Induction of complement resistance in cloned pathogenic Entamoeba histolytica

The lytic effect of complement activated through the alternative pathway (AP) was studied on pathogenic and nonpathogenic Entamoeba histolytica recently isolated from stool samples. Recent nonpathogenic isolates were nearly unaffected by exposure to AP whereas recent pathogenic stool isolates were highly susceptible to AP dependent complement-mediated lysis. Complement susceptible pathogenic stool isolates developed complement resistance in vivo during hamster liver passage and in vitro during cultivation in the presence of increasing concentrations of normal human serum (NHS). Since a clone of pathogenic HM-l.IMSS which initially was highly susceptible also acquired complement resistance during cultivation in the presence of NHS, it is concluded that complement resistance was caused by induction rather than by selection alone. Because cultivation in the presence of heat-inactivated NHS did not affect complement susceptibility of the cloned HM-l.IMSS, complement activation itself might induce complement resistance in pathogenic E. histolytica.     

Sensitivity of Entamoeba histolytica and Entamoeba dispar patient isolates to human complement

Twenty-one Entamoeba histolytica and 56 Entamoeba dispar patient isolates were investigated for their sensitivity to the classical and alternative pathway of human complement. E. histolytica and E. dispar patient isolates were differentiated by polymerase chain reaction and hexokinase isoenzyme typing. It was found that 90.3% (±12.0%) of the trophozoites of E. histolytica were lysed after 30 min by the alternative pathway of complement in the presence of 50% human serum (19 isolates showed lysis rates higher than 80%), whereas E. dispar cells were less susceptible to the alternative pathway as 68.8% (±28.2%) of lysis occurred. However, 23 of the E. dispar isolates were lysed between 100 and 80% (90.9%±9.1%), demonstrating that about half of the tested E. dispar isolates were highly sensitive to complement lysis. Only 11 of the E. dispar isolates were proven to be 'resistant' to the alternative pathway of complement and were lysed less than 40%. These results are in conflict to earlier publications, describing resistance of E. dispar to complement lysis (Hamelmann et al. 1992, 1993).     

Lysis and immobilization of Giardia murk trophozoites in vitro by immune serum from susceptible and resistant mice

To define the potential role of antibody in host defence against Giardia muris, comparative assessment of in-vitro anti-trophozoite effects of immune serum obtained from susceptible (A/J) and resistant (B10.A) mice was done. Two anti-parasite effects of serum were identified and quantified: lysis and immobilization of trophozoites. These giardicidal activities were present in the serum from both A/J and B10.A mice. Maximal lysis of trophozoites by serum from A/J or B10.A mice was 40 to 50%. The lysis of trophozoites was abolished after incubation of serum at 56 degrees C for 30 min. The addition of exogenous complement restored lytic activity of heat-inactivated serum. The immobilization of trophozoites was dependent on both the duration of incubation and concentration of serum. After 30 min of incubation, over 98% of trophozoites were immobilized by immune serum from A/J or B10.A mice. There was no apparent relationship between the capacity of immune sera from A/J and B10.A mice to kill trophozoites in vitro and the ability of these strains of mice to control the infection with G. muris.     

Luminal bacteria and proteases together decrease adherence of Entamoeba histolytica trophozoites to Chinese hamster ovary epithelial cells: a novel host defence against an enteric pathogen.

BACKGROUND: Factors that prevent colonic mucosal invasion by pathogenic Entamoeba histolytica are not understood. A key initial step in pathogenesis of injury induced by amoeba is adherence to target cells mediated by a surface glycoprotein lectin on E histolytica. Mucin degrading bacteria normally present in the colon lumen produce glycosidases that degrade soluble or cell surface glycoconjugates. AIM: To determine whether glycosidases produced by mucin degrading bacteria, alone or in combination with proteases present in colon lumen, can decrease E histolytica adherence to target epithelial cells by degrading E histolytica adherence lectin. METHODS: The effects of exposure of E histolytica trophozoites strains HM1:IMSS and 200:NIH to faecal culture supernatant fluids, culture supernatant preparations of mucin degrading bacteria, and luminal proteases on their adherence to Chinese hamster ovary (CHO) cells were determined. The amount of surface adherence lectin on E histolytica trophozoites before and after treatment with glycosidases and proteases was determined by immunofluorescence. The effect of glycosidases and proteases on purified E histolytica lectin was determined by gel electrophoresis. RESULTS: Incubation of E histolytica with culture supernatant preparations or proteases alone did not modify their CHO cell adherence. However, 24 hour incubation of trophozoites with culture supernatant preparations together with pancreatic proteases decreased CHO cell adherence of HM1:IMSS strain by 71.1% (p < 0.001) and of 200: NIH strain by 95% (p < 0.05). Incubation of trophozoites for 24 hours with faecal extracts which contain bacterial and host hydrolases decreased the adherence of the HM1:IMSS strain by 69.2% (p < 0.01) and of the 200: NIH strain by 83.0%. Reduction of trophozoite adherence to CHO cells by hydrolases was promoted by 7.5 mM cycloheximide, and was reversible on incubation in an enzyme free medium. Decrease in CHO cell adherence of trophozoites was associated with decreased lectin on trophozoites as determined by immunofluorescence using a monoclonal antibody to the lectin. Purified lectin was degraded by the mixture of faecal culture supernant preparations and proteases, but not by either alone. CONCLUSIONS: Mucin degrading bacterial glycosidases and colonic luminal proteases together, but not alone, degrade the key adherence lectin on E histolytica trophozoites resulting in decreased epithelial cell adherence. These in vitro findings suggest a potential novel host defence mechanism in the human colon wherein the invasiveness of a pathogen could be curtailed by the combined actions of bacterial and host hydrolases. This mechanism may be responsible for preventing mucosal invasion by pathogenic E histolytica.     

Differences in complement-mediated killing of Entamoeba histolytica between men and women--an explanation for the increased susceptibility of men to invasive amebiasis?

Men are more than 7 times more likely to develop amebic liver abscess or amebic dysentery caused by Entamoeba histolytica than women. Because the complement system could play a key role in controlling amebiasis, we determined whether serum from men and women differ in the ability to kill amebic trophozoites. We found that serum from women was significantly more effective in killing E. histolytica trophozoites than serum from men, and this killing was complement dependent. Our results provide a possible explanation for the differential susceptibility of men and women to amebic liver abscess and amebic colitis.     

Enhancement of virulence of Entamoeba histolytica by in vitro liver treatment.

Enhancement of the virulence of five strains of Entamoeba histolytica (three xenically maintained and two axenically maintained) was studied after in vitro incubation with normal hamster liver. Increased virulence was shown by the ability of a small number of liver-treated trophozoites to produce liver lesions in hamsters. Enhancement of virulence was positively correlated with increased resistance to normal hamster serum complement in vitro.     

cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica.

A gamma gt11 cDNA library was constructed from poly(U)-Sepharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically 35S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-125I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4 degrees C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.     

The influence of specific antibodies and complement on the motility and the viability of Entamoeba histolytica trophozoites

The distribution of peroxidase-labeled normal or specific rabbit immunoglobulin (Ig) in Entamoeba histolytica trophozoites was studied by transmission electron microscopy. Small amounts of normal Ig became attached to the cell surface but did not redistribute. Internalized specific Ig was firmly bound to the inner membranes of phagocytosis vacuoles, while aggregates of normal Ig were dispersed over the vacuolar lumen. The distribution of fluorescein isothiocyanate-labeled Ig in living amebae was correlated with the cell motility at different times. Immobilization coincided with the cellular metabolic processing of internalized material. Remobilization occurred when the Ig was degraded and antibodies again bound to the reappearing surface antigens. E. histolytica activated complement by the classical pathway. Fresh guinea pig serum alone did not produce lysis which, however, did occur when it was added together with normal rabbit Ig. Normal rabbit Ig may constitute a complement-fixing substrate and activate complement by the classical pathway. Immobilization of the amebae by cytochalasin B (10 micrograms/ml) increased the susceptibility to cytolytic effects of specific antibodies or complement, or both. Pharmacological inhibition of cell motility might augment the immunological defence of the host against amebic infection.     

An immunogenic 30-kDa surface antigen of pathogenic clinical isolates of Entamoeba histolytica

A 30-kDa surface antigen was identified by Western blots with human immune sera in all 15 isolates of E. histolytica from patients with invasive amebiasis (pathogenic) but not in 15 strains from asymptomatic patients (nonpathogenic). This antigen is highly immunogenic in naturally infected humans and was recognized by sera from 22 patients with invasive disease but not by sera from 13 patients harboring nonpathogenic strains. Its surface location is supported by its differential extraction in the detergent phase of Triton X-114 and by surface immunofluorescence of live trophozoites. Unlike previously described amebic surface antigens, this 30-kDa antigen is undetectable in axenic strains that were originally isolated from patients with invasive disease but have been adapted to grow without bacteria. Affinity-purified antibody to the 30-kDa antigen did not promote lysis of complement-resistant pathogenic strains. This surface antigen may be diagnostically important in the identification of pathogenic clinical isolates.     

Molecular basis of the enhanced susceptibility of the erythrocytes of paroxysmal nocturnal hemoglobinuria to hemolysis in acidified serum.

When incubated in acidified serum, the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) are hemolyzed through activation of the alternative pathway of complement (APC), but normal erythrocytes are resistant to this process. PNH cells are deficient in decay-accelerating factor (DAF), a complement regulatory protein that inhibits the activity of both the classical and the alternative pathways. However, deficiency of DAF alone does not account entirely for the aberrant effects of acidified serum on PNH cells. Recently, we have shown that PNH erythrocytes are also deficient in another complement control protein called membrane inhibitor of reactive lysis (MIRL) that restricts complement-mediated lysis by blocking formation of the membrane attack complex (MAC). To determine the effects of the DAF and MIRL on susceptibility to acidified serum lysis, PNH cells were repleted with the purified proteins. DAF partially inhibited acidified serum lysis by blocking the activity of the amplification C3 convertase. MIRL inhibited acidified serum lysis both by blocking the activity of the MAC and by inhibiting the activity the C3 convertase. When DAF function was blocked with antibody, normal erythrocytes became partially susceptible to acidified serum lysis. By blocking MIRL, cells were made completely susceptible to lysis, and control of C3 convertase activity was partially lost. When both DAF and MIRL were blocked, the capacity of normal erythrocytes to control the activity of the APC and the MAC was destroyed, and the cells hemolyzed even in unacidified serum. These studies demonstrate that DAF and MIRL act in concert to control susceptibility to acidified serum lysis; of the two proteins, MIRL is the more important. In addition to its regulatory effects on the MAC, MIRL also influences the activity of the C3 convertase of the APC. Further, in the absence of DAF and MIRL, the plasma regulators (factor H and factor I) lack the capacity to control membrane-associated activation of the APC.     

Interaction of serum components with the cytotoxic action of Entamoeba histolytica

Native normal human serum is capable of inhibiting the cytotoxic action of Entamoeba histolytica against K562 tissue culture target cells assessed by a 51Cr-release test. It is suggested that a part of the inhibitory activity on amoebae's cytotoxic action is represented by the complement system which is known to lyze trophozoites by activation of the alternative pathway. For the rest of the serum's inhibitory activity on amoebic cytotoxic action molecule(s) is (are) responsible which act(s) independently of Mg2+ and Ca2+. The serum components have to act before the trophozoites have come in contact with the target cells. The opsonization of trophozoites with antiamoebic antibodies led to an inhibition of amoebae's cytotoxic action (ACA) in a dose-dependent manner. The inhibitory components of normal human serum and antiamoebic antibodies potentiated each other in their capacity to inhibit ACA. It is suggested that opsonization of amoebae with C3b via its metastable binding site leads to redistribution phenomena on the amoebae's surface similar to the effects observed with antiamoebic antibodies, both events leading to inhibition of ACA.     

Pathogenicity of Entamoeba dispar under xenic and monoxenic cultivation compared to a virulent E. histolytica

Two xenic isolates and cloned cultures of Entamoeba dispar were submitted to monoxenization using Crithidia fasciculata as the associated organism. Growth in monoxenic cultivation and ability of xenic and monoxenic trophozoites to destroy VERO cells and produce lesions in hamster livers were compared to those of a virulent E. histolytica. Parental and cloned E. dispar under monoxenic cultivation showed a remarkable lower growth than the monoxenic E. histolytica and were avirulent in both in vivo and in vitro tests. When xenically cultured, trophozoites of E. dispar showed a moderate lytic activity against VERO cells (1.5 to 41.8% of destruction) but caused severe hepatic lesions in hamsters as those caused by the virulent E. histolytica (29 to 100% in prevalence and 0.86 to 4.00 in lesion degree). Although E. dispar has not been associated with invasive disease in men, the ability of xenic trophozoites to produce prominent tissue damage in experimental conditions has indicated that some strains have a considerable pathogenic potential when in presence of bacteria.     

The interaction of human T-lymphocytes and Entamoeba histolytica: killing of virulent amoebae by lectin-dependent lymphocytes

Clinical and experimental studies indicate that following invasive disease due to Entamoeba histolytica, development of human cell-mediated immune mechanisms may provide protective immunity. Activated, human monocyte-derived macrophages in vitro can kill virulent axaenic amoebic trophozoites. This study describes the interaction of lectin-stimulated T-lymphocytes and E. histolytica trophozoites (virulent strain HM1-IMSS). Amoebae progressively killed unstimulated nonimmune T-lymphocytes over 18 h incubation with no effect on amoebic viability. T-lymphocytes, stimulated with phytohaemagglutinin (PHA), were progressively cytotoxic for virulent HMI amoebae over 18 h incubation, but were also reduced in viability themselves. Lymphocyte cytotoxicity for amoebae was absent if PHA was removed before or added only during the assay. PHA-stimulated T-lymphocytes killed amoebae at cell ratios of lymphocytes to amoebae as low as 50:1 and cytotoxicity was antibody-independent. PHA-stimulated T-lymphocytes, depleted of T8-bearing cells by complement-mediated lysis, were unable to kill amoebae. Adherence of PHA-stimulated T-lymphocytes to amoebae was greater than with unstimulated T-lymphocytes. Inhibition of the amoebic adherence lectin with N-acetyl-D-galactosamine decreased lymphocyte-amoebic adherence and resulted in increased lymphocyte amoebicidal activity and lymphocyte survival. Suspension of amoebae with or without adherent PHA-stimulated T-lymphocytes in a 10% dextran solution indicated that cytotoxicity was contact dependent. In summary, PHA-stimulated T-lymphocytes of the T8-phenotype can kill virulent axaenic E. histolytica trophozoites through a contact-dependent, antibody-independent mechanism.     

Identification of genomic responses to collagen binding by trophozoites of Entamoeba histolytica

Attachment of Entamoeba histolytica trophozoites to collagen is a known stimulus for parasite activation, leading to subsequent tissue destruction and invasion. To identify cellular mechanisms of trophozoite activation, we assessed global variations in gene expression during collagen interaction with E. histolytica. A shotgun DNA microarray was constructed by use of 9600 random inserts from an E. histolytica genomic DNA library. Through differential hybridization, key differences between gene expression in collagen-activated trophozoites and that in nonactivated trophozoites were identified. Fourteen differentially regulated clones were reproducibly identified and selected for sequencing. Among the genes identified were those coding for (1) components of a signaling cascade that had been previously hypothesized to transmit responses to cell attachment, (2) adapter proteins for vesicle formation, and (3) proteins that are implicated in cytoskeletal reorganization and locomotion. Two known virulence-factor genes--those for cysteine proteinases and amebapore--also were up-regulated in response to collagen stimulation. These results provide important new clues about how a pathogen orchestrates responses to the host environment as well as a new tool for the analysis of other aspects of Entamoeba species infection and pathogenicity.     

Complement activation by antigenic fractions of Entamoeba histolytica

Complement (C) activation induced by Entamoeba histolytica in normal non-immune human serum was studied by testing in vitro the ability of different fractions of the trophozoites to cause C3 breakdown. Whole trophozoites were found to activate both alternative and classical C pathways. The antibody-independent classical pathway (MgEGTA inhibitable) C activating capacity was found to greatly increase after disruption of the cell membrane by sonication. Subsequent analysis after differential centrifugation and ion exchange chromatography of the membrane/particulate fractions showed, that this activity was not due to DNA, which has been shown to have the similar characteristics, but to other, as yet unidentified components. A rather homogenous membrane fraction obtained by elution with 0.4 M Tris-HCl, pH 8.1 (described as 4M) and some cytoplasmic constituents obtained after gel chromatography retained a moderate degree of alternative pathway C3 activating capacity seen with intact trophozoites. Thus, it seems, that serum contact to the outer surface of E. histolytica trophozoites leads to C activation via both pathways with cell death as the result and to subsequent release of more efficiently classical pathway activating components. These phenomena probably have an important role in the inflammatory process in invasive amoebiasis.     

The pathogenicity of Entamoeba histolytica is related to the capacity of evading innate immunity

The host and parasite factors that influence susceptibility to Entamoeba histolytica infection and disease are not well understood. Entamoeba histolytica pathogenicity has been considered by focusing principally on parasite rather than host factors. Thus, research has concentrated on explaining the molecular differences between pathogenic E. histolytica and non-pathogenic E. dispar. However, the amoeba molecules considered most important for host tissue destruction (amoebapore, galactose/N-acetyl galactosamine inhibitable lectin, and cysteine proteinases) are present in both pathogenic E. histolytica and non-pathogenic E. dispar. In addition, the genetic differences in pathogenicity among E. histolytica isolates are unlikely to completely explain the different outcomes of infection. Considering that the principal difference between pathogenic and non-pathogenic amoebas lies in their surface coats, we propose that pathogenicity of the amoebas is related to the composition and properties of the surface coat components (or pathogen-associated molecular patterns, PAMPs), and the ability of innate immune response to recognize these components and eliminate the parasite. According to this hypothesis, a key feature that may distinguish pathogenic (E. histolytica) from non-pathogenic (E. dispar) strains is whether or not they can overcome innate immune defences. A corollary of this hypothesis is that in susceptible individuals the PAMPs are either not recognized or they are recognized by a set of Toll-like receptors (TLRs) that leads to an inflammatory response. In both cases, the result is tissue damage. On the contrary, in resistant individuals the innate/inflammatory response, induced through the activation of a different set of TLRs, eliminates the parasite.     

Genetic control of susceptibility of mice to infection with E. histolytica

Genetic susceptibility to Entamoeba histolytica infection in nine inbred strains and one outbred strain of mice was studied. The number of E. histolytica trophozoites in the ceca of the mice was examined 5 days after intracecal inoculation of axenic amoebae. C3H/HeCr, BALB/c, NZB/BIN, B10.A, DBA/2 and C57BL/6 were susceptible whereas A/J, CE, DBA/1 and CD-1 mouse strains were relatively resistant. Examination of F1 hybrid animals derived from susceptible B10.A and resistant A/J strains of mice showed that susceptibility was dominant over resistance. Segregation analysis of backcross and F2 progeny derived from the same progenitor strains is compatible with the hypothesis that susceptibility to E. histolytica infection in mice is controlled by a single, dominant gene which has been designated Enh. No association was found between the H-2 haplotype and the trait of susceptibility to amoebiasis, indicating that the major histocompatibility complex does not play a major role in regulating the early phase of the response to infection with E. histolytica.     

Use of antibodies to characterize a 220-kilodalton surface protein from Entamoeba histolytica

Antibodies were prepared against a 220-kilodalton (kDa) protein partially purified by Sepharose 4B chromatography of Entamoeba histolytica strain HM38:IMSS homogenates, and the protein was found to have lectin properties. The antibodies specifically recognized this protein in trophozoite homogenates. Immunologically related molecules with the same molecular weight were identified by polyclonal antibodies in strains HM1:IMSS, Entamoeba invadens, and Entamoeba histolytica Laredo. Six monoclonal antibodies recognized only the 220-kDa protein present in strains HM38 and HM1, a result indicating the presence of similar epitopes in the proteins from virulent strains isolated from humans. All the antibodies against the 220-kDa protein have the following properties: (1) they bind to the plasma membrane of live or fixed trophozoites, (2) they partially inhibit the adhesion of trophozoites to erythrocytes and cultured cells, and (3) they inhibit erythrophagocytosis.     

体外纯培养的蓝氏贾第鞭毛虫滋养体异常形态观察

目的观察体外纯培养的蓝氏贾第鞭毛虫滋养体的异常形态。方法用改良TYI-S-33培养基培养贾第虫,在接种后不同时间取培养管底部虫体涂片,Giemsa’s染色,在显微镜下观察虫体形态并照相。结果除观察到典型的营二分裂法繁殖的贾第虫滋养体外,还可见到多种形态异常的虫体,包括虫体呈非典型二分裂;滋养体胀大,胞质中有团块状和(或)不规则形状的嗜酸性物质;在胀大的贾第虫细胞内含有团块状和(或)不规则形状的嗜酸性物质以及鞭毛;在胀大的贾第虫细胞内含有6或8个核状物以及鞭毛;在一个母体细胞中含有3或4个子体细胞的雏形;胞质互相融合的4个滋养体的雏形;胞质互相融合的3个滋养体;胞质互相融合的4个滋养体;1对营二分裂的滋养体与另一个滋养体互相融合;2对营二分裂的滋养体互相融合;仅有1个细胞核的滋养体。结论在体外纯培养的贾第虫滋养体中发现一些异常形态的虫体,造成这种现象的原因尚不清楚,其发育过程有待进一步研究。     

Zymodemes of Entamoeba histolytica in Dhaka, Bangladesh

Stool samples containing Entamoeba histolytica were obtained from two sources: an urban hospital and an urban slum. Using the enzyme patterns of three enzymes (E.C.2.7.1.1 hexokinase, E.C.5.3.1.9 glucose phosphate isomerase and E.C.2.7.5.1 phosphoglucomutase) stained after cellulose acetate electrophoresis, four zymodemes (I, II, XIV and XVI) were identified in 71 isolates. Zymodemes considered to be pathogenic (II and XIV) were identified from 31 of 34 isolates from the urban hospital, and these zymodemes were strongly associated with dysentery and trophozoites containing ingested red blood corpuscles. Blood visible to the naked eye was more commonly seen in stools from which zymodeme XIV was isolated than in those containing zymodeme II (P less than 0.001). Zymodemes considered to be non-pathogenic (I and XVI) were identified in 34 of 37 isolates from slum dwellers and were not associated with blood in the stools.