**2. Biology of** *Entamoeba histolytica*

*Entamoeba histolytica* trophozoites (Figure 1) live and multiply indefinitely within the mucosa of the large intestine feeding normally on starches and mucous secretions and interacting metabolically with the host's gut bacteria. However, such trophozoites commonly initiate tissue invasion when they hydrolyze mucosal cells and absorb the predigested products in order to meet their dietary provisions. Filopodia (tiny cytoplasmic extensions) that form from the surface of their trophozoites are believed to play a role in the pathogenicity of certain strains. Examples of functions related to pathogenesis include: endocytosis and/or pinocytosis, exocytosis, tissue penetration, cytotoxic substances release or contact cytolysis of host cells. Other host factors that may also influence the invasiveness of *E. histolytica* are the oxidation-reduction potential and gut contents pH both of which are largely influenced by the overall nutritional state of the host.

Once the parasites invade the intestinal wall, they reach the submucosa and the underlying blood vessels. From there, trophozoites travel in the blood to sites such as the liver, lungs or skin. These parasite forms are now considered to be dead-end course since they cannot leave the host and cause infection in others. Encystation occurs in the intestinal lumen, and cyst formation is complete when four nuclei are present. These infective cysts are passed into the environment in human feces and are resistant to a variety of physical conditions. On occasions, trophozoites may exit in the stool, but they cannot survive outside the human host. The signals leading to encystations or excystation are poorly understood, but findings in the reptilian parasite *Entamoeba invadens* suggest that ligation of a surface galactosebinding lectin on the surface of the parasite might be the one trigger for encystations (Stanley, 2003; Eichinger, 2001). Also, several previous proteomic and transcriptomic studies have shown that a few dozens of Rab genes/proteins are involved in important biological processes, such as stress response, virulence, and pathogenesis, and stage conversion (Picazarri et al., 2008; Chatterjee et al., 2009; Novick and Zerial, 1997; Stenmark, 2009; Nozaki and Nakada-Tsukui, 2006). EhRab11A was reported to be recruited to the cell surface by iron or serum starvation, and was suggested to be involved in encystation (McGugan and Temesvari, 2003). In contrast, EhRab11B is involved in cysteine protease secretion, and its overexpression enhanced the secretion of cysteine protease (Mitra et al., 2007; Nozaki and Nakada-Tsukuia, 2006).

Amoebiasis in the Tropics: Epidemiology and Pathogenesis 205

(Bardwell, 2006) - regulate a number of different cellular processes such as proliferation, and response to a variety of environmental stresses like osmotic stress, heat shock and hypoxia (Junttila, 2008). The existence of MAPK homologues has been documented in certain parasitic protozoa. For instance ERK1 and ERK2 homologues of *Giardia lamblia* have been shown to play a critical role in trophozoite differentiation into cysts (Ellis et al., 2003), Pfmap2, a MAPK homologue in *Plasmodium falciparum* is essential for the completion of the asexual phase of the parasite lifecycle (Dorin-Semblat et al., 2006) and *Leishmania major* MAPK homologues exhibit an increased phosphotransferase activity in response to pH and temperature shift (Morales et al., 2007). On the other hand, *E. histolytica* has been shown to possess a single homologue of a typical MAPK gene (EhMAPK). Activation of EhMAPK in *E. histolytica* has been found to be associated with

stress survival such as heat shock and oxidative stress response (Ghosh et al, 2010).

Fig. 2. Life cycle of *E. histolytica*/*E. dispar*. a) Mature cyst stained with 4% Lugol solution (100× magnification). b) Mature cyst without staining (100×). c) Trophozoite observed with differential interference contrast (DIC) (100×). d) Trophozoites of *E. histolytica* species with phagocyted erythrocytes (DIC 40×). Obtained with permission from Ximenez et al

(2011).

Fig. 1. *Entamoeba histolytica* trophozoites observed under the microscope stain with methylene blue (Observe that the cells did not accept the stain since they were still alive at the time the picture was taken) (Photos by Samie A)

The life cycle of *E. histolytica* is simple and consists of an infective cyst stage (10 to 15 μm in diameter) and a multiplying trophozoite stage (10 to 60 μm in diameter). Like other protozoa, *E. histolytica* appears incapable of de novo purine synthesis. Biochemical analysis has indicated that glutathione is not present. For this reason, *E. histolytica* is different from higher eukaryotes. It also uses pyrophosphate instead of ATP (McLaughlin and Aley, 1985). Mature cysts in the large intestine leave the host in large numbers and remain viable and infective in a moist, cool environment for at least 12 days. In water, cysts can live for up to 30 days. Nonetheless, they are rapidly killed by desiccation, and temperatures below 5°C and above 40°C. Mature cysts are also resistant to chlorine levels normally used to disinfect water. When swallowed, cysts pass through the stomach unharmed. In the small intestine, where conditions are alkaline and as a result of nuclear division, eight motile trophozoites are produced. These motile trophozoites then settle in the large intestine lumen, where they divide by binary fission and feed on host cells, bacteria and food particle (Figure 2). This is the first chance of the parasite making contact with the mucosa.

The organisms' biochemistry and metabolism have been reviewed by McLaughlin and Aley (1985). It has many hydrolytic enzymes, including phosphatases, glycosidases, proteinases, and an RNAse. Major metabolic end products are carbon dioxide, ethanol and acetate. *E. histolytica* is more of a metabolic opportunist which is able to exploit oxygen when it is present in the environment. Glucose is metabolized via the Embden-Meyerhof pathway exclusively, and fructose phosphate is phosphorylated, prior to lysis, by enzymatic reactions unique to *Entamoeba* spp. Pyruvate is converted mostly to ethanol, even in the presence of oxygen, via coenzyme-A, and pyruvate oxidase. Terminal electron transfers are accomplished with ferredoxinlike iron-sulphur proteins, a trait that may contribute to the efficacy of metronidazole in treatment. Similar metabolic traits in *Trichomonas vaginalis* and *Giardia lamblia* also are metronidazole targets. Mitogen Activated Protein Kinases (MAPK) – a group of proline directed serine/threonine kinases

Fig. 1. *Entamoeba histolytica* trophozoites observed under the microscope stain with

the time the picture was taken) (Photos by Samie A)

with the mucosa.

methylene blue (Observe that the cells did not accept the stain since they were still alive at

The life cycle of *E. histolytica* is simple and consists of an infective cyst stage (10 to 15 μm in diameter) and a multiplying trophozoite stage (10 to 60 μm in diameter). Like other protozoa, *E. histolytica* appears incapable of de novo purine synthesis. Biochemical analysis has indicated that glutathione is not present. For this reason, *E. histolytica* is different from higher eukaryotes. It also uses pyrophosphate instead of ATP (McLaughlin and Aley, 1985). Mature cysts in the large intestine leave the host in large numbers and remain viable and infective in a moist, cool environment for at least 12 days. In water, cysts can live for up to 30 days. Nonetheless, they are rapidly killed by desiccation, and temperatures below 5°C and above 40°C. Mature cysts are also resistant to chlorine levels normally used to disinfect water. When swallowed, cysts pass through the stomach unharmed. In the small intestine, where conditions are alkaline and as a result of nuclear division, eight motile trophozoites are produced. These motile trophozoites then settle in the large intestine lumen, where they divide by binary fission and feed on host cells, bacteria and food particle (Figure 2). This is the first chance of the parasite making contact

The organisms' biochemistry and metabolism have been reviewed by McLaughlin and Aley (1985). It has many hydrolytic enzymes, including phosphatases, glycosidases, proteinases, and an RNAse. Major metabolic end products are carbon dioxide, ethanol and acetate. *E. histolytica* is more of a metabolic opportunist which is able to exploit oxygen when it is present in the environment. Glucose is metabolized via the Embden-Meyerhof pathway exclusively, and fructose phosphate is phosphorylated, prior to lysis, by enzymatic reactions unique to *Entamoeba* spp. Pyruvate is converted mostly to ethanol, even in the presence of oxygen, via coenzyme-A, and pyruvate oxidase. Terminal electron transfers are accomplished with ferredoxinlike iron-sulphur proteins, a trait that may contribute to the efficacy of metronidazole in treatment. Similar metabolic traits in *Trichomonas vaginalis* and *Giardia lamblia* also are metronidazole targets. Mitogen Activated Protein Kinases (MAPK) – a group of proline directed serine/threonine kinases (Bardwell, 2006) - regulate a number of different cellular processes such as proliferation, and response to a variety of environmental stresses like osmotic stress, heat shock and hypoxia (Junttila, 2008). The existence of MAPK homologues has been documented in certain parasitic protozoa. For instance ERK1 and ERK2 homologues of *Giardia lamblia* have been shown to play a critical role in trophozoite differentiation into cysts (Ellis et al., 2003), Pfmap2, a MAPK homologue in *Plasmodium falciparum* is essential for the completion of the asexual phase of the parasite lifecycle (Dorin-Semblat et al., 2006) and *Leishmania major* MAPK homologues exhibit an increased phosphotransferase activity in response to pH and temperature shift (Morales et al., 2007). On the other hand, *E. histolytica* has been shown to possess a single homologue of a typical MAPK gene (EhMAPK). Activation of EhMAPK in *E. histolytica* has been found to be associated with stress survival such as heat shock and oxidative stress response (Ghosh et al, 2010).

Fig. 2. Life cycle of *E. histolytica*/*E. dispar*. a) Mature cyst stained with 4% Lugol solution (100× magnification). b) Mature cyst without staining (100×). c) Trophozoite observed with differential interference contrast (DIC) (100×). d) Trophozoites of *E. histolytica* species with phagocyted erythrocytes (DIC 40×). Obtained with permission from Ximenez et al (2011).

Amoebiasis in the Tropics: Epidemiology and Pathogenesis 207

HIV has been noted. In this study, the prevalence of *E. histolytica* in HIV/AIDS patients was 25.3% compared to 18.4% in a control HIV-group (Moran et al., 2005). Other studies in South American countries have shown no obvious association. However, a significant association between high levels of serum anti-*E. histolytica* antibodies and the presence of *E. histolytica* in the stool has been noted in studies from both Vietnam (Blessman et al., 2006) and Africa (Stauffer et al., 2006). In a South African study in the Vhembe district in the northern part of the country, a positive association between *E. histolytica* infection and HIV-positive individuals has been indicated. Among the HIV-positive individuals, those with CD4+ count less than 200 cells/µl, were relatively more likely to be seropositive for *E. histolytica* (Samie et al., 2010). In a Chinese study, a higher seroprevalence of *E. histolytica* infections was also found in HIV-infected patients (Chen et al, 2007). Furthermore, two studies conducted in

Taiwan revealed a positive association as well (Hung et al., 2005; Tsai et al., 2006).

Cuba 1.5% (*E. histolytica/dispar*) Escobedo, A. A. 1999 Bogota, Colombia *13%* (*E. histolytica*) Florez et al., 2003

Venezuela (Zulia state) *10.8%*(*E. histolytica*) Rivero et al., 2009

Northern India *7.7% (E. histolytica)* Prasad et al., 2000

India (Kolkata) *3.6% (E. histolytica)* Mukherjee et al., 2010 Sydney, Australia *3.2% (E. histolytica*/*E. dispar)* Stark et al., 2007

Ethiopia 10.3% (*E. histolytica*) Hailemariam et al., 2004 Dakar, Senegal 5.1% (*E. histolytica*) Gassama et al., 2001 South Africa 12.4% (*E. histolytica*) Samie et al., 2006 Table 1. Global prevalence of *E. histolytica* in HIV-infected and non-infected persons.

Over the past decade, there has been an increasingly reported risk of amebiasis in East Asian countries like Japan, Taiwan and South Korea particularly among men who have sex with men (MSM) probably due to oral-anal sexual contact (Hung et al., 2008; Watanabe et al., 2011). In Japan, *E. histolytica* often occur in institutions of mentally retarded individuals where outbreacks of amebiasis have been described with the prevalence rate and positive serology rate as high as 38.2% and 67.1%, respectively (Nishise et al., 2010) and has been

Uganda 1.4% (*E. histolytica*) Brink et al., 2002

before and after HAART)

contacts (*E. histolytica*)

control (*E. histolytica*)

*5.8%*(*E. histolytica*) Lindo et al., 1998

1.6% (*E. histolytica*) Daryani et al., 2009

Bachur et al., 2008

Moran et al., 2005

Matthys et al., 2011

Hung et al., 2008

Haque et al., 2009

**Country Prevalence of** *Entamoeba species* **Reference** 

Brazil 3.3% and 1% (*E. histolytica/ dispar*

Mexico 25.3% in HIV+ and 18.5% in HIV-

patients)

Bangladesh 2.1% vs. 1.4% in diarrhea and

Tajikistan 25.9% (*E. histolytica/dispar* non HIV)

Taiwan *5.8%* (*E. histolytica* in HIV

San Pedro Sula, Honduras

Mazandaran province,

Iran
