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.     

Mg2+, Mn2+, and Co2+ stimulate Entamoeba histolytica to produce chitin-like material

The mechanism of Entamoeba histolytica cyst cell wall synthesis is not well understood. Previous research has shown that cyst-like structures formed in the presence of chitin synthase cofactors (Mg2+, Mn2+, and Co2+) resist 1% sodium dodecyl sulfate lysis (RCLS), whereas those formed in the absence of cofactors (CLS) do not, and trophozoites are immediately destroyed. This suggests that E. histolytica is able to synthesize chitin, initiating a differentiation process under axenic conditions. To test this hypothesis, polysaccharide hydrolysates from E. histolytica trophozoites, CLS, or RCLS were analyzed with high-performance liquid chromatography. The major components found in all 3 preparations were N-acetylglucosamine (NAG) and glucose (GLC), with RCLS possessing 129 and 180 times more NAG and 2.4 and 2.0 more GLC than trophozoites and CLS, respectively. After 36 hr of incubation with chitinase (16 U/ml) in a hypotonic medium (50 mOsm/kg), 68% of RCLS was lysed, and 100% lost affinity for calcofluor white M2R. The RCLS polysaccharides bound wheat germ agglutinin and appeared as long and thin or short and thick fibers. Accordingly, Mg2+, Mn2+, and Co2+ stimulated E. histolytica to synthesize a chitin-like material.     

High-resolution electron microscopical study of cyst walls of Entamoeba spp

Knowledge of the fine structural organization, molecular composition and permeability properties of the cell surface of intestinal protozoan cysts is important to understand the biologic basis of their resistance. Recent studies on the biology of the cyst walls of Entamoeba histolytica and Entamoeba invadens have considerably advanced knowledge on the cellular processes involved in the transport and surface deposition of the main cyst wall components. Using transmission electron microscopy, cytochemistry, scanning electron microscopy and freeze-fracture techniques, we have obtained new information. In mature cysts the permeability of Entamoeba cysts is limited to small molecules not by the cyst wall, but by the plasma membrane, as demonstrated with the use of ruthenium red as an electron-dense tracer. Cell walls of E. histolytica cysts are made up of five to seven layers of unordered fibrils 7-8 nm thick. Alcian blue stains a regular mesh of fibrils approximately 4 nm thick, running perpendicularly to the cyst wall. In addition, abundant ionogenic groups are seen in cyst walls treated with cationized ferritin. In the mature cysts of E. histolytica and E. invadens small cytoplasmic vesicles with granular material were in close contact with the plasma membrane, suggesting a process of fusion and deposition of granular material to the cell wall. The plasma membrane of mature cysts is devoid of intramembrane particles when analyzed with the freeze-fracture technique. When viewed with scanning electron microscopy the surface of E. histolytica cysts clearly differs from that of Entamoeba coli and E. invadens.     

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: an ecto-phosphatase activity regulated by oxidation-reduction reactions

In this work, an ecto-phosphatase activity of Entamoeba histolytica was characterized using intact cells. This activity presented the following biochemical characteristics: (i) it hydrolyzes p-NPP with V(max) of 8.00+/-0.22 nmol p-NP x h(-1) x 10(-5) cells and K(m) of 2.68+/-0.25 mM; (ii) it is inhibited by acid phosphatase inhibitors, such as sodium molybdate (K(i)=1.70+/-0.24 microM) and sodium fluoride (K(i)=0.25+/-0.02 mM); (iii) it also showed high sensitivity to phosphotyrosine phosphatase inhibitors, such as sodium orthovanadate (K(i)=1.07+/-0.14 microM), bpV-PHEN (K(i)=0.38+/-0.02 microM) and mpV-PIC (K(i)=0.39+/-0.04 microM). Zn(2+), an oxidizing agent, decreased the enzymatic activity in 50%. DTT and GSH, two reducing agents, enhanced the activity twofold. The non-invasive E. histolytica and free-living E. moshkovskii were less efficient in hydrolyzing p-NPP than the pathogenic E. histolytica suggesting that this enzyme could represent a virulence marker for this cell.     

Development of simple and stable ELISA for detection of copro antigen in intestinal amoebiasis

A simple, sensitive and stable ELISA (enzyme linked immunosorbent assay) was developed using rabbit antibody to fractionated Entamoeba histolytica antigen for the detection of copro antigen in the faeces of individuals with intestinal amoebiasis. In this test none of the healthy individuals, all trophozoite positive, 40% cyst passers and 6% individuals living in endemic area showed the presence of copro antigen. ELISA using polyclonal rabbit antibody could detect 1-5 trophozoites/well and 20-50 ng protein per well of NIH-200 strain of E. histolytica and the sensitivity of the test was comparable with that using monoclonal antibody. Cross reaction was observed only with E. invadens when faeces having other parasites were screened. The reagents of ELISA were stabilized and found to be stable for more than 6 months when stored at 4 degrees C.     

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%).     

Patterns of evolution in the unique tRNA gene arrays of the genus Entamoeba

Genome sequencing of the protistan parasite Entamoeba histolytica HM-1:IMSS revealed that almost all the tRNA genes are organized into tandem arrays that make up over 10% of the genome. The 25 distinct array units contain up to 5 tRNA genes each and some also encode the 5S RNA. Between adjacent genes in array units are complex short tandem repeats (STRs) resembling microsatellites. To investigate the origins and evolution of this unique gene organization, we have undertaken a genome survey to determine the array unit organization in 4 other species of Entamoeba-Entamoeba dispar, Entamoeba moshkovskii, Entamoeba terrapinae, and Entamoeba invadens-and have explored the STR structure in other isolates of E. histolytica. The genome surveys revealed that E. dispar has the same array unit organization as E. histolytica, including the presence and numerical variation of STRs between adjacent genes. However, the individual repeat sequences are completely different to those in E. histolytica. All other species of Entamoeba studied also have tandem arrays of clustered tRNA genes, but the gene composition of the array units often differs from that in E. histolytica/E. dispar. None of the other species' arrays exhibit the complex STRs between adjacent genes although simple tandem duplications are occasionally seen. The degree of similarity in organization reflects the phylogenetic relationships among the species studied. Within individual isolates of E. histolytica most copies of the array unit are uniform in sequence with only minor variation in the number and organization of the STRs. Between isolates, however, substantial differences in STR number and organization can exist although the individual repeat sequences tend to be conserved. The origin of this unique gene organization in the genus Entamoeba clearly predates the common ancestor of the species investigated to date and their function remains unclear.     

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.     

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.     

Entamoeba invadens: in vitro axenic encystation with a serum substitute

The current media for axenic cultivation of Entamoeba histolytica and Entamoeba invadens are supplemented with bovine or equine serum, which provides several essential nutrients to amoebas. Serum has also been considered an essential component in encystation media for E. invadens. A substitute of serum, PACSR has been described as an alternative for growth of E. histolytica and also maintains growth of E. invadens. When PACSR was used instead of serum for encystation of E. invadens the efficiency was the same as for serum. Our present data show that PACSR can support the growth and induction of encystation of E. invadens strain IP-1.     

Asymptomatic cyst passers of Entamoeba histolytica but not Entamoeba dispar in institutions for the mentally retarded in Japan

Entamoeba histolytica/Entamoeba dispar was isolated from 50 asymptomatic amebic cyst passers in three institutions for the mentally retarded in Kanagawa Prefecture, Japan. To distinguish between E. histolytica and E. dispar, the isolates were analyzed by PCR, reactivity to monoclonal antibodies, and zymodemes. All isolates were identified as E. histolytica. The results lead us to conceive that, in Japan, E. histolytica is predominant even in asymptomatic cyst passers.     

Recognition of Entamoeba histolytica 115-kDa surface protein by human secretory immunoglobulin A antibodies from asymptomatic carriers

Entamoeba histolytica is a protozoan parasite that can invade the intestinal mucosa. Infection induces production of secretory immunoglobulin A (SIgA) antibodies that can diminish the adhesion between E. histolytica trophozoites and epithelial cells in vitro and reduce the rate of new infections in children. SIgA antibodies produced by asymptomatic cyst carriers could play a protective role against the damage caused by E. histolytica. To identify membrane antigens capable of inducing SIgA response in E. histolytica cyst carriers, salivary SIgA antibodies were confronted with blotted plasma membrane proteins from amebae. A surface 115-kDa ameba protein was recognized by 62% of the human SIgA antibodies tested. The 115-kDa protein is not a mannose-containing glycoprotein and has no protease activity. Rabbit anti-115-kDa protein antibodies were capable of reducing erythrophagocytosis but were unable to protect culture cells from the cytopathic damage caused by E. histolytica. However, anti-115-kDa protein antibodies induced surface receptor redistribution.     

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.     

Entamoeba histolytica extrachromosomal circular ribosomal DNA: analysis of clonal variation in a hypervariable region.

The ribosomal RNA genes of the protozoan parasite Entamoeba histolytica are highly repeated and display restriction fragment length polymorphism. Using a set of four DNA probes spanning the coding region and part of the flanking region of the E. histolytica ribosomal RNA genes, an analysis of the DNA bands generated by EcoRI digestion of Entamoeba DNA is presented. This analysis included five strains of E. histolytica, four strains of E. moshkovskii, and one strain each of E. invadens and E. terrapinae. No common bands were observed between E. histolytica and the other Entamoeba. Within E. histolytica, two bands were conserved in all strains while the others were polymorphic. Detailed analysis of DNA from independently isolated clones of the strain HM-1:IMSS of E. histolytica showed two bands to be highly polymorphic. Of these, the 4.4-kb band of clone 6 was further analyzed. Polymorphism in this band could even be demonstrated in cells of the same clone. Restriction enzyme analysis of this DNA band from two clones of HM-1:IMSS showed that the polymorphism may be due to variable numbers of DraI repeat units present in this DNA stretch.     

PCR diagnosis of Entamoeba histolytica cysts in stool samples

Amebiasis is a protozoan disease caused by Entamoeba histolytica and a potential health threat in areas where sanitation and hygiene are inappropriate. Highly sensitive PCR methods for detection of E. histolytica in clinical and environmental samples are extremely useful to control amebiasis and to promote public health. The present study compared several primer sets for small subunit (SSU) rDNA and histone genes of E. histolytica cysts. A 246 bp of the SSU rDNA gene of pure cysts contained in phosphate-buffered saline (PBS) and in stool samples was successfully amplified by nested PCR, using the 1,147-246 bp primer set, of the primary PCR products which were pre-amplified using the 1,147 bp primer as the template. The detection limit of the nested PCR using the 1,147-246 primer set was 10 cysts in both groups (PBS and stool samples). The PCR to detect histone gene showed negative results. We propose that the nested PCR technique to detect SSU rDNA can be used as a highly sensitive genetic method to detect E. histolytica cysts in stool samples.