3. Peripheral vesicles

1. Introduction

88 Current Topics in Giardiasis

1.1. Giardia and giardiasis

2. Endomembrane system

environment [10, 11].

intestinalis. Currently, the preferred name is G. intestinalis.

Giardia was observed for the first time by Antony Van Leeuwenhoek in 1681, but it was Lambl who described the cell morphological characteristics in detail and named it Cercomonas intestinalis. Subsequently, Blanchard changed the nomenclature in 1888 to Giardia lamblia [1–3]. The parasite is also known as Giardia intestinalis, Giardia duodenalis, Giardia enterica and Lamblia

The trophozoite of G. intestinalis inhabits the small intestine and causes the disease, whereas the cyst is protected by a cyst wall, can survive in adverse environmental conditions and thus is responsible for parasite transmission. Giardiasis starts when the cysts are ingested via food or contaminated water and reach the small intestine. The trophozoites emerge from the cyst wall and colonize the intestinal epithelium [4]. The emerged trophozoites adhere and spread out by binary divisions and form a monolayer over the intestinal mucosa provoking local inflammation and reduction in nutrient uptake. The parasites may reach the final portions of the intestine, becoming a cyst again, and they are liberated with the feces. The cysts can then infect new hosts [4]. Diarrhea is the main symptom of G. intestinalis infection, and giardiasis occurs in humans and several animals throughout the world. Giardial transmission between different species is frequent, and this characterizes giardiasis as a zoonotic disease [5]. The infection rates of giardiasis are associated with sanitary conditions since low rates are observed when sanitary conditions are implemented [6]. Giardiasis mainly affects children and is considered a cosmopolite disease [7]. Several factors such as geographic area, group of analysis, sensitivity of the diagnostic methods and health care accessibility influence the prevalence rates reported [8]. The disease treatment is based in nitroimidazole-derived drugs (metronida-

zole, tinidazole and ornidazole), since metronidazole is the most widely used drug [9].

The endomembrane system of higher eukaryotes comprises of a number of structures, such as the endoplasmic reticulum, nucleus, Golgi, lysosomes, peroxisomes, autophagosomes and vesicles involved in different traffic pathways. Many theories have addressed the evolutionary origin of eukaryotic membranes; the most acceptable one is the invagination of plasma membrane, which is based on the similarity between the endoplasmic reticulum (ER) lumen to the

Although Giardia belongs to the eukaryotic group, it lacks some of the typical organelles found in eukaryotes; therefore, this parasite is an interesting model to study cell evolution. Mitochondria and peroxisomes are not present in this parasite, as found in morphological and biochemical studies. In addition, Golgi complex and vesicles of the endocytic pathway are incipient. On the other hand, Giardia trophozoites exhibit membrane structures that incorporate the cationic, membrane potential-sensitive fluorophore rhodamine 123 and reduce a tetrazolium fluorogen. Based on this observation, the existence of membrane-associated sites with some similarities to G. intestinalis belongs to the Giardiinae family, and it has a unique organellar system formed by numerous small vacuoles named peripheral vesicles (PVs) (Figures 2–4, 5c, 9a). The PVs are oval, elongated and are 100–200 nm in size (Figure 2c); they are located in the cell periphery, right below the plasma membrane (Figure 2) [14, 15]. The PVs have a fundamental role in the endocytosis process as well as during the digestion and retrograde transport of the parasite [16, 17]. A number of cytochemical studies indicated resident enzymes, such as acid phosphatases, sulfur-binding proteins (SH) and glucose-6-phosphatase in the PVs. The localization of these proteins pointed that the PVs present compatible functions to those initial or late

Figure 2. Peripheral vesicles of G. intestinalis. Transmission electron microscopy (TEM) of vegetative non-encysting (a and b) and encysting (c) parasite. The peripheral vesicles (PV, arrows) are right below the plasma membrane (a–c) and present similar size, shape and location in both forms (a–c). Artificially colored (b). N, nucleus; A, axoneme; F, flagellum; ESV, encystation-specific vesicle.

endosomes [17]. The accumulation of gold-labeled macromolecules such as albumin, peroxidase, transferrin and low-density lipoprotein in PVs [15] reinforced this idea. Moreover, the cytochemical localization of acid phosphatase, a classical lysosomal marker, in these vesicles Kattenbach and colleagues [15] led to the suggestion that G. intestinalis presents an endosomallysosomal system that later on during evolution was subdivided into compartments such as early or late endosomes and lysosomes [17]. Besides the degradation function played by the PVs, to date, it is the only known organelle involved in the Giardia endocytic pathway that is capable of accumulating fluid phase and membrane-bound molecules [18]. It was

Figure 3. Endoplasmic reticulum of G. intestinalis. Electron tomography of a thick section after cytochemistry for glucose-6-phosphatase (a), 3-D reconstruction of the same cell (b) and TEM (c). (a) The reaction product is on the nuclear envelope (NE), the endoplasmic reticulum (ER) and some peripheral vesicles (PVs, arrow). An arrowhead indicates to a point near the ER and nuclear envelope (NE). (b). Distribution of ER tubules (T) (yellow) and cisternae (NE) (green) occupying the majority of space in the cytosol. PVs (blue). Cross section of the ER appears vesicular (V); ER longitudinal view: tubular (T). N, nucleus; f, flagella axonemes. (c) Note the ER profiles and its proximity to PVs. (figures a and b, from Abodeely et al. [19]); (figure c, unpublished).

demonstrated that these vesicles periodically open to the cell exterior either via a channel or by fusion with the plasma membrane and take up soluble material before closing again (Figure 4) [19]. The uptake of soluble material from the environment into PV is not selective, which is in contrast to further retrograde transport that allows only certain, yet undefined, substances to rapidly cross over into the proximal ER [19]. It has been demonstrated that three protein complexes are associated with the endocytic machinery in Giardia, showing a discrete localization in the cortical area of trophozoites by fluorescence microscopy [20, 21]. These protein complexes are the clathrin heavy chain (GlCHC), subunits of the AP2 heterotetramer (GlAP2) and Giardia dynamin-related protein (GlDRP) [20, 21]. Recently, a detailed protein interactome of GlCHC revealed all of the conserved factors in addition to a novel protein, a putative clathrin light chain [22]. However, there are no clathrin coated-vesicles in Giardia. It was claimed that the giardial clathrin is organized into static cores surrounded by dynamic interaction partners, which are most likely involved in the regulation of fusion between the plasma

endosomes [17]. The accumulation of gold-labeled macromolecules such as albumin, peroxidase, transferrin and low-density lipoprotein in PVs [15] reinforced this idea. Moreover, the cytochemical localization of acid phosphatase, a classical lysosomal marker, in these vesicles Kattenbach and colleagues [15] led to the suggestion that G. intestinalis presents an endosomallysosomal system that later on during evolution was subdivided into compartments such as early or late endosomes and lysosomes [17]. Besides the degradation function played by the PVs, to date, it is the only known organelle involved in the Giardia endocytic pathway that is capable of accumulating fluid phase and membrane-bound molecules [18]. It was

Figure 2. Peripheral vesicles of G. intestinalis. Transmission electron microscopy (TEM) of vegetative non-encysting (a and b) and encysting (c) parasite. The peripheral vesicles (PV, arrows) are right below the plasma membrane (a–c) and present similar size, shape and location in both forms (a–c). Artificially colored (b). N, nucleus; A, axoneme; F, flagellum; ESV,

encystation-specific vesicle.

90 Current Topics in Giardiasis

Figure 4. Schematic representation of the endocytic network of G. intestinalis. Active proteases reside primarily in the ER, where endocytosed proteins are degraded. PVs contain clathrin and are the site of initial uptake. Membrane fusions between PVs and between PVs and the ER are dynamic. Endocytosed proteins go from PVs to ER by dynamic fusions. ER, endoplasmic reticulum; PV, peripheral vesicles (from Abodeely et al. [19]).

membrane and the PVs in a "kiss-and-flush"-like mechanism [22]. These factors are key components of the clathrin-dependent endocytic machinery in higher eukaryotes and protozoa alike.
