphery of the cell. Two well distinguished structures were seen associated with the anterior flagella. The first one corresponds to the "dense rods", located just below the axoneme. The second one is a system of filaments located in the upper portion of the flagellum, facing the marginal plates and connecting these two structures. The thickness of the filaments is around 18 nm and they are spaced at intervals of 4-32 nm (average 18 nm). The length of the filaments may vary from 33 to 240 nm. We suggest that this filamentous structure of Giardia may help the dynamics and behavior of the anterior flagella of trophozoites during protozoan motility and adhesion, providing favorable conditions for the establishment of parasitism.

Giardia lamblia: a report of drug effects under cell differentiation.

The Giardia lamblia life cycle is characterized by two phases during which two major cell differentiation processes take place: encystation and excystation. During encystation, the trophozoites transform into cysts, the resistance form. Once ingested by a susceptible host, the cysts are stimulated to excyst in the stomach, and the excysted trophozoites adhere to the epithelium of the upper small intestine. Our work analyses the effects of four benzimidazole derivatives during Giardia differentiation into cysts and evaluates the excystation efficiency of water resistant cysts. Albendazole (AB) showed the most significant results by inhibiting encystation about 30% and a decreasing rate of excystation efficiency. The ultrastructural organization of the cyst adhesive disk was notably affected by AB treatment. Although other benzimidazoles showed some effect on encystation, they were not able to inhibit the excystation process. It is known that the benzimidazoles affect the cytoskeleton of many organisms but how it interferes in Giardia differentiation processes is our main focus. The importance of studying Giardia's differentiation under drug action is reinforced by the following arguments: (1) Cysts eliminated by hosts undergoing treatment could still be potentially infective; (2) once the host has been treated, it would be desirable that the shedding of cysts into the environment is avoided; (3) the prevention of Giardia dissemination is a question of extreme importance mainly in underdeveloped countries, where poor sanitary conditions are related to high rates of giardiasis. This report concerns the importance of keeping the environment free from infective cysts and on Giardia's drug resistance and differentiating abilities.

Effects of three benzimidazoles on growth, general morphology and ultrastructure of Tritrichomonas foetus.

Tritrichomonas foetus is a venereal pathogen of cattle, which causes infertility, early embryonic death or abortion. In order to evaluate the potential trichomonicidal activity of benzimidazoles, the effects of thiabendazole, mebendazole and albendazole were analyzed on the multiplication, general morphology and ultrastructure of T. foetus. It was found that mebendazole presented the highest IC(50%) (2.3 microM), when compared with albendazole (IC(50%)=9.4 microM) and thiabendazole (IC(50%)=142.6 microM), and that such effects were irreversible. Concerning microscopic analysis, thiabendazole- and mebendazole-treated cells presented increased volume, internalization of the flagella, disruption or multiplication of the nucleus, multiple organelles and cytoplasmic vacuolization. Albendazole-treated cells exhibited slight alterations, because the parasite became slightly rounded, its flagella were not internalized but the cytoplasm was vacuolated. Mebendazole was indeed highly effective as an in vitro trichomonicidal agent, and this might open up new possibilities for the use of mebendazole in the therapy of bovine trichomoniasis.