Crystal structure of the cysteine protease inhibitor 2 from Entamoeba histolytica: functional convergence of a common protein fold

Cysteine proteases (CP) are key pathogenesis and virulence determinants of protozoan parasites. Entamoeba histolytica contains at least 50 cysteine proteases; however, only three (EhCP1, EhCP2 and EhCP5) are responsible for approximately 90% of the cysteine protease activity in this parasite. CPs are expressed as inactive zymogens. Because the processed proteases are potentially cytotoxic, protozoan parasites have developed mechanisms to regulate their activity. Inhibitors of cysteine proteases (ICP) of the chagasin-like inhibitor family (MEROPS family I42) were recently identified in bacteria and protozoan parasites. E. histolytica contains two ICP-encoding genes of the chagasin-like inhibitor family. EhICP1 localizes to the cytosol, whereas EhICP2 is targeted to phagosomes. Herein, we report two crystal structures of EhICP2. The overall structure of EhICP2 consists of eight β-strands and closely resembles the immunoglobulin fold. A comparison between the two crystal forms of EhICP2 indicates that the conserved BC, DE and FG loops form a flexible wedge that may block the active site of CPs. The positively charged surface of the wedge-forming loops in EhICP2 contrasts with the neutral surface of the wedge-forming loops in chagasin. We postulate that the flexibility and positive charge observed in the DE and FG loops of EhICP2 may be important to facilitate the initial binding of this inhibitor to the battery of CPs present in E. histolytica.     

The unusual transhydrogenase of Entamoeba histolytica

We have expressed and purified a protein fragment from Entamoeba histolytica. It catalyses transhydrogenation between analogues of NAD(H) and NADP(H). The characteristics of this reaction resemble those of the reaction catalysed by a complex of the NAD(H)- and NADP(H)-binding subunits of proton-translocating transhydrogenases from bacteria and mammals. It is concluded that the complete En. histolytica protein, which, along with similar proteins from other protozoan parasites, has an unusual subunit organisation, is also a proton-translocating transhydrogenase. The function of the transhydrogenase, thought to be located in organelles which do not have the enzymes of oxidative phosphorylation, is not clear.     

Entamoeba histolytica and/or Entamoeba dispar: infection frequency in HIV+/AIDS patients in Mexico city

The objective of this work was to evaluate the frequency of Entamoeba histolytica/Entamoeba dispar intestinal infection in HIV+/AIDS subjects and their HIV- close relatives or sexual partners. Enteric parasites were investigated in stool samples by microscopic examination and E. histolytica and E. dispar were identified by PCR. We found by microscopic analysis in HIV+/AIDS group that the E. histolytica/E. dispar complex was present in 5.9% of the members, while in the HIV- group was 2.9%. With PCR we found that the E. histolytica prevalence was 25.3% in the HIV+/AIDS group and 18.5% in the HIV-group. The difference in the results obtained with the microscopic and PCR is due to the different sensibility of the procedures. Besides, we found patients who were infected with E. histolytica in both groups were asymptomatic cyst passers. Our results suggest that E. histolytica strains prevalent in the studied community appear to be of low pathogenic potential.     

Sulfate activation in mitosomes plays an important role in the proliferation of Entamoeba histolytica

Mitochondrion-related organelles, mitosomes and hydrogenosomes, are found in a phylogenetically broad range of organisms. Their components and functions are highly diverse. We have previously shown that mitosomes of the anaerobic/microaerophilic intestinal protozoan parasite Entamoeba histolytica have uniquely evolved and compartmentalized a sulfate activation pathway. Although this confined metabolic pathway is the major function in E. histolytica mitosomes, their physiological role remains unknown. In this study, we examined the phenotypes of the parasites in which genes involved in the mitosome functions were suppressed by gene silencing, and showed that sulfate activation in mitosomes is important for sulfolipid synthesis and cell proliferation. We also demonstrated that both Cpn60 and unusual mitochondrial ADP/ATP transporter (mitochondria carrier family, MCF) are important for the mitosome functions. Immunoelectron microscopy demonstrated that the enzymes involved in sulfate activation, Cpn60, and mitochondrial carrier family were differentially distributed within the electron dense, double membrane-bounded organelles. The importance and topology of the components in E. histolytica mitosomes reinforce the notion that they are not "rudimentary" or "residual" mitochondria, but represent a uniquely evolved crucial organelle in E. histolytica.     

Effect of phosphatidylcholine-cholesterol liposomes on Entamoeba histolytica virulence

Trophozoites of Entamoeba histolytica HM-1:IMSS become less virulent after long-term maintenance in axenic cultures. The factors responsible for the loss of virulence during in vitro cultivation remain unclear. However, it is known that in vitro cultivation of amoeba in culture medium supplemented with cholesterol restores their virulence. In this study, we analyzed the effect of adding phosphatidylcholine-cholesterol (PC-Chol) liposomes to the culture medium and evaluated the effect of this lipid on various biochemical and biological functions of E. histolytica HM-1:IMSS in terms of its virulence. The addition of PC-Chol liposomes to the culture medium maintained the virulence of these parasites against hamster liver at the same level as the original virulent E. histolytica strain, even though these amoebae were maintained without passage through hamster liver for 18 months. The trophozoites also showed increased endocytosis, erythrophagocytosis, and carbohydrate residue expression on the amoebic surface. Protease activities were also modified by the presence of cholesterol in the culture medium. These findings indicate the capacity of cholesterol to preserve amoeba virulence and provide an alternative method for the maintenance of virulent E. histolytica trophozoites without the need for in vivo procedures.     

A role for a galactose lectin and its ligands during encystment of Entamoeba

In the life cycle of Entamoeba parasites alternate between the colon-dwelling trophozoite and the infectious cyst forms. The physiologic stimuli that trigger differentiation of trophozoites into cysts remain undefined. On the surface of the human-infecting Entamoeba, parasites express a galactose/N-acetylgatactosamine (gal/galNAc)-binding lectin, which plays demonstrated roles in contact-dependent lysis of target cells and resistance to host complement. Using a reptilian parasite, Entamoeba invadens, to study cyst formation in vitro, we found that efficient encystation was dependent on the presence of gal-terminated ligands in the induction medium. Precise concentration ranges of several gal-terminated ligands, such as asialofetuin, gal-bovine serum albumin (gal-BSA), and mucin, functioned in encystation medium to stimulate differentiation. Greater than 10 mM levels of free gal inhibited the amoeba aggregation that precedes encystation and prevented formation of mature cysts. Inhibitory levels of gal also prevented the up-regulation of genes which normally occurs at 24 h of encystation. The surface of Entamoeba invadens was found to express a gal lectin which has a heterodimeric structure similar to that of Entamoeba histolytica. The 30 kDa light subunit (LGL) of the E. invadens lectin is similar in overall size and sequence to the LGL of E. histolytica. The heavy subunits, however, differ in size, have an identical spacing of cysteines in their extracellular domains, and have highly conserved C-terminal transmembrane and cytoplasmic domains. These results suggest a new role for the Entamoeba gal lectins in monitoring the concentrations of gal ligands in the colon and contributing to stimuli that induce encystment.     

Acetylation of the Entamoeba histone H4 N-terminal domain is influenced by short-chain fatty acids that enter trophozoites in a pH-dependent manner

Treatment of higher eukaryotic cells with short-chain fatty acids (SCFA) such as butyrate causes decreased levels of histone deacetylase (HDAC) activity and hyperacetylation of histones, and thereby affects gene expression, cell growth and differentiation. Entamoeba parasites encounter high levels of SCFA in the host colon, and in vitro these compounds allow trophozoite stage parasites to multiply but prevent their differentiation into infectious cysts. The Entamoeba invadens IP-1 histone H4 protein has an unusual number of lysines in its N-terminus, and these become hyperacetylated in trophozoites exposed to the HDAC inhibitors trichostatin A (TSA) or HC-toxin, but not in trophozoites exposed to butyrate. We have now found that several other commonly studied isolates of Entamoeba parasites also have an extended set of histone H4 acetylation sites that become hyperacetylated in response to TSA, but hypoacetylated in response to butyrate, suggesting an unusual sensitivity of this parasite's histone modifying enzymes to SCFA. Butyrate was found to enter trophozoites in a pH-dependent manner consistent with diffusive entry of the un-ionised form of the fatty acid into the amoebae. Transit of the Entamoeba organism through areas of the host intestine with distinct pH and SCFA concentrations would therefore result in very different levels of SCFA within the parasite. Entamoeba appears to have acquired unique alterations of its histone acetylation mechanism that may allow for its growth in the presence of varying amounts of the bacterial fermentation products.     

Characterization of the gene encoding serine acetyltransferase, a regulated enzyme of cysteine biosynthesis from the protist parasites Entamoeba histolytica and Entamoeba dispar. Regulation and possible function of the cysteine biosynthetic pathway in Entamoeba.

The enteric protist parasites Entamoeba histolytica and Entamoeba dispar possess a cysteine biosynthetic pathway, unlike their mammalian host, and are capable of de novo production of L-cysteine. We cloned and characterized cDNAs that encode the regulated enzyme serine acetyltransferase (SAT) in this pathway from these amoebae by genetic complementation of a cysteine-auxotrophic Escherichia coli strain with the amoebic cDNA libraries. The deduced amino acid sequences of the amoebic SATs exhibited, within the most conserved region, 36-52% identities with the bacterial and plant SATs. The amoebic SATs contain a unique insertion of eight amino acids, also found in the corresponding region of a plasmid-encoded SAT from Synechococcus sp., which showed the highest overall identities to the amoebic SATs. Phylogenetic reconstruction also revealed a close kinship of the amoebic SATs with cyanobacterial SATs. Biochemical characterization of the recombinant E. histolytica SAT revealed several enzymatic features that distinguished the amoebic enzyme from the bacterial and plant enzymes: 1) inhibition by L-cysteine in a competitive manner with L-serine; 2) inhibition by L-cystine; and 3) no association with cysteine synthase. Genetically engineered amoeba strains that overproduced cysteine synthase and SAT were created. The cysteine synthase-overproducing amoebae had a higher level of cysteine synthase activity and total thiol content and revealed increased resistance to hydrogen peroxide. These results indicate that the cysteine biosynthetic pathway plays an important role in antioxidative defense of these enteric parasites.     

Impact of solar radiation in disinfecting drinking water contaminated with Giardia duodenalis and Entamoeba histolytica/dispar at a point-of-use water treatment

Aims:  To determine the impact of natural sunlight in disinfecting water contaminated with cysts of Giardia duodenalis and Entamoeba histolytica/dispar using plastic containers.
Methods and Results:  Known quantities of Giardia duodenalis and Entamoeba histolytica/dispar cysts in sterile water were exposed to the sun. Containers were made of polyethylene terephthalate, eight painted black on one side, one not painted and another cut open at the top and the last was a high density polypropylene container. Viability testing was performed using vital and fluorescent dyes. The same assays were conducted under cloudy conditions. Thermal control tests were also performed using heat without ultra violet light from the sun. Results show that 99·9% of parasites was inactivated when water temperatures reached 56°C after sunlight exposure.
Conclusion:  Both solar radiation and heat produced by the sun have a synergistic effect in killing cysts of Giardia duodenalis and Entamoeba histolytica/dispar when temperatures rise above 50°C, with complete death at 56°C, using painted 2-l PET containers.
Significance and Impact of the Study:  Solar disinfection system using PET containers painted black on one side can be used to disinfect water against Giardia duodenalis and Entamoeba histolytica/dispar using natural sunlight.     

Entamoeba histolytica: observations on metabolism based on the genome sequence

The sequencing of the genome of Entamoeba histolytica has allowed a reconstruction of its metabolic pathways, many of which are unusual for a eukaryote. Based on the genome sequence, it appears that amino acids may play a larger role than previously thought in energy metabolism, with roles in both ATP synthesis and NAD regeneration. Arginine decarboxylase may be involved in survival of E. histolytica during its passage through the stomach. The usual pyrimidine synthesis pathway is absent, but a partial pyrimidine degradation pathway could be part of a novel pyrimidine synthesis pathway. Ribonucleotide reductase was not found in the E. histolytica genome, but it was found in the close relatives Entamoeba invadens and Entamoeba moshkovskii, suggesting a recent loss from E. histolytica. The usual eukaryotic glucose transporters are not present, but members of a prokaryotic monosaccharide transporter family are present.     

The salutary effect of milk on amoebiasis and its reversal by iron.

Observations among milk-drinking African nomads showed an unusual freedom from infection with Entamoeba histolytica compared with similar nomads taking a mixed diet. A controlled study among Maasai pastoralists showed that the administration of iron to correct their dietary iron deficiency sharply increased their susceptibility to amoebiasis. Examination of the milk of their Zebu cattle showed that it not only had a concentration of iron below the minimum necessary for the growth of E histolytica but also contained partly saturated lactoferrin and transferrin, which may actively compete with the parasite in the colon for ambient iron. These observations suggest the possibility of a long-standing ecological compromise between nomads, their milk diet, and E histolytica.     

Entamoeba lysyl-tRNA synthetase contains a cytokine-like domain with chemokine activity towards human endothelial cells

Immunological pressure encountered by protozoan parasites drives the selection of strategies to modulate or avoid the immune responses of their hosts. Here we show that the parasite Entamoeba histolytica has evolved a chemokine that mimics the sequence, structure, and function of the human cytokine HsEMAPII (Homo sapiens endothelial monocyte activating polypeptide II). This Entamoeba EMAPII-like polypeptide (EELP) is translated as a domain attached to two different aminoacyl-tRNA synthetases (aaRS) that are overexpressed when parasites are exposed to inflammatory signals. EELP is dispensable for the tRNA aminoacylation activity of the enzymes that harbor it, and it is cleaved from them by Entamoeba proteases to generate a standalone cytokine. Isolated EELP acts as a chemoattractant for human cells, but its cell specificity is different from that of HsEMAPII. We show that cell specificity differences between HsEMAPII and EELP can be swapped by site directed mutagenesis of only two residues in the cytokines' signal sequence. Thus, Entamoeba has evolved a functional mimic of an aaRS-associated human cytokine with modified cell specificity.     

Effect of dinitroaniline herbicides on the growth of Entamoeba histolytica

The effect of the dinitroaniline herbicides oryzalin and trifluralin on the growth of Entamoeba histolytica was examined. Oryzalin inhibited the growth of E. histolytica strain HM-1:IMSS. Trifluralin was less effective than oryzalin for this parasite. Entamoeba histolytica was more resistant to these dinitroanilines than other parasitic protozoa examined so far, including Leishmania spp., Trypanosoma brucei, Plasmodium falciparum, Toxoplasma gondii, and Cryptosporidium parvum. Colchicine, a potent microtubule inhibitor of animal cells, was much less effective for E. histolytica, even at very high concentrations. A reptilian parasite, Entamoeba invadens strain IP-1, examined for comparison, was more resistant to these dinitroanilines than E. histolytica. Accumulation of E. histolytica trophozoites in mitosis was observed after culture in 100 microM oryzalin. The inhibitory effect of oryzalin on the growth of E. histolytica trophozoites was abrogated by removal of the drug after exposure to 100 microM for 2 days. In parallel to the recovery of growth after removal of the drug, the percentage of trophozoites in mitosis was reduced to a normal level. The results indicate that treatment of trophozoites with oryzalin arrests mitosis and that its effect is reversible. Therefore, oryzalin is a useful tool for studies relating to the cell cycle of this parasite.     

Unexpected diversity and differential success of DNA transposons in four species of entamoeba protozoans

We report the first comprehensive analysis of transposable element content in the compact genomes (approximately 20 Mb) of four species of Entamoeba unicellular protozoans for which draft sequences are now available. Entamoeba histolytica and Entamoeba dispar, two human parasites, have many retrotransposons, but few DNA transposons. In contrast, the reptile parasite Entamoeba invadens and the free-living Entamoeba moshkovskii contain few long interspersed elements but harbor diverse and recently amplified populations of DNA transposons. Representatives of three DNA transposase superfamilies (hobo/Activator/Tam3, Mutator, and piggyBac) were identified for the first time in a protozoan species in addition to a variety of members of a fourth superfamily (Tc1/mariner), previously reported only from ciliates and Trichomonas vaginalis among protozoans. The diversity of DNA transposons and their differential amplification among closely related species with similar compact genomes are discussed in the context of the biology of Entamoeba protozoans.     

A simple micropore system for experimental studies on trichomonad parasites

A simple leak-free micropore chamber containing protozoan parasite species was implanted subcutaneously on the back of hamsters and evaluated for viability and multiplication of protozoan parasites. Trophozoites of defined strains of Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, and Tritrichomonas foetus were used; their survival and multiplication in the chambers formed the basis of evaluation. Entamoeba histolytica and G. lamblia did not survive more than 6 hr and succumbed due to cellular adhesion. Trichomonas vaginalis and T. foetus survived 3 and 6 days and multiplied a maximum of 3.6 and 26 times, respectively. This indicated that exchange of body fluids and cells needed for the survival and multiplication of trichomonads readily occurs. This preliminary observation showed that micropore chambers may be useful for chemotherapeutic and immunological studies on trichomonads in ectopic sites.     

Modulation of host cell intracellular Ca2+

During the course of evolution, protozoan parasites have developed strategies to subvert the immune response of their host in order to multiply, reproduce and survive. One of these inherited strategies is their capacity to modulate the host cell transductional mechanisms in their favor. Alteration of host cells Ca(2-) homeostasis following interaction and/or invasion by protozoan parasites such as Leishmania donovani, Trypanosoma cruzi, Plasmodium falciparum or Entamoeba histolytica has been reported. There is direct evidence that such disturbances are responsible for pathogenesis observed during parasitic infections. This homeostatic imbalance of Ca(2+) in the host cell is an early inducible event whose underlying mechanisms needs further investigation, as discussed here by Martin Olivier.