In vitro activity of some natural honeys against Entamoeba histolytica and Giardia lamblia trophozoites

Considering the potentiality of honey in combating diseases, the present study was carried out aiming to assess the in vitro antiprotozoal activity of several honeys (Ziziphus spina-christi, Acacia nilotica, Acacia seyal, and Cucurbita maxima) against Entamoeba histolytica and Giardia lamblia by employing the sub-culture method. All the tested honeys inhibited the growth of trophozoites, and the level of inhibition varied according to the assayed concentrations and incubation times. Acacia seyal honey had completely stopped motility of E. histolytica trophozoites at a concentration ≤ 50 µg/ml after incubation for 72 h. Ziziphus spina-christi, Acacia seyal, and Acacia nilotica honeys had completely inhibited the growth of Giardia lamblia trophozoites at concentration ≤ 200 µg/ml after 72 h. These inhibitory activities were similar to that of Metronidazole? which showed IC₅₀ = 0.27. The mammalian cytotoxicity of these honeys against normal Vero cell line which determined by applying MTT method verified the nontoxicity of the examined honeys. Also the proximate composition of the samples indicated compliance with the natural honey standards. The findings of the study indicate the need for in vivo studies and further investigations to identify active principles with antiprotozoal activities from natural honeys.     

Molecular basis of defence against oxidative stress in Entamoeba histolytica and Giardia lamblia

Gastrointestinal protozoa, viz. Entamoeba histolytica and Giardia, are able to survive in a microaerobic environment. The role of parasitic factors, particularly cysteine, cysteine-rich proteins, superoxide dismutase, and certain alternative mechanisms, has been described in the defence against oxidative stress. The role of the host-derived factors, particularly the phagocytosis of bacteria or host erythrocytes (intact or their enzymatic/non enzymatic components), in the detoxification of reactive oxygen metabolites in E. histolytica may provide novel approaches for the chemotherapy of invasive amoebiasis.     

Evidence for lateral transfer of genes encoding ferredoxins,nitroreductases, NADH oxidase,and alcohol dehydrogenase 3 from anaerobic prokaryotes to Giardia lamblia and Entamoeba histolytica

Giardia lamblia and Entamoeba histolytica are amitochondriate, microaerophilic protists which use fermentation enzymes like those of bacteria to survive anaerobic conditions within the intestinal lumen. Genes encoding fermentation enzymes and related electron transport peptides (e.g., ferredoxins) in giardia organisms and amebae are hypothesized to be derived from either an ancient anaerobic eukaryote (amitochondriate fossil hypothesis), a mitochondrial endosymbiont (hydrogen hypothesis), or anaerobic bacteria (lateral transfer hypothesis). The goals here were to complete the molecular characterization of giardial and amebic fermentation enzymes and to determine the origins of the genes encoding them, when possible. A putative giardia [2Fe-2S]ferredoxin which had a hypothetical organelle-targeting sequence at its N terminus showed similarity to mitochondrial ferredoxins and the hydrogenosomal ferredoxin of Trichomonas vaginalis (another luminal protist). However, phylogenetic trees were star shaped, with weak bootstrap support, so we were unable to confirm or rule out the endosymbiotic origin of the giardia [2Fe-2S]ferredoxin gene. Putative giardial and amebic 6-kDa ferredoxins, ferredoxin-nitroreductase fusion proteins, and oxygen-insensitive nitroreductases each tentatively supported the lateral transfer hypothesis. Although there were not enough sequences to perform meaningful phylogenetic analyses, the unique common occurrence of these peptides and enzymes in giardia organisms, amebae, and the few anaerobic prokaryotes suggests the possibility of lateral transfer. In contrast, there was more robust phylogenetic evidence for the lateral transfer of G. lamblia genes encoding an NADH oxidase from a gram-positive coccus and a microbial group 3 alcohol dehydrogenase from thermoanaerobic prokaryotes. In further support of lateral transfer, the G. lamblia NADH oxidase and adh3 genes appeared to have an evolutionary history distinct from those of E. histolytica.     

Recent advances in DNA-mediated gene transfer of Entamoeba histolytica

During the past few years, the introduction of DNA-mediated gene transfer into parasite research has permitted subtle studies on fundamental aspects of parasite biology. In this paper, Egbert Tannich describes the recent breakthrough of successful Entamoeba histolytica transfection, and the subsequent developments in this field.     

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.     

Entamoeba histolytica: collagenolytic activity and virulence

Several axenic strains of pathogenic and nonpathogenic Entamoeba histolytica were tested for their capacity to digest native radioactive type I collagen gels and to produce liver abscesses when injected into the liver of newborn hamsters. The results demonstrate that the pathogenic strains of amebas (HM1:IMSS, HM3:IMSS, HM38:IMSS and HK9) have a collagenolytic activity that closely correlates with their in vivo capacity to produce liver lesions. The nonpathogenic isolate (Laredo) did not show collagenolytic activity and failed to produce lesions in the liver of newborn hamsters. The results also demonstrate that type I collagen obtained from rodents and cats is degraded less by amebic collagenase than is bovine collagen, which is similar to human collagen. These findings suggest that species susceptibility to invasive infection may depend, among other factors, on the characteristics of the extracellular components of host tissues.     

Evolution of Base Excision Repair in Entamoeba histolytica is shaped by gene loss,gene duplication,and lateral gene transfer

During its life cycle, the protist parasite Entamoeba histolytica encounters reactive oxygen and nitrogen species that alter its genome. Base excision repair (BER) is one of the most important pathways for the repair of DNA base lesions. Analysis of the E. histolytica genome revealed the presence of most of the BER components. Surprisingly, this included a gene encoding an apurinic/apyrimidinic (AP) endonuclease that previous studies had assumed was absent. Indeed, our analysis showed that the genome of E. histolytica harbors the necessary genes needed for both short and long-patch BER sub-pathways. These genes include DNA polymerases with predicted 5′-dRP lyase and strand-displacement activities and a sole DNA ligase. A distinct feature of the E. histolytica genome is the lack of several key damage-specific BER glycosylases, such as OGG1/MutM, MDB4, Mag1, MPG, SMUG, and TDG. Our evolutionary analysis indicates that several E. histolytica DNA glycosylases were acquired by lateral gene transfer (LGT). The genes that encode for MutY, AlkD, and UDG (Family VI) are included among these cases. Endonuclease III and UNG (family I) are the only DNA glycosylases with a eukaryotic origin in E. histolytica. A gene encoding a MutT 8-oxodGTPase was also identified that was acquired by LGT. The mixed composition of BER genes as a DNA metabolic pathway shaped by LGT in E. histolytica indicates that LGT plays a major role in the evolution of this eukaryote. Sequence and structural prediction of E. histolytica DNA glycosylases, as well as MutT, suggest that the E. histolytica DNA repair proteins evolved to harbor structural modifications that may confer unique biochemical features needed for the biology of this parasite.     

Early lateral transfer of genes encoding malic enzyme, acetyl-CoA synthetase and alcohol dehydrogenases from anaerobic prokaryotes to Entamoeba histolytica

The fermentation enzymes, which enable the microaerophilic protist Entamoeba histolytica to parasitize the colonic lumen and tissue abscesses, closely resemble homologues in anaerobic prokaryotes. Here, genes encoding malic enzyme and acetyl-CoA synthetase (nucleoside diphosphate forming) were cloned from E. histolytica, and their evolutionary origins, as well as those encoding two alcohol dehydrogenases (ADHE and ADH1), were inferred by means of phylogenetic reconstruction. The E. histolytica malic enzyme, which decarboxylates malate to pyruvate, closely resembles that of the archaeon Archaeoglobus fulgidus, strongly suggesting a common origin. The E. histolytica acetyl-CoA synthetase, which converts acetyl-CoA to acetate with the production of ATP, appeared to be closely related to the Plasmodium falciparum enzyme, but it was no more closely related to the Giardia lamblia acetyl-CoA synthetase than to those of archaea. Phylogenetic analyses suggested that the adh1 and adhe genes of E. histolytica and Gram-positive eubacteria share a common ancestor. Lateral transfer of genes encoding these fermentation enzymes from archaea or eubacteria to E. histolytica probably occurred early, because the sequences of the amoebic enzymes show considerable divergence from those of prokaryotes, and the amoebic genes encoding these enzymes are in the AT-rich codon usage of the parasite.     

Characterization of the Entamoeba histolytica ornithine decarboxylase-like enzyme

Background

The polyamines putrescine, spermidine, and spermine are organic cations that are required for cell growth and differentiation. Ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the polyamine biosynthetic pathway, is a highly regulated enzyme.

Methodology and Results

To use this enzyme as a potential drug target, the gene encoding putative ornithine decarboxylase (ODC)-like sequence was cloned from Entamoeba histolytica, a protozoan parasite causing amoebiasis. DNA sequence analysis revealed an open reading frame (ORF) of ∼1,242 bp encoding a putative protein of 413 amino acids with a calculated molecular mass of 46 kDa and a predicted isoelectric point of 5.61. The E. histolytica putative ODC-like sequence has 33% sequence identity with human ODC and 36% identity with the Datura stramonium ODC. The ORF is a single-copy gene located on a 1.9-Mb chromosome. The recombinant putative ODC protein (48 kDa) from E. histolytica was heterologously expressed in Escherichia coli. Antiserum against recombinant putative ODC protein detected a band of anticipated size ∼46 kDa in E. histolytica whole-cell lysate. Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ODC, had no effect on the recombinant putative ODC from E. histolytica. Comparative modeling of the three-dimensional structure of E. histolytica putative ODC shows that the putative binding site for DFMO is disrupted by the substitution of three amino acids—aspartate-332, aspartate-361, and tyrosine-323—by histidine-296, phenylalanine-305, and asparagine-334, through which this inhibitor interacts with the protein. Amino acid changes in the pocket of the E. histolytica enzyme resulted in low substrate specificity for ornithine. It is possible that the enzyme has evolved a novel substrate specificity.

Conclusion

To our knowledge this is the first report on the molecular characterization of putative ODC-like sequence from E. histolytica. Computer modeling revealed that three of the critical residues required for binding of DFMO to the ODC enzyme are substituted in E. histolytica, resulting in the likely loss of interactions between the enzyme and DFMO.     

Synthesis of 3-tetrazolylmethyl-4H-chromen-4-ones via Ugi-azide and biological evaluation against Entamoeba histolytica,Giardia lamblia and Trichomona vaginalis

The synthesis of novel 3-tetrazolylmethyl-4H-chromen-4-ones via an Ugi-azide multicomponent reaction and their biological evaluation against Entamoeba histolytica, Giardia lamblia and Trichomona vaginalis are described. Reported yields are moderate to good and biological results show that these compounds could be considered as candidates to anti-parasitic drugs, especially against G. lamblia.     

Giardia lamblia: biochemical characterization of an ecto-ATPase activity

In this work, we describe the ability of living trophozoites of Giardia lamblia to hydrolyze extracellular ATP. In the absence of any divalent cations, a low level of ATP hydrolysis was observed (0.78 ± 0.08 nmol Pi × h⁻¹ × 10⁻⁶ cells). The ATP hydrolysis was stimulated by MgCl₂ in a dose-dependent manner. Half maximum stimulation of ATP hydrolysis was obtained with 0.53 ± 0.07 mM. ATP was the best substrate for this enzyme. The apparent K_m for ATP was 0.21 ± 0.04 mM. In the pH range from 5.6 to 8.4, in which cells were viable, this activity was not modified. The Mg²⁺-stimulated ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A₁ (V-ATPases), and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The impermeant agent DIDS and suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases, decreased the enzymatic activity in a dose-dependent manner, confirming the external localization of this enzyme. Besides ATP, trophozoites were also able to hydrolyse ADP and 5´ AMP, but the hydrolysis of these nucleotides was not stimulated by MgCl₂. Our results are indicative of the occurrence of a G. lamblia ecto-ATPase activity that may have a role in parasite physiology.     

In vivo determination of the gap2 gene promoter activity in Giardia lamblia

A shuttle vector for Escherichia coli and Giardia lamblia was modified to produce a reporter plasmid, which monitors the expression of prescribed gene in G. lamblia by measuring its luciferase activity. Promoter regions of the gap2 gene, one of the genes induced during encystation, were cloned into this plasmid, and the resultant constructs were then transfected into trophozoites of G. lamblia. Transgenic trophozoites containing one of the 3 gap2-luc reporters were induced to encystation, and characterized with respect to gap2 gene expression by measuring their luciferase activities. Giardia containing a gap2-luc fusion of 112-bp upstream region showed full induction of luciferase activity during encystation.     

Giardia lamblia: characterization of proteinase activity in trophozoites

The proteinase activity of Giardia lamblia trophozoites, Portland 1 strain, was characterized with respect to substrate specificities and inhibitor sensitivities. Proteinase activity with urea-denatured hemoglobin (UDH), alpha-N-benzoyl-DL-arginine-2-naphthylamide (BANA), and alpha-N-benzoyl-argininamide (BAA) as substrates exhibited pH optima of 5.8, 3.8, and 5.0, respectively. For BANA, the apparent Km was 0.20 mM and the Vmax was 2.56 microM. For BAA, the apparent Km was 4.0 mM and the Vmax was 8.69 microM. Dithiothreitol (DTT, 5 mM) enhanced proteinase activity threefold for UDH, fourfold for BAA, and fivefold for BANA. Iodoacetamide, L-tosylamide-2-phenylethyl chloromethyl ketone (TPCK), and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), each at 1 mM, inhibited proteinase activity by greater than 90% with BANA and BAA. Iodoacetamide inhibited proteinase activity by 35% with UDH; TPCK and TLCK inhibited activity greater than 70% with UDH. Activity on BAA was inhibited by 91% with Zn2+ and activity on UDH was inhibited by 30% with Cu2+. Virtually complete inhibition of proteinase activity on BANA and BAA was obtained with leupeptin and chymostatin at 1 microgram/ml. Pepstatin A, chelators, and other heavy metals had no apparent effect on proteinase activity. Two polypeptide bands (ca. 105 and 40 kDa) indicative of proteinase activity were visualized by sodium dodecyl sulfate-gelatin polyacrylamide gel electrophoresis. The 105 kDa band was visible over the pH range of 4 to 7, but with greater intensity from pH 5 to 7. The 40 kDa band, while present at pH 5, was most intense at pH 6 and 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of acid phosphatase activity in Giardia lamblia and Giardia muris trophozoites

Numerous membrane-bounded vacuoles are found adjacent to the plasma membrane of the pathogenic protozoan Giardia lamblia. The function of these vacuoles has been discussed by several authors. Approximately 100-400 nm in diameter with a core of low electron density, they have been suggested to be mitochondria, mucocysts, lysosomes, and endocytotic vacuoles. Enzyme cytochemical localization for acid phosphatase activity using cerium as a capturing agent demonstrates reaction product in these vacuoles as well as in the endoplasmic reticulum and nuclear envelope cisternae. The distribution of reaction product suggests the vacuoles are lysosome-like; however, their function and development remain in question.     

Entamoeba histolytica: cytopathogenicity and lectin activity of avirulent mutants

Three clones of Entamoeba histolytica (L-6, C93, C919) were isolated by mutagenesis with ethyl methanesulfonate from the axenic strain HM1:IMSS and were studied for adherence, cytolytic, and soluble galactose inhibitable lectin activity. Avirulent clones adhered to and killed fewer Chinese hamster ovary cells than HM1:IMSS (P less than 0.01). However, only C919 was deficient in adherence to red blood cells. Galactose (1.0 g) completely inhibited adherence of all the mutants to Chinese hamster ovary cells; however, adherence to erythrocytes was only partially inhibitable by galactose. Avirulent mutants were more susceptible to being killed by human neutrophils in vitro (P less than 0.01 compared to HM1:IMSS). Soluble protein preparations from all the avirulent mutants were markedly less mitogenic for human lymphocytes and had lower lectin activity for Chinese hamster ovary cells compared to the HM1:IMSS wild type (P less than 0.01 for each activity with each mutant). Indirect immunofluorescence with a monoclonal antibody (F-14) that recognizes the Gal/GalNAc lectin was positive for L-6 and C919. These findings utilizing avirulent mutants of E. histolytica further support a role for the amebic galactose inhibitable lectin in the in vivo pathogenesis of amebiasis.     

Chemoattractant activity of Entamoeba histolytica for human polymorphonuclear neutrophils

The ability of axenic Entamoeba histolytica trophozoites and amoebic protein preparations to stimulate chemotaxis of human polymorphonuclear neutrophils (PMN) was evaluated. Virulent E. histolytica (strain HM1:IMSS) stimulated chemotaxis (delta distance = 0.55 +/- 0.02 mm, P less than 0.01 vs. control medium). Sonicated (100 micrograms protein/ml) or homogenized (500 micrograms protein/ml) virulent amoebae also significantly stimulated PMN chemotaxis, whereas preparations of the nonpathogenic "Entamoeba-like" Laredo strain did not stimulate chemotaxis. Preparations of subcellular fractions of E. histolytica demonstrated maximal stimulation of PMN chemotaxis existed in nonvesiculated membranes and the supernatant from plasma membranes.     

The Giardia lamblia actin gene and the phylogeny of eukaryotes

The single-copy actin gene of Giardia lamblia lacks introns; it has an average of 58% amino acid identity with the actin of other species; and 49 of its amino acids can be aligned with the amino acids of a consensus sequence of heat shock protein 70. Analysis of the potential RNA secondary structure in the transcribed region of the G. lamblia actin gene and of the single-copy actin gene of nine other species did not reveal any conserved structures. The G. lamblia actin sequence was used to root the phylogenetic trees based on 65 actin protein sequences from 43 species. This tree is congruent with small-subunit rRNA trees in that it shows that oomycetes are not related to higher fungi; that kinetoplatid protozoans, green plants, fungi and animals are monophyletic groups; and that the animal and fungal lineages share a more recent common ancestor than either does with the plant lineage. In contrast to smalls-ubunit rRNA trees, this tree shows that slime molds diverged after the plant lineage. The slower rate of evolution of actin genes of slime molds relative to those of plants, fungi, and animals species might be responsible for this incongruent branching. Correspondence to: G. Drouin     

Evidence for adhesive activity of the ventrolateral flange in Giardia lamblia

Giardia lamblia is an intestinal protozoan that inhabits the intestinal tract of man and other mammals by attaching to the mucosal surface via the contractile activity of an attachment organelle called the ventral adhesive disk. We have investigated the presence of other attachment mechanisms in G. lamblia trophozoites by using microfabricated substrates that sterically interfere with formation of the hypothesized "negative pressure" under the ventral adhesive disk that would mediate attachment to a substratum. Pillars measuring 1 microm high and 2 microm in diam. were constructed in microarrays with spacings smaller than the diameter of the ventral adhesive disk. Using high resolution field emission scanning electron microscopy, the attachment of trophozoites to the tops of pillars in the microfabricated substrates was investigated. Firm adhesion of trophozoites was observed to be mediated by direct attachment of the ventrolateral flange membrane to the tops of microfabricated pillars. Attachment to microfabricated surfaces was 16% of that observed for attachment mediated by the ventral adhesive disk (4.4 +/- 1.5 cells/100 micro2 micropillar surface vs. 25.9 +/- 3.1 cells/100 micro2 flat substrate, p < 0.0001) This is the first report of trophozoite adhesion to a substratum by a mechanism other than the direct attachment of the ventral adhesive disk, and provides experimental evidence that the ventrolateral flange may play a role in trophozoite adhesion. A hypothesis is presented describing how the adhesive nature of the ventrolateral flange might be involved in normal attachment of G. lamblia trophozoites to a substratum.     

YI-S, a Casein-free Medium for Axenic Cultivation of Entamoeba histolytica, Related Entamoeba, Giardia intestinalis and Trichomonas vaginalis

ABSTRACT. Pancreatic digests of casein are major ingredients of media used in the axenic cultivation of lumen-dwelling parasitic protozoa, especially Entamoeba, Giardia , and trichomonads. The digest used almost exclusively in the development of these media, Medo-Peptone (Trypticase® BBL), has not been available since 1981. Moreover, none of dozens of similar type digests tested since then in our laboratory has proved equal to Medo-Peptone, and in the last two years it has become increasingly difficult to obtain new batches which will support even modest growth of Entamoeba histolytica . In response to this problem we have developed a casein-free medium, YI-S, consisting of a nutrient broth, vitamin mixture and serum. We recommend it as a replacement for the casein-dependent medium TYI-S-33, currently the most widely used for axenic culture of Entamoeba histolytica and other lumen-dwellers.