Virulence and virulence factors in Entamoeba histolytica, the agent of human amoebiasis

Microbes and Infection 14 (2012) 1428e1441 www.elsevier.com/locate/micinf

Virulence and virulence factors in Entamoeba histolytica, the agent of human amoebiasis Daniela M. Faust a,b, Nancy Guillen a,b,* a

Institut Pasteur, Cell Biology of Parasitism Unit, 75724 Paris Cedex 15, France b INSERM U786, 75724 Paris Cedex 15, France Received 8 February 2012; accepted 28 May 2012 Available online 16 June 2012

Abstract Human infections with Entamoeba histolytica sporadically become pathogenic, unknown triggers converting the parasite to its invasive phenotype. Parasite virulence results from complex hosteparasite interactions implicating multiple amoebic and host factors, eliciting host defence responses and parasite resistance to stress caused by the host reactions and changing environments during tissue invasion. Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Amoebiasis; Entamoeba; Parasiteehost interactions; Stress response; Virulence

1. Introduction Parasites are organisms that live and multiply taking resources from others, while causing variable degrees of damage to the latter. A number of species have evolved to live inside a host. In general, well-adapted parasites do not threaten host survival, implying a delicate balance between host damage and their own profit that allows progression through the life cycle and maintenance of infectivity and transmission, thus the permanence of the species. Parasites profiting from host nutritional resources without causing obvious damage are commensal organisms. In particular circumstances they are or

Abbreviations: ALA, amoebic liver abscess; AP, amoebapore; CP, cysteine protease; CRD, cysteine-rich domain; ECM, extracellular matrix; Gal/GalNAc, galactose- and N-acetylgalactosamine-inhibitable; GPI, glycosylphosphatidylinositol; GS, glucose starvation; Hsp, heat shock protein; IFN, interferon; IL, interleukin; KERP, lysine- and glutamic acid-rich protein; LPG, lipophosphoglycan; LPPG, lipophosphopeptidoglycan; NK, natural killer; NO, nitric oxide; PPG, proteophosphoglycan; ROS, reactive oxygen species; TLR, Toll-like receptor; TNF, tumour necrosis factor. * Corresponding author. Unite´ Biologie Cellulaire du Parasitisme, Institut Pasteur, 28, rue du Dr Roux, 75724 Paris Cedex 15, France. Tel.: þ33 1 45688675; fax: þ33 1 45688674. E-mail address: [email protected] (N. Guillen).

may become pathogenic, i.e. they cause a disease, by breaking down host defence mechanisms and by inhibiting host functions. Whereas pathogenicity and virulence both designate the parasite’s capacity to provoke disease, virulence is more specifically associated with the degree of disease severity (e.g. the fatal case rate), pathogen infectivity and tissue invasiveness. Pathogenic microorganisms express virulence factors, which are molecules implicated in the establishment of the pathology and generally required in the processes of adhesion and colonization, tissue invasion, evasion from and inhibition of host immune responses. The ability of pathogens to adapt to and to survive changing environments and to manipulate host responses can also be considered as virulence traits. Entamoeba histolytica is a pathogenic amoeba causing amoebiasis in humans. Its virulence is generally attributed to the capability to destroy tissues through adherence, host cell killing and extracellular matrix (ECM) proteolysis, linked to the expression of a set of virulence factors and commonly evaluated in a hamster or gerbil animal model. The outcome of an infection also critically depends on host genetic determinants and environmental factors. Experimental model systems to study E. histolytica infections are contributing to the discovery of new aspects of the biology of amoeba colonization and dissemination in humans. Here, we review current

1286-4579/$ - see front matter Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.micinf.2012.05.013

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knowledge on the strategies utilized by E. histolytica to subvert immunity and to invade tissues, particularly intestine and liver. 2. The life cycle of E. histolytica Asymptomatic infections with E. histolytica are common, whereas the symptoms of invasive amoebiasis develop in approximately 10% of the infected individuals, resulting in 50 million cases and 100,000 deaths annually [1]. Recent data on prevalence and morbidity suggest that amoebiasis is probably endemic in many less-developed countries [2,3], making this disease a permanent public health problem that needs attention from health authorities. Entamoeba has a relatively simple life cycle (Fig. 1) consisting of two stages, the dormant cyst and the vegetative trophozoite stage. The main mode of transmission of amoebiasis is ingestion of E. histolytica cysts from contaminated food or water. Excystation in the intestinal lumen produces trophozoites (Fig. 2) that colonize the large intestine (Fig. 3) by adhering to colonic mucins, feed on bacteria of the intestinal flora and divide [2]. Trophozoite populations may reach high densities and aggregate, a process expected to trigger the shift from exponential growth to encystation and which can also be envisaged as a trigger of virulence. An interesting parallel can be drawn with pathogenic bacteria such as Staphylococcus aureus, which at low cell density express proteins promoting attachment and colonization, whereas at high cell density, these traits are repressed and the bacteria initiate secretion of toxins and proteases required for dissemination [4]. However, there are no clues indicating that E. histolytica communicates through a quorumsensing like system which initiates encystation and/or pathogenicity, although it secretes numerous proteins and cell activators [5], some of which may act as (auto-) inducers of virulence. Unlike many parasite species, E. histolytica does not depend on a vector for transmission, since cysts are excreted in stools and perpetuate the life cycle by further faecaleoral spreading. Because of its life cycle, E. histolytica is considered a group III pathogen, i.e. a microorganism of potential use as an agent of bioterrorism, as classified by the National Institute of Allergy and Infectious Diseases. In addition, due to its environment

host cyst

excystation

ingestion trophozoite excretion cyst

invasion encystation

Fig. 1. Entamoeba histolytica life cycle and infection of human hosts. The life cycle of E. histolytica consists in alternating environment-resistant contaminating cysts and vegetative trophozoites. Infection occurs directly upon cyst ingestion, without intermediate hosts as a vector. Cysts differentiate into trophozoites that colonize the intestinal mucus, multiply and produce new cysts. Invasive infection occurs only in approximately 10% of the carriers.

Fig. 2. Interaction of virulent E. histolytica with human liver sinusoidal endothelial cells. Scanning electron microscopy micrograph of a trophozoite (highlighted in colour) in contact with human liver sinusoidal endothelial cells [92]. Scale bar 5 mm.

relatively easy transmission and its impact in terms of mortality and morbidity it is a class B agent, i.e. a pathogen for which monitoring and diagnosis should be improved, according to the Center for Disease Control. 3. The invasive infection by E. histolytica For unknown reasons, commensal trophozoites may become invasive, i.e. they start to destroy the muco-epithelial barrier thus inducing the overproduction of mucus, killing host cells and provoking inflammation and subsequently dysentery (Fig. 3). Breaking the intestinal barrier and the blood vessels causes the loss of water and blood in the stools. Therefore, at the macroscopic level, the detection of blood and mucus in diarrhoeal stools is an index to suspect amoebic dysentery, but a more precise diagnosis is mandatory to discriminate from bloody diarrhoea caused by infection with enteropathogenic bacteria. Having reached the vessels present in the mucosa, trophozoites may disseminate via the afferent blood flow of the portal vein system and cause damage to other organs, in particular, amoebic liver abscesses (ALAs) (see review [6]). These are the most common manifestations of extraintestinal amoebiasis. 4. Human colon colonization by different Entamoeba species Due to the presence of diverse Entamoeba species in the human colon including non-pathogenic Entamoeba dispar, Entamoeba coli and Entamoeba moshkovskii, the causative agent of amoebiasis, E. histolytica wore many names for more than 100 years [7]. Based on biochemical and immunological

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asymptomatic infection

invasive infection dissemination

C

Fe ROS

O2

NO excretion Gal/GalNAc lectin bacteria cyst trophozoite

excretion Fig. 3. Intestinal infections are mostly asymptomatic. Trophozoites adhere to the mucus (produced by epithelial cells) and feed on bacteria. Lytic enzyme activities (arrows) allow trophozoite embedding at the mucus surface to resist mechanical forces in the gut lumen. Trophozoites divide, aggregate and encyst upon environmental encystation signals (potentially cell density, altered mucus properties). Carriers continuously excrete cysts and trophozoites. Invasive infection: upon unidentified triggers, the increased lytic activity progressively degrades the mucus barrier. Trophozoites in contact with epithelium kill and phagocytose human cells, penetrate into the lamina propria. The host reaction includes infiltration and activation of neutrophils, mast cells and macrophages, and leads to the production of reactive oxygen species (ROS), nitric oxide (NO) and pro-inflammatory cytokines responsible for the inflammation. Trophozoites may progress into the underlying ECM and tissues, reach blood vessels and breach the endothelial barrier. Parasites may disseminate to the liver and are confronted with increased levels of oxygen, iron (Fe) and with the complement system (C) attack.

characteristics, Diamond and Clark [8] in 1993 proposed that two morphologically identical species colonize the human colon: E. dispar and E. histolytica, the latter being the only amoeba type pathogenic for humans, though in most cases the infected person does not develop the disease. Evidence validating the colon multispecies hypothesis was provided in 2002 by studies demonstrating the genetic differences between E. histolytica and E. dispar through the analysis of some repetitive DNA elements [9]. These findings support the evolutionary notion that phenotypic differences between pathogenic and nonpathogenic Entamoeba species result from the natural selection of mutations in genomic loci (i.e. adaptive selection) [10]. Identification of these adaptive loci, on a genome-wide scale, might shed light on the genomic basis of their phenotypic differences. Other than adaptive selection, the functional impact of a single mutation can also be estimated based on evolutionary conservation of protein domains, i.e. the patterns

of sequence conservation and divergence of the same domain in a wide range of homologues [11]. New generation sequencing for future genome-wide analyses and chromosome organization studies will provide insights into this genomics issue and elucidate the link between Entamoeba species and/or strain genotypes and pathogenic behaviour. 5. Impact of the genome organisation on E. histolytica virulence The study of the parasite E. histolytica entered a new era after the genome sequencing of the virulent strain HM1:IMSS [12], commonly used in laboratories worldwide. The genome is characterized by its high content (75.9%) in adenine and thymine and the abundance of repetitive DNA sequences, preventing chromosome assembly [13]. This 20.8 Mb genome contains 1496 scaffolds and 8201 predicted genes [14]. The genome is compact with an average gene size of 1167 bp and

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an intergenic distance of 800 bp. A phylogenetic analysis identified 96 genes potentially acquired by lateral transfer [15] from bacteria of the commensal flora of the human gut Bacteroidetes group. These genes are mainly involved in the metabolism of amino acids and nucleotides, and in the synthesis of ironesulfur clusters. 5.1. Sequences of tRNA, genetic diversity and diagnosis of virulent strains One interesting feature of the E. histolytica genome is the abundance of intrachromosomal repeated sequences. The first type consists of transfer RNA (tRNA) encoding sequences, arranged in clusters of 500e1750 bp containing multiple repetitions [16]. The estimated 4500 tRNA genes are localized within 70e250 clusters. Between them, the tRNAs are separated by short tandem sequence repeats, which have been useful for genotyping E. histolytica strains isolated from individuals who either were asymptomatic or had intestinal or extraintestinal disease [17,18]. The data suggest that specific genome rearrangements associated with invasive disease is a possible explanation for the different tRNA genotypes detected in liver abscess- and stool-derived parasites from the same infected person [19]. Note that genotypes from isolates of asymptomatic carriers were highly polymorphic, whereas in isolates from amoebiasis patients the same genotype was shared between parasites responsible for dysentery or ALA. The linkage between parasite genotype and infection outcome is unexpected, since it has been assumed that E. histolytica is a clonal organism for which meiotic recombination and rearrangement of genes should not contribute to genotype differences. Thus, as Ali and collaborators stated, a novel mechanism for generating genetic variation may exist, based on genome instability, that should continuously lead to the generation of various genotypes through intragenomic reorganization. The analysis of the E. histolytica genome revealed a set of genes necessary for meiosis, pointing to the possibility of sexual reproduction in natural populations [13].

(Hsp) 70 family, the large leucine-rich repeats BspA-like surface protein family [22] and the GTPase of immunityassociated protein AIG family of proteins, having significantly higher expression levels in virulent E. histolytica [23e25]. Interestingly, irreversible silencing of amoebapore (AP), a pore-forming protein believed to be involved in pathogenicity, in E. histolytica was obtained following transfection with a plasmid containing sequences of a SINE [26]. Further work suggests that the presence of two tRNA genes is responsible for this unusual epigenetic phenomenon [27]. These observations led to speculate that transposable elements inserted in the neighbourhood of genes could lead to the increased gene expression related to virulence [14]. This interesting, although provocative hypothesis raises the unsolved question concerning the role of small RNA in the regulation of amoebic gene expression [28], and consequently the potentiality of epigenetic phenomena to govern pathogenicity [29]. Genomes have evolved multiple layers of molecular defence systems against transposons such as DNA methylation [29] and RNA interference [30]. Both mechanisms are operative in E. histolytica, but proof for their link to pathogenicity has yet to be provided. 6. Human susceptibility to infection with E. histolytica The outcome of E. histolytica infections not only depend upon the parasites’ genotypes and phenotypes, but also upon the individual’s gender, genetic loci and environmental factors, as the availability of food (Table 1). Whereas asymptomatic infection distributes equally between men and women, amoebic dysentery and ALA are more frequent in men, for yet unidentified reasons. Interestingly, serum from women is more efficient in complementTable 1 Host influences on Entamoeba histolytica virulence. Susceptibilitya

Gender differences

Barrier responses

Genetic loci Leptin signalling Bacterial gut flora Mucus overproduction Disruption intercellular junctions Endothelial cell retraction Cell death

Innate immune response

Neutrophil, mast cell, macrophage, NKT cell activation Production ROS, NO and derivates Complement-mediated lysis Tissue damage

Immune response

Intestinal secretory IgA Antibodies to Gal/GalNAc, SREHP, M17, KERP1

Microenvironment

ECM Oxygen Glucose Iron

5.2. Link between short interspersed nuclear elements and the expression of genes involved in virulence A second type of sequence repeats consists of retrotransposon elements of the LINE (long interspersed nuclear element) and SINE (short interspersed nuclear element) family. These elements are class I transposons that encode the reverse transcriptase and endonuclease required for retrotransposition [20]. About 20% of the E. histolytica genome consists of transposable elements [14]. Within the sequence of 393 SINEs examined [21], up to four 26e27 bp repeats were detected. Some SINEs with two repeats are actively transcribed and appeared to be identical but inserted into different sites in the genome, suggesting that active SINE transposition may still be occurring, their activation may be triggered by certain environmental stresses. Some protein encoding gene families appear to be physically linked to transposable elements, as a cluster of 31 members of the Heat shock protein

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a For data sustaining the table, see reviews [2,6,43e46,67,85] and references therein.

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mediated amoeba lysis [31], and in a mouse model, the increased amounts of interferon (IFN)g and natural killer (NK) T cells in females may sustain resistance [32]. Nutritional status and IFNg levels have been linked and higher IFNg production associated with a reduced risk of amoebic diarrhoea/dysentery [33]. The correlation between malnutrition and increased susceptibility [34] led to search for genetic polymorphisms influencing nutritional status and susceptibility, and the discovery of a single amino acid polymorphism in the leptin receptor that associates in humans and mice with increased susceptibility for amoebiasis [35]. Signalling through the hormone leptin has pleiotropic effects that, depending on the target organ, contribute to the adaptation to nutrient levels, to inflammatory responses and to protection from apoptosis. Its protective role in amoebiasis was demonstrated in a receptor-deficient mouse model, the site of protective activity localized to the gut epithelium and the implication of SHP2/ERK and STAT3 signalling pathways identified [36]. Recently, Marie et al., 2012 [37] unravelled the molecular mechanism using a cell culture system: activation of leptin signalling confers to human epithelial cells protection from amoeba-induced cell death, including from caspase-3 activation, dependent on the activation of the STAT3 pathway. The leptin receptor mutant associated with higher human and murine susceptibility increases the susceptibility of cell cultures to E. histolytica cell killing activity. Concerning further genetic determinants for susceptibility, evidence exists that a particular class II human leukocyte antigen allele correlates with natural protection [38]. 7. Study of the pathophysiology of amoebic infection with in vivo and ex vivo models In the absence of an experimental model reproducing the entire parasite life cycle and all aspects of the pathogenesis of the human disease, intestinal and hepatic amoebiasis is investigated by separate in vivo approaches. They consist in the direct infection of target organs in different susceptible and resistant animals, belonging essentially to rodent species (for a detailed review [39]). More recently, alternative model systems have been developed to better represent human pathophysiology, as human [40] or pig [41] colon explants and pig cecal loops. The infections are carried out with trophozoites adapted to and grown in axenic culture and the virulent E. histolytica strain HM1:IMSS is the most widely used. Studies include the comparison with trophozoites from the same species but modified for the expression of given virulence factors or exhibiting attenuated virulence, as well as from nonpathogenic Entamoeba species (see below). The following main notions emerged from the in vivo data: (I) Trophozoites progressively loose their virulence in axenic culture and its maintenance requires regular contact with the animal host [42]. (II) Tissue destruction and survival in target organs rely on a strong adaptive response, achieved by the regulation of the expression of specific parasite genes, encoding potential virulence factors [43]. (III) The host

influences the degree of virulence and the expression of virulence “traits”, host defence responses and genetic factors are crucial determinants for the pathophysiology of experimental amoebiasis [44,45]. The initial host response is proinflammatory, the cellular component consisting in a neutrophil infiltration to sites of parasite invasion, followed by the activation of e.g. mast cells, macrophages and NKT cells [46]. (IV) The inflammation is the main factor for tissue damage thereby favouring the progression of parasite invasion [44,46]. 7.1. Mongolian gerbils (Meriones unguiculatus) Male gerbils are susceptible to experimental ALA [47] and cecal infection with E. histolytica [48]. However, only early stages of intestinal invasion can be studied, the ulcerative lesions heal spontaneously and the trophozoites are completely eliminated. Gerbils have been used to evaluate the protective effect of immunization with recombinant serine-rich E. histolytica protein (SREHP) antigen [49] and for transcriptome analyses [23]. 7.2. Mouse strains with different susceptibility to infection with E. histolytica and genetically modified mouse lines In mice, only early steps in the intestinal infection can be investigated, due to the spontaneous resorption of intestinal lesions and the clearance of parasites. The studies identified cellular (as neutrophils, mast cells, CD4þ T cells) and molecular (as interleukin (IL)-4, IL-10) players important for the immune response and pointed to the existence of host gender and genetic factors determining susceptibility (e.g. [50e52]). Infections of genetically modified animals revealed the implication of IL-10 in the resistance of C57BL/6 mice and a mouse genetics approach identified several chromosomal loci potentially conferring resistance [53]. Gender differences in the resistance of C57BL/6 mice to ALA were observed and linked to differences in early cytokine production mediated at least in part by NKT cells [32]. Amoebic lipophosphopeptidoglycans (LPPGs) (see below) were found as the possible natural NKT cell ligand inducing the protective IFNg response [54]. The role of neutrophil activation in ALA resistance of BALB/C mice seems in turn related to the production of nitric oxide (NO) killing the parasites [55]. In a pro-inflammatory host environment, trophozoites are exposed to higher NO levels, likely produced by stimulated immune cells (neutrophils, macrophages), and undergo programmed cell death [56]. E. histolytica in turn induces neutrophil and macrophage apoptosis that may contribute to dampen tissue inflammation and damage, highlighting the importance of programmed cell death in ALA pathogenesis. Finally, evidence exists that E. histolytica is able to suppress human macrophage functions and amoeba extracts inhibit tumour necrosis factor (TNF)- and IFNg-induced macrophage respiratory burst and NO production (also see review [44]).

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Severe combined immunodeficiency (SCID) mice develop ALA [57] and have been used to study E. histolytica-induced apoptosis of inflammatory cells and hepatocytes. Gene expression profiles of infected liver tissue indicate the simultaneous activation of inflammatory, regenerative and apoptotic pathways [58].

expensive and technically demanding, this model presents the advantage to allow the analysis of intestinal and hepatic infection in the same animal.

7.3. Golden hamster (Mesocricetus auratus)

SCID mice were engrafted subcutaneously with human foetal intestinal tissue [65]. Thus, the target tissue is of human origin, while inflammatory cells are from the mouse host. Grafts were allowed to develop before direct infection of the xenograft lumen. Infection with virulent E. histolytica mimics a number of features of human amoebic colitis, not observed with the non-pathogenic E. moshkovskii species, with extensive tissue damage and an early inflammatory response, composed primarily of neutrophils and an increase in human IL-1b and IL-8 during early stages of infection. Additionally, the data suggest a role for amoebic CP-A5 in xenograft damage. Adaptive immune responses cannot be investigated with this model, due to the lack of lymphocytes, and it does not account for the role of the human colonic mucus.

Hamsters do not develop intestinal lesions but are the most widely used animal model for ALA [59]. Males are highly susceptible and produce large abscesses, with histological features similar to human ALA. Recently, ex vivo infections of precision-cut liver slices have been introduced [60] as well as an approach challenging trophozoites in vivo with peritoneal pro-inflammatory responses prior to testing for ALA formation [56]. A detailed histological study of E. histolytica-infected hamster livers [59] revealed multiple inflammatory foci formed in the parenchyma, consisting of few trophozoites located in the centre surrounded by rings of inflammatory cells delimiting the necrotic tissue, while the remaining hepatic tissue appeared normal. Massive trophozoite death was observed in the first hours post-infection [61]. A critical point for success of infection is reached after 12 h when the lowest number of trophozoites is observed. The process then enters a “commitment” phase during which parasites multiply and the foci size increases. The deposit of amoebic material on the endothelial cell surface suggested that these cells are rapidly aggressed by cytotoxic parasite effectors and that their stimulation to produce pro-inflammatory signals might be the initial trigger for the inflammatory response [62]. Apoptosis occurred in neutrophils and hepatocytes upon parasite progression in the parenchyma and in endothelial cells very rapidly (1 h) after infection [62]. The latter observation suggests that disturbance of the endothelial barrier may result from trophozoite interactions which likely facilitate the passage of trophozoites between the blood circulation and the hepatic tissue. The data show further that galactose- and N-acetylgalactosamine-inhibitable (Gal/GalNAc) lectin function, a major virulence factor, is required for efficient tissue penetration and the induction of an early inflammatory and apoptotic response. Analysis of ALA development in the hamster serves to define in vivo functions of candidate virulence factors, such as the lysine- and glutamic acid-rich protein (KERP)1 [25] and cysteine protease (CP)-A5 [63] (see below).

7.5. SCID mouse-human intestinal xenografts (SCIDHU-INT)

7.6. Human colon explants To study the kinetics of parasite tissue penetration, the hosteparasite interactions and the molecular mechanisms underlying human colon invasion by E. histolytica, Bansal et al. [40] have established an adult human colon ex vivo model mimicking early steps of intestinal amoebiasis. Virulent trophozoites destroy the mucus layer, detach and lyse enterocytes, induce a pro-inflammatory reaction (IFNg, IL-1b, IL6, IL-8, TNF) and penetrate the lamina propria. They migrate along the dense collagen scaffold, reach the Lieberku¨hn’s crypts and disorganize the loose collagen scaffold before progressing into the intestinal mucosa [66]. This experimental system allows the comparison of different parasite strains with the same colon sample. E. dispar was unable to elicit the effects observed with virulent E. histolytica. Incubation with E. histolytica strains epigenetically silenced for the expression of known “major” virulence factors (see below) revealed that Gal/GalNAc lectin and APs are not required for invasion of human colon explants and that CP-A5 is not needed for crossing the mucus, but contributes to the penetration of the lamina propria and the inflammation induction [40]. 8. Well-characterized “major” virulence factors and their regulation

7.4. Pigs (Sus scrofa domesticus) Recently, a “large” animal model using outbred “mini”-pigs has been assessed [41]. The interest in using pigs to investigate the human disease relies in high similarities between these two species with respect to anatomy, physiology and genetics (for review [64]). Disruption of the mucosal architecture has been observed with infected colon explants. Moreover, abscesses were detected in the liver of one animal 7 days post-injection into the portal vein and the liver parenchyma. Although

The preceding description of E. histolytica-induced pathogenesis highlights the complexity for a definition of virulence. In fact, for successful invasion, the parasite must cross the intestinal or hepatic barrier, resist to and escape the host responses, destroy the tissue and migrate to the subsequent microenvironment. The resulting infection thus requires the combination of the capacities to defend, to attack and to migrate. These main events responsible for amoebiasis are not the result of the activity of a single virulence factor, but of

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a successively regulated and coordinated action of diverse factors during the invasion. Some important molecules have been studied for their role in tissue invasion: adherence, cytotoxicity, cell killing and phagocytosis as well as the onset of host immune responses (Table 2 and reviews [6,43,44]). 8.1. The Gal/GalNAc lectin The first step in the intestinal invasive process is the penetration of the mucus layer to which the parasite binds through its major surface component, the Gal/GalNAc lectin of 260 kDa [67]. This lectin is a heterodimer of a disulfidelinked heavy (170 kDa, HgL) and light (35/31 kDa, LgL) subunit, which is noncovalently associated with an intermediate subunit of 150 kDa. The Gal/GalNAc lectin has been identified among the amoebic surface proteins interacting with the brush border of intestinal epithelial cells [68], but its role in intestinal amoebiasis is not yet well established. The protein complex is an immunodominant antigen recognized by sera from 95% of patients with liver abscess [69] and children with stool IgA antibodies specific for the lectin appear protected from intestinal infection [70]. Therefore, the lectin is the preferred candidate for the diagnosis of E. histolytica/dispar and vaccination proposals. Resistance to complementmediated lysis favours survival of E. histolytica in the bloodstream [71]. The HgL subunit contains a cysteine-rich domain (CRD) displaying homology with CD59, a human inhibitor of the complement attack complex. A monoclonal antibody against the Gal/GalNAc lectin abrogated amoebic

resistance to complement [67]. The Gal/GalNAc lectin thus appears to participate not only in adherence and host cell killing but also in evasion from the complement system via a remarkable mimicry of human CD59. The CRD also powerfully activates bone marrow derived macrophages to produce TNF and NO, both of which show amoebicidal activity [72]. The exposure in vitro of murine macrophages to the Gal/GalNAc lectin induces increased expression of Tolllike receptor (TLR) 2, a receptor for secreted microbial molecules, and proinflammatory cytokine production [73]. The contribution of Gal/GalNAc lectin to the recruitment of inflammatory cells and cytokine production was studied in more detail with the hamster model of ALA through the analysis of HgL-dependent signalling pathways. HgL presents homology with b2-integrin within the carboxy-terminal sequence responsible for signalling involved in the reorganization of the amoebic cytoskeleton [67] and Gal/GalNAc lectin accumulates in lipid rafts sustaining membrane signal transduction platforms [74]. Signal transduction through HgL is of great importance for E. histolytica virulence since its loss (upon overexpression of the C-terminal portion of HgL; HGL2 parasites) results in drastic changes in ALA development and parasite adhesion to cells [75,76]. The HGL-2 trophozoites cause the formation of considerably smaller inflammatory foci containing twice as many trophozoites as the virulent controls [62], suggesting a reduced activation of immune cells, which correlated with the reduced number of macrophages and neutrophils within hepatic blood vessels. Moreover, TNF production was not detected 24 h after infection,

Table 2 Entamoeba histolytica processes and factors contributing to virulence. Motilitya,b Actinc Actin-BPs Adherencea Gal/GalNAc lectin KERP1 Cytotoxicity, host cell killinga CPs APs Phagocytosisa CaBP1/C2PK TMKs Vesicle traffickinga Vps4 Arf Host defence escapea CPs Peroxiredoxin Stress responsea,d Hsps Peroxiredoxin Glucose starvation responsea KRiP1 LgL1 Calcium signallinga CaBP1/C2PK URE3-BP

Myosin IB, Myosin II

Rho, Rac small GTPases

CaBP1

b1FNR

ADH112

CP-A5 (RGD)

PLs

CPADH

ROM1 protease

Myosin IB, Myosin II

Rab, Rac small GTPases

CaBPs

Rab small GTPase

PPGs Ruberythrin

STIRP

M17

PAK

SREHP

FP4

Arginase

serp

MLIF

PGE2

Thioredoxin reductase

FeSOD

p34

URE3-BP

BPs, binding proteins; CaBP, calcium-binding protein; b1FNR, beta-1 integrin-like fibronectin receptor; ADH, adhesin; STIRP, serine threonine isoleucine-rich protein; M17, immunodominant antigen M17; ROM, rhomboid; PL, phospholipase; CPADH, complex of CP112 and ADH112; C2PK, C2 domain-containing protein kinase; TMK, transmembrane kinase; PAK, p21-activated kinase; SREHP, serine-rich E. histolytica protein; FP, FYVE-domain-containing protein; Vps, vacuolar protein sorting; serp, serpin (serine protease inhibitor); Arf, ADP-ribosylation factor GTPase; MLIF, monocyte locomotion inhibitor factor; PGE2, prostaglandin E2; FeSOD, iron-superoxide dismutase; p34, NADPH:flavin oxidoreductase; KRiP, lysine-rich protein; LgL, light subunit of Gal/GalNAc lectin; URE3-BP, upstream regulatory element 3 binding protein. a Processes. b For data summerized in the table, see reviews [2,6,42e46,67,85] and references therein. c Molecules participating in the process. d Response to heat, oxygen, ROS, NO.

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corresponding to the absence of macrophages and NK cell activation. With virulent amoebae, apoptosis of sinusoidal endothelial cells was detected in the first hour of infection and at 24 h for hepatocytes and neutrophils, while apoptosis in the parenchyma was delayed by 24 h in the foci formed by HGL2. These results suggest a role of the Gal/GalNAc lectin in apoptosis signalling and amoeba-induced cell death and consequently in virulence. The data are in line with pioneer observations suggesting the implication of the Gal/GalNAc lectin in cytotoxicity and cell death [77]. In this context, it is interesting to note that E. histolytica transfers Gal/GalNAc lectin to the lateral surface of epithelial cells prior to killing them [78]. The HGL-2 trophozoites were also inhibited in migration within liver tissue [79] forming inflammatory foci close to blood vessels [62]. The restricted motility led to an abortive propagation of trophozoites, underlining the major role in hepatic invasion played by parasite adherence to host cells.

PPG2). PPGs are associated with extensively modified polypeptides with glucan side chains of various lengths. The attenuated E. histolytica strain Rahman synthesizes only one class of PPGs [80]. Anti-PPG antibodies reduce parasite adhesion and cytotoxicity suggesting an important role of PPGs in parasiteehost interaction [81]. A role for LPPG in the induction of host immune responses has been deduced from the following observations: Reduction of surface PPG (by interference with the expression of GPI biosynthetic enzyme) provokes a drastic decrease in trophozoite survival in the presence of complement and these parasites are avirulent in the hamster ALA model [82]. LPPG released from lysed trophozoites is recognized through TLR2 and TLR4/CD14 and induces the production of IL-8, IL-10, IL-12p40, and TNF by monocytes ([83] and references therein). LPPG is implicated in the protection against invasive amoebiasis and specific activation of NKT cells by LPPG conferred significant protection [54].

8.2. KERP1

8.4. Cysteine proteases and the example of CP-A5

Following migration through the mucus layer, the trophozoites enter in contact with the intestinal epithelium. A tropism of amoeba for the brush border of enterocytes has been noted, suggesting the existence of cellular components that may function as signals for tissue invasion [68]. To find new surface compounds involved in the pathogenesis of E. histolytica, proteins binding to the brush border were purified [68]. In addition to Gal/GalNAc lectin, two novel proteins enriched in lysine (K) and glutamic acid (E) were identified and named KERP1 and KERP2. Subsequent studies focused on the role of KERP1 in the infectious process because, unlike KERP2, it does not present homology with any protein known to date, including those of E. dispar. KERP1 is localized at the trophozoite plasma membrane and in internal vesicles and binds to host cell surfaces. Using experimentally induced ALA in hamsters demonstrated the link between kerp1 gene expression and hepatic amoebiasis [25]. The use of antisense RNA-mediated inhibition of kerp1 gene expression suggested an intricate gene regulation, that leads under stress conditions to a reduction of KERP1 abundance, correlated with the reduced ability of modified trophozoites to provoke liver abscesses. Thus, KERP1 is a key factor for the establishment of amoeba-cell contacts and the progression of liver abscesses and, consequently, for amoebic virulence.

In the progression of tissue invasion, one remarkable feature of E. histolytica is its ability to lyse human cells and to destroy the ECM. The genome of E. histolytica comprises 80 genes encoding proteases, including 50 CPs [84] of the papain superfamily [85]. Activation of CPs (Fig. 4) includes the removal of propeptide regions for the proper folding of the enzyme, the inactivation of the peptidase domain and the stabilisation of the enzyme against denaturation. In axenic E. histolytica cultures, only CP-A1, CP-A2, CP-A5 and CP-A7 are highly expressed and account for over 90% of the proteolytic activity in trophozoite extracts [86]. CPs are directly involved in tissue invasion through their ability to degrade ECM proteins as well as mucin 2, the major component of colon mucus [87]. They are also very important for immune evasion by degrading host antibodies and complement [85]. Among the E. histolytica CPs participating in the pathogenic process, CP-A5 is the prime candidate, since it is unique to E. histolytica, localizes at the amoebic surface [88], is involved in human colon invasion [40] and ALA formation [63]. CP-A5 contains an arginineeglycineeaspartic acid (RGD) integrin binding motif in its propeptide region which has also been found in the proregion of cathepsin X from higher eukaryotes. In cell adhesion proteins like fibronectin, RGD motifs serve as ligand recognition sites for cell surface receptors such as the integrins. CP18 and CP112 also contain RGD, localized within the catalytic domain but are not highly expressed or secreted by E. histolytica. Binding of CP-A5 RGD to avß3 integrin on enterocytes triggers PI3 kinase/ AKT signalling and induces NFkB proinflammatory responses [89]. CP-A5 was shown to damage enteric neurons in vitro and in a mouse model of intestinal amoebiasis [44]. In human colon explants in contact with E. histolytica, CP-A5 is not required for mucus degradation (although CP activity is necessary) [40] but seems to be involved in further steps of intestinal invasion that rely on ECM destruction and human

8.3. The role of LPPG in virulence and immune responses to E. histolytica In addition to proteins, the most abundant surface molecules of E. histolytica trophozoites are glycosylphosphatidylinositol (GPI)-anchored lipophosphoglycans (LPGs) that form a densely packed glycocalyx on the entire trophozoite surface, ensuring a protective physical barrier. Virulent E. histolytica synthesizes two types of LPG, forming a family of GPI-anchored proteophosphoglycans (PPGs) with unusual properties, named LPG (or PPG1) and LPPG (or

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A

pre(13-20 aa) pro(72-80 aa)

mature (216-225 aa) C CC

C

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C

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-COOH DWR ERFNIN

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catalytic domain 50 amino acids (aa)

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Fig. 4. Dependence on CP-A5 activity of human colon explant pro-inflammatory responses to Entamoeba histolytica. A. Domains present in E. histolytica subfamily A members of the C1 papain superfamily, from which CP-A1, -A2, -A5 and -A7 are the most expressed in culture. (Brackets give the domain length variation of the different CP-As). Cathepsin pro-domains function to maintain the enzyme in an inactive form until it reaches a site appropriate for protease function and as a structural template for proper protein folding. CP-A5 contains an RGD motif (amino acids 92e94) within the pro-domain, mature CP-A5 spans amino acids 94e318. B. Abrogation of CP-A5 activity by epigenetic silencing leads to parasites unable to induce inflammatory responses and human cell lysis in the colon explant model of intestinal amoebiasis, measured as the release of cytokines and lactate dehydrogenase (LDH) respectively, upon incubation with either virulent or modified trophozoites and controls without parasites [40].

components induced or activated in the presence of CP-A5 [66]. The conversion of pre-IL-1b into its active form depends on CP-A5 activity. Active IL-1b in turn induces the production of nitrogen compounds by IFNg-sensitized cells [90]. Amoeba whose cp-A5 gene expression was diminished by antisense RNA or epigenetic silencing have reduced pathogenicity in the SCID-HU-INT model, revealing a link between the pro-inflammatory activity of CP-A5 and intestinal invasion ([90] and also Fig. 4). The direct involvement of CPs in ALA formation was demonstrated by inhibiting their function with a CP-specific inhibitor, trans-epoxysuccinyl-Lleucyl-amido-4-guanidino-butane (E-64), and by interfering with cp-a5 gene expression [63]. In conclusion, CPs and CPA5 in particular are major factors involved in pathogenicity, but the relation between their activity and that of other (i.e. metallo- and serine-) proteases is yet not explored.

SAPLIP domain proteins (such as NK-lysine and granulysine) are capable of forming pores. The E. histolytica genome contains 16 SAPLIP domain-encoding genes, but only AP-A, -B and -C exhibit the predicted pore forming activity. The APs are essential for the phagocytosis of bacteria by E. histolytica and E. dispar. Toxicity to prokaryotic and eukaryotic cells suggested a role in ALA formation, subsequently demonstrated by the absence of abscesses in SCID mice or hamsters infected with trophozoites whose AP expression was downregulated by antisense RNA or epigenetic silencing. However, in the SCID-HU-INT model, trophozoites not expressing AP-A do induce inflammation and tissue damage [91] and APs are not involved in human colon explant invasion [40] or in virulent amoeba-induced responses and death of human liver sinusoidal endothelial cells [92], suggesting distinct pathogenic processes depending on the target organ.

8.5. AP proteins

9. Phenotypic differences of E. histolytica strains with distinct virulence

Amoebapores [26] are proteins of 77 amino acids, stored in vesicles and containing the “saposin-like protein” (SAPLIP) domain found in saposin B, a physiological detergent. Other

An interesting fact is the necessity of regular passages in the liver of hamsters or gerbils for E. histolytica to preserve its

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virulence, as long-term cultivated parasites lose their ability to form liver abscesses [93]. Virulent and long-term culturederived, so-called virulence-attenuated, trophozoites both exhibit adherence, host cell killing and proteolytic properties when tested in vitro or ex vivo with mammalian cell cultures. Attenuated virulence is thus best defined as the loss of the capacity to produce experimental liver abscesses in susceptible animals, in which these parasites disappear 48e72 h after injection [93]. The need of virulence exacerbation in an animal calls for the existence of specific transcriptional adaptive mechanisms essential for E. histolytica survival in different environments and the challenge is to determine the complex phenotype enabling parasites to survive (I) in toxic levels of redox-active compounds including oxygen, NO, reactive oxygen species (ROS) and nitroxy radicals, and (II) upon depletion of glucose, iron and upon global metabolic changes during tissue destruction. Distinct levels of virulence are observed among E. histolytica field strains: while HM1:IMSS is virulent, Rahman is naturally attenuated, showing reduced activity in various in vitro assays and decreased virulence in animal models [94]. Comparison of gene expression profiles between the virulent HM1:IMSS and the attenuated Rahman strain [95] identified genes associated with the virulent phenotype, including a gene encoding a serine-, threonine- and isoleucine-rich protein (STIRP), implicated in amoebic interaction with target cells [96]. STIRP thus is a candidate for further analysis in experimental animal models. Several genes with potential roles in stress responses and some SINE transcripts have decreased expression levels in the Rahman strain. A proteomic study has identified higher levels of peroxiredoxin in HM1:IMSS [94]. Peroxiredoxin is crucial for protection against oxidative stress, as decreased gene expression causes the accumulation of ROS and a decrease in parasite viability as well as in its cytotoxicity [97]. Overall, these experiments allow the conclusion that despite the similar genomes of isolates belonging to the same species and their comparable expression of “major” virulence factors, differential gene expression that confers the capacity to respond to host and environmental challenges accounts for differences in virulence. Comparison of the transcriptional profile of virulent E. histolytica HM1:IMSS harvested from hamster liver abscesses and of an attenuated HM1:IMSS strain grown in culture for over 5 years [25] led to the conclusion that virulence correlates with upregulation of key genes involved in stress responses, including molecular chaperones, SSP1 and peroxiredoxin, as well as unknown genes encoding a group of lysine-rich proteins. Seven of these have lysine contents higher than 25 percent. Among them KERP1 was identified. Two other genetically very similar E. histolytica lines (A and B) derived from HM1:IMSS differ also in their virulence properties [23]. Microarray analysis of cultured parasites of both lines indicated that members of the AIG1 family of GTPases were overexpressed in the virulent trophozoites. In plants AIG1 proteins are believed to regulate cell death and to participate in defence against bacteria. Interestingly, in

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HM1:IMSS trophozoites isolated from the mouse model of amoebic colitis, aig1 gene expression was highly up-regulated [24], as well as the expression of genes encoding the chaperones Hsp70 and Hsp90, both involved in stress responses and belonging to multigene families in which some members map close to transposable elements in the genome [14]. Extreme stress responses have also been observed when parasites were in contact with NO [98,99]. Glycolysis and amino acid catabolism are the major pathways for energy production in E. histolytica. In the presence of NO, glycolytic enzymes are inactivated provoking low ATP levels and parasite death [93,99,100] and extensive ER fragmentation appears as a protective mechanism at early stages of stress responses [99]. Taken together, these results support the idea that in colon or liver an important stress response is induced in virulent strains upon contact with components of either the environment (ECM, cells) or the immune response. In contrast, avirulent trophozoites are unable to induce these responses and thus unable to survive. Virulence in the host is thus sustained by the ability of E. histolytica to activate a transcriptional and cellular program enabling it to resist and to survive to the stresses and to successively move to a new tissue microenvironment. The effect of the tissue response on amoebic motility is not discussed here but it is relevant to note that TNF is chemoattractant for trophozoites [101] and that mechanical forces exerted by virulent trophozoites were proposed to participate in liver tissue destruction [92]. In axenic culture, E. histolytica grows in the presence of 1% glucose, whereas the human large intestine is a lowglucose environment due to the absorption of simple sugars in the small intestine. E. histolytica relies solely on glycolysis and fermentation and lacks the tricarboxylic acid cycle and the mitochondrial electron chain reactions. Glucose starvation (GS) is an extensively studied metabolic stress that activates amoebic virulence [102]. In E. histolytica, GS did not modify Hsp abundance. Instead, GS increases the levels of transcription factor URE3-BP whose overexpression leads to higher virulence and increased cell motility [103]. Increased levels were further found for the lysine-rich protein KRiP1 that has a role in liver abscess formation [25,102], and for the Gal/ GalNAc lectin LgL1 subunit. It will now be of interest to determine whether the specific parasite response to short term GS reflects the gene expression pattern and protein levels in the low-glucose environment of the large intestine. 10. Concluding remarks During infection pathogens need to adapt to host responses and distinct tissue-specific environments, including “chemical” (e.g. cytokines, ROS) and mechanical challenges, such as crossing biological barriers. The commensal flora and the availability of oxygen, glucose and iron represent important parameters affecting the intestinal infection process. The enteric pathogen E. histolytica survives during infection, essentially by responding rapidly to changes in the microenvironment in intestine, blood and liver and escaping host defence

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mechanisms. Virulent E. histolytica activates a gene expression program enabling to react to stresses originated by host inflammatory and immune factors. The set of virulence factors, essential to sustain the invasive process, includes wellcharacterized adhesion molecules, cysteine proteases and factors involved in cell killing. The finding of a linkage of the E. histolytica genotype and its genome organization with the infection outcome is an interesting issue for further investigation. Components of the host immune response eliciting a predominant innate reaction were identified, using animal and ex vivo models. Inflammatory tissue damage appeared as essential for invasion. Host genetics determine susceptibility, the best characterized example being the resistance to E. histolytica conferred by leptin signalling activity. All together, data provide a new integrative and exciting view of the mechanisms underlying infection during amoebiasis. Characterizing the molecular bases of E. histolytica-host cross-talks and amoebic stress responses should provide information on (I) the nature of parasite activities that distinguish virulent and attenuated strains and (II) the stressinducing components from the tissue environment, opening unprecedented opportunities for diagnosis and control of amoebiasis which remains an important infectious disease affecting large populations of humans.

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Acknowledgements [15]

The project of the Cell Biology of Parasitism Unit is supported by grants from the French National Agency for Research (ANR), the Pasteur-Weizmann Research Council and the ECOS-Nord program (France-Mexico). We thank Chung-Chau Hon, Nora Hernandez Cuevas and Doranda Perdomo for critical reading of the manuscript and MarieRe´gine Lambrecht for help in its preparation.

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