Abstract
Giardia intestinalis is a protozoan that colonizes the small intestine of virtually all mammals, adhering to the mucosal epithelial cells. It is a cosmopolitan parasite and agent of giardiasis, which can lead to human diarrheal diseases. The Giardia life cycle presents two forms—the trophozoite and the cyst—which are responsible for infection and transmission, respectively. This cell has been considered an excellent model for evolutionary studies, even though there are controversial hypotheses as to whether this parasite is an early eukaryote or not. G. intestinalis has a unique and very basic endomembrane system. The trophozoite gathers a very small pack of membrane-bounded structures: nuclei, endoplasmic reticulum (ER), peripheral vesicles (PV) and mitosomes. These organelles are involved in many functions from regulatory aspects in gene expression as well as membrane traffic events. Two functional nuclei are observed in the parasite; they are always located symmetrically in the anterior region of the trophozoite. The ER and PV commonly share and accumulate functions in the secretory pathway, they are responsible for endocytosis and digestion processes. The mitosome is a mitochondria-related organelle that does not produce ATP and lacks several mitochondrial characteristics. During the parasite differentiation into cyst, different types of vesicles appear into the cell body: the encystation specific vesicles (ESVs) and the encystation carbohydrate-positive vesicles (ECVs). These vesicles work together to form the parasite’s cyst wall in order to ensure that the cell reaches the cyst stage. Interestingly, Giardia does not present a morphologically recognized Golgi apparatus. It has been claimed that during the encystation process, the ESVs could represent a Golgi-like structure, because this organelle presents some characteristics of that high eukaryotic Golgi apparatus. In this book chapter, we highlight the G. intestinalis endomembrane system, emphasizing their morphology, proteins involved in its organization as well as their functional role.
Keywords
- parasite
- morphology
- giardiasis
- ultrastructure
1. Introduction
1.1. Giardia and giardiasis
The trophozoite of
2. Endomembrane system
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 environment [10, 11].
Although
The membrane system of
Below, we will discuss each of the membrane-bounded structures that compose the endomembrane system of
3. Peripheral vesicles
4. Endoplasmic reticulum
Although electron microscopy revealed similar structures to endoplasmic reticulum (ER), there is still a controversy concerning the real presence of this organelle in
Although
It was proposed that the
5. Nuclear envelope
One of the most intriguing features of
An inner and an outer membrane compose the nuclear envelope of higher eukaryote cells. The outer membrane is continuous with the ER membrane, which presents ribosomes engaged in protein synthesis. The inner nuclear membrane contains, in addition to the trilaminar membrane, filamentous proteins that form the nuclear lamina, which provides structural support for this membrane. The nuclear envelope of all eukaryotes is perforated by elaborated structures known as nuclear pore complexes [31].
The
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 nuclei poles. This type of division is named semi-open mitosis, because only the nuclear poles are open. The spindle microtubules penetrate into the nuclei by these open poles. (2) Each nucleus moves to the central portion of the parasite, and one of them is located in the dorsal region and another in the ventral region and (3) the spindle is observed in the telophase [34]. Moreover, the parasite does not synchronize the nuclei division, and thus it is possible to find cells with three or four nuclei [35]. During the encystation process, the parasite mitosis still occurs; this is similar to what happens in the trophozoite vegetative form [36]. The nuclear division starts in the initial stages of encystation process through a semi-open mitosis. Bridges that originate by the nuclear membrane fusion connect the parental daughter nuclei. This interconnection between the nuclei remains intact while the parasite is in the cyst form; this is a characteristic of this stage in the
Encysting cells show intranuclear inclusions that are morphologically similar to the ESVs and the ER membranes (Figure 5c and d), which may be a result of nuclear envelope folding. The presence of these inclusions could indicate intense ER activity since it forms from the outer nuclear membrane [33].
6. Encystment
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
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].
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 post-translational 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].
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
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 mitochondria-related organelles as the hydrogenosomes found in
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].
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.
8. Golgi complex
There is still controversy regarding the presence of a Golgi complex in
Some groups proposed a similarity between the ESVs and the Golgi complex [47, 52, 63–65] supported by: (1) COPI and COPII association with the ESVs [47]; (2) the ESVs are sensitive for Brefeldin A, a drug known to inhibits the anterograde Golgi cisternae movement [63]; (3) the ESVs dependence of GTPases Sar1 and Arf1 for biogenesis and maturation, respectively [64]. However, the ESVs present some characteristics that do not fit with those presented by a classical Golgi: (1) the ESVs appear only during the encystation process; (2) no classical Golgi markers such as GM130, galactosyl transferases or the trans-Golgi network marker Rab6 are present in the parasite; (3) the ESVs do not present morphological characteristics that define this organelle as a Golgi, in accordance with parameters that have been well defined for many years. This is considered a strong argument for the absence of a typical Golgi in
9. Final remarks
The endomembrane system of
There are several questions to be answered regarding the biology of Giardia—for example, the right pathway of endocytic and exocytic materials, the formation of the cyst wall, each protein segregation and polymerization in the formation of the cyst wall, the glycosylation phenomena of secreted proteins, the role of each nucleus in the whole process of the Giardia life cycle, among others.
Acknowledgments
This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Programa de Núcleos de Excelência (PRONEX).
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