Entamoeba Species, Including Amebic Colitis and Liver Abscess

Species of Entamoeba

Many Entamoeba species infect humans, but of greatest significance because of their prevalence are the four morphologically identical amebae E. histolytica, E. dispar, E. bangladeshi, and E. moshkovskii. Only E. histolytica is a cause of invasive amebiasis, whereas E. moshkovskii is associated with a noninvasive diarrhea and other two are nonpathogenic. All of these species are in the quadrinucleated cyst clade of Entamoeba. The E. histolytica and E. dispar genomes share 90% identity in genic regions, and E. moshkovskii is also closely genetically related. In most industrialized countries, E. dispar is 10 times more common than E. histolytica, and E. histolytica and E. dispar can be equally prevalent even in a developing country. The presence of ingested erythrocytes was the sole morphologic characteristic of some usefulness in identifying E. histolytica, but in one study, this characteristic was present in only 68% of cases of E. histolytica but also in 16% of cases of E. dispar. E. moshkovskii is also prevalent and geographically widely distributed. In preschool children from an urban slum, E. moshkovskii was present in 21%, E. histolytica in 16%, and E. dispar in 36%. In another study in Tanzania, among approximately 100 human immunodeficiency virus (HIV)–infected individuals with diarrhea, E. histolytica was present in 4%, E. moshkovskii in 13%, and E. dispar in 5%. In Sydney, Australia, 50% of cases of Entamoeba identified by stool O&P examination were E. moshkovskii. E. moshkovskii has recently been shown to be a cause of diarrhea.

E. hartmanni is also in the quadrinucleated cyst clade, but it is smaller than E. histolytica, having trophozoites of 3 to 12 µm in diameter and cysts of 10 µm in diameter, whereas E. histolytica trophozoites are 12 to 60 µm in diameter, and cysts are 10 to 20 µm in diameter. E. coli cysts have up to eight nuclei, with trophozoites of size similar to those of E. histolytica. Entamoeba gingivalis does not form a cyst and is not an inhabitant of the intestine; instead, it is observed in the mouth in gingival scrapings. Human infection with the uninucleated cyst amebae E. polecki, E. chattoni, and E. suis is generally rare. E. polecki and E. suis infections are linked to contact with pigs, and E. chattoni infection is linked to contact with monkeys. Other nonpathogenic amebae include I. bütschlii, which has characteristic glycogen vacuoles in the cysts; E. nana, which has a characteristic nuclear structure that lacks peripheral chromatin; and D. fragilis, which is more closely related to the flagellates than to the ameba with binucleate trophozoites.

Genotypes of Entamoeba histolytica

In addition to the genetic differences between the three morphologically identical amebae E. histolytica, E. dispar, and E. moshkovskii, genetically distinct strains or genotypes also exist within E. histolytica. Genotypes have been distinguished by the use of isoenzymes, polymorphisms in protein-coding DNA, and polymorphisms in short tandem repeat loci linked to transfer RNA genes (reviewed by Ali and colleagues ). Important findings from genotyping include the following: E. histolytica contains many genotypes ; patients may be infected with more than one genotype at a time ; and certain genotypes are associated with diarrhea, others with colonization, and others with amebic liver abscess formation. In contrast to this high level of diversity in repetitive noncoding DNA, individual protein-encoding genes such as the galactose and N -acetyl- d -galactosamine (Gal/GalNAc) lectin are conserved between genotypes. An additional nuance to genotypes is that in a study of patients with amebic liver abscess, comparison of the amebae in the intestine and liver demonstrated in every case that each patient had different genotypes in the two locations. This surprising result suggested that there is a genetic bottleneck between the intestine and liver, and only a subset of intestinal isolates is capable of causing extraintestinal disease.

Life Cycle

The life cycle of E. histolytica begins with an infectious quadrinucleated cyst and continues with an invasive uninucleated trophozoite. The cyst is ingested from fecally contaminated food or water or through oral-anal sexual practices and, in the intestine, excysts to eight trophozoites ( Fig. 272.1 ). The environmental stability of the cyst and relative resistance to chlorine has resulted in waterborne outbreaks caused by contamination of municipal water supplies. In most laboratory-based studies of the cyst, the reptilian parasite Entamoeba invadens has been used because E. histolytica does not encyst in culture. Studies of the process of encystation in E. invadens have demonstrated the role of quorum sensing through a surface Gal/GalNAc lectin to initiate encystation after an initial environmental signal such as osmotic shock, low glucose level, or interaction with colonic mucins. Later steps in formation of the cyst require signaling through β-adrenergic receptors and autophagy. The cyst wall of E. invadens contains a chitin-binding lectin. Transcriptional networks associated with encystation were identified in cultures of clinical isolates of E. histolytica that contain encysting organisms. A total of 672 cyst-specific transcripts were identified, including chitin synthetase, some of the transmembrane kinases, and cysteine proteinases, as well as genes involved in transcription, such as those for chromodomain proteins and an myb -like protein, EhMyb.

Metabolism

No tricarboxylic acid cycle or oxidative phosphorylation exists for E. histolytica. Many metabolic enzymes appear to have been acquired through lateral gene transfer from bacteria. Functional mitochondria, or any other compartmentalized energy generation system, are lacking, although there is a remnant of the mitochondria called the mitosome. Glycolysis is the major pathway for adenosine triphosphate generation and occurs in the cytosol. Catabolism of amino acids is a second energy source. Pyruvate ferredoxin oxidoreductase is essential in both glycolysis and amino acid catabolism and also serves to activate metronidazole through its reduction of ferredoxin, which suggests that metronidazole resistance would probably not develop. Energy stores are predominantly in glycogen that occurs in cytoplasmic granules.

Pathways for the biosynthesis of amino acids, with the exception of serine and cysteine, are lacking. Cysteine is of special importance because it is the major thiol. Synthetic enzymes for cholesterol fatty acid and phospholipids also have been identified. In contrast, de novo purine synthesis is lacking. More than 100 transporters have been identified in the genome, but their characterization is incomplete at this time.

Cell Biology

Vesicular trafficking is of paramount importance to the parasite, inasmuch as endocytosis and phagocytosis serve as mechanisms of nutritional uptake. Exocytosis of the cysteine proteinases and ameba pores implicated in virulence and cyst wall components are also important functions of vesicular trafficking, in addition to the more typical roles in transport to and from the endoplasmic reticulum to the Golgi complex and cell surfaces. This complexity of function is reflected in the presence of 91 Rab genes (in comparison with 11 in Saccharomyces cerevisiae ) involved in vesicle fusion. Lysosomal acidification is key in cell amebic trogocytosis and cell killing. N-linked glycosylation of proteins is unusual in that mannose-5 N -acetylglucosamine-2 is the most abundant N-linked glycan, whereas in other eukaryotes this would typically be processed by the addition of branching sugars.

Transmembrane kinases are present and extraordinarily diverse ( Fig. 272.2 ). These kinases number more than 100 and are part of the family of Gal/GalNAc lectin-related proteins that share the extracellular CXXC and CXC motifs of the lectin intermediate (Igl) subunit. The kinase activity (Ser/Thr vs. Tyr) of the transmembrane kinases, as well as their substrates, ligands, and biologic functions, are all yet to be determined. The immediate downstream effectors of the transmembrane kinases are also yet to be identified, although more than 100 protein phosphatases are known to be present in the genome. An unusual feature of these phosphatases is the presence of leucine-rich repeat domains implicated in protein-protein interactions. There are numerous seven-transmembrane G protein–coupled receptors and trimeric G proteins. A G protein–regulated adenylyl cyclase that functions downstream of an adrenergic ligand receptor has been biochemically identified. Cytosolic proteins involved in signal transduction include Ras, Rac, Rab, Rho, and Arf and their exchange factors: EF-hand motif calcium-binding proteins, phosphatidylinositol 3-kinase, and protein kinase C and mitogen-activated protein kinases. It seems likely that this complex signaling system is required for the adaptation of the parasite to its host.

Some of the unique aspects of the cytoskeleton include the lack of dependence on microtubules for motility, which is instead mediated by actin-myosin motors, and the lack of intermediate filament proteins such as keratins, desmin, and vimentin. Polymerization of actin into polymers of F-actin leads to microfilament assembly, which, through the myosin family of molecular motors, provides vesicular transport and motility by means of pseudopods.

Genome Structure

The original genome sequence of E. histolytica was from the HM-1:IMSS strain originally isolated from the rectal biopsy sample of a Mexican man with amebic dysentery. Because of a high content of repetitive DNA, the genome has not been completely assembled but instead exists in approximately 1800 fragments, with the average of 12 sequencing reads per fragment. The incomplete nature of the genome ensures that some genes will be missing and that some misassembly will occur. The E. histolytica genome is estimated to be 14 chromosomes, 8000 genes, and 24 million base pairs of DNA, a size that is approximately comparable with those of Plasmodium and Trypanosoma spp. The average gene length of 389 amino acids, however, is approximately half that of Plasmodium. Approximately 50% of the genome is noncoding DNA, including 20% of the genome that is dedicated to ribosomal RNA genes that are encoded in extrachromosomal circles, and another 10% that encodes transfer RNA genes organized in repetitive linear arrays (probably at chromosome ends). Additional repetitive DNA in the genome includes long interspersed repeated sequence (LINE) and short interspersed repeated sequence (SINE) transposable elements.

The DNA content of Entamoeba appears to vary under different growth conditions. The nuclear DNA content of E. histolytica was shown to be 10-fold higher in axenic (bacteria-free) than xenic culture. In addition, 40-fold increases in DNA content were observed as trophozoites emerged from E. invadens cysts. The most plausible explanation for these observations is that the ploidy of E. histolytica varies through a growth-dependent process of DNA replication without nuclear division.

Control of messenger RNA (mRNA) expression in E. histolytica shares similarities with later branching eukaryotes: The parasite transcribes mRNA monocistronically by using RNA polymerase II under the control of upstream regulatory elements. Thirty percent of genes are predicted to contain introns, and the pre-mRNA splicing machinery includes conserved U2, U4, and U5 small nuclear RNAs. Most of the protein subunits of RNA polymerase II are also conserved, but not all general transcription factors are identified yet. Other differences from yeast and metazoans include the presence of a third core promoter regulatory element for RNA polymerase II, altered histone code, and unique aspects of mRNA silencing through small RNAs.

Adherence

The initial contact of parasite to host is mediated by the parasite's Gal/GalNAc lectin, which binds to carbohydrate determinants on the host. Adherence to human colonic mucin glycoproteins, human neutrophils and erythrocytes, certain bacteria, and a variety of cell culture lines is inhibited by up to 90% by Gal or GalNAc. Blockade of lectin activity with Gal or GalNAc prevents contact-dependent cytolysis, and glycosylation-deficient mutant cell lines lacking terminal Gal/GalNAc residues on N- and O-linked sugars are nearly totally resistant to amebic adherence and cytolytic activity.

The colonic mucin layer of the large intestine is the first receptor encountered by the trophozoite lectin. Binding of the lectin to colonic mucins inhibits Gal/GalNAc and is of very high affinity (dissociation constant of 8.2 × 10 ^–11 M ^–1 ). The mucin layer may protect the host from the parasite's contact-dependent cytolysis by binding to and neutralizing the lectin, while serving as a site of attachment for the parasite to colonize the large bowel. Interaction of trophozoites with colonic mucins appears to be a dynamic process, whereby trophozoites both induce the secretion of colonic mucins and degrade them.

The Gal/GalNAc lectin is composed of a 260-kDa heterodimer of disulfide-linked heavy (170-kDa) and light (35- to 31-kDa) subunits that is noncovalently associated with an intermediate subunit of 150 kDa. The 170-kDa subunit contains a carboxyl-terminal cytoplasmic and transmembrane domain adjacent to a cysteine-rich extracellular domain. Five distinct genes (termed hgl1 to hgl5 ) encoding the lectin's heavy subunit have been identified, sequenced, and shown to be expressed in trophozoites. The sequence of the hgl genes is nearly completely conserved in isolates of E. histolytica from different continents, an important consideration for vaccine design. The carbohydrate recognition domain is located within the cysteine-rich domain of the heavy subunit. The lectin localizes to lipid rafts in the plasma membrane. The lectin can be specifically released from the cell surface through the action of an amebic rhomboid protease, which probably explains earlier observations of both membrane-bound and soluble forms of the lectin.

The Gal/GalNAc lectin appears to have other biologic functions in addition to adherence. Interference with lectin activity blocks chemotaxis in response to tumor necrosis factor-α (TNF-α). The function of the light subunits may include lateral mobility of the Gal/GalNAc lectin, as evidenced by defects in capping of the lectin in amebae silenced for one of the genes that encodes the light subunits ( lgl genes). In an animal model, disruption of lectin function by expression of a dominant negative mutant blocked the ability of the parasite to cause liver abscess. The lectin may serve as an organizing site for cytoplasmic proteins, with cytoplasmic proteins of the amebae that bind directly or indirectly to the lectin, including a thiol-specific antioxidant, spectrin, actin, myosin, talin, calreticulin, and cysteine proteinase 2. The lectin has a role in evasion of serum lytic activity by inhibiting the formation of the complement membrane attack complex through blockade of C5b-9 assembly. As mentioned previously, the lectin also appears to play an initiating role in cyst production.

A recent study has shown that E. histolytica invades through the intestinal epithelium via a unique cell biologic process called trogocytosis.

Cytolysis

Cytolysis occurs after adherence in a process that involves phagocytic ingestion of the host cells in bites, a process called “trogocytosis-like” (see Fig. 272.3 ). Apposition of amebic and target cell plasma membranes, as can be achieved by centrifugation of target cells and amebae into a pellet, does not lead to cytolysis if the amebic lectin is inhibited with Gal/GalNAc or if the target cell lacks Gal and GalNAc on its surface. This is consistent with the fact that lectin not only mediates adherence but also participates in the trogocytosis-like cytolytic event. Anti-lectin monoclonal antibody directed against epitope 1 of the lectin heavy subunit blocked cytotoxicity but not adherence, which implicates the lectin directly in the cytotoxic event. Killing occurs after the lectin engages GalNAc on O-linked target cell surface oligosaccharides: lectin-mediated capping of the O-linked structures (sialic acid-Gal–[sialic acid]–Gal-NAc) could be deduced to mediate killing.

Killing of host cells is not caused by an isolated toxin, inasmuch as parasite extracts have no cytotoxic activity. Cytolysis does require an intact parasite cytoskeleton, as demonstrated by inhibition of Rho, by cytochalasin disruption of the cytoskeleton, and by expression of dominant-negative myosin II. The earliest observed event in a dying cell is a rise in intracellular calcium within seconds of direct contact by an amebic trophozoite; this event is associated with membrane blebbing. Extracellular ethylenediaminetetraacetic acid and treatment of the target cells with the slow sodium-calcium channel blockers verapamil and bepridil significantly reduce amebic killing of target cells in suspension. Isolation of amebic pore-forming proteins similar in function to pore-forming proteins of the immune system has been reported by a number of investigators. A purified 5-kDa amebapore and a synthetic peptide based on the sequence of its third amphipathic α-helix have cytolytic activity for nucleated cells at high concentrations (10–100 µM). Silencing of amebapore A blocked the ability of amebae to release monolayer tissue culture cells from a plastic well, although cell death was not specifically measured. The optimal pH of amebapore is 5.3, and amebapore is inactive at a pH of 7, which may be of some significance in view of the inhibition of cytotoxicity with weak base treatment of amebae. Interestingly, no DNA degradation was observed in cells lysed in vitro by the purified amebapore, which is suggestive of a different mechanism of cell killing by the purified amebapore than by the intact parasite.

Cells killed by the parasite undergo nuclear chromatin condensation, membrane blebbing, and internucleosomal DNA fragmentation. There is evidence of a nonclassical mechanism of apoptotic killing by E. histolytica. Overexpression of the Bcl-2 protein that inhibits apoptosis caused by a variety of cellular stresses (e.g., serum starvation, ultraviolet radiation) did not prevent murine cell DNA fragmentation after exposure to E. histolytica. Furthermore, E. histolytica caused hepatocyte apoptosis in mice deficient in the Fas/Fas ligand and tumor necrosis factor receptor-1 signaling pathways. Caspase 8–deficient cells, resistant to killing by Fas ligand, were readily killed by E. histolytica. Caspase 8–deficient cells treated with a caspase 9 inhibitor (Ac-LEHD-fmk) (at a level sufficient to inhibit apoptosis through etoposide) were readily killed as well. Together, these data suggest that E. histolytica initiates host cell apoptosis by directly activating the host cell's distal apoptotic machinery. Caspase 3 was activated within minutes of E. histolytica adherence, and the caspase 3 inhibitor Ac-DEVD-CHO at 100 µmol (sufficient to block killing through actinomycin D) blocked E. histolytica killing, as measured both by DNA fragmentation and by Cr51 release; this outcome indicates that both apoptotic death phenotype and necrosis were necessary. In conclusion, amebic killing of the host is a result of parasite nibbling (trogocytosis) on the human cell, which results in apoptosis in the host cell at the level of caspase 3 activation.

Phagocytosis

In multicellular organisms, phagocytosis is the final step in the apoptotic pathway and serves to limit inflammation by preventing spillage of toxic intracellular contents of dead cells. Although amebic killing of cells by contrast involves phagocytosis followed by death, phagocytosis could similarly limit the host inflammatory response and enable E. histolytica to establish a persistent infection. Inhibition of endocytosis by galactose and phosphatidylserine is additive, consistent with the Gal/GalNAc lectin and an as-yet unidentified phosphatidylserine receptor acting as coreceptors for ingestion. Understanding the molecular mechanisms of engulfment of and subsequent death of the host promises to reveal much about pathogenesis, inasmuch as amebae that are defective in phagocytosis are also defective in virulence.

Role of Bacteria

The effect of the gut bacteria on the biologic properties of E. histolytica may be profound. As mentioned previously, the genome content of Entamoeba is lower when the parasite is grown in the presence of bacteria. In addition, amebae cultured with bacteria are better able to destroy monolayers of tissue culture cells and resist oxidative stress. Recently it was shown that the gut microbiome is enriched in Prevotella copri in children with amebic diarrhea, and that antibiotic-induced alterations of the gut microbiome act via alterations in neutrophil trafficking to the gut in the mouse model of amebic colitis. Thus ultimate susceptibility to amebic colitis may in part be due to dysbiosis limiting neutrophil migration to the colon.

Cysteine Proteinases

E. histolytica encodes at least 44 genes that are cysteine proteinases, some of which are membrane bound and others predicted to be soluble. These activities are implicated in a number of potentially important activities and include degradation of colonic mucin glycoproteins, digestion of hemoglobin and villin, inactivation of interleukin (IL)-18, and digestion of extracellular matrix.

Role of Leptin in Host Resistance

Amebiasis is more common in malnourished children (a physiologic state of leptin deficiency). This suggests that the nutritional hormone leptin could play a protective role in amebiasis. In fact, a mutation in the leptin receptor was discovered that was associated with amebiasis susceptibility in children. The mutation is a nonconservative substitution (Q223R) in the extracellular cytokine receptor homology domain 1. The mutation accounted for the majority of susceptibility to amebiasis in children because it conferred a 3.9-fold greater risk of intestinal amebiasis and was present in almost half the children. The purely nutritional role of leptin did not explain protection because the Q223R mutation was not associated with children's nutritional status. Experiments in mice validated the human study, with leptin-deficient ( ob / ob ), leptin receptor–deficient ( db / db ), and 223R leptin receptor knock-in mice highly susceptible to intestinal E. histolytica infection. The site of leptin action was localized to the gut, because an intestinal epithelial cell–specific deletion of the leptin receptor rendered mice susceptible, whereas lack of the leptin receptor in the central nervous system or bone marrow–derived cells did not. Leptin receptor signal transducer and activator of transcription 3 (STAT3) and Src homology 2 domain–containing phosphatase 2 (SHP2) signaling were required for protection in vivo because susceptibility was conferred by mutation of tyrosine 985 or 1138, which mediate leptin signaling through the SHP2/extracellular signal–regulated kinase and STAT3 pathways, respectively. The importance of leptin signaling in prevention of amebic killing of the host could even be seen in single-cell studies, where transfection of human embryonic kidney cells with the leptin receptor rendered them resistant to amebic killing.

Macrophage Migration Inhibitory Factor and Inflammation

Colonic inflammation is a hallmark of E. histolytica infection, hence the term amebic colitis. E. histolytica, like several pathogenic protozoans, secretes a protein homologue of the proinflammatory cytokine macrophage migration inhibitory factor (MIF). A recent study found a positive correlation between E. histolytica MIF levels and intestinal inflammation in persons with amebic colitis. In the same study, the researchers demonstrated a causal relationship between E. histolytica MIF and gut inflammation using cellular and mouse models of amebic colitis. E. histolytica MIF-induced mucosal inflammation resulted in increased matrix metalloproteinases (MMPs) production. MMPs are enzymes that break down extracellular matrix proteins and promote cell migration. MMPs were shown recently to promote E. histolytica tissue invasion in a human colon explant model. This potential link between E. histolytica MIF, inflammation, and tissue invasion might explain why a parasite would encode a proinflammatory cytokine.