Pathogenicity of amoebiasis
E. histolytica causes intestinal and extraintestinal amoebiasis based on the site of infection. Though most infections do not harm the host (asymptomatic infections), establishment in the colonic mucosa via the Galactose/N-acetyl Galactosamine inhibitable lectin (Gal-lectin) is a pre-requisite for the disease (Chadee et al., 1987). Pathogenic forms of the parasite are known to secrete enzymes that facilitate their invasion into the mucosa and sub-mucosa causing deep-flask shaped ulcers (Figure 3), and in some cases entering the circulation and reaching internal organs like the liver, lungs, skin, etc. The disease in the colon is the most common with acute diarrhoea and dysentery accounting for 90% of the clinical amoebiasis cases (Espinosa-Cantellano and Martínez-Palomo, 2000) and only 1% involve the liver (Haque et al., 2003) (Figure 4).
Asymptomatic colonization
Asymptomatic infections are characterized by the parasite living in perfect harmony within the host. E. histolytica trophozoites have developed elusive tactics to prevent them from being purged from the host.. By modulating signals by intestinal epithelial cells (IEC), trophozoites direct anti-inflammatory host responses leading to a tolerogenic/hyporesponsive immune state favourable to their survival (Kammanadiminti and Chadee, 2006). Furthermore, products secreted by non-pathogenic E. histolytica strains normally disrupt and suppress NFkB signaling and as a result diminish pro-inflammatory responses normally detrimental to the parasite (Artis, 2008). Interleukin 10 (IL-10), an anti-inflammatory cytokine, has been shown to play a significant role in maintaining this hyporesponsive state. On the other hand, a deficiency of IL-10 more often than not predisposes the host to develop the clinical amoebiasis (Hamano et al., 2006)
Intestinal amoebiasis
After an incubation period of 1-4 weeks, the parasite invade the colonic mucosa, producing characteristic ulcerative lesions and a profuse bloody diarrhea (amoebic dysentery). Amoebic invasion through the mucosa and into the submucosa is the hallmark of amoebic colitis. Contact of the trophozoites via the Gal/ GalNAc lectin triggers a signaling cascade initiating the death of the host cell through different mechanisms such as phagocytosis, cytotoxicity and caspase activation instigating the invasive (intestinal and/or extraintestinal) stages of the disease. Other molecules involved in the disease process include: a serine-rich E. histolytica protein (SREHP), amoebapores, and cysteine proteases (Boettner at al., 2002; Mortimer and Chadee, 2010). Activation of damaging inflammatory and non- inflammatory responses following contact of the trophozoites to the gut wall induces a massive neutrophil infiltration across the epithelium into the underlying tissues resulting in weakening of epithelial cells and the mucous layer and allowing trophozoites to invade the intestinal epithelium and disseminating to other bodily sites (Ackers and Mirelman, 2006). The ulcers formed may be generalized involving the whole length of the large intestine or they may be localized in the ileo-caecal or sigmoido-rectal regions. Ulcers are normally disconnected with sizes varying from pin-head size to more than 2.5 cm in diameter. They may be deep or superficial. Base of the deep ulcers is generally formed by the muscularis layer. Nonetheless, superficial ulcers do not extend beyond the muscularis layer. A large number of fatalities results from perforated colons with concomitant peritonitis. E. histolytica also causes amoebomas. These are pseudotumoural lesions, whose formation is associated with necrosis, inflammation and oedema of the mucosa and submucosa of the colon. These granulomatous masses may obstruct the bowel.
Fig. 3. "Flask-shaped" ulcer of invasive intestinal amebiasis (hematoxylin-eosin, original magnification x50).
While the serine rich E. histolytica protein (SREHP) have been shown to promote adhesion of the trophozoites to host cells, cysteine proteases (CP), are known for their virulence in other protozoa as well as in tumour metastasis. Five E. histolytica proteins (EhCP1, 2, 3, 5 and 112) have been identified. All are alleged to play a role in the destruction of host cells, phagocytosis, together with the recruitment of neutrophils and macrophages and the induction of intestinal inflammation (Mortimer and Chadee, 2010). Moreover, EhCP5 has also been shown to perform a variety of functions such as evasion of the host complement and immune system by preventing the activation of the classical complement system via the inactivation of IgG and the degradation of IgA (Laughlin and Temesvari, 2005).
Equally important in the pathogenesis and virulence of E. histolytica is the role of the phagosome-associated proteins. Many have been identified and their function in endocytosis and pathogenesis has been established. Examples include: EhRacA, EhRacG, EhPAK, actin and several Rab7-related GTPases (Laughlin and Temesvari, 2005). Cytokines such as IL-1ß, IL-1a, IL-8 and TNF-α are suspected of aggravating the disease process and driving the immunopathogenesis mechanism (Kammanadiminti et al., 2003). Although neutrophils are known to cause intestinal tissue damage they are nevertheless critical for controlling the infection. Nonetheless, host and/or parasite factors normally play a role in determining whether the parasite is cleared or the disease becomes established (Asgharpour et al., 2005).
Although most intestinal invasions heal following an acute inflammatory response, E. histolytica evades destruction in a modest number of individuals and a chronic state is established. This chronic state is associated with the development of a non-protective adaptive immune response. Human data, in vitro and in vivo models support a paradigm that Th1 responses in the gut clear E. histolytica, while Th2 responses through the production of IL-4 are anti-protective, likely through suppressing IFN-γ. It is not yet clear what signals drive an anti-protective Th2 immune response instead of an effective protective Th1 response towards the infection. Evidence suggesting that genetics, the MHC restriction, nutrition and bacterial flora might play a role in directing the immune response towards E. histolytica infection exists. For example, the MHC class II allele DQBl*0601 was reported to be associated with resistance to E. histolytica (Mortimer and Chadee, 2010). Susceptibility to ALA has been found to be associated with HLA-DR3 and complotype SC01 in some Mexican populations; this association is not seen for amoebic colitis or asymptomatic colonization with E. histolytica (Stanley, 2003).
Extraintestinal amoebiasis
About 5% individuals with intestinal amoebiasis, 1-3 months after the disappearance of the dysenteric attack, develop extraintestinal amoebiasis. Once in the blood, the parasite uses many different strategies to avoid elimination by the host and reaches other sites in the body (such as the liver, lungs, brain, etc). The most common extraintestinal site affected by the parasite is the liver and an Amoebic liver abscess (ALA) is its most common manifestation, predominantly seen in adult males. This chronic stage of ALA is characterized by defective cell-mediated immunity and the suppression of T cells and their defective proliferative responses (Campbell et al., 1999). E. histolytica trophozoites reaching the liver create their unique abscesses, which are well circumscribed regions of cytolysed liver cells, liquefied cells, and cellular debris. The lesions are surrounded by connective tissue enclosing few inflammatory cells and trophozoites. Parenchymal cells adjacent to the lesion are often unaffected. However, lysis of neutrophils by E. histolytica trophozoites might release mediators that lead to the death of liver cells, and extend damage to hepatocytes not in direct contact with the parasite. Studies have shown that in ALA in mice, most hepatocytes die from apoptosis, but necrosis is also present. In ALA from humans, the small numbers of amoebas relative to the size of the abscess suggests that E. histolytica can kill hepatocytes without direct contact (Stanley 2003). From the liver, E. histolytica trophozoites may enter into the general circulation and reach other organs (Figure 4).
Fig. 4. Amoebic Liver abscess. Gross specimen of liver tissue with an abscess (white) that formed due to infection of the organ with Entamoeba histolytica.
Role of genetic characteristics of the infecting strains in the pathogenesis of amoebiasis
The outcome of an infection may depend on several factors among which the genetic characteristics of the specific pathogen have been identified as an important one. Few polymorphic genetic loci have been identified and targeted to aid in the study of the population structure of E. histolytica strains and their possible relationships with the parasite’s virulence and disease outcome (Clark, 2006; Paul et al., 2007). Examples of these genetic markers include protein coding genes (serine – rich E. histolytica protein, [SREHP] and Chitinase) and non-coding DNA (Strain Specific Gene and tRNA gene linked short tandem repeats [STR]) of PCR-amplified genes (Haghighi et al., 2003; Samie et al., 2008). In a study in Bangladesh, the tRNA-linked STR genotyping system has provided evidence that the parasite genome does influence the outcome of infection. tRNA-linked STR genotyping was also behind the recent observation of differences between parasite genotypes in the intestine and the liver abscess of same patients (Ali et al., 2007). Few studies, albeit inconclusive, using the polymorphic SREHP marker have indicated that certain SREHP profiles might be responsible for the presentation of intestinal amoebic symptoms (Ayeh-kumi et al., 2001; Samie et al., 2008). Yet, all studies with SREHP marker did support previous findings of extensive genetic diversity among E. histolytica isolates from the same geographic origin (Ayeh-kumi et al., 2001; Simonishvili et al., 2005; Samie et al., 2008; Tanyuksel et al., 2008). Thus, it seems that the parasite genotype does play a role in the outcome of infection in humans thus linking parasite diversity and virulence. Other approaches, such as SNP identification coupled with microarray-based analysis of gene expression or proteomic comparisons among parasites will be needed to identify the actual genes responsible for these results and to help us understand the mechanism of parasite virulence and pathogenesis (Ali et al., 2008).
Conclusions
Up to date, there are still large gaps in our knowledge of species prevalence rates in different regions of the world particularly in the African continent where very few studies are being conducted using molecular methods. In order to address this limitation, there is need to implement species-specific diagnosis of E. histolytica, E. dispar and E. moshkovskii, particularly in countries where these organisms are endemic. Based on the limited information available to date it appears that molecular and genomic studies are still needed combined to molecular epidemiology studies in order to advance our understanding of amoebiasis. The currently available genome sequence is very useful in better understanding the biology of the parasite, however, E. histolytica strains from Africa still need to have the genome sequenced. Comparative genomics will probably allow the understanding of the pathogenicity of some strains of E. histolytica compared to non-pathogenic strains as well as better understanding of E. dispar in relation to E. histolytica. Further collaborations between scientists from developed countries and those from developing countries is essential in answering questions on the epidemiology, pathogenesis and biochemistry of E. histolytica which is the causing agent of amoebiasis.
