Purification and identification of major soluble 40-kDa antigenic protein from Entamoeba histolytica: its application for serodiagnosis of asymptomatic amebiasis.

One of the major soluble antigenic proteins of Entamoeba histolytica was purified to homogeneity and identified on a molecular basis. Its recombinant protein was expressed in Escherichia coli as a fusion protein with Shistosoma japonicum glutathione S-transferase. Apparent molecular weight of the purified antigenic protein was estimated to be 40-kDa and molecular-based analysis indicated that the purified protein was NADP+-dependent alcohol dehydrogenase (EhADH1). The application of the purified protein for the serodiagnosis of amebiasis was evaluated using an enzyme-linked immunosorbent assay applied to sera obtained from patients with amebiasis and healthy human controls. The purified protein was well recognized by the sera from asymptomatic amebiasis humans (22/22, 100%), whereas, it was less recognized by the sera from symptomatic amebiasis patients (5/16, 31%) with amebic colitis or liver abscess. To confirm the antigenicity of EhADH1, the recombinant glutathione S-transferase-EhADH1 fusion protein was also evaluated by the enzyme-linked immunosorbent assay using the same sera. The recombinant protein was also recognized by the sera from asymptomatic amebiasis humans (14/22, 64%) and less recognized by the sera from symptomatic amebiasis patients (2/16, 13%). These results suggest that the purified protein is applicable antigen for serodiagnostic screening of asymptomatic amebiasis humans.     

Identification of Immunogenic Epitopes of the 170-kDa Subunit Adhesin of Entamoeba histolytica in Patients with Invasive Amebiasis

ABSTRACT. Entamoeba histolytica causes amebic dysentery (AD) and liver abscess (ALA). Little is known about protective immunity to amebiasis, and studies in this area have been complicated by the paucity of defined ameba antigens. We examined the proliferative responses of peripheral blood mononuclear cells (PBMC) from patients with AD and ALA to a recombinant protein containing a portion of the 170 kDa adhesin of E. histolytica (170CR), and to two synthetic peptides (1 and 2) derived from the 170 kDa sequence that were predicted to contain T cell epitopes. A significant number of patients with AD and ALA had PBMC that proliferated to 170CR molecule, and several individuals with ALA and AD had T cells that recognized one or both peptides. Contrarily, individuals from a non-endemic region for amebiasis did not respond to 170CR protein, or to both peptides. In regard to antibody response, nine of fifteen patients with ALA showed antibodies to 170CR protein. These same patients had antibodies to peptide 2. We identified peptides from 170-kDa adhesin that may contain both T and B cell epitopes recognized by some patients with invasive amebiasis. These peptides may be valuable reagents in studies of the immune response to amebiasis.     

The plasma membrane of Entamoeba histolytica: structure and dynamics.

The plasma membrane components of the parasitic protozoan Entamoeba histolytica, the causative agent of human invasive amebiasis, have been biochemically and immunologically characterized during the last decade. In addition, genes coding for certain surface proteins have been cloned. In spite of these advances, a unified characterization of plasma membrane antigenic components of the parasite is still required for badly needed advancements in the design of useful diagnostic, epidemiologic, and immunoprophylactic tools. Here we review current knowledge on this issue and address the problem of the considerable variation in the electrophoretic profiles of plasma membrane proteins obtained by different groups. In addition, the differences in the degree of recognition of reported membrane antigens with human immune sera, and the diverse interpretations concerning the possible functions of the surface molecules characterized are discussed. A comparative analysis of plasma membrane proteins of E histolytica trophozoites using three different isolation methods revealed that it is possible to select for specific membrane proteins, depending on the lysis conditions. In our view, the method of Calderón and Avila preserves more proteins than other methods tested. Using sera from recent cases of invasive amebiasis studied by several laboratories in various geographical areas, a basic antigenic pattern of 11 principal proteins with molecular weights of 220, 170, 150, 125, 97, 80, 60, 45, 20 and 9 kDA was established for the pathogenic E histolytica strain HM1:IMSS, used by most research groups.     

Partial characterization of a 36-kDa antigen of Entamoeba histolytica and its recognition by sera from patients with amoebiasis

A 36-kDa antigen of axenically grown pathogenic Entamoeba histolytica (HM1-IMSS) was eluted from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-resolved crude amoebic extract antigens. The immunoreactivity of this partially purified 36-kDa antigen with monoclonal antibody (MoAb) 3D(10) altered significantly (P<0.01) after heat and trypsin treatment but remained unaltered after treatment with sodium metaperiodate (P0.5), thereby indicating the protein nature of the epitope recognized by MoAb 3D(10). The epitope was found to be localized on the surface as well as in the cytoplasm of the E. histolytica trophozoites with the majority of it in the cytoplasm. In addition, this epitope was also found to be present on the cyst form of the parasite. The 36-kDa molecule was recognized by the sera from 29 (85%) of the 34 patients with amoebic liver abscess and five (83%) of the six patients with amoebic colitis. No serum samples from asymptomatic cyst passers, from patients with non-amoebic hepatic or intestinal disorders and apparently healthy subjects had antibodies that reacted with this 36-kDa molecule. The immune responses in man to this 36-kDa amoebic molecule indicate a potential specific role for this molecule in invasive amoebiasis.     

Monoclonal antibodies directed against the galactose-binding lectin of Entamoeba histolytica enhance adherence.

The Entamoeba histolytica galactose-binding lectin is a surface glycoprotein composed of 170- and 35-kDa subunits. Inhibition of this lectin with galactose or anti-170 kDa subunit polyclonal antibody blocks amebic adherence to target cells and colonic mucin glycoproteins. We describe the properties of 10 mAb with specificity for the 170-kDa subunit. Based on competitive binding studies, six nonoverlapping antigenic determinants on the lectin were identified. The effect of the mAb on adherence of amebic trophozoites to both Chinese hamster ovary (CHO) cells and human colonic mucins was measured. Antilectin antibodies directed against epitopes 1 and 2 enhanced adherence, with the number of amebae having at least three adherent CHO cells increasing with the addition of epitope 1 mAb from 26 +/- 9 to 88 +/- 2% and the binding of colonic mucins increasing from 34 +/- 1 to 164 +/- 3 pg/10(5) amebae. Antibody-enhanced adherence remained 90 to 100% galactose inhibitable, occurred at 4 degrees C and was not Fc mediated. Univalent Fab fragments of epitope 1 mAb augmented mucin binding by 238% and CHO cell adherence by 338%. The binding of purified lectin to CHO cells was increased from 1.1 +/- 0.1 to 2.4 +/- 0.3 ng/10(3) CHO cells by mAb directed to epitope 1, demonstrating that enhanced adherence was due to direct activation of the lectin. mAb to epitope 3 bound to the lectin only upon its solubilization from the membrane and had no effect on adherence. Adherence to CHO cells and mucins was inhibited from 50 to 75% by mAb to epitopes 4 and 5; epitope 6 mAb inhibited amebic adherence to CHO cells but not mucins. The pooled sera from 10 patients with amebic liver abscess blocked the binding to the 170-kDa subunit of mAb directed to all six epitopes. Striking individual variations in the effects of immune sera on adherence were observed. Although the sera of all 44 South African patients with amebic liver abscess had high titer anti-lectin antibodies, 16 patients' sera significantly (more than 3 SEM) enhanced adherence whereas 25 patients' sera significantly inhibited adherence. Antilectin antibodies exert profound functional effects on the interaction of E. histolytica with target cells and human colonic mucins. Exploration of the clinical consequences of adherence-enhancing and inhibitory antibody responses may give insight into the role of antilectin antibodies in immunity to invasive amebiasis.     

Molecular cloning of a member of the Fasciola hepatica saposin-like protein family

A 436-bp complementary DNA (cDNA) was isolated from an adult Fasciola hepatica cDNA expression library by screening with the serum from a rabbit infected with F. hepatica for 4 wk. The deduced amino acid sequence encoded by this cDNA is an 11.5-kDa polypeptide that has significant homology to F. hepatica NK-lysin protein, to several members of saposin-like or NK-lysin protein families, as well as 3 amoebapore precursors of Entamoeba histolytica. The most striking feature observed within this protein, denoted FhSAP-2, is the presence of 6 conserved cysteine residues arranged within 5 amphipathic alpha-helical domains and the presence of 7 hydrophobic residues in strictly conserved positions. Using enzyme-linked immunosorbent assay it was found that rFhSAP-2 is highly reactive with sera from rabbits infected with F. hepatica for 2-14 wk as well as with sera from humans with chronic fascioliasis. An anti-rFhSAP-2 rabbit antiserum reacted with F. hepatica excretory-secretory antigens by Western blot, revealing a major 11.5-kDa and 2 minor 46- and 67-kDa antigenic polypeptides. This suggests that FhSAP-2 may be an antigen released from cytoplasmic storage granules present within F. hepatica parasites. rFhSAP-2 also exhibits a strong lytic activity on human erythrocytes and peripheral blood mononuclear cells. This suggests that cell lysis could be 1 of the biological functions of this protein.     

Amebiasis in HIV-1-infected Japanese men: clinical features and response to therapy

Invasive amebic diseases caused by Entamoeba histolytica are increasing among men who have sex with men and co-infection of ameba and HIV-1 is an emerging problem in developed East Asian countries. To characterize the clinical and epidemiological features of invasive amebiasis in HIV-1 patients, the medical records of 170 co-infected cases were analyzed retrospectively, and E. histolytica genotype was assayed in 14 cases. In this series of HIV-1-infected patients, clinical presentation of invasive amebiasis was similar to that described in the normal host. High fever, leukocytosis and high CRP were associated with extraluminal amebic diseases. Two cases died from amebic colitis (resulting in intestinal perforation in one and gastrointestinal bleeding in one), and three cases died from causes unrelated to amebiasis. Treatment with metronidazole or tinidazole was successful in the other 165 cases. Luminal treatment was provided to 83 patients following metronidazole or tinidazole treatment. However, amebiasis recurred in 6 of these, a frequency similar to that seen in patients who did not receive luminal treatment. Recurrence was more frequent in HCV-antibody positive individuals and those who acquired syphilis during the follow-up period. Various genotypes of E. histolytica were identified in 14 patients but there was no correlation between genotype and clinical features. The outcome of metronidazole and tinidazole treatment of uncomplicated amebiasis was excellent even in HIV-1-infected individuals. Luminal treatment following metronidazole or tinidazole treatment does not reduce recurrence of amebiasis in high risk populations probably due to amebic re-infection.     

Cytopathologic and genetic diagnosis of pulmonary amebiasis: a case report

BACKGROUND: Amebiasis is a parasitic infection with Entamoeba histolytica. Pulmonary amebiasis is rare since the infection is commonly manifested as amebic colitis or liver abscess. Most pleuropulmonary amebiasis is seen in patients with amebic liver abscesses. A pulmonary amebic lesion without either a liver abscess or amebic colitis is extremely rare. Thus, reported cases of sputum cytologic diagnosis of a pulmonary amebic lesion from a patient without a liver abscess are also very rare. CASE: A 53-year-old man presented with a dry cough and mild fever. Chest radiography revealed an abnormal solitary mass lesion in the right upper lung field. The clinical diagnosis was a bacterial lung abscess. Sputum cytologic examination demonstrated many trophozoites of E. histolytica. Following sputum cytodiagnosis, serologic tests revealed a slightly high but almost normal titer of IgG antibodies to E. histolytica, indicating the possible presence of the pathogen. Polymerase chain reaction (PCR) using E. histolytica-specific primers for DNA extracted from the sputum sample revealed specific DNA product. CONCLUSION: Pulmonary amebiasis without either a liver abscess or amebic colitis must be distinguished from bacterial abscesses and neoplastic disease. A sputum cytologic examination combined with PCR for DNA extracted from a sputum sample is a good approach to the diagnosis of a pulmonary amebic abscess.     

Entamoeba histolytica disturbs the tight junction complex in human enteric T84 cell layers.

Entamoeba (E.) histolytica trophozoites initiate amebiasis through invasion into the enteric mucosa. It was our aim to understand the molecular interactions between amebic trophozoites and enterocytes during the early steps of invasion. Trophozoites of E. histolytica strain HM1:IMSS were seeded on the apical side of enteric T84 cell layers, which were established on filters in two-compartment culture chambers. Cocultures were analyzed for paracellular permeability by measurement of transepithelial electrical resistance (TER) and for the tight junction proteins ZO-1, ZO-2, occludin, and cingulin by immunocytochemistry and immunoprecipitation. On direct contact with the apical side of the enteric cells, trophozoites caused an increase in paracellular permeability as evidenced by a decrease of TER associated with an increase in [(3)H]mannitol flux. Immunoprecipitation of cocultures revealed dephosphorylation of ZO-2, loss of ZO-1 from ZO-2, and degradation of ZO-1 but less so of ZO-2 and none of occludin or E-cadherin. In conclusion, trophozoite-associated increase in paracellular permeability of enteric cell layers is ascribed to disturbance of the molecular organization of tight junction proteins.     

Progress towards an amebiasis vaccine

Amebiasis (infection by Entamoeba histolytica) remains a major health problem in much of the developing world. Morbidity and mortality from amebic dysentery and amebic liver abscess have persisted despite the availability of effective anti-amebic therapy, suggesting a need for alternative measures of disease control. Through the application of recombinant DNA technology, several E. histolytica antigens have now been expressed in prokaryotic systems and tested in animal models as vaccines to prevent invasive amebiasis. In this review, Sam Stanley Jr discusses why a vaccine for amebiasis may be feasible, and describes the recent development of several promising recombinant E. histolytica antigen-based parenteral and oral vaccine candidates.     

Cleavage of C3 by a neutral cysteine proteinase of Entamoeba histolytica

The major secreted proteinase of Entamoeba histolytica, a 56-kDa neutral cysteine proteinase, activates C by cleaving C3. The action of the proteinase is similar to C-derived C3 convertases because it produces a single cleavage of the alpha-chain in a dose- and time-dependent manner and cleaves C3 between residues 78 and 79, only one amino acid residue distal to the natural site acted on by the C3 convertases. C3a generation was detected by RIA. The 105-kDa fragment produced by the cleavage of the alpha-chain was structurally and functionally equivalent to the alpha'-chain of C3b, as demonstrated by susceptibility to the action of factors I and H and participation in the activation of the alternative pathway of C. Activation of C by the 56-kDa neutral cysteine proteinase may play a role in the early inflammatory response in amebic lesions and thus contribute to the pathogenesis of invasive amebiasis.     

Putative terminase subunits of herpes simplex virus 1 form a complex in the cytoplasm and interact with portal protein in the nucleus

Herpes simplex virus (HSV) terminase is an essential component of the molecular motor that translocates DNA through the portal vertex in the capsid during DNA packaging. The HSV terminase is believed to consist of the UL15, UL28, and UL33 gene products (pUL15, pUL28, and pUL33, respectively), whereas the HSV type 1 portal vertex is encoded by UL6. Immunoprecipitation reactions revealed that pUL15, pUL28, and pUL33 interact in cytoplasmic and nuclear lysates. Deletion of a canonical nuclear localization signal (NLS) from pUL15 generated a dominant-negative protein that, when expressed in an engineered cell line, decreased the replication of wild-type virus up to 80-fold. When engineered into the genome of recombinant HSV, this mutation did not interfere with the coimmunoprecipitation of pUL15, pUL28, and pUL33 from cytoplasmic lysates of infected cells but prevented viral replication, most nuclear import of both pUL15 and pUL28, and coimmunoprecipitation of pUL15, pUL28, and pUL33 from nuclear lysates. When the pUL15/pUL28 interaction was reduced in infected cells by the truncation of the C terminus of pUL28, pUL28 remained in the cytoplasm. Whether putative terminase components localized in the nucleus or cytoplasm, pUL6 localized in infected cell nuclei, as viewed by indirect immunofluorescence. The finding that the portal and terminase do eventually interact was supported by the observation that pUL6 coimmunoprecipitated strongly with pUL15 and weakly with pUL28 from extracts of infected cells in 1.0 M NaCl. These data are consistent with the hypothesis that the pUL15/pUL28/pUL33 complex forms in the cytoplasm and that an NLS in pUL15 is used to import the complex into the nucleus where at least pUL15 and pUL28 interact with the portal to mediate DNA packaging.     

Re-oxygenation of hypoxic simian virus 40 (SV40)-infected CV1 cells causes distinct changes of SV40 minichromosome-associated replication proteins.

Hypoxia interrupts the initiation of simian virus 40 (SV40) replication in vivo at a stage situated before unwinding of the origin region. After re-oxygenation, unwinding followed by a synchronous round of viral replication takes place. To further characterize the hypoxia-induced inhibition of unwinding, we analysed the binding of several replication proteins to the viral minichromosome before and after re-oxygenation. T antigen, the 34-kDa subunit of replication protein A (RPA), topoisomerase I, the 48-kDa subunit of primase, the 125-kDa subunit of polymerase delta, and the 37-kDa subunit of replication factor C (RFC) were present at the viral chromatin already under hypoxia. The 70-kDa subunit of RPA, the 180-kDa subunit of polymerase alpha, and proliferating cell nuclear antigen (PCNA) were barely detectable at the SV40 chromatin under hypoxia and significantly increased after re-oxygenation. Immunoprecipitation of minichromosomes with T antigen-specific antibody and subsequent digestion with micrococcus nuclease revealed that most of the minichromosome-bound T antigen was associated with the viral origin in hypoxic and in re-oxygenated cells. T antigen-catalysed unwinding of the SV40 origin occurred, however, only after re-oxygenation as indicated by (a) increased sensitivity of re-oxygenated minichromosomes against digestion with single-stranded DNA-specific nuclease P1; (b) stabilization of RPA-34 binding at the SV40 minichromosome; and (c) additional phosphorylations of RPA-34 after re-oxygenation, probably catalysed by DNA-dependent protein kinase. The results presented suggest that the subunits of the proteins necessary for unwinding, primer synthesis and primer elongation first assemble at the SV40 origin in form of stable, active complexes directly before they start to work.     

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.     

Comparison of two methods (microscopy and enzyme-linked immunosorbent assay) for the diagnosis of amebiasis

Diagnosis of amebiasis is usually performed on a clinical basis alone in most endemic countries having limited economic resources. This epidemiological study was conducted using modern diagnostic tests for amebiasis in the southeastern region of Turkey, an endemic area for amebiasis. The population of this study included patients with symptomatic diarrhea/dysentery attending both Yuzuncu Yil University, Van and Harran University, Sanliurfa, Turkey. A total of 380 stool specimens were collected and examined for Entamoeba by light microscopy (fresh, lugol, and trichrome staining) and stool antigen detection based- enzyme-linked immunosorbent assay (EIA) test (TechLab Entamoeba histolytica II). 24% (91/380) of stool specimens were positive for E. histolytica/Entamoeba dispar trophozoites/cysts microscopically using trichrome staining. 13% (51/380) of the stool specimens were found to be positive for E. histolytica by the EIA test, including 15% (14/91) of microscopy (+) stool specimens and 13% (37/289) of microscopy (-) stool specimens. Enteric parasites were common in these populations with 66% (251/380) of the study population harboring more than one parasite. In addition to the 13% (51/380) of patients determined to have E. histolytica by EIA, eighty-six patients (22.6%) had Blastocystis hominis, 54 (14.2%) Entamoeba coli, 44 (11.5%) Giardia lamblia, 16 (4.2%) Chilomastix mesnili, 15 (3.9%) Iodamoeba bütschlii, 12 (3.1%) Hymenolepis nana, 9 (2.3%) Endolimax nana, 9 (2.3%) Dientamoeba fragilis, and 8 (2.1%) had Ascaris lumbricoides. We concluded that E. histolytica infection was found in 13% of the patients presenting with diarrhea in Van and Sanliurfa Turkey.     

An outbreak of amebiasis in an institution for the mentally retarded in Japan

The results of an epidemiological survey in a 190-patient institution for mentally retarded were reported. Twenty percent of the patients had either cysts or trophozoites of Entamoeba histolytica in their stools, and 38% were positive serologically. The amebic outbreak revealed a sex-independent but age-dependent distribution; younger patients had more serious symptoms in cases invasive amebiasis. A high prevalence of amebic infection was found in the heavily retarded patients, and the positive cases tended to concentrate in certain training classes. Further demographic analysis suggests that the amebic infection was possibly caused by abnormal behavior of heavily retarded patients.     

Cytologic demonstration of Entamoeba histolytica using immunoperoxidase techniques. Report of two cases

An indirect immunoperoxidase technique to detect Entamoeba histolytica in cell samples from patients suspected to have amebiasis is described. Using a rat antiserum specific for E. histolytica, the organism was clearly identified both in smears and in cell blocks. This immunoperoxidase technique seems to offer great possibilities for a specific, accurate and rapid identification of amebic infestation in diagnostic cytology.     

Immunoenzyme assay using micro Dot on nitrocellulose (Dot-ELISA) in the diagnosis of Chagas' disease. I. Comparative study of 2 antigenic preparations of Trypanosoma cruzi

Using the Dot-ELISA technique, two antigenic preparations of Trypanosoma cruzi epimastigote forms have been compared for the diagnosis of Chagas' disease: (1) The cytoplasmic fraction (cytoplasmic antigen) and (2) whole formalin fixed epimastigotes (integral antigen). There was been used sera from 95 chagasic patients with chronic cardiomyopathy, positive conventional serology and either positive or negative xenodiagnosis; 74 subjects with negative conventional serology, and either clinically normal or presenting cardiomyopathy; 74 patients with different diseases including syphilis, toxoplasmosis, leishmaniasis or autoantibodies such as rheumatoid factor and antinuclear antibodies. By defining the diagnostic titers (cut off): 1:512 for cytoplasmic antigen and 1:128 for the integral antigen, a sensitivity of 100% has been obtained with both antigenic preparations, being the specificity of 96% for the former and 100% for the latter when leishmaniasis sera were not included. A comparative study with conventional serology was carried out using 147 sera from a Laboratory of Chagas' diagnosis; Dot-ELISA with cytoplasmic antigen showed co-positivity index of 1.0, co-negativity 0.989 and efficiency of 0.993, and Dot-ELISA with integral antigen 1.0, 0.979 and 0.986 respectively. According to this evaluation, Dot-ELISA using whole formalin fixed epimastigotes might be a practical alternative for the serological diagnosis of Chagas' disease.     

Human monoclonal anti-La antibodies. The La protein resides on a subset of Ro particles

We have addressed the problem of anti-La autoimmune responses by defining the specific binding sites of human mAb to the La protein. Two human anti-La mAb were developed; one an IgM (kappa) (designated 8G3) and the second an IgG1 (kappa) (9A5) isotype. The mAb 8G3 immunoprecipitated the La RNA and La protein from crude human cell lysates; bound the 50-kDa La protein and a 28-kDa digestion fragment in immunoblots, and recognized a small defined internal segment from the cloned La protein. In contrast, the IgG isotype (9A5) failed to precipitate native La from cell lysates but bound the same segment of digested La protein and the same polypeptide of 131 amino acids in length from the cloned La protein. Immunoprecipitation experiments performed with these mAb demonstrated that the La protein is a component of a subset of Ro particles. The data suggest that the La protein is not present on the hY RNA in the absence of the Ro polypeptide. These observations may define functional subsets or maturation states of hY RNA based on their association with Ro or Ro and La polypeptides.