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Entamoeba histolytica: Five facts about modeling a complex human disease in rodents

Entamoeba histolytica: Five facts about modeling a complex human disease in rodents

  • Carolina Mendoza Cavazos, 
  • Laura J. Knoll
PLOS
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Fact 1: Rodent models do not mimic the entire life cycle of E. histolytica

Entamoeba histolytica is an extracellular enteric eukaryotic parasite. Globally, an average of 50 million cases and 55,000 to 100,000 deaths are due to E. histolytica infection each year, primarily impacting the developing world [1,2]. The world is widely unprepared for an outbreak of E. histolytica due to the lack of a vaccine and the use of a single drug type as treatment (reviewed in [3]). E. histolytica is the causative agent of the diarrheal disease known as amebiasis, but it can sometimes penetrate the intestinal wall, enter the circulation, and cause abscesses throughout the body, most commonly in the liver. E. histolytica has 2 main stages during its life cycle: the trophozoite and the cyst stage. The infectious agent is the cyst and is transmitted through the oral–fecal route via contaminated food or water. Animal models are not available for this stage interconversion, which is essential for disease propagation and pathogenesis. Excystation is the transition from the cyst form to the rapidly dividing trophozoite stage. Once a trophozoite, E. histolytica can either undergo invasive (5% to 10% of symptomatic infections) or noninvasive disease progression. As the parasite replicates during a noninvasive infection, it undergoes a second developmental change known as encystation, which involves the synthesis of a cyst wall to endure the exterior environment until a suitable host is encountered.

Because the cyst stage of E. histolytica cannot be induced in culture or rodents, Entamoeba invadens has served as an excellent model for the study of excystation and encystation in vitro. Some examples of insights from the study of E. invadens include the identification of stage-specific promoters [4], cholesteryl sulfate impact on encystation efficiency [5], the negative regulation of encystation by heat shock protein 90 (Hsp90) in vitro [6], and the discovery of various transcription factor that regulate stage conversion [7,8]. A rodent model that produces E. histolytica cysts could be used to generate new treatments or vaccines for amebiasis by targeting parasite development. However, tremendous advances have been accomplished using the current infection model including vaccine development (reviewed in [9]), microbiota–parasite interactions (reviewed in [10]), host innate and adaptive immune response to infection (reviewed in [11]), and molecular mechanisms responsible for tissue damage (reviewed in [12]).

Fact 2: Trophozoites can initiate infection in rodent models

A robust way to culture trophozoites has been developed in the laboratory setting [13] and has been the primary resource to perform in vivo infection. Trophozoites are surgically delivered into the target organ within the animal models. For intestinal studies, the trophozoites are injected into a surgically exposed cecum or an artificial colonic loop. While this procedure is invasive and bypasses the oral portion of the infection, the murine model has provided insight into many parasite–host interactions. These discoveries include, but are not limited to, alteration to Paneth cell function by E. histolytica infection [14], the genetic predisposition to disease progression due to host polymorphism in leptin receptors [15] or intergenic insertion previously linked to inflammatory bowel disease [16], the ability to nibble on alive host cells (known as trogocytosis) [17], the discovery of pathogenicity of Entamoeba moshkovskii [18], and microbiome-mediated immune cell recruitment during E. histolytica infection [19,20]. To model invasive disease such as amoebic liver abscesses, trophozoites are delivered via intraportal inoculation to either hamsters or guinea pigs (reviewed in [21]). Physiochemical factors encountered throughout the gastrointestinal tract can influence parasite development but are bypassed using these methodologies [22]. To our knowledge, there are 3 studies conducted in the 1980s in which oral inoculation was performed with either cysts [23] or trophozoites [24,25]. E. histolytica strain SAW 408 trophozoites were orally inoculated into 3 types of rats pretreated with an antihistamine called cimetidine that blocks the production of stomach acid [24]. The rats displayed the expected pathology in the intestinal tract 21 days postinfection. However, mice that were previously described as SAW 760– (Entamoeba dispar) or SAW 408–sensitive [26] did not display pathology by histology or shedding by wet mounts. A follow-up study [27] tracked the health of rats 12 months after oral infection, concluding that infections can persist long term. These studies highlight that rodents can be useful for studying the complex biology of E. histolytica but that more research is needed.

Fact 3: E. histolytica invasive disease can be modeled in rodents

E. histolytica disease outcomes range widely in humans: death, sepsis, liver abscesses, ulcers, dysentery, abdominal pain and mucoid diarrhea with occasional blood, and asymptomatic shedding. The field’s focus on invasive disease is due to the pathogenicity of the trophozoite stage within the host and the morbidity and mortality that invasive disease causes. One of the most widely used strains, HM-1:IMSS, was isolated from a male patient admitted into a public hospital in Mexico City in 1967. Although the virulence of the initial isolate was high, this strain undergoes continuous culture passaging, so virulence has changed over the years. Some laboratories pass trophozoites through the cecum or liver of animal models to retain virulence [28,29]. Maintaining virulence in this strain has allowed modeling of invasive disease. Frontline researchers in the amebiasis community have contributed immeasurable knowledge using intestinal pathology and development of liver abscess as a measure of disease progression. Hamsters and guinea pigs are used for the study of invasive disease, especially for the development of liver abscesses, and are the preferred model for vaccine development [9]. Various murine strains have been studied to determine what dictates susceptibility to E. histolytica infection, including but not limited to mice with a deletion in the secretory mucin of the gastrointestinal tract (MUC2) to determine the effect of an induced dysbiosis state on infection outcome [30] and mice with a deletion in the leptin receptor (Lepob) to determine the molecular mechanism by which leptin modulates mucosal protection [31]. However, whether cysts are present in the feces is an outstanding question that has not been addressed.

Fact 4: Disease modeling in rodents has shown microbiome–host–parasite interactions

Recent literature suggests that the host microbiome is a modifier of disease outcome and parasite development. The microbiota serves as an immune response trainer [32], processor of carbon sources unavailable to the parasite [33], sustenance to the parasite via phagocytosis [34], can produce metabolites that inhibit encystation [35], and together with a healthy mucus barrier, is the first line of defense against parasite infection (reviewed in [36]). Moreover, the microbiota is required for E. histolytica pathogenicity, as germ-free mice have an attenuated response to parasite infection [30]. The importance of the microbiome is also observed in human cohorts and case studies, demonstrating a correlation between dysbiotic state, a well-known niche for opportunistic pathogens, and E. histolytica infection outcome [37,38]. Recently, the microbiome has been shown to have an effect on disease severity via the recruitment of neutrophils [19,20]. Additionally, halting of encystation has been observed in E. invadens via microbiome metabolites [35]. The oral infection route is a modulator of disease progression and host immunological response, based on the impact pathogen delivery has in other parasites. For Toxoplasma gondii, immune murine knockout strains only displayed an increased susceptibility to infection when parasites were delivered through the natural route of infection [39]. For Trypanosoma cruzi, oral versus gastrointestinal delivery of trypomastigotes displays distinct patterns of disease progression in BALB/c mice [40]. Lastly, vaccine administration factors are evidence of how the route of delivery of antigens can lead to distinct immune responses, for example, mucosal inoculation leading to immunoglobulin A (IgA) production [41]. While bacterial microbiome changes have been correlated with various parasitic infections in numerous cohort studies (reviewed in [42]), it is unclear if the parasitic infection causes a state of dysbiosis or vice versa. Animals colonized with fixed microbial communities and orally challenged with either parasite stage will provide insight regarding the effect of the microbiome on parasite development.

Fact 5: Developing a rodent model that produced cysts would be beneficial for the parasitology field

Trophozoites are the metabolically active form of E. histolytica, but the transmissible form is the cyst. These 2 forms of the parasites are quite different in terms of morphology, protein content, and ploidy [43,44]. Some scholars argue that the field has moved beyond the lack of cyst stage as the current animal model induces disease and has provided a significant understanding of parasite virulence, host susceptibility, and disease progression [45]. Currently, mechanisms for the induction of E. histolytica developmental changes and cyst production are not available. Researchers can obtain cysts from human patients in the clinical setting or nonhuman primates in captivity, which limits their access to most laboratories [46]. Targeting parasite’s developmental changes is a strategy that can result in transmission halting, as only the parasites that are equipped to surviving in the environment, protected by a cyst wall, are infectious to a new host. Approaches focused on encystation are being considered as potential avenues to decline transmission of enteric protozoa (reviewed in [47]). In the 1980s, wet mounts of fecal samples were routinely conducted [24]. Today, there are some diagnostic methods for the identification of the cyst-like structures that are strain specific. The monoclonal antibody 1A4 targets the Jacob2 lectin, while avoiding cross-reactivity with xenic cultures of E. dispar isolates [48]. Excystation attempts found histamine and glucose availability affect these metabolic processes [49]. The most recent attempt to determine the presence of cysts used a colitis mouse model but found no cysts in tissue histology, cecum, or stool [50]. Having a mouse model that produces infectious cysts that remain stable in storage would be beneficial for the parasitic community for the following reasons: (1) reduces the number of animals used to maintain parasite virulence; (2) alleviates the need for the continuous passage of parasites in culture; (3) improves methods of detection for food safety and patient diagnostics; (4) provides a new platform for antiparasitic drug screening; (5) allows for the study of the host response to developmental changes of the parasite; and (6) permits the examination of E. histolytica’s interactions with other pathogenic and nonpathogenic protozoans (Fig 1). For drug screening and host immune response studies, a rodent model that can be orally challenged with either trophozoite or cyst stages of the parasite, while displaying invasive colitis, would be the most useful. Particular challenges that would need to be addressed are (1) consistency in the number or cysts used to initiate infection; (2) the extent of invasive disease; and (3) the number of cysts recovered from the colon or fecal samples.

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Fig 1. Ideal animal model that can support the full life cycle of Entamoeba histolytica.

From top left to bottom right: (1) fewer animals used to maintain virulence; (2) less need for continuous passaging; (3) new detection methods for food safety and patient diagnostics; (4) model for drug screening; (5) focus on host response to developmental changes of the parasite in vivo; and (6) could serve as potential model to pioneer in vivo studies of E. histolytica with other pathogenic and nonpathogenic protozoans. Figure created with BioRender.com.

https://doi.org/10.1371/journal.ppat.1008950.g001

Acknowledgments

Due to space limitations, we were unable to include all the work of colleagues but would like to thank them for their contributions to the Entamoeba histolytica field. We would also like to thank Sarah Wilson and Apoorva Maru for their critical read and editing of this manuscript.

References

  1. 1. WHO. Entamoeba taxonomy. Bull World Health Organ. 1997;75(3):291–294. pmid:9277015
  2. 2. Carrero JC, Reyes-López M, Serrano-Luna J, Shibayama M, Unzueta J, León-Sicairos N, et al. Intestinal amoebiasis: 160 years of its first detection and still remains as a health problem in developing countries. Int J Med Microbiol. 2020;310(1):151358. pmid:31587966
  3. 3. Shirley DT, Watanabe K, Moonah S. Significance of amebiasis: 10 reasons why neglecting amebiasis might come back to bite us in the gut. PLoS Negl Trop Dis. 2019;13(11):e0007744. pmid:31725715
  4. 4. Manna D, Ehrenkaufer GM, Singh U. Regulation of gene expression in the protozoan parasite Entamoeba invadens: identification of core promoter elements and promoters with stage-specific expression patterns. Int J Parasitol. 2014;44(11):837–845. pmid:25075445
  5. 5. Mi-ichi F, Miyamoto T, Takao S, Jeelani G, Hashimoto T, Hara H, et al. Entamoeba mitosomes play an important role in encystation by association with cholesteryl sulfate synthesis. Proc Natl Acad Sci U S A. 2015;112(22):E2884–E2890. pmid:25986376
  6. 6. Singh M, Sharma S, Bhattacharya A, Tatu U. Heat Shock Protein 90 regulates encystation in Entamoeba. Front Microbiol. 2015;6:1125. pmid:26528271
  7. 7. Manna D, Lentz CS, Ehrenkaufer GM, Suresh S, Bhat A, Singh U. An NAD +-dependent Novel Transcription Factor Controls Stage Conversion in Entamoeba. Elife. 2018;7. pmid:30375973
  8. 8. Manna D, Singh U. Nuclear Factor Y (NF-Y) Modulates Encystation in Entamoeba via Stage-Specific Expression of the NF-YB and NF-YC Subunits. MBio. 2019;10(3). pmid:31213550
  9. 9. Quach J, St-Pierre J, Chadee K. The future for vaccine development against Entamoeba histolytica. Hum Vaccin Immunother. 2014;10(6):1514–1521. pmid:24504133
  10. 10. Burgess SL, Petri WA. The Intestinal Bacterial Microbiome and E. histolytica Infection. Curr Trop Med Rep. 2016;3:71–74. pmid:27525214
  11. 11. Nakada-Tsukui K, Nozaki T. Immune Response of Amebiasis and Immune Evasion by Entamoeba histolytica. Front Immunol. 2016;7:175. pmid:27242782
  12. 12. Ghosh S, Padalia J, Moonah S. Tissue destruction caused by entamoeba histolytica parasite: cell death, inflammation, invasion, and the gut microbiome. Curr Clin Microbiol Rep. 2019;6(1):51–57. pmid:31008019
  13. 13. Diamond LS. Axenic cultivation of Entamoeba hitolytica. Science. 1961;134(3475):336–337. pmid:13722605
  14. 14. Cobo ER, Holani R, Moreau F, Nakamura K, Ayabe T, Mastroianni JR, et al. Entamoeba histolytica Alters Ileal Paneth Cell Functions in Intact and Muc2 Mucin Deficiency. Infect Immun. 2018;86(7).
  15. 15. Mackey-Lawrence NM, Guo X, Sturdevant DE, Virtaneva K, Hernandez MM, Houpt E, et al. Effect of the leptin receptor Q223R polymorphism on the host transcriptome following infection with Entamoeba histolytica. Infect Immun. 2013;81(5):1460–1470. pmid:23429533
  16. 16. Wojcik GL, Marie C, Abhyankar MM, Yoshida N, Watanabe K, Mentzer AJ, et al. Genome-Wide Association Study Reveals Genetic Link between Diarrhea-Associated Entamoeba histolytica Infection and Inflammatory Bowel Disease. MBio. 2018;9(5). pmid:30228239
  17. 17. Ralston KS, Solga MD, Mackey-Lawrence NM, Somlata, Bhattacharya A, Petri WA. Trogocytosis by Entamoeba histolytica contributes to cell killing and tissue invasion. Nature. 2014;508(7497):526–530. pmid:24717428
  18. 18. Shimokawa C, Kabir M, Taniuchi M, Mondal D, Kobayashi S, Ali IK, et al. Entamoeba moshkovskii is associated with diarrhea in infants and causes diarrhea and colitis in mice. J Infect Dis. 2012;206(5):744–751. pmid:22723640
  19. 19. Burgess SL, Leslie JL, Uddin MJ, Oakland DN, Gilchrist CA, Moreau GB, et al. Gut microbiome communication with bone marrow regulates susceptibility to amebiasis. J Clin Invest. 2020. pmid:32369444
  20. 20. Watanabe K, Gilchrist CA, Uddin MJ, Burgess SL, Abhyankar MM, Moonah SN, et al. Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis. PLoS Pathog. 2017;13(8):e1006513. pmid:28817707
  21. 21. Santi-Rocca J, Rigothier MC, Guillén N. Host-microbe interactions and defense mechanisms in the development of amoebic liver abscesses. Clin Microbiol Rev. 2009;22(1):65–75. pmid:19136434
  22. 22. Mitra BN, Pradel G, Frevert U, Eichinger D. Compounds of the upper gastrointestinal tract induce rapid and efficient excystation of Entamoeba invadens. Int J Parasitol. 2010;40(6):751–760. pmid:20018192
  23. 23. Chauhan D. Simultaneous caecal and liver infections in hamster, mouse and guinea-pig by oral feeding of Entamoeba histolytica cysts. Curr Sci. 1987;56(23):1223–1224.
  24. 24. Owen DG. Attempts at oral infection of rats and mice with trophozoites of Entamoeba histolytica. Trans R Soc Trop Med Hyg. 1984;78(2):160–164. pmid:6087507
  25. 25. Quadri GSA, Saleem Y, Ishaq M, Habibullah CM. Experimental hepatic amoebiasis in immunosuppressed mice fed orally with cysts of Entamoeba histolytica. IRCS Medical Science. 1985;13(7):590–591.
  26. 26. Owen DG. A mouse model for Entamoeba histolytica infection. Lab Anim. 1985;19(4):297–304. pmid:4068657
  27. 27. Owen DG. The effect of orally administered Entamoeba histolytica on Wistar and athymic (rnu rnu) rats observed during a 12-month period. Trans R Soc Trop Med Hyg. 1987;81(4):621–623. pmid:2895512
  28. 28. Lushbaugh WB, Kairalla AB, Loadholt CB, Pittman FE. Effect of hamster liver passage on the virulence of axenically cultivated Entamoeba histolytica. Am J Trop Med Hyg. 1978;27(2 Pt 1):248–254. pmid:206160
  29. 29. Bos HJ, van de Griend RJ. Virulence and toxicity of axenic Entamoeba histolytica. Nature. 1977;265(5592):341–343. pmid:189211
  30. 30. Leon-Coria A, Kumar M, Moreau F, Chadee K. Defining cooperative roles for colonic microbiota and Muc2 mucin in mediating innate host defense against Entamoeba histolytica. PLoS Pathog. 2018;14(11):e1007466. pmid:30500860
  31. 31. Guo X, Roberts MR, Becker SM, Podd B, Zhang Y, Chua SC, et al. Leptin signaling in intestinal epithelium mediates resistance to enteric infection by Entamoeba histolytica. Mucosal Immunol. 2011;4(3):294–303. pmid:21124310
  32. 32. Partida-Rodríguez O, Serrano-Vázquez A, Nieves-Ramírez ME, Moran P, Rojas L, Portillo T, et al. Human Intestinal Microbiota: Interaction Between Parasites and the Host Immune Response. Arch Med Res. 2017;48(8):690–700. pmid:29290328
  33. 33. Sicard JF, Le Bihan G, Vogeleer P, Jacques M, Harel J. Interactions of Intestinal Bacteria with Components of the Intestinal Mucus. Front Cell Infect Microbiol. 2017;7:387. pmid:28929087
  34. 34. Iyer LR, Verma AK, Paul J, Bhattacharya A. Phagocytosis of Gut Bacteria by Entamoeba histolytica. Front Cell Infect Microbiol. 2019;9:34. pmid:30863724
  35. 35. Byers J, Faigle W, Eichinger D. Colonic short-chain fatty acids inhibit encystation of Entamoeba invadens. Cell Microbiol. 2005;7(2):269–279. pmid:15659070
  36. 36. Leon-Coria A, Kumar M, Chadee K. The delicate balance between Entamoeba histolytica, mucus and microbiota. Gut Microbes. 2020;11(1):118–125. pmid:31091163
  37. 37. Verma AK, Verma R, Ahuja V, Paul J. Real-time analysis of gut flora in Entamoeba histolytica infected patients of Northern India. BMC Microbiol. 2012;12:183. pmid:22913622
  38. 38. Yanagawa Y, Arisaka T, Kawai S, Nakada-Tsukui K, Fukushima A, Hiraishi H, et al. Case Report: Acute Amebic Colitis Triggered by Colonoscopy: Exacerbation of Asymptomatic Chronic Infection with. Am J Trop Med Hyg. 2019;101(6):1384–1387. pmid:31595870
  39. 39. Pittman KJ, Cervantes PW, Knoll LJ. Z-DNA Binding Protein Mediates Host Control of Toxoplasma gondii Infection. Infect Immun. 2016;84(10):3063–3070. pmid:27481249
  40. 40. Barreto-de-Albuquerque J, Silva-dos-Santos D, Pérez AR, Berbert LR, de Santana-van-Vliet E, Farias-de-Oliveira DA, et al. Trypanosoma cruzi Infection through the Oral Route Promotes a Severe Infection in Mice: New Disease Form from an Old Infection? PLoS Negl Trop Dis. 2015;9(6):e0003849. pmid:26090667
  41. 41. Zimmermann P, Curtis N. Factors That Influence the Immune Response to Vaccination. Clin Microbiol Rev. 2019;32(2). pmid:30867162
  42. 42. Burgess SL, Gilchrist CA, Lynn TC, Petri WA. Parasitic Protozoa and Interactions with the Host Intestinal Microbiota. Infect Immun. 2017;85(8). pmid:28584161
  43. 43. Luna-Nácar M, Navarrete-Perea J, Moguel B, Bobes RJ, Laclette JP, Carrero JC. Proteomic Study of Entamoeba histolytica Trophozoites, Cysts, and Cyst-Like Structures. PLoS ONE. 2016;11(5):e0156018. pmid:27228164
  44. 44. Mukherjee C, Clark CG, Lohia A. Entamoeba shows reversible variation in ploidy under different growth conditions and between life cycle phases. PLoS Negl Trop Dis. 2008;2(8):e281. pmid:18714361
  45. 45. Marie C, Petri WA. Regulation of virulence of Entamoeba histolytica. Annu Rev Microbiol. 2014;68:493–520. pmid:25002094
  46. 46. Regan CS, Yon L, Hossain M, Elsheikha HM. Prevalence of Entamoeba species in captive primates in zoological gardens in the UK. PeerJ. 2014;2:e492. pmid:25097822
  47. 47. Aguilar-Díaz H, Carrero JC, Argüello-García R, Laclette JP, Morales-Montor J. Cyst and encystment in protozoan parasites: optimal targets for new life-cycle interrupting strategies? Trends Parasitol. 2011;27(10):450–458. pmid:21775209
  48. 48. Spadafora LJ, Kearney MR, Siddique A, Ali IK, Gilchrist CA, Arju T, et al. Species-Specific Immunodetection of an Entamoeba histolytica Cyst Wall Protein. PLoS Negl Trop Dis. 2016;10(5):e0004697. pmid:27152855
  49. 49. Nayeem MA, Habibullah CM, Saleem Y, Quadri GS, Ishaq M. In vitro encystation and excystation of Entamoeba histolytica trophozoites. Indian J Exp Biol. 1993;31(6):562–563. pmid:8406604
  50. 50. Houpt E, Vines R, Camerini V, Lockhart L, Petri W. The mucosal immune response in a mouse model of amebic colitis. Arch Med Res. 2000;31(4 Suppl):S89.