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Entamoeba histolytica is a protozoan parasite that infects humans and causes the disease amebiasis. The spectrum of intestinal amebiasis varies from colonization without symptoms to fulminating diarrhea and intestinal hemorrhage. The dissemination of the parasite via invasion of the intestinal epithelium allows the trophozoites to invade extra-intestinal sites, most usually the liver. Without treatment, the amebic liver abscesses may continue to enlarge and, if ruptured, cause mortality owing to acute peritonitis. Cases of clinical amebiasis have been reported worldwide, in particular in under-developed and developing counties in Africa, South America, the Indian subcontinent, and Mexico. It has been estimated that approximately 500 million individuals are infected with E. histolytica and about 100,000 people die of invasive amebiasis annually, making it the third leading parasitic cause of death, after malaria and schistosomiasis.1 The host-parasite interaction in human amebiasis is very complicated, and different aspects of innate immunity of the human host against the parasite still are unknown. New insights into the pathogenesis of amebic infections have come from development of in vitro and in vivo models of disease, new molecular and genetic approaches, the identification of key factors in E. histolytica pathogenesis, recognition of the mechanisms of evasion from the host’s harmful responses, and detection of crucial elements of the host immune responses both innate and acquired. In this chapter, we discuss the innate immunity of human hosts against the parasite and the most important parasite virulence factors and survival strategies that are implicated in pathogenesis.
Ameba encounter natural “barriers” in both the intestine and systemic circulation after extra-intestinal invasion. Although the exact role of these “natural barriers” and their potential roles are still undefined, in the gut, for example, these innate “barriers” prevent potential pathogens and antigens from gaining access to the underlying epithelium, a process called non-immune exclusion.
Intestinal mucins are highly glycosylated molecules and consist of a core protein (apomucin) joined to oligosaccharides. Mucin glycoproteins line the surface epithelium of respiratory, urogenital, and digestive tracts from the nasal cavity/oropharynx to rectum.2 Member of the mucin family can differ considerably in size. Some are small, whereas others contain several thousands of residues and are among the largest known.3 However, gastrointestinal mucins consist solely of high molecule mass glycoproteins and their size varies from 0.5 × 106 to 25 × 106 Dalton.4 Mucins consist of a peptide backbone containing alternating glycosylated and nonglycosylated domains, with O-linked glycosylated regions comprising 70–80% of the polymer. N-Acetylglucosamine, N-acetylgalactosamine, fucose, and galactose are the 4 primary mucin oligosaccharides.5,6 Mucin oligosaccharide chains are often terminated with sialic acid or sulfate groups, which account for the polyanionic nature of mucins at a neutral pH.5 Gastrointestinal mucins are the first line of host defense against enteric pathogens, including E. histolytica. Binding sites of mucins have been shown to compete with those of underlying epithelium, preventing attachment of pathogens to the intestinal wall. To establish colonization, E. histolytica needs to bind to colonic mucin oligosaccharides via the 170kDa heavy subunit of the parasite’s Gal/GalNAc lectin. The Gal/GalNAc lectin binds to rat and human colonic mucins with a very high affinity.7 The ability of rat and human colonic mucins to inhibit amebic attachment and cytolysis of target epithelial cells demonstrate the protective role of mucin against E. histolytica. In the gerbil model of amebic colitis, it has been shown that goblet cell mucin stores has been depleted before invasion of E. histolytica to colonic epithelium.8 Although the cause of such depletion is still unknown, it is speculated that parasite-derived secretagogues are responsible.9 In addition to the direct protective role of intestinal mucins, intestinal bacterial flora compete for attachment to mucin and may prevent amebic lodgment.10 The protective key role of mucus blanket in natural immunity against E. histolytica infections has been verified both in vitro and in vivo. For example, the mucus–producing colonic cell line, LS174T, inhibits amebic adherence to Chinese hamster ovary (CHO) cells, showing the ability of these mucus-producing cells to compete with attachment sites that are of the utmost importance for E. histolytica colonization.11. Also, the protective role of the mucus layer has been detected in vitro by the finding that E. histolytica trophozoites obliterate epithelial cell monolayers without a mucus barrier more quickly and easily than those protected by a mucus cover.11 Additionally, studies by using the mouse model of intestinal amebiasis showed E. histolytica induced the expression of cyclooxygenase-2 in epithelial cells and macrophages, and the resultant prostaglandins enhance epithelial permeability, mediating neutrophils responses.12
The human complement system is an important early host defense against amebic infection. E. histolytica that disseminates from the bowl through the blood stream is exposed to complement, another component of innate mechanism of host resistance against invading pathogens. Earlier, it had been demonstrated that non-immune sera lysed axenic strains of E. histolytica, implying a role of complement against the parasite in blood circulation.13 Axenic E. histolytica isolates activate both the alternative and classical pathways.14,15 Additionally, this event has been shown to occur in patients with ALA by finding the high concentrations of serum or plasma concentrations of components of the classical (C1q, C4) and alternative (C3, factor B) pathways, regulatory protein factor H, and one of the C3 products of degradation, C3d, in patients with amebic liver abscess.16 It has been shown that the major E. histolytica extracellular proteinase, a 56kDa neutral cysteine proteinase, activates complement by E. histolytica in the fluid phase.17 It is generally believed that both pathogenic E. histolytica and nonpathogenic E. dispar are susceptible to human complement. It was shown that more than 90% of E. histolytica trophozoites were lysed after exposure to the alternative pathway components.18 However, even though human complement-mediated cytolysis of E. histolytica has an effective amebicidal activity in vitro, a considerable number of ameba can survive and escape human complement activity in vivo (see below). Reed et al.19, for the first time, showed that both serum-sensitive and serum-resistant stains of the parasite could activate complement. The increased frequency, rate, and severity of amebic liver abscess (ALA) in complement-depleted hamsters treated with cobra venom factor (CoF) reinforced the fact that complement components may play an important role in innate immunity against amebic infections.20 The Gal/GalNAc lectin molecule of E. histolytica has sequence homology and antigenic cross-reactivity with CD59, a membrane inhibitor of C5b-9 in human erythrocytes, suggesting the E. histolytica adhesin has both molecular mimicry and shared complement-inhibitory functions.21 The Gal/GalNAc lectin bound C8 and C9 and efficiently, preventing membrane attack complex (C5b-9) formation and subsequent cell lysis. A definite role for the Gal/GalNAc lectin was confirmed by abrogation of amebic complement resistance following treatment with a monoclonal antibody to Gal/GalNAc lectin molecule.21
Several factors contribute to the pathogenicity of E. histolytica, and some may still await identification. However, three pathogenic factors of the parasite have been investigated extensively and characterized at molecular levels. These three virulence factors are: the Gal/GalNAc adhesin, mediating adherence to host cells and contributing to amebic resistance to complement, the amoebapores, small peptides that produce pores in target cell membranes, and the cysteine proteinases that play a key role in E. histolytica tissue invasion, evasion of host defenses, and parasite induction of gut inflammation.
The Gal/GalNAc lectin is a novel multifunctional virulence factor of E. histolytica, participating in adherence, cytolysis, invasion, resistance to human complement, and also perhaps encystation.22 Perhaps, the most important part in amebic pathogenesis and pathology is to adhere to the colonic wall. Adhesion of the parasite occurs mainly through the Gal/GalNAc lectin, which binds to exposed terminal Gal/GalNAc residues of target cell glycoproteins.23 Other molecules thought to be involved in part in adhesion of the parasite are: a 220-kDa lectin, a 112-kDa adhesin, and a surface lipophosphoglycan.24
The Gal/GalNAc adhesin is a novel multifunctional protein composed of a heterodimer of heavy (170-kDa), light (35/31 kDa) 23, with a non-covalently-linked intermediate (150-kDa) subunit.24 The 170-kDa heavy subunit (hgl) is a type I transmembrane protein with a small intracellular domain and a carbohydrate recognition domain (CRD) contained in its extracellular domain.25 The 30-kDa light subunit (lgl) is covalently attached to the heavy subunit through disulfide linkages. The light subunit has been shown to have several isoforms 23,26, although the significance of the different isoforms is not clear yet. The intermediate subunit (igl) of the Gal/GalNAc has been cloned and characterized recently, and it has been shown that it lacks a carbohydrate recognition domain (CRD) 24. The Gal/GalNAc lectin mediates adherence of trophozoites to human colonic glycoproteins, human colonic epithelium, human neutrophils, and erythrocytes, and to certain bacteria.27,28 Evidence for the participation of this molecule in the adhesion event of the parasite has been detected by decreased amebic adherence to target cells when the lectin is inhibited by galactose29, by inhibition of adherence with monoclonal antibodies (mAbs) directed against the carbohydrate recognition domain (CRD) of the lectin, and finally by the lack of amebic adherence to Chinese hamster ovary (CHO) cell mutants lacking Gal/GalNAc30. In addition to its role in adherence, the Gal/GalNAc lectin also participates in the cytolytic events, since contact-dependent target cell lysis is reduced in the presence of galactose as well as by a monoclonal antibody against the heavy subunit is able to inhibit cytolysis in part without blocking adherence.31,32 Interestingly, the purified lectin, even at high concentrations, has no cytotoxic effect, suggesting that this protein may be involved in signaling of cytolysis most likely via stimulation of active polymerization.32,33 Furthermore, it has been suggested that this lectin is implicated in amebic resistance to human complement. The adhesin binds to purified c8 and c9 components of complement and prevents formation of the membrane-attack complex (C5b-9).21
Once E. histolytica establishes contact with mammalian cells, a rapid cytolytic event takes place that result in swelling, surface blebbing, and lysis of the target cell and leaving the parasite intact. The similarity of this phenomenon to perforin-mediated lysis of target cells by cytotoxic T lymphocytes34 suggested the possible presence of a channel-forming protein, amoebapore, in E. histolytica.35,36 Amoebapores are a family of small peptides contained in cytoplasmic vesicles in the trophozoites with maximum activity at acidic pH. The amebapore of E. histolytica is a channel-forming peptide of 77 amino acid residues; these proteins have now been purified, sequenced, and the relevant genes have been cloned.37 Three amoebapore isoforms, A, B, and C, at a ratio of 35:10:1, respectively, have been characterized; these peptides showing 35 to 57% deduced amino acids sequence identity and are encoded by a family of three genes.37,38
All of these three peptides have a common six cysteine residues at identical positions and also a histidine residue near the C terminus. Significant similarities have been determined between these peptides in both structural and functional basis and NK-lysin, pore-forming peptide occurring in natural killer (NK) cells and porcine T cells.39 Structural modeling with the use of genetic algorithm, suggests a compact tertiary structure composed of four α-helix bundles stabilized by three disulfide bonds, a structure that is also present in NK-lysin.40
Amebapores are now believed that aggregate through the arrangement of their amphipathic α-helices and finally they form a channel within the plasma membrane through which water, ions, and other small molecules pass and thus the target cell lyses. Amebapores have cytolytic activity against several human cell lines including human Jurkat T cells;40 these peptides also are effective in forming pores in gram-positive bacterial membrane.38,40 However, causing damage to thick gram-negative bacterial shield requires high concentrations of amebapore or removal of thick wall in advance with lysosome.40
A homologous peptide to E. histolytica has been determined in non-pathogenic E. dispar trophozoites.41 This peptide has significant shared structural and functional properties to that of E. histolytica, including highest activity at acidic pH, presence in cytoplasmic granules, and a 95% identity of primary structures. However, irrespective of these similarities, the activity of E. dispar amebapore is 60% lower than that from E. histolytica. It has been suggested that this lower activity may be related to shortened putative amino-terminal α-helix of the E. dispar porin.41 Irrespective of all advances in the molecular biology and biochemistry of amebapores, their exact roles in E. histolytica cytolytic events have not been yet known. Amebapores are not continuingly secreted from viable trophozoites in vitro,42 suggesting that these peptides may be secreted upon stimulus, including target cell contact, and play a role in lysis of host cells during invasion.39 However, more recently, transcriptional silencing of the gene encoding amoebapore isoform A (ap-a) was detected in E. histolytica when trophozoites were transfected with a hybrid plasmid construct containing the ap-a gene flanked by the upstream and downstream segments of the original Ehap-a gene.43 According to invitro investigations, it also proposed that trophozoites without amoebopore A were unable to produce pathological effects as well as the to adhere to lyse bacteria.43 In vivo, it has been demonstrated that the G3 strain of E. histolytica, an avirulent strain of the parasite derived from virulent strain HM1:IMSS that lacks amoebapore A, had behaviors comparable to avirulent Rahman avirulent strain of the parasite and it may be consider as a vaccine candidate in the future.44 The presence of pore-forming activity in non-pathogenic E. dispar suggests that the primary role of these peptides is likely to destroy phagocyted bacteria, the main source of Entamoeba sp. in the gut39,45.
Cysteine proteinases occur in a wide range of organism including bacteria, plants, invertebrates, and vertebrates.46 In mammals, these enzymes are involved in protein turnover within lysosomes. In addition, extracellular cysteine proteases have been implicated in various physiological and pathophysiological processes, including tumor invasion and metastasis.47 In protozoan parasites, different types of cysteine proteases have been characterized widely and shown to have a variety of functions (including evasion from the host immune responses and roles in developmental cycle of the parasite) and cytopathic effects (including induction of apoptosis).48
Several cysteine proteinases with molecular weight between 16 to 96 kDa have been observed in E. histolytica extracts. Previously, investigators purified two distinct cysteine proteinases from E. histolytica and designated them as amoebapain (now know as ehcp3) 49 and histolysin (now known as ehcp1).50 However, three different main isoforms of cysteine proteinases are produced in E. histolytica of about 30kDa (27-to 30-kDa) and are encoded by a family of more than 30 genes. 51,52 E. histolytica trophozoites show strong proteolytic activity and release large amounts of cysteine proteinases into growth media.50,53 Previous studies showed that E. histolytica secreted 10- to 1,000-fold more cysteine proteinase activity than did E. dispar isolates.54 It appears that ehcp5 is the only cysteine proteinase knows that is present on ameba surface.55 In addition to ehc5, it has been suggested that ehp1 is also important in E. histolytica-induced pathogenesis.51 The non-pathogenic E. dispar has four cysteine proteinase genes (edcp2, edcp3, edcp4, edcp6), with the highest expression belonging to edcp3. Using ehcp sequences as cross-hybridizing probes, it was revealed that functional orthologs corresponding to ehcp1 and ehcp5 are absent in E. dispar.56,57 Apparently, only two of E. dispar cysteine proteinase genes are expressed,56 and this may explain why E. dispar has low levels of cysteine proteinase activity. Additionally, E. dispar lacks several of the most important E. histolytica cysteine proteinases, and this again might explain in part its non-invasive nature.
Cysteine proteinases have been shown to be involved in host invasion by some other parasites including Trypanosoma cruzi, Plasmodium falciparum, Cryptosporidium parvum, and Toxoplasma gondii. 58 Cysteine proteinases have been implicated in the cytopathic effects of E. histolytic upon target cells, resulting in the release of adherent cells from monolayers, presumably by degradation of the components of the extra-cellular matrix, including fibronectin, laminin, and collagens as well as an extra-cellular matrix from vascular smooth muscles.59 These cytopathic effects correspond with the amount of CP activity secreted and can be inhibited by some specific peptide inhibitors.60 For example, using exogenous laminin or cysteine proteinase-specific inhibitor L-trans-epoxysuccinyl-leucylamido- (4-guanidino) butane (E-64) to neutralize cysteine proteinase reduced the formation of ALA significantly in the severe combined immunodeficient (SCID) mouse.61 In the recent years, new generations of cysteine proteinase-specific inhibitors have been introduced which have been effective against E. histolytica CPs, 60 suggesting the potential of these specific inhibitors as novel antiamebic chemotherapy.62
Interaction of E. histolytica trophozoites with diverse external stimuli, such as exposure to extracellular matrix (ECM) proteins, appears to activate signaling pathways through G-protein-coupled receptors. In vitro studies indicate that the parasite releases proteases and/or toxin-like molecules when in contact with several types of cultured cells.56 (Meza, 2000). Adhesion to fibronectin (FN) and its proteolytic fragments is known to induce a wide variety of cellular responses, such as expression of genes encoding proteases, secretion of proteins, activation of lymphocytes and differentiation of neural, endothelial, myoblastic and many other types of cells.63,64; an early reaction to fibronectin binding is formation of actin adherence plates and focal contact in trophozoites. It has been shown that FN action is mainly dependent on the influx of external Ca2+. 65 It has been shown that the protein kinase C (PKC) pathways are activated in amebas by information transduced as a result of trophozoite binding to FN.66 Adherence of E. histolytica trophozoites to another extracellular component, collagen, is a known stimulus for parasite activation, leading to subsequent tissue destruction and invasion. In vitro interaction of E. histolytica with collagen induces intracellular formation and release of electron-dense granules (EDGs) and stimulation of collagenolytic activity.67 Additionally, results of one study showed that tyrosine phosphorylation is involved in collagen signaling in amoebas and that pp125FAK and p42MAPK homologs may play an active role in turning on the genetic program that enables the parasite to invade its host.68 There is also evidence suggesting that adhesion to collagen and activation of EDGs secretion are integrin-dependent events and the involvement of actin, vimentin, and tubulin in restructuring cytoskeleton during EDGs secretion are evident. 69 More recent experimental studies attempting to identify and characterize the gene(s) that are upregulated by the human collagen type I and Ca2+ interactions showed that interaction of E. histolytica with human collagen type I and Ca2+ triggers the transcriptional activation of at least two important genes responsible for pathogenesis of amebiasis.70 It was demonstrated that there were similarities between mechanisms of phagocytosis of bacteria and erythrocytes by ameba and macrophages, support the idea of coincidental selection of amebic genes encoding proteins that mediate destruction of host cells.71 Rho family GTPases regulates many features of the cell behavior in eukaryotic cells and the members of the rac superfamily have critical role in regulating a wide range of cellular processes such as cellular growth, differentiation, vesicle transport, nuclear transport, and actin cytoskeleton regulation. 72 Genes for these components of signaling pathway have been identified in E. histolytica.73 More recently, a family of over 80 putative transmembrane kinases (TMKs) has been recognized in E. histolytica.74 In addition to their role in antigenic variations, it seems that they are involved in signal transduction via sensing the E. histolytica ambient.
Amebic granules contain copious amounts of tissue-destructive activities, such as hydrolytic enzymes and strong cysteine proteinases, whose secretion contributes to the damage of host cells and tissues. These proteases, mainly cysteine proteinases, can escape the antibody-mediated humoral immune responses by degrading IgA and IgG antibodies. It has been well documented that human serum and secretory IgA was degraded completely when exposed to viable axenic trophozoite of E. histolytica (HM1:IMSS strain), parasite lysates, and medium that had been conditioned by incubation with viable trophozoites.75 This phenomenon could be more important for ameba since E. histolytica trophozoites must conquer the destructive act of secretory IgA (sIgA) and the serum IgG antibodies during intestinal colonization and extra-intestinal dissemination.
Interaction of E. histolytica trophozoites with sera from patients with invasive amebiasis results in initial clustering of bound antibodies into small patches, followed by a rapid mobilization of these patches to the posterior pole of the cell, where the uroid is formed.76 It is believed that the uroid, the posterior appendix that forms during the movement of trophozoite, plays a role in the escape of ameba from the host immune response. In one study, it was shown that myosin II is of the utmost importance in capping and formation of uroid in E. histolytica parasite, in such a way that myosin II was three times more concentrated within the uroid compared with the rest of the cell, suggesting that the release of caps may depend upon mechanical contraction driven by myosin II activity.77 This results in the remarkable ability of E. histolytica to rapidly regenerate substantial amounts of plasma membrane. The properties of surface receptor redistribution (capping), liberation of caps, and plasma membrane regeneration, may contribute to the survival of the parasite in the host during infection.
Tissue invasion by E. histolytica has been associated with suppression of cell-mediated immunity. It has been known that E. histolytica exerts different modulatory effects on macrophages, and T cells especially T helper type 1(Th1).78 T cells are important sources of macrophages-activating cytokines, and can be directly cytotoxic to amoeba, although the mechanisms involved are unknown. There is some evidence that amebic infections are associated with T cell modulation. Reduced delayed-type hypersensitivity reaction during the acute phase of the disease is one index of functional suppression of T cell during human amebiasis.79 Similarly, in the mice models, intestinal amebiasis has been associated with a cyclic suppression in immune responses, and it has been proposed that these alterations, observed at the cellular level, might facilitate invasion of the host by the parasite.80 The cytotoxic ability of macrophages and their potential as antigen-presenting and cytokines-releasing elements are reduced during the acute phase of ALA. Exposure of human mononuclear phagocytes to the monocyte locomotion-inhibitory factor produced by E. histolytica led to a swift increase in the intracellular concentration of adenosine 3':5' cyclic monophosphate (cAMP) and inhibits the respiratory burst in human macrophages.81 This study suggests that like other leukotactic inhibitors, the monocyte locomotion-inhibitory factor produced by E. histolytica operates through modulations of intracellular cAMP. In addition, the parasite exerts suppression that appears to be a local event mediated by direct exposure of macrophages to E. histolytica directly or its by-products. For examples, the treatment of murine macrophages with amebic antigens in vitro reduces the production of molecules associated to the I region of the MHC induced by INF-γ.82 This study also showed that E. histolytica subverted critical macrophage accessory function in part via prostaglandin E2 (PGE2) biosynthesis. Similarly, amebic liver abscess-derived macrophages produced low basal levels of TNF in response to stimulation with lipopolysaccharide (LPS), whereas peritoneal and spleen macrophages as well as Kupffer cells from infected animals did not release TNF constituently in vitro.83
It has been shown that ameba develop complement resistance after repeated exposure to active human serum.84 In this study, it also was shown that susceptibility to complement-dependent lysis was regained 6 weeks after serum treatments were terminated, suggesting that resistance to lysis was an acquired rather than a genetic property. It was also shown that both complement resistant and complement sensitive strains of E. histolytica could activate complement but only the latter could survive.19 It has been suggested that complement resistance is necessary for E. histolytica tissue invasion. Braga et al. 21 showed that the Gal/lectin molecule had sequence homology and antigenic cross reactivity with CD59, a membrane inhibitor of C5b-9 in human erythrocytes, suggesting that the Gal/lectin also has complement-inhibitory functions. It seems that Gal/lectin bound C8 and C9, preventing formation of the membrane attack complex and subsequent cell lysis.21 Recently, it has been shown that different strains of the mouse model of intestinal amebiasis and also genetically deficient mice for IL-12, IFN-gamma, or inducible NO synthase are resistance against intracecal inoculation of E. histolytica.85 This study also demonstrated the important role of host immune responses to resistance to infection as well as the fact that depletion of CD4+ cells significantly diminished both parasite burden and host’s inflammatory responses.