6. Encystment

areas exist between the two nuclear membranes that become parallel but are distinct from the diaphragms found in nuclear envelopes of the eukaryotic cells [33]. Interestingly, the parasite pore complex distribution and clustering is different in each nucleus. Giardia nuclei are not identical; they seem to be either in different phases of chromosome condensation or they have different metabolic activity. Dividing nuclei displayed very few pore complexes, which is a characteristic of low metabolic activity and/or low nucleus-cytoplasm transport. The pore complexes in G. intestinalis are very uniform in size and shape, and they contain annular

Figure 5. Nuclei of G. intestinalis. Transmission electron microscopy (a, c and d) and freeze-fractured (figure b) images in non-encysting cells (a and b) and under process of encystation (c and d). (a) Both nuclei are similar in size; basal bodies and axonemes are just between the nuclei. (b) Pore complexes (arrowheads) similar in size and shape, with annular substructures. (c) In early stages of encystation (10 h) a nascent ESV (arrow) is close to nuclear envelope. (d) After 21 h of encystation the ESVs are closer to the peripheral vesicles (asterisks) and plasma membrane. N, nuclei; VD, ventral disc; ESV, encystation specific vesicle. (figure a: from Benchimol [35]; figure b: from Benchimol [34]; figures c and d: Midlej V,

The parasite mitosis is not similar to other organisms, presenting different characteristics: (1) the nuclear envelope does not fragment completely during mitosis, leaving open places on the

substructures (Figure 5b) that are similar to those of higher eukaryotic cells [33].

De Souza W and Benchimol, unpublished).

94 Current Topics in Giardiasis

The encystment (or encystation) is the given name for the parasite differentiation process of a trophozoite into a cyst (Figure 6). This process consists of several events and occurs in response to environmental or chemical stimuli. The chemical stimulus is a set of an alkaline pH, an increase of bile concentration and the presence of lactic acid released by bacteria that live in the gut [37]. The encystation process is a key for the parasite virulence mechanism and is responsible for the change to a resistant form that can survive in the outside environment for subsequent infection of a new host. This process also promotes the parasite immune evasion and is target to vaccine and drugs development [38, 39].

The encystation process is characterized by a gradual transformation of a flagellated trophozoite—which looks like a cut half pear—into a different structure called the cyst (Figure 6). The trophozoites lose their abilities to adhere, and there is a folding of the ventral disc, followed by its fragmentation [40]. The cell becomes rounded, internalizes the flagella as in an endocytic process and finally a filamentous layer involving the parasite creating the cyst wall (CW). Its superficial filaments connect cyst clusters [40]. Two layers form the CW: a filamentous layer and a membranous layer [41]. Biochemical analyses have focused on the filamentous layer, which is composed by 57% of proteins and 43% of carbohydrates [42].

The main protein components are the cyst wall proteins 1, 2 and 3 (CWPs 1, 2 and 3) and the HCNCp that belongs to a new class of Giardia's proteins, known as cysteine-rich protein, differs from the variant surface proteins (VSP) [29]. The β-1,3-N-acetyl-D-galactosamine polymer (GalNAc) makes up almost 86% of the carbohydrates that compose the filaments layer of CW [42]. The GalNAc polymer forms the CW fibrils that are covered by protein clusters, such as the CWPs [43].

Figure 6. Encystation process of G. intestinalis. (a) Scheme of changes from trophozoite to a rounded oval cyst. (b) Scanning electron microscopy of encystation process showing the cell differentiation: the trophozoite internalizes the flagella, becoming oval. During encystation, the caudal flagella form a tail. At the end, the mature cyst presents a cyst wall and flagella are not seen anymore (unpublished).

#### 6.1. Encystation vesicles

#### 6.1.1. Encystation-specific vesicles (ESVs)

Before the formation of CW, in the beginning of the encystation process, large 1-μm vesicles known as encystation specific vesicles (ESVs) appear (Figures 2c, 5d, 7–10) [44]. The protein content of the ESVs is basically CWPs 1–3 (Figures 7a and b, 10) that originate in the endoplasmic reticulum; afterwards, the encystation vesicles emerge from endoplasmic reticulum points (Figures 7c and 9a) [45]. This mechanism is not fully understood; however, the available data points to two hypotheses: (1) the CW material concentrates in a specialized endoplasmic reticulum sub-compartment, and afterwards, a lateral segregation occurs [46] and/or (2) the CWPs transport to the ESVs through vesicles containing COPII followed by a homotypic fusion [47].

Figure 7. Encystation-specific vesicles (ESVs) during differentiation of G. intestinalis by confocal microscopy (a and b) and transmission electron microscopy (TEM) (c). (a and b) Immunofluorescence of the parasite induced to encyst in vitro for 21 h and labeled with an anti-CWP1 antibody against the cyst wall. In (c) ESVs are electron-dense, membrane-bounded vesicles. Note that some ESVs are close to the nuclei and endoplasmic reticulum (arrows). N, nuclei; A, axonemes (unpublished).

6.1. Encystation vesicles

96 Current Topics in Giardiasis

fusion [47].

6.1.1. Encystation-specific vesicles (ESVs)

and flagella are not seen anymore (unpublished).

Before the formation of CW, in the beginning of the encystation process, large 1-μm vesicles known as encystation specific vesicles (ESVs) appear (Figures 2c, 5d, 7–10) [44]. The protein content of the ESVs is basically CWPs 1–3 (Figures 7a and b, 10) that originate in the endoplasmic reticulum; afterwards, the encystation vesicles emerge from endoplasmic reticulum points (Figures 7c and 9a) [45]. This mechanism is not fully understood; however, the available data points to two hypotheses: (1) the CW material concentrates in a specialized endoplasmic reticulum sub-compartment, and afterwards, a lateral segregation occurs [46] and/or (2) the CWPs transport to the ESVs through vesicles containing COPII followed by a homotypic

Figure 6. Encystation process of G. intestinalis. (a) Scheme of changes from trophozoite to a rounded oval cyst. (b) Scanning electron microscopy of encystation process showing the cell differentiation: the trophozoite internalizes the flagella, becoming oval. During encystation, the caudal flagella form a tail. At the end, the mature cyst presents a cyst wall

> The ESVs maturation is less controversial: about 15–24 h post-encystment induction, before the CWP secretion, the ESVs recruit sequentially membrane peripheral proteins [48]. Thus, the ESVs and their content enter in a maturation way in which the CWPs are post-translationally modified. The presence of the protein disulfide isomerase 2 (PDI2) in ESVs indicates a posttranslational mechanism [49] as well the CWP2 C terminal region cleavage by a specific encystation protease [50] and by the phosphorylation of newly synthesized CWPs [51].

Figure 8. Three-dimensional reconstruction of encysting G. intestinalis. (a–f) Dual-beam microscopy and 3D reconstruction of 21-h encysted parasites. Seven parasites were reconstructed; ESVs are yellow (arrows) (Figs. a-b) and are distributed nearby the nuclei colored by light blue (N) (Figs. e–f). Cell membranes are in different colors (big contours) (unpublished).

#### 6.1.2. Encystation carbohydrate-positive vesicles (ECVs)

For a long time, the understanding of how glycopolymers are transported to build the sugar portion of the CW remained an open question. This was mainly due to the lack of a marker that could track the carbohydrate portion of Giardia's CW with strong specificity [52]. Some researchers used the Dolichos biflorus agglutinin (DBA) lectin, which has specificity to the GalNAc glycopolymer, to label the cyst wall of other parasites such as Toxoplasma gondii [53, 54]. Midlej and collaborators [55] used the DBA lectin as a tool to track the sugar moieties of G. intestinalis CW and were able to identify the encystation carbohydrate-positive vesicles

Figure 9. TEM images of G. intestinalis in process of encystation. (a) Immunolabeling with CWP1 antibody. Note the presence of two types of vesicles: electron dense (ESVs) and electron lucent (ECVs) (arrows). The ESVs (inset), which are juxtaposed to the ECVs (asterisks), present an intense labeling with anti-CWP1, whereas the ECVs present no labeling. (b) Cytochemistry for carbohydrates in encysted parasites: cell membranes, glycogen granules, the peripheral lumen and contents of the ECVs show a positive reaction (arrows), whereas the ESVs are negative (from Midlej et al. [55]).

6.1.2. Encystation carbohydrate-positive vesicles (ECVs)

98 Current Topics in Giardiasis

For a long time, the understanding of how glycopolymers are transported to build the sugar portion of the CW remained an open question. This was mainly due to the lack of a marker that could track the carbohydrate portion of Giardia's CW with strong specificity [52]. Some researchers used the Dolichos biflorus agglutinin (DBA) lectin, which has specificity to the GalNAc glycopolymer, to label the cyst wall of other parasites such as Toxoplasma gondii [53, 54]. Midlej and collaborators [55] used the DBA lectin as a tool to track the sugar moieties of G. intestinalis CW and were able to identify the encystation carbohydrate-positive vesicles

Figure 8. Three-dimensional reconstruction of encysting G. intestinalis. (a–f) Dual-beam microscopy and 3D reconstruction of 21-h encysted parasites. Seven parasites were reconstructed; ESVs are yellow (arrows) (Figs. a-b) and are distributed nearby the nuclei colored by light blue (N) (Figs. e–f). Cell membranes are in different colors (big contours) (unpublished).

> (ECVs) (Figures 9 and 10). The ECVs are 0.2–2 μm membrane-bounded organelles (Figure 9). By electron microscopy, they are electron-lucent, whereas ESVs are electron dense (Figure 9). Moreover, the ECVs do not react with antibodies against CWPs (Figure 10) [55]. These vesicles are only in those encysting cells and are involved in the Giardia's CW biogenesis. The origin of ECVs seems to be related to the rough endoplasmic reticulum, because a budding vesicle was detected from this organelle in a similar way to what happens with the ESVs [56]. Thus, both

Figure 10. Immunolocalization of ECVs in encysting Giardia. DBA lectin was used to track the ECVs by immunofluorescence with confocal microscopy (a–c) and by cryo-immunogold in TEM microscopy (d and e). The ESVs were labeled with an anti-CWP1 antibody (a, c, d). The ESVs are red (a), while the ECVs are green (b). Note that the ECVs do not colocalized with the ESVs (c). The nuclei are blue. (d and e) Cryo-immunogold labeling with an anti-CWP1 antibody (gold with 5 nm) and the DBA lectin (gold with 10 nm). ECVs show a specific labeling for DBA (arrow)(Figs. c-e), whereas the ESVs (e) are with the anti-CWP1 antibody, with no labeling for the DBA lectin (inset). In the inset of figure e, an intense labeling for DBA is seen in ECV juxtaposed to the ESV (from Midlej et al. [55]).

secretion products are synthesized in the endoplasmic reticulum, budded together and are later separated and transported to the protozoan periphery to be secreted via exocytosis [55].

#### 7. Mitosomes

Mitosomes are organelles described by Tovar and collaborators [57]. This name means "crypton" and was used to indicate it as reduced mitochondria. It is part of the mitochondriarelated organelles as the hydrogenosomes found in Trichomonas [58]. Although the mitosomes are related to mitochondria, it lacks several mitochondrial characteristics and functions, such as ATP synthesis, the citric acid cycle, oxidative phosphorylation, heme biosynthesis, presence of DNA, lipid metabolism and the amino acid and urea cycles [59]. On the other hand, mitosomes present mitochondrial characteristics, such as biosynthesis of Fe-S clusters, presence of a TOM and TIM protein family transport machinery and a double membrane (Figure 11a–c) [60].

The mitosomes are small organelles, 200 nm in size, distributed over the cytoplasm, although some of them are placed between the flagellar axonemes. Because of that, they are divided into two distinct groups: the peripheral and central mitosomes (Figure 11d and e), which are dispersed in the cell and between nuclei, respectively [57]. The presence of an iron-sulfur complex (IscS and IscU proteins) makes its identification and characterization easier [61]. Mitosomes are also present, besides the IscS and IscU proteins, chaperones, such as Cnp60 and HSP70 [62]. During the encystation process, the mitosomes change their behavior, modulating Cpn60 and HSP70, and also alter their shape (Figure 11d and e) [62].

secretion products are synthesized in the endoplasmic reticulum, budded together and are later separated and transported to the protozoan periphery to be secreted via exocytosis [55].

labeling for DBA is seen in ECV juxtaposed to the ESV (from Midlej et al. [55]).

Figure 10. Immunolocalization of ECVs in encysting Giardia. DBA lectin was used to track the ECVs by immunofluorescence with confocal microscopy (a–c) and by cryo-immunogold in TEM microscopy (d and e). The ESVs were labeled with an anti-CWP1 antibody (a, c, d). The ESVs are red (a), while the ECVs are green (b). Note that the ECVs do not colocalized with the ESVs (c). The nuclei are blue. (d and e) Cryo-immunogold labeling with an anti-CWP1 antibody (gold with 5 nm) and the DBA lectin (gold with 10 nm). ECVs show a specific labeling for DBA (arrow)(Figs. c-e), whereas the ESVs (e) are with the anti-CWP1 antibody, with no labeling for the DBA lectin (inset). In the inset of figure e, an intense

Mitosomes are organelles described by Tovar and collaborators [57]. This name means "crypton" and was used to indicate it as reduced mitochondria. It is part of the mitochondriarelated organelles as the hydrogenosomes found in Trichomonas [58]. Although the mitosomes are related to mitochondria, it lacks several mitochondrial characteristics and functions, such as ATP synthesis, the citric acid cycle, oxidative phosphorylation, heme biosynthesis, presence of DNA, lipid metabolism and the amino acid and urea cycles [59]. On the other hand, mitosomes present mitochondrial characteristics, such as biosynthesis of Fe-S clusters,

7. Mitosomes

100 Current Topics in Giardiasis

Figure 11. Mitosomes of G. intestinalis. Electron tomography (figures a–c) and super-resolution-structured illumination microscopy (SR-SIM) (figures d and e) of peripheral and central mitosomes. (a–c) Mitosomes in a non-encysted parasite are identified by their double-bound membrane (black arrows). Some mitosomes are elongated or ovoid organelles. (b) Three-dimensional reconstruction of mitosomes in green. (c) After reconstruction, the 3D model was placed on electron tomography micrograph. VD, ventral disc; M, mitosome; Mt., microtubules. (d and e) SR-SIM, vegetative cell (d) and cyst (e). Mitosomes are labeled using an antibody anti-IscU (green fluorescence). The super-resolution microscopy revealed that the central mitosome (white arrow) is seen as a unique tiny organelle between the nuclei (d). The spots of the central mitosome are better visualized in the inset, presenting a different axis angle (d). (e) Cyst mitosomes observed by SR-SIM. Nuclei are labeled with DAPI (blue) (from Midlej et al. [62]).

Thus, the current knowledge concerning mitosomes is still limited. There are a number of unanswered questions related to the biology of this organelle and its proteins as well as related to the importance of mitosomes in the parasite life cycle.
