Effect of iron on adherence and cytotoxicity of Entamoeba histolytica to CHO cell monolayers

Iron is an essential element for almost all living organisms. The possible role of iron for growth, adherence and cytotoxicity of Entamoeba histolytica was evaluated in this study. The absence of iron from TYI-S-33 medium stopped amebic growth in vitro. However, iron concentrations in the culture media of 21.4-285.6 microM did not affect the growth of the amebae. Although growth was not retarded at these concentrations, the adhesive abilities of E. histolytica and their cytotoxicities to CHO cell monolayer were correlated with iron concentration. Amebic adhesion to CHO cell monolayers was significantly reduced by low-iron (24.6 +/- 2.1%) compared with 62.7 +/- 2.8 and 63.1 +/- 1.4% of amebae grown in a normal-iron and high-iron media, respectively. E. histolytica cultured in the normal- and high-iron media destroyed 69.1 +/- 4.3% and 72.6 +/- 5.7% of cultured CHO cell monolayers, but amebae grown in the low-iron medium showed a significantly reduced level of cytotoxicity to CHO cells (2.8 +/- 0.2%). Addition of divalent cations other than iron to amebic trophozoites grown in the low-iron medium failed to restore levels of the cytotoxicity. However, when E. histolytica grown in low-iron medium were transferred to normal-iron medium, the amebae showed completely restored cytotoxicity within 7 days. The result suggests that iron is an important factor in the adherence and cytotoxicity of E. histolytica to CHO cell monolayer.     

Entamoeba histolytica: comparison of the role of receptors and filamentous actin among various endocytic processes

Entamoeba histolytica is the causative agent of amoebic dysentery. Uptake of iron is critical for E. histolytica growth and iron-bound human transferrin (holo-transferrin) has been shown to serve as an iron source in vitro. Although a transferrin-binding protein has been identified in E. histolytica, the mechanism by which this iron source is taken up by this pathogen is not well understood. To gain insight into this process, the uptake of fluorescent-dextran, -holo-transferrin, and human red blood cells (hRBCs) was compared. Both dextran and transferrin were taken up in an apparent receptor-independent fashion as compared to hRBCs, which were taken up in a receptor-mediated fashion. Interestingly, the uptake of FITC-dextran and FITC-holo-transferrin differentially relied on an intact actin cytoskeleton suggesting that their internalization routes may be regulated independently.     

Ehhmbp45 is a novel hemoglobin-binding protein identified in Entamoeba histolytica

Hemoglobin-binding proteins are necessary for pathogens to obtain iron from Hb. Entamoeba histolytica can grow using Hb as source of iron, but the underlying mechanism has not previously been established. In this work, we identified a 45 kDa Hb-binding protein of E. histolytica, which we named Ehhmbp45. In silico analysis showed that Ehhmbp45 contains the conserved domains needed for Hb-binding, while overlay assays demonstrated that Ehhmbp45 is able to bind Hb. In addition, we found that Ehhmbp45 mRNA levels were up-regulated under iron starvation conditions and were subsequently restored to basal levels when Hb was added to the cell cultures. These findings provide the first insights on the role of Ehhmbp45 in iron acquisition from Hb.     

The adverse effect of iron repletion on the course of certain infections.

The incidence of infections was studied in 137 iron-deficient Somali nomads, 67 of whom were treated with placebo and 71 with iron. Seven episodes of infection occurred in the placebo group and 36 in the group treated with iron; these 36 episodes included activation of pre-existing malaria, brucellosis, and tuberculosis. This difference suggested that host defence against these infections was better during iron deficiency than during iron repletion. Iron deficiency among Somali nomads may be part of an ecological compromise, permitting optimum co-survival of host and infecting agent.     

Influence of goat and cow milk on the digestive and metabolic utilization of calcium and iron

The effects of goat and cow milk on the digestive and metabolic utilization of calcium and iron were studied in rats using a standard (non-milk) control diet. The digestive utilization of calcium is greater when the animals consume the goat-milk-based diet rather than that based on cow milk or the standard diet. The digestive utilization of iron, however, is similar for the goat-milk diet and the standard diet, and in both cases superior to that based on cow milk. The calcium content in the femur, sternum and Longissimus dorsi muscle (L.D. muscle) provides an indication of what happens during the utilization of the mineral; more is deposited when the rats consume a milk-based diet, particularly one based on goat milk. The iron content in the reserve organs, namely the liver and the spleen, is greater with the standard diet and the goat milk diet than with that containing cow milk. There is an obviously beneficial effect of goat milk on the metabolism of calcium and iron, which minimizes any interaction between the two minerals.     

Iron bioavailability from fortified fluid milk and petit suisse cheese determined by the prophylactic-preventive method

In this research, we measure the iron bioavailability of micronized ferric orthophosphate when it is used to fortify low-fat fluid milk enriched with calcium and petit suisse cheese using the prophylactic-preventive method in rats. Four groups of male weaned rats received a basal diet (control diet; 6.5 ppm Fe), a reference standard diet (SO₄Fe; 18.2 ppm Fe), a basal diet using iron-fortified fluid milk as the iron source (milk diet; Fe ppm 17.9), and a basal diet using iron-fortified petit suisse cheese as the iron source (cheese diet; 18.0 ppm Fe) for 22 d. The iron bioavailability of the different sources was calculated as the ratio between the mass of iron incorporated into hemoglobin during the experiment and the total iron intake per animal. The relative biological values with regard to the reference standard (RBV%) were 61% and 69% for the milk and cheese diet, respectively. These results show that according to this method, the iron bioavailability in both fortified foods can be considered as medium bioavailability rates.     

Indirect haemagglutination (IHA) test in the serodiagnosis of amoebiasis

Among 72 patients clinically suspected of Entamoeba histolytica (E. histolytica) infections, 39 positive cases (54%) were detected serologically by the indirect hemagglutination (IHA) test. Parasitologically, microscopic examination of three consecutive stool specimens from all these patients indicated positivity for E. histolytica cysts and or trophozoites in 10 of the patients with IHA antibody titers greater than or equal to 1:128, which is of clinical significance. Another 2 patients were parasitologically positive but showed low IHA antibody titres (1:32-1:64); follow up indicated response to treatment with metronidazole. The highest serological positivity (100%) were detected in patients with liver abscess, all were clinically proven cases of extra-intestinal amoebiasis. IHA antibody levels of clinical significance were seen in all four patients with chronic dysentery with parasitological evidence of E. histolytica in their stools. In a group of patients with abdominal pain nine positives were detected serologically, four of which were positively diagnosed concurrently by parasitology; the remaining five patient's sera showed high IHA antibody titres with absence of cysts or trophozoites in stools, indicative possibly of persistence of antibodies from past infection. The serologic determination of E. histolytica IHA antibodies in a control group consisting of normal healthy school children and adults of both sexes without any clinical evidence of amoebiasis showed the absence of any positive titres of clinical significance; low titres (1:32-1:64) were detected in 5.2% of 232 sera tested. Parasitological examination of three consecutive stool specimens from all individuals in the control group showed the presence of cysts of E. histolytica in just two among 232 tested (0.9%).     

Iron-binding Proteins in Milk and Resistance to Escherichia coli Infection in Infants

Human milk contains large quantities of iron-binding protein, of which the greater proportion is lactoferrin, though small amounts of transferrin are also present. Three samples of human milk with unsaturated iron-binding capacities of between 56 and 89% had a powerful bacteriostatic effect on Escherichia coli O111/B4. The bacteriostatic properties of milk were abolished if the iron-binding proteins were saturated with iron. Purified human lactoferrin, in combination with specific E. coli antibody, strongly inhibited the growth of E. coli, and this effect was also abolished by saturating the lactoferrin with iron.Guinea-pig milk also contains lactoferrin and transferrin. Newly born guinea-pigs fed on an artificial diet and dosed with E. coli O111 had higher counts of E. coli O111 in the intestine than suckled animals. The apparent suppressive effect of guinea-pig milk on E. coli in the intestine could be reversed by feeding the iron compound haematin. It seems that iron-binding proteins in milk may play an important part in resistance to infantile enteritis caused by E. coli.     

Entamoeba histolytica: genetic diversity of clinical isolates from Bangladesh as demonstrated by polymorphisms in the serine-rich gene.

The varied organ tropisms and clinical presentations of infection by Entamoeba histolytica have stimulated interest in the role of parasite genetic diversity in virulence. We investigated genetic diversity among 54 E. histolytica isolates from Bangladesh by analyzing polymorphism in the serine-rich gene by nested PCR on DNA extracted from stool and liver aspirate pus. We detected both size and restriction site polymorphisms among the isolates within this endemic area. A combination of the nested PCR results and the AluI digestion of the PCR products examined yielded 25 distinct DNA banding patterns among the 42 stool isolates and an additional 9 distinct patterns among the 12 liver abscess isolates. Approximately half of the isolates had unique polymorphisms. Interestingly, the majority of E. histolytica from the liver had polymorphisms which were not present in intestinal isolates from the same geographic area. These data are consistent with the existence of genetic differences between E. histolytica which cause intestinal and those which cause hepatic disease. We conclude that there is genetic diversity within E. histolytica isolates from an endemic population as reflected in serine-rich E. histolytica protein gene polymorphism. The correlation of genetic differences with the pathogenic potential of E. histolytica strains and the implications of genetic diversity for the immunoprophylaxis of amebiasis require further study.     

Comparative genomic hybridizations of Entamoeba strains reveal unique genetic fingerprints that correlate with virulence

Variable phenotypes have been identified for Entamoeba species. Entamoeba histolytica is invasive and causes colitis and liver abscesses but only in approximately 10% of infected individuals; 90% remain asymptomatically colonized. Entamoeba dispar, a closely related species, is avirulent. To determine the extent of genetic diversity among Entamoeba isolates and potential genotype-phenotype correlations, we have developed an E. histolytica genomic DNA microarray and used it to genotype strains of E. histolytica and E. dispar. On the basis of the identification of divergent genetic loci, all strains had unique genetic fingerprints. Comparison of divergent genetic regions allowed us to distinguish between E. histolytica and E. dispar, identify novel genetic regions usable for strain and species typing, and identify a number of genes restricted to virulent strains. Among the four E. histolytica strains, a strain with attenuated virulence was the most divergent and phylogenetically distinct strain, raising the intriguing possibility that genetic subtypes of E. histolytica may be partially responsible for the observed variability in clinical outcomes. This microarray-based genotyping assay can readily be applied to the study of E. histolytica clinical isolates to determine genetic diversity and potential genotypic-phenotypic associations.     

Entamoeba histolytica: the serine-rich gene polymorphism-based genetic variability of clinical isolates from Georgia

It is generally accepted that a majority of individuals infected by Entamoeba histolytica do not develop symptomatic disease. However, the parasite and the host factors contributing to the development of the disease, remain undetermined. It is also unclear why certain individuals develop extra-intestinal amebiasis without exhibiting apparent intestinal symptoms. An outbreak of amebic liver abscess in Tbilisi, Georgia in 1998-1999 suggested that the causative E. histolytica strain had an unusual propensity for extra-intestinal spread. To correlate the genetic differences with pathogenic potential of the parasite, we have examined the SREHP gene polymorphisms among Georgian E. histolytica isolates. Comparison of polymorphic patterns revealed the presence of several different genotypes of E. histolytica, thus preventing an association of a single genotype with hepatic disease, but supporting the previous finding of extensive genetic diversity among E. histolytica isolates from the same geographic origin.     

Entamoeba histolytica: transferrin binding proteins

Entamoeba histolytica trophozoites depend on iron for their growth; thus, they must use some host iron-containing molecules to fulfill this requirement. In this work we report that amoebas are able to utilize human holo-Tf as iron source and to recognize it through transferrin binding proteins. By use of an anti-human transferrin antiserum in an immunoblotting assay, two main polypeptides with apparent molecular masses of 70 and 140 kDa were found in total extract of trophozoites cultured in vitro. However, when a monoclonal anti-human transferrin receptor antibody was used, only one band with molecular mass of 140 kDa was observed. Both the human transferrin and the monoclonal antibody recognized a protein on the amoebic surface, demonstrated by confocal microscopy. Furthermore, the complex transferrin-transferrin binding protein was internalized by an endocytic process and probably dissociated inside the cell. This mechanism could be one manner in which E. histolytica acquires iron from the human host transferrin.     

Entamoeba histolytica: genetic diversity of African strains based on the polymorphism of the serine-rich protein gene

The polymorphism of the serine-rich Entamoeba histolytica protein (SREHP) among isolates obtained from different geographic regions was analyzed by a nested PCR followed by restriction analysis. Thirteen different profiles were generated from 23 E. histolytica isolates from Cameroon, Zimbabwe and South Africa while 20 others were generated from 38 E. histolytica PCR positive stool samples from South Africa. One of the profiles was common to isolates from Cameroon, Zimbabwe and South Africa and constituted the most prevalent (26.1%) of all the profiles. However, profiles unique to each country were also observed amongst the samples. A non-significant difference was observed between isolates from diarrheic and non-diarrheic samples. Of interest, of the five HIV positive stool samples three had the same profile indicating the possibility that some E. histolytica strains might be more common/pathogenic in immuno-compromised individuals. The results obtained showed that African isolates of E. histolytica may possess extremely complex genetic structures independent of geographic location. This study indicates that certain profiles might be responsible for the presentation of intestinal amoebic symptoms. However, more extended studies need to be performed in order to confirm these observations.     

Molecular differentiation of Entamoeba histolytica and Entamoeba dispar from Tunisian food handlers with amoeba infection initially diagnosed by microscopy

The purpose of the study was to obtain more reliable epidemiological data concerning Entamoeba (E.) histolytica infection in Tunisian food handlers using established molecular tools able to differentiate E. histolytica from E. dispar. From 2002 to 2005, 4,266 fresh stools specimens received in the setting of the National program of food handlers' control were analysed by optical microscopy. Twelve (2.8 per thousand) were positive for the presence of four nuclei cysts identified as E. histolytica/E. dispar. Extraction of DNA from the 12 samples, followed by specific amplifications of E. histolytica and E. dispar SSU rDNA, showed that 11 samples (92%) were positive for E. dispar and negative for E. histolytica. Sequencing analysis of 8 PCR products permitted to verify the results obtained with conventional PCR. The remaining sample was negative by PCR amplifying E. histolytica DNA or E. dispar DNA specifically, although it did not show any inhibition. It probably contains protozoan cysts genetically distinct from these two species but morphological similar. Estimation of relative proportions between E. histolytica and E. dispar in cyst carriers showed that all explored individuals harboured the non pathogenic E. dispar strains. This result highlights the need of use in this population of complementary tests that allow specific diagnosis and obviate unnecessary chemotherapy.     

Entamoeba histolytica: rapid identification and differentiation of Indian isolates by riboprinting

Entamoeba histolytica infection still remains one of the major public health problem for developing countries like India. A rapid and accurate detection of this parasite is essential for prevention and control of amoebiasis. In this study, using the method of 'riboprinting' (PCR-RFLP of rRNA genes from amoeba) we have analysed 15 stool samples from symptomatic patients of amoebiasis. All 15 patients of clinical amoebiasis had E. histolytica in their stool and two of the samples also showed mixed infection of E. dispar. Apart from the known restriction enzyme sites within the amoeba SSU-rRNA genes, a new Sau3A site having a discriminatory value is identified in these E. histolytica isolates from India. Hence, it is possible to rapidly identify E. histolytica DNA and differentiating it from E. dispar using minute amounts of clinical stool samples, thus eliminating the laborious parasite culturing process. Thus, riboprinting is advantageous for clearcut identification of E. histolytica in order to decide an effective antiamoebic therapy.     

Outcome of untreated infection with Entamoeba histolytica in homosexual men with and without HIV antibody.

Among homosexual men the prevalence of infection with Entamoeba histolytica is high. To determine the clinical importance of this infection 55 homosexual men carrying the parasite were investigated in detail. No clinical, serological, or histological evidence of invasive amoebiasis was found in any of them. The patients were not treated and were followed up for 12 to 29 months (mean 21.6 months), during which period none developed symptoms that could be attributed to E histolytica. Spontaneous loss of the parasite occurred in 17 patients, some of whom later became reinfected. Sixteen patients had antibody to human immunodeficiency virus, and infection with E histolytica showed the same benign course in them as in the patients who did not have antibody. Throughout the study classification of the isolates of E histolytica consistently showed that they belonged only to non-pathogenic zymodemes. The findings provide further evidence that E histolytica in homosexual men is a commensal organism.     

Purine salvage in Entamoeba histolytica

Pulse-labeling of the nucleotide pool in Entamoeba histolytica with radioactive precursors, and subsequent high performance liquid chromatographic (HPLC) analysis of the radiolabeled nucleotides, indicate that E. histolytica is incapable of de novo synthesis of purine nucleotides. Hypoxanthine, inosine and xanthine could not be converted to nucleotides in E. histolytica, which suggests the absence of interconversion between adenine nucleotides and guanine nucleotides through formation of IMP. Adenosine was actively incorporated into nucleotides at an initial rate of 130 pmoles per minute per 10(6) trophozoites. Adenine, guanosine and guanine were also incorporated at much lower rates. The rate of adenine incorporation was enhanced by the presence of guanosine; the rate of guanine incorporation was significantly increased by adenosine. These stimulatory effects suggest that the ribose moiety of adenosine or guanosine can be transferred to another purine base to form a new nucleoside, and that the purine nucleosides are the immediate precursors of E. histolytica nucleotides. HPLC results showed that the radiolabel in adenine was exclusively incorporated into adenine nucleotides and that guanine was found only among guanine nucleotides, whereas the radioactivity associated with the ribose moiety of adenosine or guanosine was distributed among both adenine and guanine nucleotides.     

Detection of Entamoeba histolytica antigen in stool samples in Mersin, Turkey

Entameoba histolytica, 1 of the 2 Entamoeba species with similar morphology that infect humans, causes invasive intestinal and extraintestinal diseases, whereas Entamoeba dispar is found commensally and is noninvasive. Because of their morphologic similarity, E. histolytica and E. dispar cannot be differentiated microscopically. The antigens of E. histolytica and E. dispar, however, may be detected by the ELISA method. Previous studies have found that the detection of antigens in the stool is as sensitive and specific as cultures and isoenzyme analyses. Stool samples from 272 patients with diarrhea in the province of Mersin, Turkey, were examined for the presence of Entamoeba species microscopically and for Entamoeba (E. histolytica/E. dispar) antigens using the ELISA method. An E. histolytica-specific ELISA test was used to examine 29 E. histolytica/E. disparpositive samples. Twenty-four (8.82%) of the samples tested positive for E. histolytica/E. dispar by trichrome staining, and 29 (10.6%) of the samples tested positive for E. histolytica/E. dispar by the Entamoeba screening test. Entamoeba histolytica was positive in 21 (7.72%) and E. dispar positive in 8 (2.94%) samples. The detection of true E. histolytica infection is possible with the use of E. histolytica-specific antigen ELISA tests. Thus, real cases of amoebiasis can be detected and treated, and overtreatment of the patients with E. dispar, which is the nonpathogenic species, will be prevented.