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Entamoeba Histolytica and Non‑Virulent Entamoeba Moshkovskii in a Mouse Model Narumol Khomkhum1, Somphob Leetachewa2, Aulia Rahmi Pawestri1 and Saengduen Moonsom1*

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Entamoeba Histolytica and Non‑Virulent Entamoeba Moshkovskii in a Mouse Model Narumol Khomkhum1, Somphob Leetachewa2, Aulia Rahmi Pawestri1 and Saengduen Moonsom1* Khomkhum et al. Parasites Vectors (2019) 12:101 https://doi.org/10.1186/s13071-019-3363-5 Parasites & Vectors RESEARCH Open Access Host-antibody inductivity of virulent Entamoeba histolytica and non-virulent Entamoeba moshkovskii in a mouse model Narumol Khomkhum1, Somphob Leetachewa2, Aulia Rahmi Pawestri1 and Saengduen Moonsom1* Abstract Background: Despite similarities in morphology, gene and protein profles, Entamoeba histolytica and E. moshko- vskii show profound diferences 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. Methods: To study diferences in host-antibody response profles between E. histolytica and E. moshkovskii, mice were immunized intraperitoneally with diferent sets of Entamoeba trophozoites as single species, mixed species and combinations. Results: 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 sig- nifcantly higher levels and long-lasting antibody responses than those challenged with E. histolytica alone. Interest- ingly, E. histolytica-specifc anti-sera promoted the cytopathic ability of E. histolytica toward Chinese hamster ovarian (CHO) cells, but showed no efect on cell adhesion. There was no signifcant efect 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-specifc mAbs produced from mice targeted cytoplasmic and cytoskeletal proteins, whereas 73% of E. moshkovskii-specifc mAbs targeted plasma membrane proteins. Conclusions: The present fndings suggest that infection with mixed Entamoeba species or E. moshkovskii efectively induces an antibody response in mice. It also sheds light on roles of host antibody response in the pathogenic difer- ence of E. histolytica and E. moshkovskii trophozoites, and cell surface protein modifcations of the amoebic parasites to escape from host immune system. Keywords: Amoebiasis, Entamoeba histolytica, Entamoeba moshkovskii, Pathogenicity, Antibody dependent enhancement Background [2]. After invading the intestinal lamina propria, this Entamoeba histolytica is an anaerobic pathogenic proto- parasite enters the blood stream to reach other organs zoan parasite that causes approximately 100,000 global and results in abscesses, most commonly in the liver [3], deaths annually due to amoebiasis [1]. Disease symptoms and rarely in lungs [4] and brain [5]. Te pathogenesis of range from mild diarrhea to severe bloody diarrhea with E. histolytica starts with parasite adhesion at the large mucus as the parasite invades the intestinal epithelium intestinal epithelium and secretion of cysteine proteases, leading to the degradation of host tissues. Te secreted *Correspondence: [email protected] cysteine proteases play important roles in degrading gut 1 Department of Protozoology, Faculty of Tropical Medicine, Mahidol mucosal IgA and circulating IgG, resulting in the inef- University, Ratchawithi, Bangkok 10400, Thailand Full list of author information is available at the end of the article fectiveness or failure of host immunity, thus inversely © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Khomkhum et al. Parasites Vectors (2019) 12:101 Page 2 of 11 promote extra-intestinal infection of E. histolytica [6, by Professor Tomoyoshi Nozaki, Department of Biomed- 7]. In addition, the parasite-gut adhesion was shown to ical Chemistry, Graduate School of Medicine, Univer- trigger host signal transductions through caspases 3-like sity of Tokyo, Japan, were axenically cultured in bis-iron cascade and caspases 8- and 9-independent manner [8]. serum (BIS) medium at 37 °C and 26.5 °C, respectively. Tese lead to apoptotic cell death, which were preferen- Cells were harvested by placing culture tubes on ice for tially phagocytosed by the parasite. Te interaction also 10 min to detach the cells, followed by centrifugation at stimulates production of pro-infammatory cytokines, 200× g for 3 min at 4 °C with three washes using cold including interleukin (IL)-1, IL-6, IL-8, IFN-γ and tumor phosphate-bufered saline (PBS). Viable amoeba cells necrosis factor (TNF)-α, which consequentially promote were counted using a hemocytometer by trypan blue tissue damages and severity of the disease [9, 10]. Inhi- exclusion (0.2% trypan blue). For studies of host-anti- bition of TNF-α has been proved to signifcantly reduce body response, BALB/c mice (3 mice/group; 12 mice in 6 the infammation and tissue destruction [11], while the total) were immunized with 2 × 10 cells of mixed spe- absence of the anti-infammatory cytokine IL-10 has cies (1 106 cells each of E. histolytica and E. moshko- × 6 been shown to result in increased severity of intestinal vskii) or 2 × 10 cells of individual species (E. histolytica amoebiasis [12]. Tus, the manifestation of amoebiasis or E. moshkovskii) according to 4 immunization designs apparently happens through the parasite’s ability to acti- (group 1 mice received E. histolytica cells for 4 doses; vate cytokine-mediated cell deaths and manipulate the group 2 mice received E. moshkovskii cells for 4 doses; host immune system. group 3 mice received E. histolytica and E. moshkovskii Entamoeba moshkovskii was previously considered as cell mixture for 2 doses, followed by E. histolytica cells a non-pathogenic protozoan parasite, which was com- for 2 doses; group 4 mice received E. histolytica and monly found to co-occur in human stools collected from E. moshkovskii cell mixture for 2 doses followed by E. E. histolytica endemic areas, often leading to misdiagno- moshkovskii cells for 2 doses). Immunization was per- sis of E. histolytica due to their mostly identical morphol- formed intraperitoneally (IP) with two-week intervals. ogy [13, 14]. Despite being considered non-pathogenic, Whole blood was collected from the ventral tail vein E. moshkovskii has been gradually reported as associated before each immunization [19] and after the 4th boost for with diarrhea in humans and mice [15–17]. Recently, 2 weeks (B4: bleed 4) and 8 weeks (B5: bleed 5). Serum E. moshkovskii was reported to cause subcutaneous collected before the frst immunization (pre-immunized abscess in Indonesia [18]. Shimokawa et al. [16] showed serum) was used as a negative control for the baseline that E. moshkovskii was able to cause symptoms, includ- antibody level of each mouse. ing weight loss, diarrhea and colitis in susceptible mice as is the case for E. histolytica. Furthermore, damages of Monoclonal antibody (mAb) production the intestinal epithelium of E. moshkovskii-infected mice BALB/c mice (2 mice per set) were immunized with 6 were observed due to host IFN-γ mediated cell apoptosis 2 × 10 cells of E. histolytica and E. moshkovskii tropho- [17]. zoite for 3 doses followed by 2 doses of mixed Entamoeba 6 In the present study, we aimed to investigate the immu- cells (1 × 10 cells each of E. histolytica and E. moshkovs- nogenicity of E. histolytica and E. moshkovskii tropho- kii) in two-week intervals. Blood samples were collected zoites through host-antibody response profles as well from the tail vein before each immunization and tested as efect of the immunized sera on Entamoeba patho- for Entamoeba-specifc antibodies. Te mice were sacri- genicity. We found that mouse immunization with mixed fced and splenic B cells were fused with mouse myeloma Entamoeba species was able to induce both specifc IgA cells using the standard hybridoma technique described and IgG higher levels than single species. Te efect of by Moonsom et al. [19]. Hybridoma cells secreting Enta- the immunized sera on cytopathic activity and host cell moeba-specifc antibodies were screened by enzyme- adhesion were investigated and the possible immune eva- linked immunosorbent assay (ELISA) with Entamoeba sion and cell manipulating mechanisms by E. histolytica cell lysate proteins. Limiting dilutions were performed to are discussed. Our fndings may shed more light on E. obtain the mAb-producing cells. histolytica pathogenicity, which can be of further beneft in the development of diagnosis modalities, treatment Preparation of cellular protein compartments and vaccines for this parasite. Axenically cultivated E. histolytica and E. moshkovskii trophozoite cells were harvested and washed with PBS, Methods pH 7.4. Total cell lysate proteins were solubilized using Mouse immunization with Entamoeba cells mammalian protein extraction reagent (M-PER) (Termo Trophozoite cells of E. histolytica strain HM1: IMSS and Fisher Scientifc, Waltham, MA, USA). Cells were frac- E. moshkovskii strain Laredo, which were kindly
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