Host-antibody inductivity of virulent Entamoeba histolytica and non-virulent Entamoeba moshkovskii in a mouse model
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Despite similarities in morphology, gene and protein profiles, Entamoeba histolytica and E. moshkovskii show profound differences in pathogenicity. Entamoeba histolytica infection might result in amoebic dysentery and liver abscess, while E. moshkovskii causes only mild diarrhea. Extensive studies focus on roles of host immune responses to the pathogenic E. histolytica; however, evidence for E. moshkovskii remains scarce.
To study differences in host-antibody response profiles between E. histolytica and E. moshkovskii, mice were immunized intraperitoneally with different sets of Entamoeba trophozoites as single species, mixed species and combinations.
Mice prime-immunized with E. histolytica and E. moshkovskii combination, followed by individual species, exhibited higher IgG level than the single species immunization. Mice immunized with E. moshkovskii induced significantly higher levels and long-lasting antibody responses than those challenged with E. histolytica alone. Interestingly, E. histolytica-specific anti-sera promoted the cytopathic ability of E. histolytica toward Chinese hamster ovarian (CHO) cells, but showed no effect on cell adhesion. There was no significant effect of immunized sera on cytopathic activity and adhesion of E. moshkovskii toward both CHO and human epithelial human colonic (Caco-2) cell lines. Monoclonal-antibody (mAb) characterization demonstrated that 89% of E. histolytica-specific mAbs produced from mice targeted cytoplasmic and cytoskeletal proteins, whereas 73% of E. moshkovskii-specific mAbs targeted plasma membrane proteins.
The present findings suggest that infection with mixed Entamoeba species or E. moshkovskii effectively induces an antibody response in mice. It also sheds light on roles of host antibody response in the pathogenic difference of E. histolytica and E. moshkovskii trophozoites, and cell surface protein modifications of the amoebic parasites to escape from host immune system.
KeywordsAmoebiasis Entamoeba histolytica Entamoeba moshkovskii Pathogenicity Antibody dependent enhancement
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
enzyme-linked immunosorbent assay
horse radish peroxidase
ethylene diamine tetraacetic acid
Roswell Park Memorial Institute
bovine serum albumin
Chinese hamster ovary
amoebic liver abscess
antibody dependent enhancement
tumor necrosis factor alpha
Entamoeba histolytica is an anaerobic pathogenic protozoan parasite that causes approximately 100,000 global deaths annually due to amoebiasis . Disease symptoms range from mild diarrhea to severe bloody diarrhea with mucus as the parasite invades the intestinal epithelium . After invading the intestinal lamina propria, this parasite enters the blood stream to reach other organs and results in abscesses, most commonly in the liver , and rarely in lungs  and brain . The pathogenesis of E. histolytica starts with parasite adhesion at the large intestinal epithelium and secretion of cysteine proteases, leading to the degradation of host tissues. The secreted cysteine proteases play important roles in degrading gut mucosal IgA and circulating IgG, resulting in the ineffectiveness or failure of host immunity, thus inversely promote extra-intestinal infection of E. histolytica [6, 7]. In addition, the parasite-gut adhesion was shown to trigger host signal transductions through caspases 3-like cascade and caspases 8- and 9-independent manner . These lead to apoptotic cell death, which were preferentially phagocytosed by the parasite. The interaction also stimulates production of pro-inflammatory cytokines, including interleukin (IL)-1, IL-6, IL-8, IFN-γ and tumor necrosis factor (TNF)-α, which consequentially promote tissue damages and severity of the disease [9, 10]. Inhibition of TNF-α has been proved to significantly reduce the inflammation and tissue destruction , while the absence of the anti-inflammatory cytokine IL-10 has been shown to result in increased severity of intestinal amoebiasis . Thus, the manifestation of amoebiasis apparently happens through the parasite’s ability to activate cytokine-mediated cell deaths and manipulate the host immune system.
Entamoeba moshkovskii was previously considered as a non-pathogenic protozoan parasite, which was commonly found to co-occur in human stools collected from E. histolytica endemic areas, often leading to misdiagnosis of E. histolytica due to their mostly identical morphology [13, 14]. Despite being considered non-pathogenic, E. moshkovskii has been gradually reported as associated with diarrhea in humans and mice [15, 16, 17]. Recently, E. moshkovskii was reported to cause subcutaneous abscess in Indonesia . Shimokawa et al.  showed that E. moshkovskii was able to cause symptoms, including weight loss, diarrhea and colitis in susceptible mice as is the case for E. histolytica. Furthermore, damages of the intestinal epithelium of E. moshkovskii-infected mice were observed due to host IFN-γ mediated cell apoptosis .
In the present study, we aimed to investigate the immunogenicity of E. histolytica and E. moshkovskii trophozoites through host-antibody response profiles as well as effect of the immunized sera on Entamoeba pathogenicity. We found that mouse immunization with mixed Entamoeba species was able to induce both specific IgA and IgG higher levels than single species. The effect of the immunized sera on cytopathic activity and host cell adhesion were investigated and the possible immune evasion and cell manipulating mechanisms by E. histolytica are discussed. Our findings may shed more light on E. histolytica pathogenicity, which can be of further benefit in the development of diagnosis modalities, treatment and vaccines for this parasite.
Mouse immunization with Entamoeba cells
Trophozoite cells of E. histolytica strain HM1: IMSS and E. moshkovskii strain Laredo, which were kindly provided by Professor Tomoyoshi Nozaki, Department of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, Japan, were axenically cultured in bis-iron serum (BIS) medium at 37 °C and 26.5 °C, respectively. Cells were harvested by placing culture tubes on ice for 10 min to detach the cells, followed by centrifugation at 200× g for 3 min at 4 °C with three washes using cold phosphate-buffered saline (PBS). Viable amoeba cells were counted using a hemocytometer by trypan blue exclusion (0.2% trypan blue). For studies of host-antibody response, BALB/c mice (3 mice/group; 12 mice in total) were immunized with 2 × 106 cells of mixed species (1 × 106 cells each of E. histolytica and E. moshkovskii) or 2 × 106 cells of individual species (E. histolytica or E. moshkovskii) according to 4 immunization designs (group 1 mice received E. histolytica cells for 4 doses; group 2 mice received E. moshkovskii cells for 4 doses; group 3 mice received E. histolytica and E. moshkovskii cell mixture for 2 doses, followed by E. histolytica cells for 2 doses; group 4 mice received E. histolytica and E. moshkovskii cell mixture for 2 doses followed by E. moshkovskii cells for 2 doses). Immunization was performed intraperitoneally (IP) with two-week intervals. Whole blood was collected from the ventral tail vein before each immunization  and after the 4th boost for 2 weeks (B4: bleed 4) and 8 weeks (B5: bleed 5). Serum collected before the first immunization (pre-immunized serum) was used as a negative control for the baseline antibody level of each mouse.
Monoclonal antibody (mAb) production
BALB/c mice (2 mice per set) were immunized with 2 × 106 cells of E. histolytica and E. moshkovskii trophozoite for 3 doses followed by 2 doses of mixed Entamoeba cells (1 × 106 cells each of E. histolytica and E. moshkovskii) in two-week intervals. Blood samples were collected from the tail vein before each immunization and tested for Entamoeba-specific antibodies. The mice were sacrificed and splenic B cells were fused with mouse myeloma cells using the standard hybridoma technique described by Moonsom et al. . Hybridoma cells secreting Entamoeba-specific antibodies were screened by enzyme-linked immunosorbent assay (ELISA) with Entamoeba cell lysate proteins. Limiting dilutions were performed to obtain the mAb-producing cells.
Preparation of cellular protein compartments
Axenically cultivated E. histolytica and E. moshkovskii trophozoite cells were harvested and washed with PBS, pH 7.4. Total cell lysate proteins were solubilized using mammalian protein extraction reagent (M-PER) (Thermo Fisher Scientific, Waltham, MA, USA). Cells were fractioned into cytoplasmic, membranous, nuclear and cytoskeletal portions using a Qproteome cell compartment kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Protein profiles of cell fractions were analyzed on a 10%-gel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and protein concentration was determined using Bradford’s assay following the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA).
ELISA of anti-serum and mAbs with cellular fractions
Cellular protein fractions were immobilized onto the ELISA microplate for overnight at 4 °C. After blocking with 2% skim milk, culture supernatant or the immunized mouse serum containing primary antibodies; anti-E. histolytica (anti-Eh4), anti-E. moshkovskii (anti-Em6) and anti-pan-human Entamoeba; E. histolytica, E. moshkovskii and E. dispar (anti-Ehmd4) mAb or 1:5000 diluted pre-immunized serum (a control) were applied. The reactions were incubated for 1 h at room temperature. Entamoeba binding partner-mAb complexes were detected with 1:5000 diluted anti-mouse immunoglobulins (Thermo Fisher Scientific), anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA) and anti-mouse IgA (Sigma-Aldrich) conjugated with horse radish peroxidase (HRP) enzyme (Thermo Fisher Scientific) for 1 h. The colorimetric signal was developed using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific) and quenched with 1N hydrochloric acid (HCl). The color intensity was recorded at a wavelength of 450 nm using a Sunrise microplate reader (Tecan, Männedorf, Switzerland). The measurement was performed in triplicate and presented as means of optical density (OD) ± standard error of the mean (SEM).
Immunofluorescent assay of specific anti-sera with Entamoeba cells
Axenically cultivated trophozoites of E. histolytica and E. moshkovskii were harvested and attached onto cell imaging slides (Eppendorf, Hamburg, Germany). The immobilized cells were fixed with 3.7% paraformaldehyde at room temperature for 10 min and permeabilized with 0.2% Triton X-100 in 1% bovine serum albumin (BSA)-PBS for 10 min. Non-permeabilized (incubated in 1% BSA-PBS) and permeabilized cells were reacted with the immunized mouse serum (1:1000) or culture supernatant containing mAb (1 mg/ml mAb) for 1 h. Antibody positive cells were detected with anti-mouse immunoglobulin G conjugated with FITC (1:60) and viewed by confocal microscopy (LSM 700; Carl Zeiss, Jena, Germany). All immunological reactions were performed in 1% BSA-PBS.
CHO cytopathic and cell-adhesion assays of E. histolytica and E. moshkovskii
Chinese hamster ovary (CHO) cell lines were cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a CO2 incubator. Cells were harvested by 0.1 mM ethylene diamine tetraacetic acid (EDTA)-PBS, pH 7.4 and washed 3 times with cold PBS. Viable trophozoite cells of E. histolytica (1 × 105 cells) and E. moshkovskii (2 × 105 cells) were pre-incubated with pre-immunized or immunized serum (1:5000 dilution in 10% FBS-RPMI medium) at 4 °C for 30 min. The Entamoeba cell-serum mixtures were then incubated with 2 × 105 of CHO-cell suspension at a ratio of 1:5 for Entamoeba and CHO cells at 4 °C for an additional 1 h. CHO cells incubated with Entamoeba cells in culture medium or serum alone were used as controls. CHO cell adhesion was defined as an Entamoeba cell formed with at least three CHO cells [20, 21]. CHO-Entamoeba cell mixtures were stained with 500 nM propidium iodine (PI). PI positive CHO cells were counted under fluorescence microscopy (Observer Z1; Carl Zeiss) to assess the killing ability of Entamoeba cells. The assay was performed in triplicate and presented as percent amoeba cell adhesion ± standard error of the mean (SEM) or percent cell death with 95% confidence interval (CI).
Entamoeba histolytica and E. moshkovskii adhesion to human epithelial Caco-2 cell line
Entamoeba histolytica or E. moshkovskii cells (2 × 105) were pre-mixed separately with a pre-immunized and dose-4 immunized serum at a final dilution of 1:5000 in 250 μl of 10% FCS-Iscove’s Modified Dulbecco’s Medium (IMDM). The Entamoeba cell-serum mixture was incubated with a monolayer of Caco-2 cells (provided by Professor Kris Chadee, Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Canada) in a 24-well plate at the ratio of 1:1 of Entamoeba:Caco-2 cells at 37 °C for 15 min. The culture supernatant was removed and then the cells were washed twice with PBS. The adhered E. histolytica/E. moshkovskii cells were counted from at least 5 fields per well with 20× magnification using inverted microscopy (Observer Z1; Carl Zeiss). The experiment was performed in triplicate and presented as percent cell adhesion ± SEM.
Statistical analyses of cytopathic and cell-adhesion assays were performed using GraphPad Prism version 7.0 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance of differences between groups was determined using a one-way ANOVA with Tukey’s comparison of means as a post-hoc test. P-values < 0.05 were considered significant. Results presented in graphs are mean values ± 95% confidence intervals for cell death plus standard errors of the mean for cell adhesion of at least 1000 cells per group/experiment and from three independent experiments.
Mouse immunization with mixed Entamoeba species induced higher and faster antibody response than single species
Species-specific IgG antibodies promoted cytopathicity of E. histolytica toward CHO cells
Interestingly, E. moshkovskii showed cytopathic activity of 15 ± 3.4% (Fig. 2a, cytopathic assay, E. moshkovskii) and 88 ± 1.8% adhesion to CHO cells (Fig. 2b, adhesion assay, E. moshkovskii). However, there was no difference in cytopathic ability (ANOVA: F(12,91) = 0.74, P = 0.70) and CHO cell adhesion (ANOVA: F(12,99) = 1.61, P = 0.10) of E. moshkovskii in serum pre-treated and un-treated conditions. Immunized sera were tested further for their effect on amoeba adhesion to Caco-2 cells. Despite inhibition of E. moshkovskii adhesion to Caco-2 cells, there was no significant difference (ANOVA: F(5,30) = 1.91, P = 0.12) in parasitic adhesion among the immunized, pre-immunized sera and serum un-treated amoeba (Fig. 3b).
Cellular localization of mAb binding partners in E. moshkovskii and E. histolytica trophozoite cells
Both humoral and cell-mediated immune responses have been demonstrated to play roles in E. histolytica infection and disease manifestation . However, few details exist on host immune response to E. moshkovskii. In the present study, we found that E. moshkovskii trophozoites induced species-specific antibodies in mice faster than E. histolytica, suggesting a difference in host immune response to these closely related species. Furthermore, mice pre-challenged with mixed trophozoite cells of E. histolytica and E. moshkovskii followed by single Entamoeba species produced species-specific IgG and IgA antibodies to both invasive E. histolytica and non-invasive E. moshkovskii faster and higher than the single species immunizations. These high responses among Entamoeba-specific antibodies may explain the ability of people in endemic areas to tolerate Entamoeba infection, where mixed infections of Entamoeba species are always common [23, 24]. On the other hand, a high level or titer of E. histolytica-specific IgG has been widely used as a diagnostic key for amoebic liver abscess (ALA), a severe infection outcome of E. histolytica . Here, upon intra-peritoneal inoculation with Entamoeba trophozoite cells, we found that levels of E. histolytica and E. moshkovskii-specific IgG in mouse sera corresponded to doses/numbers of immunizations and dropped sharply after the last immunization. This result firstly reveals the association of a level of species-specific IgG of mice and the presence of these two Entamoeba species in the stomach. Measurement of serum IgG may be applied for monitoring intestinal infections by E. histolytica and E. moshkovskii trophozoites. Mice experienced with a E. histolytica and E. moshkovskii cell mixture prior to secondary immunization with either E. histolytica or E. moshkovskii, produced IgG antibodies to the latter species higher and faster than those mice immunized with single Entamoeba species. This may have resulted from the immunological memory of the mouse adaptive immune system . IgA antibodies produced by plasma cells within the lamina propria serve as physical barrier to prevent intestinal mucus from adhesion and invasion of pathogens . Antigens/proteins of E. histolytica were injected via the oral, intradermal, intramuscular and subcutaneous routes together with adjuvants to elicit strong IgA antibody-, cell-mediated responses and protection of the host . In this study, without any adjuvant, mixed E. histolytica and E. moshkovskii trophozoite cells followed by E. moshkovskii were inoculated in the intraperitoneal route, and could elicit IgA antibody response of mice more than the mixed E. histolytica and E. moshkovskii cells followed by E. histolytica or with E. moshkovskii alone. Furthermore, the E. moshkovskii-specific IgA antibodies were produced faster and lasted longer than those with E. histolytica. This may be due to the strong immunogenicity of E. moshkovskii and corresponds to previous reports that E. moshkovskii was eliminated from the host faster than E. histolytica [17, 29].
Pathogenicity of E. histolytica trophozoites is mediated by parasite adhesion with gut mucin through parasitic Gal/GalNAc lectin  and actions of cysteine proteinases as mentioned previously. IgG antibody responses of patients have been shown to associate with infection susceptibility and pathogenicity and re-infection of E. histolytica . In this study, sera of mice immunized with E. histolytica inversely promoted cytopathicity of E. histolytica towards CHO cells. Most sera of the E. histolytica immunized mice were found to significantly enhance parasitic adhesion to colonic Caco-2 cells. However, mice immunized with E. histolytica and E. moshkovskii cells followed by E. histolytica promoted parasite adhesion to CHO cells. It was likely that increased in adhesion and cytopathic activity of E. histolytica to these cells are associated with a level of specific IgG antibodies in the immunized sera with the exception of B5-immunized sera. It should be noted that B5 sera were collected two months after the last immunization, where a level of E. histolytica specific IgG antibodies have decreased. Therefore, there remains no explanation for the positive effect of these sera on adhesion and cytopathic activity of the parasite. However, it might be due to the sum of affinity/avidity of polyclonal antibodies found in the immunized sera after repeated immunizations. The present study also reflects a difference in susceptibility of tissue cells for E. histolytica infection. In parasitic E. histolytica, the role of specific IgG antibodies in pathogenicity is not clear; however, it has been suggested to depend on IgG subclasses. IgG2 and IgG4 have been reported as markers to follow-up ALA and recurrent infection of the parasite, respectively, whereas IgG1 and IgG3 were found to associate with parasite clearance by immune cells [25, 31]. Genetic background and family history have also been considered as factors contributing to difference in isotypes of E. histolytica specific IgG . Antibody-mediated pathogenicity and disease severity known as antibody-dependent enhancement (ADE) has been exclusively studied in dengue virus and found to occur during the secondary infection with different virus serotypes [32, 33]. During ADE, virus recognition antibodies promote virus entry into dendritic cells and macrophages through their Fc receptor and suppress host immune response, resulting in survival and high viral load and severe disease manifestation of the host [33, 34]. It is possible that E. histolytica-specific IgG might promote pathogenicity of E. histolytica through ADE. For E. moshkovskii-specific IgG and IgA antibodies, despite having moderate to high antibody levels, there is no profound effect of the immunized sera on adhesion and pathogenicity of E. moshkovskii towards CHO cells as well as parasitic adhesion to human epithelial Caco-2 cells. It might imply an alternative host immune response and different pathogenicity of E. moshkovskii from its closely related species E. histolytica.
MAbs have been produced to E. moshkovskii and E. histolytica trophozoite cells and their characterizations revealed different localization of mAb binding targets in the virulent E. histolytica, non-virulent E. moshkovskii as well as non-pathogenic E. dispar. Most of the E. histolytica mAbs target to the cytoplasmic components, whereas most of the E. moshkovskii mAbs and cross-species (anti-Ehm and anti-Ehd2) and pan-human Entamoeba (anti-Ehmd) mAbs mostly target to the cell membrane. Although most E. histolytica-specific mAbs were shown to react to cytoplasm of the E. histolytica trophozoite, its membrane proteins, including Gal/GalNAc lectin, have been recognized by polyclonal antibodies in sera of immunized mice or humans. This would explain the enhancing effect of the immunized sera on host-cell adhesion and cytopathic abilities of the E. histolytica trophozoites. Compared to non-virulent E. moshkovskii, E. histolytica trophozoites might possess an evolutional design to conceal its surface molecules from the host immune system. It has been shown that E. histolytica possesses a thick and complicated cell surface containing lipopeptidophosphoglycan to protect the amoeba from the host complement system . In addition, E. histolytica has been reported to modify its surface antigenic proteins in response to surrounding bacteria and growth conditions . Furthermore, E. histolytica has been shown to cleave, translocate and release antibody-surface molecule complexes from the parasite cell surface by the parasite protease and actin rearrangement [37, 38]. These events were not observed in the non-pathogenic E. dispar . Likewise, influenza virus causes reinfection by drifting its antigenic surface protein hemagglutinin, and further shifting of this protein also enables the virus to cause the cross-species infection [40, 41]. Modification of surface proteins in order to escape host immunity and prolong infection have also been reported in the sleeping sickness causative agent, Trypanosoma brucei, and the cattle protozoan parasite, Babesia bovis [42, 43].
Antigen variation is a crucial virulent feature that pathogens adopt to survive from the host immune system . Here, we found that anti-Ehm2 mAb reacts to the inner membrane of E. histolytica trophozoite cells but is observed on the outer membrane of E. moshkovskii cells. Likewise, anti-Ehmd4 mAb binds to inner membrane and cytoplasmic components of E. histolytica and E. moshkovskii, trophozoites but is additional observed on the cell surface of non-pathogenic E. dispar. Therefore, it is likely that variation of mAb binding molecules might occur in these three closely related Entamoeba species. It has also been shown that E. histolytica has strain-specific antigens, which contribute to their virulence . Plasmodium falciparum protozoans are the best model for altering its var gene family to escape from host immune recognition and prolong its infection .
A mixed infection of Entamoeba stimulated a higher IgA and IgG antibody response of the host compared to multi-infection by a single species of human-infecting Entamoeba. Entamoeba moshkovskii induced the host antibody response faster and higher than E. histolytica. Entamoeba-specific anti-sera promoted host cell adhesion and cytopathicity of E. histolytica, but showed no effect to those activities of E. moshkovskii. However, functional roles of the induced antibodies as well as levels of mucosal IgA in the mouse models with amoebic dysentery, colitis and liver abscess need to be further elucidated. Characterization of E. histolytica, E. moshkovskii, cross-species and pan-Entamoeba mAbs revealed profound surface antigen manipulation and translocation to conceal or escape from host immune recognition by E. histolytica over non-virulent E. moshkovskii and non-pathogenic E. dispar. These findings may be useful for further vaccine development and better understanding and pathogenicity and host immune modulation by E. histolytica in the future.
We would like to thank Professor Tomoyoshi Nozaki, Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Japan, for his supervision and providing axenic Entamoeba trophozoite cells. Our sincere appreciation also goes to Professor Kris Chadee Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Canada, for providing the Caco-2 cell line. We would like to acknowledge Mr Irwin F. Chavez, Department of Tropical Hygiene, Faculty of Tropical Medicine, Mahidol University, Thailand, for reviewing the manuscript for grammar and syntax.
This work was supported by a grant from the Thailand Research Fund and Office of Higher Education Commission (grant no. MRG5480083) and Dean’s Research Fund-2011, Faculty of Tropical Medicine, Mahidol University.
Availability of data and materials
The data supporting the conclusions of this article are included within the article.
SM designed and planned the experiments and revised the manuscript. NK conducted the laboratory experiments, data analysis, and wrote the manuscript. SL conducted data analysis. ARP reviewed and edited the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The animal housing, immunization protocol and animal processing were approved by the Faculty of Tropical Medicine, Animal Care and Use Committee (permit number: FTM-ACUC 016/2012).
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