Bacterial Expression of a Human Monoclonal Antibody-Alkaline Phosphatase Conjugate Specific for Entamoeba histolytica

We previously produced human monoclonal antibody Fab fragments specific to Entamoeba histolytica in Escherichia coli. In order to use these Fab fragments for diagnostic purposes, an expression vector to produce a fusion protein of Fab and alkaline phosphatase (PhoA) in E. coli was designed and constructed. The E. coli PhoA gene was fused to the 3′ terminus of the gene encoding the heavy-chain Fd region. The kappa and Fd genes from a previously prepared antibody clone, CP33, which is specific for the 260-kDa lectin of E. histolytica, were used as human antibody genes. When the fusion protein of CP33 and PhoA was incubated with paraformaldehyde-fixed trophozoites of E. histolytica and developed with a substrate, the trophozoites appeared to be stained. These results demonstrate the feasibility of bacterial expression of a human monoclonal antibody-PhoA conjugate specific for E. histolytica and that the antibody can be used to detect E. histolytica antigen without the use of chemically conjugated secondary antibodies.     

Characteristic protein electrophoretic patterns of fourEntamoeba strains

Summary Total proteins from trophozoites of four axenized strains of the histolytica group ofEntamoeba (twoE. histolytica, oneE. invadens, and oneE. moshkovskii) were analyzed by disc-electrophoresis in polyacrylamide sodium dodecyl sulphate slab gels and by radial immunodiffusion. Each strain had a characteristic and reproducible electrophoretic pattern with about 30 major protein bands distributed between molecular weights 12,000 and 110,000. A striking similarity was found between the protein patterns of bothE. histolytica strains. Immunodiffusion also resulted in characteristic patterns, in which the greatest resemblance occurred in the same strains. The sensitivity and high resolution of the electrophoretic method for protein analysis and its correlation with antigenic characterization suggests its use as a taxonomic criterion of the histolytica group of the genusEntamoeba.     

Differential detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii in fecal samples by nested PCR in the United Arab Emirates (UAE)

Amoebiasis is one of the most important infectious diseases afflicting mainly tropical and subtropical countries. This study was carried out in the Sharjah Emirate, UAE in order to accurately detect and differentiate Entamoeba histolytica, Entamoeba dispar and E. moshkovskii in fecal samples collected from the Sharjah municipality public health clinic by ELISA and nested polymerase chain reaction (PCR). One hundred and twenty specimens were examined and the PCR was positive for E. histolytica, E. dispar and E. moshkovskii (collectively referred to as Entamoeba complex) in 19.2% (23 out of 120). Of those, 10% (12/120) were mono — infection with E. histolytica; 2.5% (3/120) with E. dispar; and 2.5% (3/120) E. moshkovskii. The nested PCR also detected mixed infections by both E. histolytica and E. dispar in 3.3% (4/120) and E. dispar and E. moshkovskii in 0.8% (1/120). The TechLab ELISA kit failed to detect E. histolytica in any of the E. histolytica PCR positive samples. Overall, the percentage of E. histolytica including those found in mixed infections was 13.3% (16/120). Compared to nested PCR, microscopy was found to have an overall sensitivity of 52.2% and a specificity of 75.2% for detection of Entamoeba complex. The present study indicates that E. histolytica is present in the UAE with an average incidence rate of 13.3%. However, larger studies need to be conducted in order to confirm these findings. We propose the use of PCR in both the routine diagnosis of amoebiasis and epidemiological survey in the UAE.     

Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells

Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process of Entamoeba histolytica host cell engulfment.     

Trophozoites of <Emphasis Type="Italic">Entamoeba histolytica</Emphasis> express a CD59-like molecule in human colon

In vitro studies have proved the presence of epitopes of CD59 in the surface of trophozoites of Entamoeba histolytica (E. histolytica). However, it has not been proved if CD59 molecules are expressed in the surface during the trophozoites’ tissue invasion. The aim of the present study was to determine whether the complement-regulatory protein CD59 is present on trophozoites of E. histolytica in human colon. Eleven specimens of amoebic colitis were studied by immunohistochemistry and electron microscopy techniques with a monoclonal antibody against human CD59 molecule. Our results show that a CD59-like molecule is expressed in trophozoites of E. histolytica found in colonic amebic lesions. Also, a CD59-like molecule was detected by western blot analysis in whole lysate of E. histolytica as well as on the plasma membrane by immunocytochemistry. These results suggest that E. histolytica can use CD59-like protein against the lytic action of membrane attack complex.     

Trophozoite and nuclear size,DNA base composition,and nucleotide sequence homology of several Entamoeba strains in axenic culture

We carried out a comparative study of nuclear and trophozoite diameters and of DNA thermal denaturation in eight Entamoeba strains cultured axenically (four of them E. histolytica, two initially designated as E. invadens, one E. moshkovskii, and one E. histolytica-like), as well as an analysis of the overall DNA sequence homology of the non-E. histolytica strains. The average nuclear (N) and trophozoite (T) diameters (in m) were, respectively: global averages ±SD for E. histolytica strains, 6.5±2.5 and 28.8±3.7; E. invadens IP101, 5.8 and 27.5, and PZ, 7.8 and 33.6; E. moshkovskii FIC, 4.1 and 12.9; E. histolytica- like Laredo strain, 5.0 and 20.6. The GC content of DNA, estimated by thermal elution in hydroxyapatite, was around 23% in HK9 and its clone HK9-1 and around 27% in the HM2 and HM3 E. histolytica strains; it was 37% in the Laredo strain, 26% in IP101, 35% in PZ, and 33% in FIC. The reassociation kinetics of PZ strain DNA showed that it consists of 40% repeated sequences and 60% unique sequences. By means of DNA association experiments in which one of each pair of DNAs tested had been labeled in vitro with ¹²⁵I, we found the following overall sequence homology among the strains tested: PZ-FIC, 38%; IP101-Laredo, 38%; IP101-FIC, 47%; PZ-Laredo, 49%; Laredo-FIC, 69%; and IP101-PZ, 83%. We conclude that trophozoites of different E. histolytica strains have similar nuclear size and GC content, whereas these parameters and the nucleotide sequences are clearly different in every other Entamoeba species. Our data also suggest that PZ and IP101 strains do not belong to the same species.     

Interaction between trophozoites ofEntamoeba histolytica and the human intestinal cell line HT-29 in the presence or absence of leukocytes

Studies on the interaction between trophozoites ofEntamoeba histolytica of pathogenic or nonpathogenic origin and epithelial cells of the human intestine can contribute to the understanding of the pathogenesis of invasive amoebiasis. We have examined the interaction of virulentE. histolytica with the human colonic carcinoma cell line HT-29. Differentiated HT-29 cells are comparable to the mucosa cells to whichE. histolytica attaches physiologically. Adherence betweenE. histolytica trophozoites and HT-29 cells was effectively inhibited by glycoconjugates containing galactose, indicating the importance of the 170-kDa lectin ofE. histolytica in binding to intestinal cells. Adherence was not significantly inhibited by glycoconjugates containingN-acetyl-glucosamine, indicating that the 220-kDa lectin ofE. histolytica is not involved in binding to HT-29 cells. The destruction of HT-29 cells by pathogenicE. histolytica was dependent on adherence. The destruction was enhanced when polymorphonuclear granulocytes were added to theE. histolytica trophozoites.     

Lectin-induced agglutination of trophozoites of different species and strains ofEntamoeba

In vitro agglutinability of trophozoites of threeEntamoeba histolytica strains, cultivated under axenic conditions in the presence of concanavalin A (Con A), was shown to be related to the degree of their pathogenicity for experimental animals and of the concentration of Con A. Seven strains ofE. invadens tested also agglutinated in the presence of Con A, and the degree of agglutination was proportional to the concentration of the lectin. Three strains ofE. histolytica-like Laredo-type amebae, a strain ofE. terrapinae, and a strain ofE. moshkovskii agglutinated very slightly, only in the presence of the highest concentration of Con A tested.     

Role of leukocytes and amebic proteinases in experimental rat testicular necrosis produced byEntamoeba histolytica

To investigate the role of amebic proteinases and host leukocytes, we studied amebiasis experimentally in the rat testis. The degree of inflammation and necrosis produced by different strains was correlated with proteinase activity and with zymograms. Intratesticular injection of axenically grown trophozoites of a pathogenic strain (HM-1 ofEntamoeba histolytica) produced indistinguishable lesions in normal animals and leukopenic rats (<1000 leukocytes/mm³), suggesting that granulocytes do not contribute to the formation of lesions in this model. Testicular lesions produced by five different strains ofE. histolytica ranging from highly virulent to almost nonpathogenic were proportional to the proteinase activity of each amebic strain. Inhibition of amebic proteinases in vitro and subsequent injection into the rat testis markedly reduced the inflammatory lesions resulting from highly virulentE. histolytica. The pathogenicity of three other amebae (E. laredo, E. moshkovskii, andE. invadens) was generally proportional to their proteinase activity; however,E. laredo showed high proteinase activity and caused minimal tissue damage. These results suggest that the pathogenic potential ofEntamoeba spp. in the rat testis may be related to the type as well as the level of their proteinase activity.     

Evaluation of a new multiplex PCR assay (ParaGENIE G-Amoeba Real-Time PCR kit) targeting Giardia intestinalis,Entamoeba histolytica and Entamoeba dispar/Entamoeba moshkovskii from stool specimens: evidence for the limited performances of microscopy-based approach for amoeba species identification

ObjectivesBesides the potential to identify a wide variety of gastrointestinal parasites, microscopy remains the reference standard in clinical microbiology for amoeba species identification and, especially when coupled with adhesin detection, to discriminate the pathogenic Entamoeba histolytica from its sister but non-pathogenic species Entamoeba dispar/Entamoeba moshkovskii. However, this approach is time-consuming, requires a high-level of expertise that can be jeopardized considering the low prevalence of gastrointestinal parasites in non-endemic countries. Here, we evaluated the CE-IVD-marked multiplex PCR (ParaGENIE G-Amoeba, Ademtech) targeting E. histolytica and E. dispar/E. moshkovskii and Giardia intestinalis.MethodsThis evaluation was performed blindly on a reference panel of 172 clinical stool samples collected prospectively from 12 laboratories and analysed using a standardized protocol relying on microscopy (and adhesin detection by ELISA for the detection of E. histolytica) including G. intestinalis (n = 37), various amoeba species (n = 55) including E. dispar (n = 15), E. histolytica (n = 5), as well as 17 other gastrointestinal parasites (n = 80), and negative samples (n = 37).ResultsThis new multiplex PCR assay offers fast and reliable results with appropriate sensitivity and specificity for the detection of G. intestinalis and E. dispar/E. moshkovskii from stools (89.7%/96.9% and 95%/100%, respectively). Detection rate and specificity were greatly improved by the PCR assay, highlighting several samples misidentified by microscopy, including false-negative and false-positive results for both E. dispar/E. moshkovskii and E. histolytica.ConclusionGiven the clinical relevance of amoeba species identification, microbiologists should be aware of the limitations of using an algorithm relying on microscopy coupled with adhesin detection by ELISA.     

Entamoeba histolytica Induces Host Cell Death in Amebic Liver Abscess by a Non-Fas-Dependent, Non-Tumor Necrosis Factor Alpha-Dependent Pathway of Apoptosis

Amebic liver abscess is characterized by extensive areas of dead hepatocytes that form cavities surrounded by a thin rim of inflammatory cells and few Entamoeba histolytica trophozoites. E. histolytica produces pore-forming proteins and proteinases, but how trophozoites actually kill host cells has been unclear. Here, we report that E. histolytica induces apoptosis in both inflammatory cells and hepatocytes in a severe combined immunodeficient (SCID) mouse model of amebic liver abscess. By studying infection in C57/BL6.lpr and C57/BL6.gld mice, we found that E. histolytica-induced apoptosis does not require the Fas/Fas ligand pathway of apoptosis, and by using mice with a targeted deletion of the tumor necrosis factor receptor I gene, we have shown that E. histolytica-induced apoptosis is not mediated by tumor necrosis factor alpha. Our data indicate that apoptosis plays a prominent role in the host cell death seen in amebic liver abscess in a mouse model of disease and suggest that E. histolytica induces cell death without using two common pathways for apoptosis.     

Interaction of antibodies with Entamoeba histolytica trophozoites from experimental amebic liver abscess: an immunocytochemical study

Using immunocytochemical techniques, we studied the interaction of antibodies with Entamoeba histolytica trophozoites present during the development of amebic liver abscess. Hamsters were intrahepatically inoculated with HM1-IMSS axenic amebas and sacrificed at different days post-inoculation. IgG of rabbit anti-E. histolytica and IgG of rabbit anti-IgG of hamster were used, both labeled with peroxidase. With the rabbit anti-E. histolytica, all trophozoites present in hepatic lesions from 1–7 days post-inoculation were highly labeled. The IgG of rabbit anti-IgG of hamster intensively stained only those trophozoites present in lesions from 1–2 days post-inoculation. From day 3, the intensity and number of labeled trophozoites decreased progressively. The results suggest that the interaction between the amebas and the IgG of hamster is non-specific during the first 2 days. The absence of labeling in the chronic stages could be due to changes in the membrane antigens of the parasite or to alterations in the bloodstream around necrosis. Also, the anti-E. histolytica antibodies produced in the serum during the development of the hepatic disease are apparently incapable of reaching and interacting with the trophozoites present on the liver abscess. This can explain in part why antibodies do not have an important role in the defense of the host. Received: 26 September 1999 / Accepted: 24 November 1999     

Entamoeba invadens: Identification of a SERCA protein and effect of SERCA inhibitors on encystation

Calcium has an important role on signaling of different cellular processes, including growth and differentiation. Signaling by calcium also has an essential function in pathogenesis and differentiation of the protozoan parasites Entamoeba histolytica and Entamoeba invadens. However, the proteins of these parasites that regulate the cytoplasmic concentration of this ion are poorly studied. In eukaryotic cells, the calcium-ATPase of the SERCA type plays an important role in calcium homeostasis by catalyzing the active efflux of calcium from cytoplasm to endoplasmic reticulum. Here, we reported the identification of SERCA of E. invadens (EiSERCA). This protein contains a putative sequence for endoplasmic reticulum retention and all domains involved in calcium transport identified in mammalian SERCA. By immunofluorescence assays, an antibody against SERCA of E. histolytica detected EiSERCA in a vesicular network in the cytoplasm of E. invadens trophozoites, co-localizing with calreticulin. Interestingly, EiSERCA was redistributed close to plasma membrane during encystation, suggesting that this pump could participate in regulate the calcium concentration during this process. In addition, thapsigargin and cyclopiazonic acid, both specific inhibitors of SERCA, affected the number and structure of cysts, supporting the hypothesis that calcium flux mediated by SERCA has an important role in the life cycle of Entamoeba.     

Immunization with the Entamoeba histolytica Surface Metalloprotease EhMSP-1 Protects Hamsters from Amebic Liver Abscess

Diarrhea and amebic liver abscesses due to invasive Entamoeba histolytica infections are an important cause of morbidity and mortality in the developing world. Entamoeba histolytica adherence and cell migration, two phenotypes linked to virulence, are both aberrant in trophozoites deficient in the metallosurface protease EhMSP-1, which is a homologue of the Leishmania vaccine candidate leishmanolysin (GP63). We examined the potential of EhMSP-1 for use as a vaccine antigen to protect against amebic liver abscesses. First, existing serum samples from South Africans naturally infected with E. histolytica were examined by enzyme-linked immunosorbent assay (ELISA) for the presence of EhMSP-1-specific IgG. Nine of 12 (75%) people with anti-E. histolytica IgG also had EhMSP-1-specific IgG antibodies. We next used a hamster model of amebic liver abscess to determine the effect of immunization with a mixture of four recombinant EhMSP-1 protein fragments. EhMSP-1 immunization stimulated a robust IgG antibody response. Furthermore, EhMSP-1 immunization of hamsters reduced development of severe amebic liver abscesses following intrahepatic injection of E. histolytica by a combined rate of 68% in two independent animal experiments. Purified IgG from immunized compared to control animals bound to the surface of E. histolytica trophozoites and accelerated amebic lysis via activation of the classical complement cascade. We concluded that EhMSP-1 is a promising antigen that warrants further study to determine its full potential as a target for therapy and/or prevention of invasive amebiasis.     

Differentiation of Entamoeba histolytica from E. dispar facilitated by monoclonal antibodies against a 150-kDa surface antigen

Murine monoclonal antibodies (mAbs) were produced against an n-octyl-β-D-glucopyranoside- extracted fraction of trophozoites of Entamoeba histolytica HM-1:IMSS. Four of the mAbs were reactive with a 150-kDa surface antigen characterized by Western-immunoblot analysis under nonreducing conditions. When the reactivity of the four mAbs with nine reference strains of E. histolytica was examined by an indirect fluorescence antibody test, two of the mAbs (EH3015 and EH3023) were found to react with all nine strains and the other two mAbs (EH3056 and EH3126) reacted with seven strains. The four mAbs did not react with any E. dispar reference strain or with other enteric protozoan parasites. The reactivity of EH3015 and EH3023 with numerous isolates of E. histolytica and E. dispar collected in our laboratories was also examined. The 2 mAbs reacted with all of the 37 E. histolytica isolates tested but did not react with any of the 33 isolates of E. dispar. These results indicate that common antigenic epitopes of E. histolytica are on the 150-kDa surface molecule and that mAbs can distinguish between E. histolytica and E. dispar. Received: 12 October 1996 / Accepted 13 December 1996     

Adherence of pathogenic and non-pathogenicEntamoeba histolytica strains to neutrophils

The adherence of polymorphonuclear leukocytes (PMNs) to eight pathogenic and nine nonpathogenic strains ofEntamoeba histolytica was examined. No difference between pathogenic and nonpathogenic strains was found. The addition of different carbohydrates confirmed the importance of the 170-kDa lectin ofE. histolytica in binding to PMNs, corroborated by the finding that treatment of PMNs with galactosidase inhibited adherence. Inhibition of the microfilament system ofE. histolytica using cytochalasin B resulted in a loss of adherence to PMNs. Inhibition of the microtubule system using nocodazole did not affect adherence. Preincubation of the trophozoites with serum resulted in enbanced adherence, but the serum factor responsible for this effect could not be identified. Fibronectin, vitronectin, integrins (CD11/CD18 molecules), complement, and mannose-binding protein did not seem to mediate adherence betweenE. histolytica and PMNs. In summary, these results indicate that defective adherence mechanisms are not a common feature of nonpathogenicE. histolytica strains.     

Effects of Panmede and various horse serum concentrations on the axenic cultivation ofEntamoeba histolytica strains in TPS-1 medium

We analyzed the influence of Panmede and horse serum concentrations on the growth of fiveEntamoeba histolytica strains (HK9, HM1, HM2, HM3 and HM38) axenically cultivated in TPS-1 medium. Panmede was evaluated by comparing the growth of strain HM1 in medium prepared with each of 15 Panmede lots; the yields ofE. histolytica trophozoites depended on the lot quality of Panmede, and their maximal values ranged from 8×10³ to 8.9×10⁴ amoebae/ml. The growth-promoting effect of eight lots of horse serum on strains HK9 and HM1 were studied using a single Panmede lot of good quality. Yields obtained with strain HK9 ranged from 8×10⁴ to 1.8×10⁵ amoebae/ml, whereas yields obtained with HM1 ranged from 3×10⁴ to 1.2×10⁵ amoebae/ml. Thus, the optimal serum concentration in TPS-1 medium that caused maximal growth ofE. histolytica cultures depended on the quality of the serum lot and proved to be specific for each of the fiveE. histolytica strains investigated. It ranged from 18% (v/v) for strain HM2 to 28% (v/v) for strain HM1. Our results reveal that the growth ofE. histolytica trophozoites in TPS-1 medium can be distinctly improved by selecting appropriate lots of Panmede and horse serum and using optimal serum concentrations.     

Participation of the serine-rich Entamoeba histolytica protein in amebic phagocytosis of apoptotic host cells

Entamoeba histolytica is an intestinal ameba that causes dysentery and liver abscesses. Cytotoxicity and phagocytosis of host cells characterize invasive E. histolytica infection. Prior to phagocytosis of host cells, E. histolytica induces apoptotic host cell death, using a mechanism that requires contact via an amebic galactose-specific lectin. However, lectin inhibition only partially blocks phagocytosis of already dead cells, implicating at least one additional receptor in phagocytosis. To identify receptors for engulfment of apoptotic cells, monoclonal antibodies against E. histolytica membrane antigens were screened for inhibition of phagocytosis. Of 43 antibodies screened, one blocked lectin-independent uptake of apoptotic cells, with >90% inhibition at a dose of 20 μg/ml (P < 0.0003 versus control). The same antibody also inhibited adherence to apoptotic lymphocytes and, to a lesser extent, adherence to and killing of viable lymphocytes. The antigen recognized by the inhibitory antibody was purified by affinity chromatography and identified by liquid chromatography-mass spectrometry as the serine-rich E. histolytica protein (SREHP). Consistent with this, the inhibitory antibody bound to recombinant SREHP present in bacterial lysates on immunoblots. The SREHP is an abundant immunogenic surface protein of unclear function. The results of this unbiased antibody screen strongly implicate the SREHP as a participant in E. histolytica phagocytosis and suggest that it may play an important role in adherence to apoptotic cells.