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Pathogenesis of amebiasis William A Petri Jr

It is an exciting time in the study of Entamoeba histolytica. Over the past two years, the natural history and burden of disease in humans has been redefined, mucosal immune responses associated with protection identified, and the developmental regulation of encystation outlined. The number of genes sequenced has increased from a few hundred to a few thousand, and study of the genome structure is revealing unusual repetitive elements and plasticity. DNA microarrays promise the first ability to examine global patterns of mRNA abundance. The mechanism of transcriptional control via histone modifications and sequence-specific DNA-binding proteins are to be delineated. Advances in cell biology are providing new insights into invasion through the intestinal epithelium.
Addresses Division of Infectious Diseases, Room 2115, MR4 Building, Lane Road, PO Box 801340, University of Virginia Health System, Charlottesville, Virginia 22908-1340, USA; e-mail: wap3g@virginia.edu Current Opinion in Microbiology 2002, 5:443447 1369-5274/02/$ see front matter 2002 Elsevier Science Ltd. All rights reserved. Published online 20 June 2002 Abbreviations PFGE pulsed-field gel electrophoresis URE upstream regulatory element

amebiasis. Two pieces of evidence were consistent with inheritance of this susceptibility. First, family aggregation studies demonstrated that the serum anti-lectin IgG antibodies clustered in families. Secondly, prospective study showed that the absence of serum anti-lectin IgG antibodies was not due to lack of exposure to E. histolytica infection. The demonstration of acquired resistance and inherited susceptibility to amebiasis is an important step towards understanding the contribution of the innate and acquired immune systems to protection from amebiasis. I apologize to the readers of this review and my colleagues that many advances have not even been mentioned because of lack of space.

Determinants of parasite invasiveness

Inroads are being made on the contributions of environmental factors such as intestinal flora or malnutrition that affect disease progression in amebiasis. Mirelmans group [12,13] has shown that Escherichia coli serotype 055 is phagocytosed via the parasite Gal/GalNAc lectin. Uptake of the bacteria and long-term (day-long) culture of the bacteria with the parasite results in an 8090% decrease in adherence, cytotoxicity and capability of the parasite to form hamster liver abscess. After ingestion of E. coli strain 055, there is a downregulation in the amount of the Gal/GalNAc lectin lgl1 gene. The Rahman strain of E. histolytica, which has little ability to kill cells in vitro, was also found by differential display cDNA approaches to have decreased amounts of lgl1 mRNA. The expression of lgl1 mutants in E. histolytica is an approach that is being taken now to further investigate the function of the protein in cytotoxicity. The amino terminus of the mature protein has been deleted in the Mirelman lab [12,13], removing a site of potential phosphorylation and myristoylation as well as a CXXC motif, but leaving the amino-terminal signal sequence and the rest of the carboxy terminus of the molecule intact. This protein, when expressed in E. histolytica, was assembled with hgl to form the lectin heterodimer, and caused a 30% decrease in adherence and a 50% decrease in cytotoxicity. Further studies to include point mutations in the area deleted are underway, with this preliminary data pointing to a role of the lectin light subunit in cytotoxicity. Tachibana and colleagues have identified the 150 kDa intermediate subunit of the Gal/GalNAc lectin [14,15]. All three subunits of the Gal/GalNAc lectin (Igl, Hgl and Lgl) co-localized on the surface membrane of trophozoites. Two unlinked copies of genes encoding Igl were sequenced and found to encode cysteine-rich proteins with amino- and carboxy-terminal hydrophobic signal sequences characteristic of glycosylphosphatidyl-inositol (GPI)-anchored membrane proteins. The igl genes lacked carbohydrate recognition domains but were members of a large family of amebic genes containing CXXC and CXC

Introduction: amebiasis in humans

The availability of accurate diagnostic tests for Entamoeba histolytica infection has enabled a fresh look at the epidemiology and natural history of amebiasis [1,2,3,4,510,11]. Haque and colleagues are examining the natural history of amebiasis in pre-school children from an urban slum of Dhaka. New E. histolytica infections were detected in half of the children during two years of observation [4]. Of children with asymptomatic E. histolytica infection, one in 10 went on to develop diarrhea within two months of the onset of infection. Overall, 10% of the children had diarrhea associated with E. histolytica infection, and 4% suffered from amebic colitis during the two-year study period. Amebiasis was, thus, a significant cause of morbidity in these young children. Acquired immunity to amebiasis in Bangladeshi children was associated with the production of mucosal IgA against the parasite Gal/GalNAc lectin. (The Gal/GalNAc lectin is a virulence factor required for trophozoite adherence to the host.) An intestinal IgA response against the lectin carbohydrate recognition domain was associated with a 70% reduction in infections in the Dhaka children during two years of prospective observation. In contrast, children who developed serum anti-amebic IgG antibodies in response to E. histolytica infection were more susceptible to

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motifs with sequence similarity to the Giardia lamblia variant surface proteins. The function of Igl in adherence or antigenic variation is unknown.

The amoebapore and cytotoxicity

Leippes laboratory has pioneered investigation of the amoebapore gene family [16,17,18]. Amoebapore proteins migrate as 5 kDa molecules on sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) and are 77 amino acids long with six cysteines. There are three amoebapore proteins (A, B and C): A and B have 57% identity; A and C have 47% identity; and B and C have 35% identity. The proteins appear to form pores in cell membranes by a barrel-stave mechanism similar to that of complement. Amoebapore C is the most potent for cytotoxicity and kills bacteria at concentrations of 0.210 M, depending on the organism, with Gram-positive bacteria being more readily killed than Gram-negative bacteria. Amoebapores are only active at acidic pH with an optimum of 5.2, and have no activity above pH 6. A primary function may be to kill bacteria, and Leippe has observed colocalization of endocytosed bacteria with amoebapores in E. histolytica. Amoebapores are located in cytoplasmic granules, which also contain lysozyme, phospholipase A2, cysteine proteinase and calcium-binding proteins. The role of amoebapores in killing of eukaryotic cells is becoming clearer. A 5 M amoebapore caused 60% release of the intracellular stain BCECF at 12 hours from eukaryotic cells, with the first detected molecule to be released being ATP. A decrease in mitochondrial membrane potential was seen with a 1 M amoebapore, as measured by MitoTracker Red. Amoebapores do not bind to E. histolytica membranes but do bind to membranes of the human Jurkat T cell culture lines. Also, amoebapores do not bind to liposomes made from purified membrane lipids of E. histolytica, but do bind to soybean phospholipid liposomes, suggesting that the membrane lipids of E. histolytica protect the parasite from amoebapore insertion. Thus, the mechanism by which the amebae safely store these cytotoxic molecules appears to be explained.

polarized Caco2 cells upon transfer. It is not clear what portions or subunits of the lectin are transferred, as not all antilectin monoclonal antibodies recognize the transferred lectin. There is, thus, the question of processing or conformational changes in the lectin during the transfer process. Guillens laboratory has focused on the role of amebic RacG, myosin II and PAK for maintenance of polarity, surface receptor capping and locomotion of the parasite [2022]. The pseudopod (the leading edge of the ameba) is where adhesion of the organism occurs with the uroid (the tailing edge of the ameba) containing the actinmyosin motor that pushes the amebae forward. Overproduction of light meromyosin (LMM) caused a decrease in motility and capping, whereas expression of the cytoplasmic tail of the Gal/GalNAc lectin resulted in a lack of uroid formation. Despite this, the amebae containing the lectin cytoplasmic tail construct had 50% greater mobility on Caco2 cells, whereas the LMM overexpressors had a greatly diminished mobility. Myosin 1B and its partners have important functions in cytoskeleton dynamics during phagocytosis in E. histolytica. There are several unconventional myosins in metazoan cells, including myosin V (involved in intracellular vesicle trafficking), myosin X (involved in cortical tension), myosin III (involved in signal transduction) and myosin I (involved in endocytosis, phagocytosis and cell motility). Myosin I is the only unconventional myosin identified in the E. histolytica genome to date. Myosin Ib is found in the early phagosome. Overexpression of myosin Ib resulted in a threefold drop in early erythrophagocytosis, a delay in phagocytic cup closure, but no change in motility, adherence or pinocytosis.

RAB proteins
Between 40 and 45 Rab GTPases in E. histolytica regulate vesicular trafficking [2325]. Nozaki and colleagues [23] are concentrating on Rab5 and Rab7 and deciphering their role in regulation of vesicular fusion in the endocytic/ phagocytic pathway. The role of these proteins in E. histolytica appears to be quite different from that in metazoan cells. In resting cells, Rab5 and Rab7 are not in the same location in the cell, but are individually localized to small vesicles. Within five minutes of contact of an amebae with a red blood cell, the two Rab proteins, together with a third unidentified Rab family member, are involved in formation of a large intracellular vesicle, with Rab5 and Rab7 co-localized on the cytoplasmic membrane side. Vesicles containing the amoebapores are then recruited to the large vesicle, resulting in the amoebapores residing in an amorphous, yet distinct, location within the large vesicle. This vesicle then fuses with the endosome containing the ingested red cell. The Rab5-inactive mutant, Q67L, prevents Rab5 from co-localizing with Rab7, affects the transport efficiency of the amoebapore to phagosomes, and decreases red cell ingestion by 60%.

Interaction with intestinal epithelium

Leroy [19] is studying the initial steps in the interaction of E. histolytica with polarized intestinal epithelial cells. Invasion through the intestinal epithelium initiates disease. Leroy has previously shown that the amebae disturb the enterocyte epithelial tight junctions early in the interaction. Sonicates of the amebae had no effect. Inhibition of the Gal/GalNAc lectin blocked tight junction disruption, demonstrating the requirement for contact of the intact parasite with the epithelium. The tight junction proteins ZO-1 and ZO-2 were proteolytically degraded during the interaction and ZO-2 was also dephosphorylated. Transfer of the Gal/GalNAc lectin accompanied tight junction disruption to the enterocytes. Lectin transfer occurs by a yet to be understood process that requires viable trophozoites. The transferred lectin localizes to the basolateral surface of

Genomics
Progress is rapid in the understanding of the genome structure of E. histolytica [2631]. At metaphase, 3050

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chromosomes can be resolved, and with pulsed-field gel electrophoresis (PFGE), one can separate the chromosomes of the parasite, although they are not as distinct as the chromosome bands of yeast similarly separated by PFGE. There is plasticity in the size of chromosomes between isolates of E. histolytica. Hybridization of cDNAs to PFGE by Tannichs laboratory [2628] has identified 14 linkage groups, with 14 chromosomes per linkage group per nucleus, consistent with the organism being polyploid at 4N or more. Whereas the linkage groups of E. histolytica are conserved between strains, the sizes of the chromosomes are not. This has been observed not only between isolates but with prolonged cultivation of the prototypic HM1:IMSS strain. The differences in chromosome size are as large as 1 MB. One potential explanation for size variation is changes in the numbers of tandemly repeated tRNA genes, which Clark has tentatively identified at the subtelomeric regions of the chromosomes. The size of the haploid genome is <20 Mb. Circular DNA is common in the parasite, with the circles apparently intertwined and running as a 2 Mb band in PFGE. The circles contain not only the 24 kB rDNA plasmid but other plasmids of unknown composition between 2 and 60 kB. Genes are densely packed with short intergenic regions of <100 bp. Introns may be more common than previously believed (at least 1020% of genes) but are also short (45128 bp with an average of 65 bp), and are bounded by a 5 sequence of GTTGTA and 3 TAG sequence. Conserved eukaryotic branch-point sequences in the ehrp 127a-1 intron have not proven important in splicing when mutated, with another sequence (WYTWAY, in which W is adenine or thymine and Y is cytosine or thymine) that is conserved in E. histolytica introns and closer to the 3 spliced site shown to be required. Comparison of the E. histolytica and Entamoeba dispar genomes is of interest, because these are the two most closely related of the Entamoeba genera and they are both able to parasitize humans. Only E. histolytica infection, however, is recognized to cause disease. Limited sequence comparisons demonstrate 95% sequence identity in coding regions and 80% in non-coding regions. Where it has been studied, the order of the genes on the chromosomes of the two parasites has been found to be identical. One proteinencoding E. histolytica gene that is missing in E. dispar encodes the plasma membrane surface cysteine proteinase CP5. The gene for CP5 is present, yet degenerated, in E. dispar, and has multiple stop codons. Much more will be known when the joint TIGRSanger Center genome project reaches 910 times coverage of the genome this spring, with closure achieved for at least one chromosome. At this time, TIGR has 80 000 sequences from a 23 kb genomic insert library, and plans to complete an additional 100 000 sequences from 6.5 kb and 23 kb genomic insert libraries by August 2002. The Sanger Center has completed 200 000 sequences from that library, as well as produced a 15 kb bacterial artificial chromosome

(BAC) library, by the time of writing (May 2002). The goals are to have 27 BAC ends, and 40 of the 6 kb clones and 700 of the 23 kb clones sequenced per 100 kb of DNA.

Transcription regulation
The first sequence-specific transcription factors binding to upstream regulatory elements of E. histolytica proteinencoding gene promoters were identified in 2001 [32,33]. The proteins that recognize upstream regulatory element (URE) 4 in the lectin hgl5 promoter contain RNA recognition motifs, whereas the URE3-binding protein lacks a classical DNA-binding domain but contains two EF-hand calciumbinding motifs. Gilchrist [32] has discovered that calcium controls the in vitro ability of the URE3-binding protein to recognize the URE3 DNA sequence. Studies are underway to test the importance of intracellular calcium in transcriptional repression or activation mediated by the URE3-binding protein.

Encystation and excystation

An area that has seen profound advances in understanding is the regulation of the developmental change of trophozoite to cyst. Encystment of the reptilian parasite Entamoeba invadens is being studied in Eichingers laboratory [34,35,36]. Upon placement in encystment media, E. invadens trophozoites aggregate within two hours. It is from these aggregates that cysts develop within 22 hours. Eichinger has discovered that galactose blocks, and galactose terminal proteins such as mucin glycoproteins and asialofetuin, enhance aggregation and subsequent encystment [34]. It is an attractive hypothesis that interactions of the amebae with mucin glycoproteins lining the intestine promote encystment of the parasite and thereby prevent invasion. This hypothesis is consistent with the clinical observation that people who pass E. histolytica cysts are asymptomatic. These data suggested that E. invadens contained a galactose lectin that mediates the encystment process, and Eichinger has identified a heterodimer lectin consisting of 125 kDa and 29 kDa subunits with sequence identity to the E. histolytica Gal/GalNAc lectin. The cysteines of the heavy subunits are conserved in spacing with the lectin of E. histolytica, and the carboxy terminus of the heavy subunit is almost completely conserved, starting with the 11 amino acids immediately 5 to the transmembrane domain. The sequence conservation may reflect conserved signaling functions for the E. histolytica and E. invadens lectins [34,35,36]. Encystment is stimulated by epinephrine at 15 M levels. More specifically, -1 agonists such as dobutamine stimulate encystment, and antagonists such as alprenolol and propranolol block encystation. If encystment is blocked with alprenolol, one can overcome the inhibition by adding dibutryl cAMP. A model is proposed that engagement of the lectin by mucins leads to release of epinephrine, and stimulation of G-coupled receptors activate adenylate cyclase, leading to the production of cAMP.

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Makioka et al. are investigating the excystation of E. invadens [37,38]. A four-nucleated metacyst emerges from the cyst wall and then undergoes division into eight uninuclear trophozoites. The tubulin inhibitor oryzalin inhibited excystation in a dose-dependent manner. This data implicated tubulin and cytoskeleton in the littleunderstood process of excystation.

12. Ankri S, Padilla-Vaca F, Stolarsky T, Koole L, Katz U, Mirelman D: Antisense inhibition of expression of the light subunit (35 kDa) of the Gal/GalNac lectin complex inhibits Entamoeba histolytica virulence. Mol Microbiol 1999, 33:327-337. 13. Padilla-Vaca F, Ankri S, Bracha R, Koole LA, Mirelman D: Downregulation of Entamoeba histolytica virulence by monoxenic cultivation with Escherichia coli O55 is related to a decrease in expression of the light (35-kilodalton) subunit of the Gal/GalNAc lectin. Infect Immun 1999, 67:2096-2102. 14. Cheng X-J, Tachibana H: Protection of hamsters from amebic liver abscess formation by immunization with the 150- and 170-kDa surface antigens of Entamoeba histolytica. Parasitol Res 2001, 87:126-130. 15. Cheng X-J, Hughes MA, Huston CD, Loftus B, Gilchrist CA, Lockhart LA, Ghosh S, Miller-Sims V, Mann BJ, Petri WA Jr, Tachibana H: Intermediate subunit of the Gal/GalNAc lectin of Entamoeba histolytica is a member of a gene family containing multiple CXXC sequence motifs. Infect Immun 2001, 69:5892-5898. The authors of this paper provide further definition of the structure of the amebic Gal/GalNAc lectin by identification of the non-covalently associated light subunit, which shares sequence identity with the variant surface proteins of Giardia lamblia. 16. Bruhn H, Leippe M: Novel putative saposin-like proteins of Entamoeba histolytica different from amoebapores. Biochim Biophys Acta 2001, 1514:14-20. 17. Bruhn H, Leippe M: Membrane-permeabilizing polypeptides of amoebae: constituents of an archaic antimicrobial system. Zool Anal Complex Syst 2001, 104:3-11. This is a review of the multiple pore-forming granule proteins of E. histolytica. 18. Nickel R, Jacobs T, Urban B, Scholze H, Bruhn H, Leippe M: Two novel calcium-binding proteins from cytoplasmic granules of the protozoan parasite Entamoeba histolytica. FEBS Letts 2000, 486:112-116. 19. Leroy A, Lauwaet T, De Bruyne G, Cornelissen M, Mareel M: Entamoeba histolytica disturbs the tight junction complex in human enteric T84 cell layers. FASEB J 2000, 14:1139-1146. 20. Tavares P, Sansonetti P, Guillen N: Cell polarization and adhesion in a motile pathogenic protozoan: role and fate of the Entamoeba histolytica Gal/GalNAc lectin. Microbes Infect 2000, 2:643-649. 21. Voigt H, Guillen N: New insights into the role of the cytoskeleton in phagocytosis of Entamoeba histolytica. Cell Microbiol 1999, 1:195-203. 22. Voigt H, Olivo JC, Sansonetti P, Guillen N: Myosin IB from Entamoeba histolytica is involved in phagocytosis of human erythrocytes. J Cell Sci 1999, 112:1191-1201. 23. Saito-Nakano Y, Nakazawa M, Shigeta Y, Takeuchi T, Nozaki T: Identification and characterization of genes encoding novel Rab proteins from Entamoeba histolytica. Mol Biochem Parasitol 2001, 116:219-222. 24. Juarez P, Sanchez-Lopez R, Stock RP, Olvera A, Ramos MA, Alagon A: Characterization of the Ehrab8 gene, a marker of the late stages of the secretory pathway of Entamoeba histolytica. Mol Biochem Parasitol 2001, 116:223-228. 25. Temesvari LA, Harris EN, Stanley SL Jr, Cardellia JA: Early and late endosomal compartments of Entamoeba histolytica are enriched in cysteine proteases, acid phosphatase and several Ras-related Rab GTPases. Mol Biochem Parasitol 1999, 103:225-241. 26. Willhoeft U, Campos-Gongora E, Touzni S, Bruchhaus I, Tannich E: Introns of Entamoeba histolytica and Entamoeba dispar. Protist 2001, 152:149-156. 27. Willhoeft U, Tannich E: Fluorescence microscopy and fluorescence in situ hybridization of Entamoeba histolytica nuclei to analyse mitosis and the localization of repetitive DNA. Mol Biochem Parasitol 2000, 105:291-296.

Conclusions
One in 10 children in Bangladesh dies before his or her fifth birthday. One third of these deaths are due to diarrheal diseases such as amebiasis. It is heartening to see the application of molecular biology to the problem of amebic colitis. The joint efforts of scientists from the developing and developed worlds reviewed here promise to provide solutions for one of the great neglected diseases of mankind.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

of special interest of outstanding interest

1. Amano K, Takeuchi T: Amebiasis in acquired immunodeficiency syndrome. Intern Med 2001, 40:563-564. Although data is scanty, there is no suggestion at present that HIV exacerbates amebiasis. 2. Braga LL, Gomes ML, Da Silva MW, Facanha FE Jr, Fiuza L, Mann BJ: Household epidemiology of Entamoeba histolytica infection in an urban community in northeastern Brazil. Am J Trop Med Hyg 2001, 65:268-271. Evangelopoulos A, Legakis N, Vakalis N: Microscopy, PCR and ELISA applied to the epidemiology of amoebiasis in Greece. Parasitol Int 2001, 50:185-189.

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Haque R, Ali IM, Sack RB, Farr BM, Ramakrishnan G, Petri WA Jr: Amebiasis and mucosal IgA antibody against the Entamoeba histolytica adherence lectin in Bangladeshi children. J Infect Dis 2001, 183:1787-1793. Studying 300 Bangladeshi children for two years revealed that acquired immunity exists and is linked to an intestinal IgA antibody response against the Gal/GalNAc lectin. 5. Lee MB, Keystone JS, Kain KC: Nonpathogenic protozoa: laboratory reporting practices in Canada and the United States. Lab Med 2001, 32:455-456. Liu CJ, Hung CC, Chen MY, Lai YP, Chen PJ, Huang SH, Chen DS: Amebic liver abscess and Human Immunodeficiency Virus infection: a report of three cases. J Clin Gastroenterol 2001, 33:64-68. Mortele KJ, Ros PR: Cystic focal liver lesions in the adult: differential CT and MR imaging features. Radiographics 2001, 21:895-910. Nuez YO, Fernandez MA, Torres-Nuez D, Silva JA, Montano I, Maestre JL, Fonte L: Multiplex polymerase chain reaction amplification and differentiation of Entamoeba histolytica and Entamoeba dispar DNA from stool samples. Am J Trop Med Hyg 2001, 64:293-297. Schunk M, Jelinek T, Wetzel K, Nothdurft HD: Detection of Giardia lamblia and Entamoeba histolytica in stool samples by two enzyme immunoassays. Eur J Clin Microbiol Infect Dis 2001, 20:389-391.

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10. Sharp SE, Suarez CA, Duran Y, Poppiti RJ: Evaluation of the Triage Micro Parasite Panel for detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum in patient stool specimens. J Clin Microbiol 2001, 39:332-334. 11. Stanley SL Jr: Protective immunity to amebiasis: new insights and new challenges. J Infect Dis 2001, 184:504-506. This is a concise review of innate and acquired immunity to amebiasis.

28. Willhoeft U, Tannich, E: The electrophoretic karyotype of Entamoeba histolytica. Mol Biochem Parasitol 1999, 99:41-53. 29. Bhattacharya A, Bhattacharya S, Joshi A, Ramachandran S, Ramaswamy R: Identification of parasitic genes by computational methods. Parasitol Today 2000, 16:127-131. 30. Bhattacharya A, Satish S, Bagchi A, Bhattacharya S: The genome of Entamoeba histolytica. Int J Parasitol 2000, 30:401-410.

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31. Som I, Azam A, Bhattacharya A, Bhattacharya S: Inter- and intra-strain variation in the 5.8S ribosomal RNA and internal transcribed spacer sequences of Entamoeba histolytica and comparison with Entamoeba dispar, Entamoeba moshkovskii and Entamoeba invadens. Int J Parasitol 2000, 30:723-728. 32. Gilchrist CA, Holm CF, Hughes MA, Schaenman JM, Mann BJ, Petri WA Jr: Identification and characterization of an Entamoeba histolytica Upstream Regulatory Element 3 sequence-specific DNA-binding protein containing EF-hand motifs. J Biol Chem 2001, 276:11838-11843. EF-hand domains suggest a role for intracellular calcium in control of gene expression via this transcription factor. 33. Schaenman JM, Gilchrist CA, Mann BJ, Petri WA Jr: Identification of two Entamoeba histolytica sequence-specific URE4 enhancer-binding proteins with homology to the RNA-binding motif RRM. J Biol Chem 2001, 276:1602-1609.

34. Eichinger D: A role for a galactose lectin and its ligands during encystment of Entamoeba. J Eukaryot Microbiol 2001, 48:17-21. One of the most exciting developments in amebiasis is demonstration of the role of the Gal lectin in encystment. 35. Eichinger D: Encystation in parasitic protozoa. Curr Opin Microbiol 2001, 4:421-426. 36. Coppi A, Eichinger D: Regulation of Entamoeba invadens encystation and gene expression with galactose and N-acetylglucosamine. Mol Biochem Parasitol 1999, 102:67-77. 37. Makioka A, Kumagai M, Ohtomo H, Kobayashi S, Takeuchi T: Effect of jasplakinolide on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens. J Parasitol 2001, 87:399-405.

38. Makioka A, Kumagai M, Ohtomo H, Kobayashi S, Takeuchi T: Effect of calcium antagonists, calcium channel blockers and calmodulin inhibitors on the growth and encystation of Entamoeba histolytica and E. invadens. Parasitol Res 2001, 87:833-837.