Entamoeba histolytica and Giardia lamblia: effects of cysteine and oxygen tension on trophozoite attachment to glass and survival in culture media

Attachment of Entamoeba histolytica and of Giardia lamblia trophozoites to glass was monitored during the culture cycle. Attachment of each parasite was greatest during the exponential phase of axenic growth. The effects of l-cysteine upon the kinetics of attachment of trophozoites to glass were determined quantitatively. Attachment in complex growth media required cysteine, even under N₂, atmosphere. With cysteine, the rates of attachment were greatest for the first 2 hr, then continued more slowly. The numbers of attached trophozoites decreased immediately upon exposure to medium without cysteine. The role of cysteine in protecting trophozoites of both species from the lethal effects of oxygen was assessed using clonal growth in agar or agarose medium to determine viability following exposure to varying oxygen tensions in liquid medium. Cysteine was required for viability of trophozoites. Without cysteine, decreasing the oxygen tension prolonged survival. Under increased oxygen tension, cysteine delayed the onset of exponential killing. Although it has no thiol reducing group, l-cystine similarly protected E. histolytica.     

The Relation between Photosynthesis, Respiration, and Crassulacean Acid Metabolism in Leaf Slices of Aloe arborescens Mill

Leaves and leaf slices from Aloe arborescens Mill. were used to study the interrelations between Crassulacean acid metabolism, photosynthesis, and respiration. Oxygen exchange of leaf slices was measured polarographically. It was found that the photosynthetic utilization of stored malic acid resulted in a net evolution of oxygen. This oxygen production, and the decrease in acid content of the leaf tissue, were completely inhibited by amytal, although the rate of respiratory oxygen uptake was hardly affected by the presence of this inhibitor of mitochondrial electron transport. Other poisons of respiration (cyanide) and of the tricarboxylic acid cycle (trifluoroacetate, 2-diethyl malonate) also were effective in preventing acid-dependent oxygen evolution. It is concluded that the mobilization of stored acids during light-dependent deacidification of the leaves depends on the operation of the tricarboxylic acid cycle and of the electron transport of the mitochondria.     

Attachment of Entamoeba histolytica to Glass in a Defined Maintenance Medium: Specific Requirement for Cysteine and Ascorbic Acid*

SYNOPSIS. Cysteine and ascorbic acid were previously shown to be required by Entamoeba histolytica trophozoites for attachment to glass, elongation, and ameboid movement as well as for short-term (12–24 h) survival in a balanced salt solution containing bovine serum albumin and a vitamin solution (Maintenance Medium 1). If the only function of cysteine and ascorbate was to decrease the redox potential, other reducing agents should be effective. However, the requirement for cysteine in the presence of ascorbic acid was highly specific. Equally effective were D- and L-cysteine; however, of many other compounds tested, only thioglycolic acid, ascorbic acid, or L-cystine (in decreasing order) were somewhat active. Under N₂ atmosphere, cysteine and ascorbic acid were still required, although their concentrations could be halved. The ability to attach in the maintenance medium was irreversibly lost after only 5 min of cysteine-ascorbic acid deprivation; however, there was no decrease in viability when the amebae were transferred to growth medium within 30 min. Cysteine thiol groups in the medium were oxidized rapidly regardless of the concentration of ascorbic acid or the presence of amebae; however, ascorbic acid prolonged attachment of amebae.     

Protection against murine intestinal amoebiasis induced by oral immunization with the 29 kDa antigen of Entamoeba histolytica and cholera toxin

Entamoeba histolytica antigens recognized by salivary IgA from infected patients include the 29 kDa antigen (Eh29), an alkyl hydroperoxide reductase. Here, we investigate the potential of recombinant Eh29 and an Eh29-cholera toxin subunit B (CTxB) fusion protein to confer protection against intestinal amoebiasis after oral immunization. The purified Eh29-CTxB fusion retained the critical ability to bind ganglioside GM₁, as determined by ELISA. Oral immunization of C3H/HeJ mice with Eh29 administered in combination with a subclinical dose of whole cholera toxin, but not as an Eh29-CTxB fusion, induced elevated levels of intestinal IgA and serum IgG anti-Eh29 antibodies that inhibited trophozoites adherence to MDCK cell monolayers. The 80% of immunized mice seen to develop IgA and IgG immune responses showed no evidence of infection in tissue sections harvested following intracecal challenge with virulent E. histolytica trophozoites. These results suggest that Eh29 is capable of inducing protective anti-amoebic immune responses in mice following oral immunization and could be used in the development of oral vaccines against amoebiasis.     

Entamoeba histolytica and Entamoeba invadens: Effects of temperature and oxygen tension on growth and survival

The temperature ranges for axenic growth of Entamoeba histolytica strain HM-1 and Entamoeba invadens strain 165 clone II in TYI-S-33 medium were: 32 to 40 C for E. histolytica with an optimum of 35.5 to 37 C; whereas the range for E. invadens 165 II was 20 to 30 C, optimum 24 to 27 C. Growth of strain HM-1 at 40 C was dependent upon the cell density of the culture used as a source of the inoculum. Clonal growth in agar was used to assay survival of Entamoeba spp. trophozoites after exposure to deleterious physical conditions. The lowest temperature for thermal killing of E. histolytica HM-1 was 41.5 C and for E. invadens 165, 35.5 C. Both were killed rapidly at 42 C, but slowly at 0 C. Killing of E. histolytica HM-1 trophozoites by exposure to increased oxygen tensions was a function of temperature and time. At 24 C and 35.5 C, there was little loss of viability during the first 7 hr exposure, then killing was quite rapid. The cells were killed sooner if the medium was preexposed to air. In contrast, at 0 C there was less killing by oxygen. E. invadens 165 II appeared to be more oxygen sensitive at 24 than at 0 C. Other E. histolytica strains tested were similar to HM-1 in their responses to temperature and air.     

Resveratrol Induces Apoptosis-Like Death and Prevents In Vitro and In Vivo Virulence of Entamoeba histolytica

Entamoeba histolytica causes amoebiasis, an infection that kills 100,000 individuals each year. Metronidazole and its derivatives are currently used against this protozoan, but these drugs present adverse effects on human health. Here, we investigated the effect of resveratrol (a natural compound) on E. histolytica trophozoites viability, as well as its influence on the parasite virulence. Trophozoites growth was arrested by 72 μM resveratrol and the IC₅₀ was determined as 220 μM at 48 h. Cells appeared smaller, rounded and in clusters, with debris-containing vacuoles and with abnormally condensed chromatin. Resveratrol triggered reactive oxygen species production. It caused lipid peroxidation and produced phosphatidylserine externalization and DNA fragmentation this latter evidenced by TUNEL assays. It also provoked an increase of intracellular Ca²⁺ concentration, activated calpain and decreased superoxide dismutase activity, indicating that an apoptosis-like event occurred; however, autophagy was not detected. Cytopathic activity, phagocytosis, encystment and in vivo virulence were diminished dramatically by pre-incubation of trophozoites with resveratrol, evidencing that resveratrol attenuated the trophozoite virulence in vitro. Interestingly, after the inoculation of virulent trophozoites, animals treated with the drug did not develop or developed very small abscesses. Our findings propose that resveratrol could be an alternative to contend amoebiasis.     

Formation of oxidised (OX) proteins in Entamoeba histolytica exposed to auranofin and consequences on the parasite virulence

Metronidazole (MNZ), the first line drug for amoebiasis and auranofin (AF), an emerging antiprotozoan drug, are both inhibiting Entamoeba histolytica thioredoxin reductase. The nature of oxidised proteins (OXs) formed in AF‐ or MNZ‐treated E. histolytica trophozoites is unknown. In order to fill this knowledge gap, we performed a large‐scale identification and quantification of the OXs formed in AF‐ or MNZ‐treated E. histolytica trophozoites using resin‐assisted capture coupled to mass spectrometry (MS). We detected 661 OXs in MNZ‐treated trophozoites and 583 OXs in AF‐treated trophozoites. More than 50% of these OXs were shared, and their functions include hydrolases, enzyme modulators, transferases, nucleic acid binding proteins, oxidoreductases, cytoskeletal proteins, chaperones, and ligases. Here, we report that the formation of actin filaments (F‐actin) is impaired in AF‐treated trophozoites. Consequently, their erythrophagocytosis, cytopathic activity, and their motility are impaired. We also observed that less than 15% of OXs present in H₂O₂‐treated trophozoites are also present in AF‐ or MNZ‐treated trophozoites. These results strongly suggest that the formation of OXs in AF‐ or MNZ‐treated trophozoites and in H₂O₂‐treated trophozoites occurred by two different mechanisms.     

Antineoplastic kinase inhibitors: A new class of potent anti-amoebic compounds

Entamoeba histolytica is a protozoan parasite which infects approximately 50 million people worldwide, resulting in an estimated 70,000 deaths every year. Since the 1960s E. histolytica infection has been successfully treated with metronidazole. However, drawbacks to metronidazole therapy exist, including adverse effects, a long treatment course, and the need for an additional drug to prevent cyst-mediated transmission. E. histolytica possesses a kinome with approximately 300–400 members, some of which have been previously studied as potential targets for the development of amoebicidal drug candidates. However, while these efforts have uncovered novel potent inhibitors of E. histolytica kinases, none have resulted in approved drugs. In this study we took the alternative approach of testing a set of twelve previously FDA-approved antineoplastic kinase inhibitors against E. histolytica trophozoites in vitro. This resulted in the identification of dasatinib, bosutinib, and ibrutinib as amoebicidal agents at low-micromolar concentrations. Next, we utilized a recently developed computational tool to identify twelve additional drugs with human protein target profiles similar to the three initial hits. Testing of these additional twelve drugs led to the identification of ponatinib, neratinib, and olmutinib were identified as highly potent, with EC₅₀ values in the sub-micromolar range. All of these six drugs were found to kill E. histolytica trophozoites as rapidly as metronidazole. Furthermore, ibrutinib was found to kill the transmissible cyst stage of the model organism E. invadens. Ibrutinib thus possesses both amoebicidal and cysticidal properties, in contrast to all drugs used in the current therapeutic strategy. These findings together reveal antineoplastic kinase inhibitors as a highly promising class of potent drugs against this widespread and devastating disease.     

SPORE GERMINATION AND CARBON METABOLISM IN FUSARIUM SOLANI. II. ENDOGENOUS RESPIRATION IN RELATION TO GERMINATION

Cochrane , V. W., Jean C. Cochrane , C. b . Collins , and F. G. Serafin . (Wesleyan U., Middletown, Conn.) Spore germination and carbon metabolism in Fusarium solani. II. Endogenous respiration in relation to germination. Amer. Jour. Bot. 50(8): 806–814. Illus. 1963.—Endogenous oxygen uptake by ungerminated macroconidia of Fusarium solani f. phaseoli is more than doubled by exogenous ammonium ion and is increased about 7-fold by germination. The respiratory quotient is halved by the provision of ammonia, which has essentially no effect on the endogenous formation of carbon dioxide. Stimulation by azide and 2,4-dinitrophenol suggests that the supply of phosphate acceptors regulates the rate of endogenous respiration. Mercurials poison the endogenous respiration, cyanide accelerates it, and iodoacetate, arsenite, fluoride, and chelating agents have little effect. Respiration is little affected by changes in pH, external phosphate, oxygen concentration, and spore density, within the limits tested. Spore lipid concentration is increased by cultivation in a high-glucose medium, but this variation in lipid content of spores docs not affect the endogenous Qo₂, nor does a high lipid content abolish the requirement for exogenous carbon for germination. Lipid utilization during endogenous respiration accounts for 37% of the loss in dry weight; lipid is also utilized during germination, but such utilization amounts to only about 5% of the carbon requirement. Neither mannitol nor nitrogenous compounds are significant substrates of endogenous respiration. The oxidation of the exogenous substrates tested does not measurably affect the concomitant rate of endogenous respiration. It is proposed that endogenous respiration can contribute to the synthetic processes of spore germination, although quantitatively insufficient to support germination without exogenous carbon. It is also questioned whether the respiratory quotient is an adequate index of the substrate of endogenous respiration.     

The effect of light on the tricarboxylic Acid cycle in green leaves: I. Relative rates of the cycle in the dark and the light

Excised green leaves of mung bean (Phaseolus aureus L. var. Mungo) were used to determine the effect of light on the rate of endogenous respiration via the tricarboxylic acid cycle. Illumination with white light at an intensity of 0.043 gram calories cm⁻²min⁻¹ (approximately 8600 lux) of visible radiation (400-700 nm) gave a rate of apparent photosynthesis, measured as net CO₂ uptake, of 21 mg CO₂ dm⁻²hr⁻¹ which was about 11-fold greater than the rate of dark respiration. The feeding of ¹⁴CO₂ or ¹⁴C-labeled acids of the tricarboxylic acid cycle in the dark for 2 hours was established as a suitable method for labeling mitochondrial pools of cycle intermediates.     

Localisation of thiamine pyrophosphatase within the cytoplasmic fine structure of trophozoites of Entamoeba histolytica and E. invadens

McCaul T.F. and Bird R.G. 1978. Localisation of thiamine pyrophosphatase within the cytoplasmic fine structure of trophozoites of Entamoeba histolytica and E. invadens. International Journal for Parasitology8: 501–506. The distribution of thiamine pyrophosphatase (TPPase) activity was studied in both formaldehyde and glutaraldehyde fixed trophozoites of Entamoeba histolytica and E. invadens. The activity was localised within certain vacuoles. No dense deposits for TPPase activity were seen in the small vesicles, elongated smooth-walled lacunae equated with endoplasmic reticulum, or the nucleus. The demonstration of small vesicles surrounding the larger vacuoles indicated that the Golgi-like vacuoles might be involved in the production of cell coat materials and primary lysosomes.     

Entamoeba histolytica: Virulence enhancement of isoenzyme-stable parasites

Pathogenic Entamoeba histolytica isolated from patients with clinical amoebiasis can be differentiated from nonpathogenic E. histolytica obtained from asymptomatic carriers on the basis of the electrophoretic pattern of their isoenzymes. Virulence of different strains of axenically grown trophozoites of Entamoeba histolytica, as determined by various laboratory tests, such as damage to tissue culture monolayers, or their ability to cause an hepatic abscess in a hamster, are known to vary considerably. Reassociation of trophozoites of strain HK-9 with certain Escherichia coli strains for short periods of time markedly augmented their virulence, as tested by the above-mentioned methods. The bacterial association, however, did not cause any change in the electrophoretic pattern of amoebic isoenzymes (zymodeme).     

Effect of the sesquiterpene lactone incomptine A in the energy metabolism of Entamoeba histolytica

Entamoeba histolytica is the causative agent of human amoebiasis, which mainly affects developing countries. Although several drugs are effective against E. histolytica trophozoites, the control of amoebiasis requires the development of new and better alternative therapies. Medicinal plants have been the source of new molecules with remarkable antiprotozoal activity. Incomptine A isolated from Decachaeta incompta leaves, is a sesquiterpene lactone of the heliangolide type which has the major in vitro activity against E. histolytica trophozoites. However the molecular mechanisms involved in its antiprotozoal activity are still unknown. Using a proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry (ESI-MS/MS) analysis, we evidenced that 21 E. histolytica proteins were differentially expressed in response to incomptine A treatment. Notably, three glycolytic enzymes, namely enolase, pyruvate:ferredoxin oxidoreductase and fructose-1,6-biphosphate aldolase, were down-regulated. Moreover, ultrastructural analysis of trophozoites through electronic microscopy showed an increased number of glycogen granules. Taken together, our data suggested that incomptine A could affect E. histolytica growth through alteration of its energy metabolism.     

Entamoeba histolytica: genetic control of susceptibility in chicken eggs.

Five strains of axenized Entamoeba histolytica were established in eggs from chickens of different genetic backgrounds. The eggs of outbred and inbred chickens varied in the proportion of embryos from which the strains of E. histolytica were recovered and there were variations in the numbers of trophozoites recovered from individual positive eggs. It was possible, however, to find individual, single-mated hens which laid only uniformly susceptible or resistant eggs. Susceptibility and resistance to E. histolytica could be demonstrated in embryonated eggs derived both from outbred heterogenous and in highly inbred chickens but eggs from a genetically stable source were relatively constant in the distribution of susceptible and resistant embryos. Except for gross hydrocephalus, tissue reactions were not observed in the chick embryos or in the chorioallantoic, amniotic, or yolk sac membranes. The yolk sac inoculation route and 37 C incubation temperature provided the optimal survival conditions for the amoebae. Infection with avian leukosis virus and interferon production did not seem to play a role in susceptibility and resistance. Neither the presence of E. histolytica cross-reacting, maternal antibody from the hen transmitted transovarially via the yolk sac, or other antiamoebic substances appeared to play a role in the resistance observed in nonsusceptible eggs. Multiple alternating passages of trophozoites in hen's eggs/TPS-1 broth led to an increase in the number of positive embryos and a decrease in embryo deaths.     

Studies on the mechanism of acetate oxidation by bacteria. V. evidence for the participation of fumarate,malate, and oxalacetate in the oxidation of acetic acid by Escherichia coli

1. Simultaneous oxidation of C¹⁴-methyl-labeled acetate, and unlabeled malate or fumarate and α-ketoglutarate results in entrapment of labeled carbon in the C₄-dicarboxylic acids, but not in α-ketoglutarate, although all substrates are utilized at comparable rates. 2. A large endogenous reduction of all C₄-dicarboxylic acids (fumarate, oxalacetate, and malate) to succinate is observed under aerobic conditions, and when vigorous oxidation is proceeding. This effect occurs with both freshly harvested young (18 hour) cells and stored (2 week) cells. 3. This reduction can be considerably minimized under high oxygen tensions. 4. The quantitative concordance of these results with a Thunberg-Knoop cyclic mechanism for acetate oxidation is shown. Possible alternative C₄ products formed prior to succinate are not completely excluded, but it appears that the cells can utilize the succinate condensation as a major pathway in acetate oxidation.     

State of liver mitochondrial respiratory chain in rats with experimental toxic hepatitis

Polarographical determination of oxygen concentration has shown that in rats with experimental hepatitis induced by combined ethanol and CCl₄ administration for 4 weeks, the functioning of the hepatocyte mitochondrial respiratory chain is impaired. Development of liver pathology was accompanied by adipose dystrophy, fibrosis, and an increase of triglycerides and lipid peroxidation products in the liver tissue. The endogenous respiration rate in hepatocytes isolated from the pathologically altered liver was 34% higher than in the control. Cell respiration was not stimulated by the addition of the substrates malate and pyruvate with digitonine. An uncoupler of oxidation and phosphorylation, 2,4-dinitrophenol, increased the hepatocyte oxygen consumption rate by 37%, while addition of the inhibitor of the I complex, rotenone, decreased cell respiration in pathologically altered hepatocytes by 27%. The states 3 (V₃) and 4 (V₄) of mitochondrial respiration with malate + glutamate as substrates were found to be higher by 70% and 56%, respectively, as compared with the control level. When using malate + glutamate or succinate as substrates, V₃ and Vd (dinitrophenol respiration) in the toxic hepatitis hepatocyte mitochondria did not differ from the control, which indicates no uncoupling occurred of the oxidation and phosphorylation processes. Cytochrome c oxidase activity was elevated (+80%) as compared with the control. Administration of the hypolipidemic agent symvastatin simultaneously with ethanol and CCl₄ resulted in a reduction of the degree of liver adipose dystrophy, prevented activation of lipid peroxidation, and decreased the hepatocyte endogenous respiration rate. Addition of malate + pyruvate, dinitrophenol or rotenone produced oxygen consumption changes similar to those in the control. However, in mitochondria isolated from the pathologically altered liver, symvastatin induced an uncoupling effect on the respiratory chain in the presence of the substrates malate + glutamate, but did not change the cytochrome c oxidase activity. We suggest that functioning of the NCCR complex in the hepatocyte mitochondria of animals with experimental toxic hepatitis is impaired, which leads to an intensive superoxide anion production at the level of this complex. Under these conditions, the defect of the NADH-coenzyme Q-oxidoreductase is compensated by functioning of other complexes of the respiratory chain (SCCR, coenzyme Q-cytochrome c-reductase, cytochrome c oxidase, and ATP-synthase activities).     

The Aetiology of Vascular Discoloration in Cassava Roots after Harvesting: Development of Endogenous Resistance in Stored Roots

The susceptibility of cassava roots, Manihot esculenta Crantz, to vascular discoloration beneath two types of injury site, transverse cuts and periderm injuries, was compared for freshly harvested and stored roots. Susceptibility beneath transverse cuts changed rapidly, so that roots stored at ambient temperature for 5–9 days were largely resistant to vascular discoloration beneath this type of injury. Susceptibility to localized deterioration beneath periderm injuries changed more slowly, but significant decreases were observed in roots stored at ambient temperature for 10–16 days. Changes in susceptibility were observed in all cultivars tested, seven in Colombia and one in Jamaica. These changes were retarded but not prevented by storage at 2°C and by storage in sealed polyethylene bags. Pruning plants 1 to 3 weeks prior to harvesting, which has been shown to reduce the rate of post-harvest deterioration of roots, was also found to reduce the susceptibility of roots to vascular discoloration beneath injuries made immediately after harvesting. Water loss through injuries caused a respiratory response as well as vascular discoloration. This respiratory response was as large in stored (resistant) roots as in freshly harvested (susceptible) ones. The potential of cassava roots to develop endogenous resistance to vascular discoloration either before or after harvesting is discussed in relation to the problems of storage of harvested cassava roots.     

Characterization of cytosine methylated regions and 5-cytosine DNA methyltransferase (Ehmeth) in the protozoan parasite Entamoeba histolytica

The DNA methylation status of the protozoan parasite Entamoeba histolytica was heretofore unknown. In the present study, we developed a new technique, based on the affinity of methylated DNA to 5-methylcytosine antibodies, to identify methylated DNA in this parasite. Ribosomal DNA and ribosomal DNA circles were isolated by this method and we confirmed the validity of our approach by sodium bisulfite sequencing. We also report the identification and the characterization of a gene, Ehmeth, encoding a DNA methyltransferase strongly homologous to the human DNA methyltransferase 2 (Dnmt2). Immunofluorescence microscopy using an antibody raised against a recombinant Ehmeth showed that Ehmeth is concentrated in the nuclei of trophozoites. The recombinant Ehmeth has a weak but significant methyltransferase activity when E.histolytica genomic DNA is used as substrate. 5-Azacytidine (5-AzaC), an inhibitor of DNA methyltransferase, was used to study in vivo the role of DNA methylation in E.histolytica. Genomic DNA of trophozoites grown with 5-AzaC (23 µM) was undermethylated and the ability of 5-AzaC-treated trophozoites to kill mammalian cells or to cause liver abscess in hamsters was strongly impaired.     

Entamoeba histolytica Induces Cell Death of HT29 Colonic Epithelial Cells via NOX1-Derived ROS

Entamoeba histolytica, which causes amoebic colitis and occasionally liver abscess in humans, is able to induce host cell death. However, signaling mechanisms of colon cell death induced by E. histolytica are not fully elucidated. In this study, we investigated the signaling role of NOX in cell death of HT29 colonic epithelial cells induced by E. histolytica. Incubation of HT29 cells with amoebic trophozoites resulted in DNA fragmentation that is a hallmark of apoptotic cell death. In addition, E. histolytica generate intracellular reactive oxygen species (ROS) in a contact-dependent manner. Inhibition of intracellular ROS level with treatment with DPI, an inhibitor of NADPH oxidases (NOXs), decreased Entamoeba-induced ROS generation and cell death in HT29 cells. However, pan-caspase inhibitor did not affect E. histolytica-induced HT29 cell death. In HT29 cells, catalytic subunit NOX1 and regulatory subunit Rac1 for NOX1 activation were highly expressed. We next investigated whether NADPH oxidase 1 (NOX1)-derived ROS is closely associated with HT29 cell death induced by E. histolytica. Suppression of Rac1 by siRNA significantly inhibited Entamoeba-induced cell death. Moreover, knockdown of NOX1 by siRNA, effectively inhibited E. histolytica-triggered DNA fragmentation in HT29 cells. These results suggest that NOX1-derived ROS is required for apoptotic cell death in HT29 colon epithelial cells induced by E. histolytica.