Daniel J Eichinger, Ph.D.

Compounds of the upper gastrointestinal tract induce rapid and efficient excystation of Entamoeba invadens
Mitra, Biswa Nath; Pradel, Gabriele; Frevert, Ute; Eichinger, Daniel
2010 May;40(6):751-760, International journal for parasitology
The infective stage of Entamoeba parasites is an encysted form. This stage can be readily generated in vitro, which has allowed identification of stimuli that trigger the differentiation of the parasite trophozoite stage into the cyst stage. Studies of the second differentiation event, emergence of the parasite from the cyst upon infection of a host, have been hampered by the lack of an efficient means to excyst the parasite and complete the life cycle in vitro. We have determined that a combination of exposures to water, bicarbonate and bile induces rapid excystment of Entamoeba invadens cysts. The high efficiency of this method has allowed the visualization of the dynamics of the process by electron and confocal microscopy, and should permit the analysis of stage-specific gene expression and high-throughput screening of inhibitory compounds
— id: 109037, year: 2010, vol: 40, page: 751, stat: Journal Article,

CHARACTERIZATION OF THE IMMUNE RESPONSE TO THE LARVAL PHASE OF ASCARIS SUUM INFECTION AND IDENTIFICATION OF ASCARIS ANTIGENS EXPRESSED DURING THAT PHASE
Fernandez, MAG; Pollack, HJ; Eichinger, D; Hotez, PJ
2009 NOV ;81(5):134-134, American journal of tropical medicine & hygiene
— id: 106986, year: 2009, vol: 81, page: 134, stat: Journal Article,

Analysis of commercial Entamoeba histolytica ELISA kits for the detection of Entamoeba invadens in reptiles
Brewer, Laurie A; Denver, Mary C; Whitney, Meredith; Eichinger, Daniel J
2008 Sep;39(3):493-495, Journal of zoo & wildlife medicine
Entamoeba invadens is pathogenic in multiple reptile species and has caused severe outbreaks in zoos and other facilities worldwide. Infections can be difficult to diagnose and to differentiate from other reptilian Entamoeba species. The goal of this study was to determine if kits developed to identify the human pathogen Entamoeba histolytica could be used to detect E. invadens in reptile species at the Maryland Zoo. The E. histolytica II antigen enzyme-linked immunosorbent assay and the ProSpecT E. histolytica microplate assay did not react with cultured E. invadens controls or with fecal samples from multiple reptiles, demonstrating the need for a sensitive and specific test for E. invadens
— id: 95552, year: 2008, vol: 39, page: 493, stat: Journal Article,

Acetylation of the Entamoeba histone H4 N-terminal domain is influenced by short-chain fatty acids that enter trophozoites in a pH-dependent manner
Byers, Jennifer; Eichinger, Daniel
2008 Jan;38(1):57-64, International journal for parasitology
Treatment of higher eukaryotic cells with short-chain fatty acids (SCFA) such as butyrate causes decreased levels of histone deacetylase (HDAC) activity and hyperacetylation of histones, and thereby affects gene expression, cell growth and differentiation. Entamoeba parasites encounter high levels of SCFA in the host colon, and in vitro these compounds allow trophozoite stage parasites to multiply but prevent their differentiation into infectious cysts. The Entamoeba invadens IP-1 histone H4 protein has an unusual number of lysines in its N-terminus, and these become hyperacetylated in trophozoites exposed to the HDAC inhibitors trichostatin A (TSA) or HC-toxin, but not in trophozoites exposed to butyrate. We have now found that several other commonly studied isolates of Entamoeba parasites also have an extended set of histone H4 acetylation sites that become hyperacetylated in response to TSA, but hypoacetylated in response to butyrate, suggesting an unusual sensitivity of this parasite's histone modifying enzymes to SCFA. Butyrate was found to enter trophozoites in a pH-dependent manner consistent with diffusive entry of the un-ionised form of the fatty acid into the amoebae. Transit of the Entamoeba organism through areas of the host intestine with distinct pH and SCFA concentrations would therefore result in very different levels of SCFA within the parasite. Entamoeba appears to have acquired unique alterations of its histone acetylation mechanism that may allow for its growth in the presence of varying amounts of the bacterial fermentation products
— id: 78787, year: 2008, vol: 38, page: 57, stat: Journal Article,

Trichostatin A effects on gene expression in the protozoan parasite Entamoeba histolytica
Ehrenkaufer, Gretchen M; Eichinger, Daniel J; Singh, Upinder
2007 ;8:216-216, BMC genomics
BACKGROUND: Histone modification regulates chromatin structure and influences gene expression associated with diverse biological functions including cellular differentiation, cancer, maintenance of genome architecture, and pathogen virulence. In Entamoeba, a deep-branching eukaryote, short chain fatty acids (SCFA) affect histone acetylation and parasite development. Additionally, a number of active histone modifying enzymes have been identified in the parasite genome. However, the overall extent of gene regulation tied to histone acetylation is not known. RESULTS: In order to identify the genome-wide effects of histone acetylation in regulating E. histolytica gene expression, we used whole-genome expression profiling of parasites treated with SCFA and Trichostatin A (TSA). Despite significant changes in histone acetylation patterns, exposure of parasites to SCFA resulted in minimal transcriptional changes (11 out of 9,435 genes transcriptionally regulated). In contrast, exposure to TSA, a more specific inhibitor of histone deacetylases, significantly affected transcription of 163 genes (122 genes upregulated and 41 genes downregulated). Genes modulated by TSA were not regulated by treatment with 5-Azacytidine, an inhibitor of DNA-methyltransferase, indicating that in E. histolytica the crosstalk between DNA methylation and histone modification is not substantial. However, the set of genes regulated by TSA overlapped substantially with genes regulated during parasite development: 73/122 genes upregulated by TSA exposure were upregulated in E. histolytica cysts (p-value = 6 x 10(-53)) and 15/41 genes downregulated by TSA exposure were downregulated in E. histolytica cysts (p-value = 3 x 10(-7)). CONCLUSION: This work represents the first genome-wide analysis of histone acetylation and its effects on gene expression in E. histolytica. The data indicate that SCFAs, despite their ability to influence histone acetylation, have minimal effects on gene transcription in cultured parasites. In contrast, the effect of TSA on E. histolytica gene expression is more substantial and includes genes involved in the encystation pathway. These observations will allow further dissection of the effects of histone acetylation and the genetic pathways regulating stage conversion in this pathogenic parasite
— id: 78788, year: 2007, vol: 8, page: 216, stat: Journal Article,

Identification of developmentally regulated genes in Entamoeba histolytica: insights into mechanisms of stage conversion in a protozoan parasite
Ehrenkaufer, Gretchen M; Haque, Rashidul; Hackney, Jason A; Eichinger, Daniel J; Singh, Upinder
2007 Jun;9(6):1426-1444, Cellular microbiology
Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. The protozoan parasite Entamoeba histolytica converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Identification of genes involved in the developmental pathway has been severely hindered by the inability to generate E. histolytica cysts in vitro. Using parasite strains derived from recent human infections and whole-genome transcriptional profiling, we determined that 1439 genes (approximately 15% of annotated genes) were potentially developmentally regulated. Genes enriched in cysts (672 in total) included cysteine proteinases and transmembrane protein kinases, which may be involved in signal transduction. Genes enriched in trophozoites (767 in total) included genes typically thought of as important in tissue invasion by trophozoites, including the Gal/GalNAc lectin light subunit and cysteine protease 1. Putative regulators of differentiation including possible G-protein coupled receptors, signal transduction proteins and transcription factors were identified. A number of E. histolytica stage-specific genes were also developmentally regulated in the reptilian parasite E. invadens, indicating that they likely have conserved functions in Entamoeba development. These advances lay the groundwork for dissection of the molecular signals that initiate stage conversion and development of novel diagnostic and therapeutic measures targeting E. histolytica cysts
— id: 78789, year: 2007, vol: 9, page: 1426, stat: Journal Article,

A strategy for identifying serodiagnostically relevant antigens of Leishmania or other pathogens in genetic libraries
Teixeira, Marcia C A; Oliveira, Geraldo G S; Silvany, Marco A; Alcantara-Neves, Neuza M; Soares, Milena B P; Ribeiro-Dos-Santos, Ricardo; Jeronimo, Selma M B; Costa, Carlos H; dos-Santos, Washington L C; Eichinger, Daniel; Pontes-de-Carvalho, Lain
2007 Mar;35(1):51-54, Biologicals
Different individuals, when infected with the same parasite, rarely produce antibodies against the same set of antigens. Indeed, unless a particular antigen happens to be recognized by antibodies in all individuals, the use of a single antigen in the serodiagnosis of parasitic diseases leads, invariably, to false-negative results. A straightforward method for pin-pointing, in genetic libraries, the precise antigens that would increase serodiagnostic assay sensitivities is presented. The method is based on the utilization of sera that produced false-negative results against previously available antigens. Employing this false-negative serum-selection methodology for the identification of new Leishmania infantum recombinant antigens (rAgs), the sensitivity of a dipstick assay for anti-Leishmania antibodies in a panel of sera from patients with visceral leishmaniasis was increased from 83.3% to 98.1%, without affecting its specificity, by the inclusion of a fifth and a sixth L. infantum rAg
— id: 78790, year: 2007, vol: 35, page: 51, stat: Journal Article,

Entamoeba invadens: the requirement for galactose ligands during encystment
Turner, N A; Eichinger, D
2007 Aug;116(4):467-474, Experimental parasitology
During periods of stress, trophozoites of Entamoeba invadens (strain IP-1) undergo a process of differentiation (encystment) that results in a dormant cyst with a chitin-containing cyst wall. Encystment can be induced by resuspension of trophozoites from growth medium into a diluted glucose-free medium (47% LG) containing 5% adult bovine serum (ABS). ABS is thought to be a source of gal-terminated ligands that are required for high levels of encystment. After resuspension of trophozoites in 47% LG, encystment cultures were examined every 2h for responses to the (i) addition of 10mM free-galactose, (ii) resuspension of cells to serum-free medium, (iii) and dilution of encysting cultures to cell densities below that known to support full encystment (from 5 x 10(5) to 1 x 10(4)cells/ml). The role of serum components (and the gal-terminated ligand asialofetuin; ASF) adsorbed onto the surface upon which encystment proceeds, and their effect on the multi-cellular aggregation patterns formed during encystment, were also investigated. The addition of free-galactose reduced the levels of encystment (compared with the control) even when added at 10h after resuspension of trophozoites in 47% LG. The requirement for the presence of ABS during encystment was lost within 6h, with levels of encystment of cells washed free of serum reaching 80% of the control. The ability of cells to encyst when diluted to a cell density below that normally thought to support encystment reached over 50% by 8h. Efficient encystment could be obtained in 47% LG in the absence of ABS or ASF using pre-treated glass culture tubes. Encystment (47% LG; 5% ABS) using ultra low attachment plates was poor, suggesting attachment of cells to a surface via gal-terminated ligands was important for efficient encystment. The results suggest that ABS is probably not the only source of gal-terminated ligands necessary for high levels of encystment in 47% LG. While serum may provide a source of ligands which enhance the levels of encystment initially, other gal-terminated ligands possibly released by the encysting cells are still required for the completion of the encystment process and the formation of mature cysts. In addition, the gal-terminated ligands necessary for encystment efficiency may be adsorbed onto the glass surface of culture tubes and aid the initial aggregation process, as well as be involved in cell signaling during the encystment process
— id: 73292, year: 2007, vol: 116, page: 467, stat: Journal Article,

Entamoeba invadens: restriction of ploidy by colonic short chain fatty acids
Byers, Jennifer; Eichinger, Daniel
2005 Jul;110(3):203-206, Experimental parasitology
The DNA content of Entamoeba parasites appears to be regulated by an unusual mechanism. This conclusion, however, was based on experiments that examined parasites grown in media that did not contain short chain fatty acids (SCFAs) normally found in the colonic lumen. Since one of these SCFAs, butyrate, is known to affect DNA replication in eukaryotic cells, we examined the effect of SCFAs on Entamoeba trophozoite DNA content. Similar to reports from others, we found that Entamoeba invadens trophozoite cultures grown in conventional medium (TYI-S-33) contained cells with 2N, 4N, 8N, and 16N amounts of DNA. In contrast, cultures grown in TYI medium containing colonic SCFAs added in place of glucose contained a minor population with 2N, a major population with 4N, and very few cells with higher amounts of DNA. SCFAs also prevented the normal increase in the number of nuclei per cell in trophozoites that were induced to encyst. These results suggest that E. invadens trophozoite stage parasites growing in the intestine in the presence of high amounts of SCFAs have a ploidy range restricted to 2N/4N. Axenic growth of trophozoites in the absence of SCFAs, however, appears to allow trophozoites to increase the amount of DNA per cell, which they must do during the normal encystment process
— id: 78791, year: 2005, vol: 110, page: 203, stat: Journal Article,

Colonic short-chain fatty acids inhibit encystation of Entamoeba invadens
Byers, Jennifer; Faigle, Wolfgang; Eichinger, Daniel
2005 Feb;7(2):269-279, Cellular microbiology
Entamoeba parasites multiply as trophozoites in the layer of mucus that overlies the colonic epithelium. In response to stimuli that are not understood, trophozoites stop multiplying and differentiate into cysts that are released to infect another host. In the colon, Entamoeba trophozoites are exposed to the large variety of biochemicals that are carried into or are produced within this organ. The normal bacterial population of the colon releases large amounts of short-chain fatty acids (SCFAs). These compounds have effects on the growth, differentiation and repair of the colonic epithelium that correlate with de-creased activity of a Class I/II histone deacetylase (HDAC). We found that the formation of cysts, but not the growth of trophozoite-stage Entamoeba invadens parasites, was inhibited by physiologic concentrations of SCFAs. Variable levels of cyst formation did occur if SCFA concentrations were lowered. Specific inhibitors of Class I/II-type HDACs also prevented encystation, and trophozoites exposed to these compounds had increased levels of acetylation of histone H4 and other nuclear proteins. These results suggest that production of the infectious cyst stage of Entamoeba parasites is regulated in part by the levels of SCFAs made by the bacterial population of the colon
— id: 56028, year: 2005, vol: 7, page: 269, stat: Journal Article,

Parasite-derived trans-sialidase binds to heart tissue in Trypanosoma cruzi-infected animals
Alcantara-Neves, Neuza M; Ribeiro-dos-Santos, Ricardo; Amor, Ana Lucia Moreno; Uemura, Haruki; Silva-Neto, Samuel J; Eichinger, Daniel; Pontes-de-Carvalho, Lain
2004 Nov;37(5):273-278, Microbial pathogenesis
Trypanosoma cruzi is an obligate intracellular protozoan parasite that actively penetrates into non-phagocytic mammalian cells. To accomplish this, the parasite relies on the binding of cell surface ligands. It is reported herein that the T. cruzi trans-sialidase (TS), which is exposed on the parasite surface, binds to mouse heart cells, and should therefore be further studied as a possible cell penetration-related ligand. In addition, as has been proposed elsewhere, the binding of T. cruzi to tissues may turn them into targets for parasite-specific immune reactions. Washed heart sections from T. cruzi-infected mice were subjected to immunoenzymatic staining with antisera against whole T. cruzi and with polyclonal or monoclonal antibodies against TS. The anti-TS antibodies stained both parasites and uninfected heart cells in the vicinity of T. cruzi nest remains/trypomastigotes. On the other hand, an anti-T. cruzi serum, which did not recognize TS, only stained the parasites. In addition, normal heart sections from uninfected nude mice were shown to react with both enzymatically active and inactive recombinant TS molecules, probably through their amino-terminal region, since a recombinant TS lacking this region failed to bind
— id: 78792, year: 2004, vol: 37, page: 273, stat: Journal Article,

Entamoeba invadens contains the components of a classical adrenergic signaling system
Frederick, Jesse; Eichinger, Dan
2004 Oct;137(2):339-343, Molecular & biochemical parasitology
Epinephrine (Epi) was previously found to bypass the need for galactose ligands during early steps in the initiation of Entamoeba encystment. Epinephrine is presumed to act on amoebae through a classical adrenergic signaling pathway that results in the increased production of cyclic adenosine monophosphate (cAMP). The object of this study was to verify the existence of an adrenergic like pathway and its response to Epi in both whole Entamoeba trophozoites and purified plasma membrane preparations. Whole trophozoite and purified membrane preparations from Entamoeba invadens responded to the presence of Epi by increasing the production of cAMP. The modulators of heterotrimeric G protein signaling, forskolin (FK), pertussis toxin (PTX) and cholera toxin (CTX), also increased cAMP levels in whole cells and membrane fragments. All of these increases in cAMP were inhibited by specific inhibitors of adenylyl cyclase (AC). Treatment of membrane fragments with epinephrine caused an increased binding of non-hydrolysable GTP analogs. Entamoeba trophozoites therefore appear to contain G-protein-regulated adenylyl cyclase that functions downstream of an adrenergic ligand receptor
— id: 51385, year: 2004, vol: 137, page: 339, stat: Journal Article,

Gene discovery in the Entamoeba invadens genome
Wang, Zheng; Samuelson, John; Clark, C Graham; Eichinger, Daniel; Paul, Jaishree; Van Dellen, Katrina; Hall, Neil; Anderson, Iain; Loftus, Brendan
2003 Jun;129(1):23-31, Molecular & biochemical parasitology
Entamoeba invadens, a parasite of reptiles, is a model for the study of encystation by the human enteric pathogen Entamoeba histolytica, because E. invadens form cysts in axenic culture. With approximately 0.5-fold sequence coverage of the genome, we were able to get insights into E. invadens gene and genome features. Overall, the E. invadens genome displays many of the features that are emerging from ongoing genome sequencing efforts in E. histolytica. At the nucleotide level the E. invadens genome has on average 60% sequence identity with that of E. histolytica. The presence of introns in E. invadens was predicted with similar consensus (GTTTGT em leader A/TAG) sequences to those identified in E. histolytica and Entamoeba dispar. Sequences highly repeated in the genome of E. histolytica (rRNAs, tRNAs, CXXC-rich proteins, and Leu-rich repeat proteins) were found to be highly repeated in the E. invadens genome. Numerous proteins homologous to those implicated in amoebic virulence, (Gal/GalNAc lectins, amoebapores, and cysteine proteinases) and drug resistance (p-glycoproteins) were identified. Homologs of proteins involved in cell cycle, vesicular trafficking and signal transduction were identified, which may be involved in en/excystation and cell growth of E. invadens. Finally, multiple copies of a number of E. invadens genes coding for predicted enzymes involved in core metabolism and the targets of anti-amoebic drugs were identified
— id: 78793, year: 2003, vol: 129, page: 23, stat: Journal Article,

The enteric parasite Entamoeba uses an autocrine catecholamine system during differentiation into the infectious cyst stage
Coppi, Alida; Merali, Salim; Eichinger, Daniel
2002 Mar 8;277(10):8083-8090, Journal of biological chemistry
Enteric amoebae of the genus Entamoeba travel from host to host in an encysted form. We previously showed that in vitro cyst development of Entamoeba invadens requires the addition of defined amounts of multivalent galactose-terminated molecules, such as mucin, to the cultures. The amoeba surface lectin that binds mucin is presumed to convey transmembrane signals when clustered by the ligand, but the signaling molecules that function downstream of the lectin are not known. We report here that Entamoeba encystation was induced in the absence of galactose ligand when catecholamines were added to the encystation medium. Micromolar amounts of both epinephrine and norepinephrine induced encystation. Of a variety of synthetic catecholamine agonists tested, only beta(1)-adrenergic receptor agonists supported encystation, whereas alpha- and beta(2)-adrenergic receptor agonists did not. Only beta(1)-adrenergic receptor antagonists inhibited encystation, and did so even when exogenous catecholamines were not added, indicating that catecholamine binding is required for encystation and suggesting an endogenous source of the ligand. High performance liquid chromatography analysis of Entamoeba extracts showed that the amoebae themselves contain catecholamines and at least one of these is released when the cells are stimulated to encyst with galactose-terminated ligands. The presence of catecholamine binding sites on the surface of amoeba trophozoites was confirmed using radiolabeled catecholamine antagonist. Amoeba encystment was inhibited by addition of beta(1)-adrenergic receptor antagonist to cells that were stimulated to differentiate with either galactose ligand or catecholamines, but not with dibutyryl cAMP. This suggests that the amoeba catecholamine receptor functions downstream of the galactose lectin and upstream of adenylyl cyclase. This enteric protozoan parasite, therefore, contains the components of an autocrine catecholamine ligand-receptor system that may act in conjunction with a galactose lectin to regulate differentiation into the infectious cyst stage
— id: 39730, year: 2002, vol: 277, page: 8083, stat: Journal Article,

Catecholamines in Entamoebae: recent (re)discoveries
Eichinger, D; Coppi, A; Frederick, J; Merali, S
2002 NOV ;27(6):589-593, Journal of biosciences
Free-living and enteric amoebae have similar two-stage life cycles, and both organisms depend on being able to monitor environmental conditions to determine whether to continue multiplying as trophozoites, or to differentiate into dormant or transmissible cysts. Conditions that support high trophozoite densities might also be expected to select for mechanisms of information exchange between these cells. We recently determined that trophozoites of at least one species of Entamoeba release and respond to catecholamine compounds during differentiation from the trophozoite stage into the cyst stage. It turns out that this is not an entirely novel finding, as a number of previous studies have demonstrated parts of this story in free-living or enteric amoebae. We briefly review here major points of the previous studies and describe some of our recent results that have extended them
— id: 33282, year: 2002, vol: 27, page: 589, stat: Journal Article,

A role for a galactose lectin and its ligands during encystment of Entamoeba
Eichinger D
2001 Jan-Feb;48(1):17-21, Journal of eukaryotic microbiology
In the life cycle of Entamoeba parasites alternate between the colon-dwelling trophozoite and the infectious cyst forms. The physiologic stimuli that trigger differentiation of trophozoites into cysts remain undefined. On the surface of the human-infecting Entamoeba, parasites express a galactose/N-acetylgatactosamine (gal/galNAc)-binding lectin, which plays demonstrated roles in contact-dependent lysis of target cells and resistance to host complement. Using a reptilian parasite, Entamoeba invadens, to study cyst formation in vitro, we found that efficient encystation was dependent on the presence of gal-terminated ligands in the induction medium. Precise concentration ranges of several gal-terminated ligands, such as asialofetuin, gal-bovine serum albumin (gal-BSA), and mucin, functioned in encystation medium to stimulate differentiation. Greater than 10 mM levels of free gal inhibited the amoeba aggregation that precedes encystation and prevented formation of mature cysts. Inhibitory levels of gal also prevented the up-regulation of genes which normally occurs at 24 h of encystation. The surface of Entamoeba invadens was found to express a gal lectin which has a heterodimeric structure similar to that of Entamoeba histolytica. The 30 kDa light subunit (LGL) of the E. invadens lectin is similar in overall size and sequence to the LGL of E. histolytica. The heavy subunits, however, differ in size, have an identical spacing of cysteines in their extracellular domains, and have highly conserved C-terminal transmembrane and cytoplasmic domains. These results suggest a new role for the Entamoeba gal lectins in monitoring the concentrations of gal ligands in the colon and contributing to stimuli that induce encystment
— id: 26778, year: 2001, vol: 48, page: 17, stat: Journal Article,

Encystation in parasitic protozoa
Eichinger D
2001 Aug;4(4):421-426, Current opinion in microbiology
Giardia and Entamoeba parasites encase themselves in a carbohydrate-rich cyst for travel from host to host. Both parasites upregulate their Golgi apparatus during this process, yielding organelles that are now found to be similar to those of higher eukaryotes. In fact, unusual enzymes and structural proteins used for cyst wall synthesis, the complexity of the secretory pathways used to transport materials to the developing cyst walls, as well as unexpected mechanisms of gene regulation and parasite-host and parasite-parasite information exchange, are revealing a high level of sophistication in these organisms that occupy low branch points in the eukaryotic lineage
— id: 26705, year: 2001, vol: 4, page: 421, stat: Journal Article,

Characterization of the cell adhesion site of Trypanosoma cruzi metacyclic stage surface glycoprotein gp82
Manque PM; Eichinger D; Juliano MA; Juliano L; Araya JE; Yoshida N
2000 Feb;68(2):478-484, Infection & immunity
The surface glycoprotein gp82, expressed in the insect-stage metacyclic trypomastigotes of Trypanosoma cruzi, has been implicated in mammalian cell invasion. Here we have characterized the cell adhesion site of gp82 by using recombinant proteins and synthetic peptides based on gp82. The recombinant protein Del-4/8, lacking 65 amino acids of gp82 central domain (at positions 257 to 321), was virtually devoid of cell-binding activity and lacked the ability to inhibit parasite invasion, in contrast to J18, the construct containing the full-length gp82 sequence (amino acids 1 to 516). Constructs with shorter deletions, i.e., Del-4 (deleted from 257 to 271) and Del-8 (deleted from 293 to 321), bound to target cells to a significantly lesser degree than did J18. The sites deleted in recombinant proteins Del-4 and Del-8 contained acidic amino acids critical for cell adhesion. Thus, the cell-binding capacity of protein Del-E/D, lacking the glutamic acid (259/260) and aspartic acid (303/304) pairs, was negligible, as was its capacity to inhibit parasite internalization. Of a set of synthetic peptides spanning the gp82 central domain, a 22-mer hybrid peptide, p4/8, formed by two noncontiguous sequences (at positions 257 to 273 and 302 to 306) and containing the four acidic residues, competed with the binding of J18 protein to target cells and significantly inhibited ( approximately 60%) the penetration of parasites. This peptide, generated by the juxtaposition of sequences that are separated by a hydrophobic stretch in the linear molecule, appears to be mimicking a conformation-dependent cell-binding site of gp82. Experiments of antibody competition with a set of 20-mer overlapping peptides mapped the epitope for 3F6, a monoclonal antibody directed to gp82 that inhibits parasite invasion, to the sequence represented by peptide p3 (244 to 263), which has a partial overlap with the cell adhesion site
— id: 57565, year: 2000, vol: 68, page: 478, stat: Journal Article,

Regulation of Entamoeba invadens encystation and gene expression with galactose and N-acetylglucosamine
Coppi A; Eichinger D
1999 Jul 30;102(1):67-77, Molecular & biochemical parasitology
Encystation of Entamoeba invadens parasites is prevented by the presence of free galactose or N-acetylglucosamine in the encystation medium. Galactose prevents the formation of amoeba cellular aggregates which develop during the early phase of encystation, suggesting the presence of functional cell surface galactose-binding molecules, whereas N-acetylglucosamine allows aggregation to occur and prevents cyst formation at a later point. While studying sugar inhibition of amoeba encystation, it was found that high efficiency encystation required the inclusion in encystation medium of precise amounts of polyvalent galactose-terminated molecules, and these molecules could be supplied by serum or by defined glycoconjugates, including mucin. Addition of free galactose to encystation medium prevented the accumulation of three transcripts which are normally upregulated during encystation, and N-acetylglucosamine prevented accumulation of one of the transcripts. These results suggest the presence of distinct sugar-sensitive pathways that regulate differentiation of the amoeba trophozoite into infectious cysts
— id: 6253, year: 1999, vol: 102, page: 67, stat: Journal Article,

Proteasome-dependent cyst formation and stage-specific ubiquitin mRNA accumulation in Entamoeba invadens
Gonzalez J; Bai G; Frevert U; Corey EJ; Eichinger D
1999 Sep;264(3):897-904, European journal of biochemistry
Proteases play an important role in the pathogenic mechanisms and differentiation events of protozoan parasites; the proteasome/ubiquitin system is essential for maintaining the differentiation state of many cell types. A single input of the specific inhibitor of proteasomes, lactacystin, prevented encystation of the protozoan parasite Entameoba invadens, whereas a cysteine protease inhibitor, E64, only delayed encystation. The ameba target of lactacystin was purified and it displayed the features typical of eukaryotic 20S proteasome complexes. In addition, transcripts encoding ubiquitin were detectable in trophozoites stage cells, disappeared immediately following transfer of amoebae to encystation induction medium, and reappeared at the same time during encystation as other encystation-specific transcripts. These results demonstrate that proteasome function is required during the conversion of the disease-causing trophozoite into the infectious cyst stage of Entamoeba parasites, and that ubiquitin transcript levels undergo an unusual decrease during the early stages of this differentiation process
— id: 8485, year: 1999, vol: 264, page: 897, stat: Journal Article,

Crithidia fasciculata induces encystation of Entamoeba invadens in a galactose-dependent manner
Cho J; Eichinger D
1998 Aug;84(4):705-710, Journal of parasitology
The ability of the flagellate Crithidia fasciculata to induce encystation of the reptile pathogen, Entamoeba invadens, was studied in vitro. A specific ratio of flagellate to amoeba was required; both live and heat-killed C. fasciculata induced amoebic encystation. The interaction between the Crithidia and Entamoeba cells was found to be galactose-mediated because the addition of galactose to the culture medium, or the pretreatment of the flagellate with galactosidase, eliminated its ability to induce encystation. Galactose was also found to prevent the amoeba amoeba aggregation that normally occurs in axenic cultures of encystation-induced E. invadens. Both galactose and glcNAc completely inhibited cyst formation of these induced cultures, although the latter sugar did not prevent cell aggregation. These results indicate that a galactose-mediated interaction between E. invadens cells is an early step in the in vitro encystation pathway
— id: 12080, year: 1998, vol: 84, page: 705, stat: Journal Article,

Encystation of entamoeba parasites
Eichinger D
1997 Jul;19(7):633-639, Bioessays
Entamoeba histolytica is a protozoan parasite of humans, and the causitive agent of intestinal amebiasis. The disease-causing stage of the parasite is an osmotically sensitive ameboid form, which differentiates into a thick-walled cyst for transmission from person to person. The conditions within the human intestine that induce encystment of the amoeba are unknown, but studies using an amoebic parasite of reptiles are now yielding information about the molecules and host:parasite interactions involved in the process. An understanding of the amoeba's obligatory encystment pathway should provide an approach for interrupting the transmission of this parasite, for which there is currently no vaccine
— id: 8216, year: 1997, vol: 19, page: 633, stat: Journal Article,

Proteasome function is required for encystation of Entamoeba invadens
Gonzalez J; Frevert U; Corey EJ; Nussenzweig V; Eichinger D
1997 ;28 Spec No:139-140, Archives of medical research (Mexico)
— id: 12421, year: 1997, vol: 28 Spec No, page: 139, stat: Journal Article,

Temperature differences for trans-glycosylation and hydrolysis reaction reveal an acceptor binding site in the catalytic mechanism of Trypanosoma cruzi trans-sialidase
Ribeirao, M; Pereira-Chioccola, V L; Eichinger, D; Rodrigues, M M; Schenkman, S
1997 Dec;7(8):1237-1246, Glycobiology
Trypanosoma cruzi, the agent of Chagas disease, expresses on its surface a trans-sialidase that catalyzes preferentially the transference of alpha-2,3-linked sialic acid to acceptors containing terminal beta-galactosyl residues, instead of the typical hydrolysis reaction, found in most sialidases. The trans-sialidase is responsible for the acquisition of the host sialic acid by this protozoan parasite, which does not synthesize sialic acids. Here, we have studied some kinetic properties of a recombinant trans-sialidase expressed in Escherichia coli. We found that it has sequential-type kinetics for the transferase reaction, as shown for the parasite-derived enzyme. The rates of sialic acid transfer to water (hydrolysis), and to beta-galactosyl residues have a unique behavior with respect to the reaction temperature. While the hydrolysis rate of sialyllactose increases continuously up to 35 degrees C, the temperature for the maximal rate of trans-glycosylation depends on the acceptor concentration. At low acceptor concentrations the rate of trans-glycosylation is maximal at 13 degrees C and independent of the amount of sialic acid donors. With increasing acceptor concentrations, maximal rates of trans-glycosylation are shifted to higher temperatures. This finding is explained by an 8-fold increase in the Km for the acceptor from 13 degrees C to 33 degrees C. Differences in hydrolysis and transfer rates were also obtained by using 4-methylumbelliferyl-N-acetyl-neuraminic acid. However, its hydrolysis rate is much higher than the rate of transference to lactose, suggesting that a long-lived enzyme-sialosyl intermediate is not formed. In addition, lactose does not increase the rate of methyl-umbelliferone release at any temperature, indicating that the rate limiting step is the aglycon release. Based on these results we propose that transglycosylation in T. cruzi sialidase is favored by the existence of a binding site for beta-galactosyl residues, which accepts the new glycosidic bond as sialic acid is released from the donor. With increasing temperature the affinity for the acceptor decreases, resulting in a concomitant increase in the rate of transfer to water, which, in turn, can be suppressed by increasing the acceptor concentration
— id: 78794, year: 1997, vol: 7, page: 1237, stat: Journal Article,

Single immunizing dose of recombinant adenovirus efficiently induces CD8+ T cell-mediated protective immunity against malaria
Rodrigues EG; Zavala F; Eichinger D; Wilson JM; Tsuji M
1997 Feb 1;158(3):1268-1274, Journal of immunology
The immunogenicity of a recombinant replication defective adenovirus expressing a major malaria Ag, the circumsporozoite (CS) protein (AdPyCS), was determined using a rodent malaria model. A single immunizing dose of this construct induced a large number of CS-specific CD8+ and CD4+ T cells in the spleens of these animals, particularly when given by the s.c. or i.m. route. A single dose of AdPyCS also induced high titers of Abs to Plasmodium yoelii sporozoites in mice. No other form of presentation of the CS protein given as a single immunizing dose, i.e., irradiated sporozoites, recombinant vaccinia, or influenza virus, etc., elicits comparably high numbers of CS-specific CD8+ T cells. The high concentration of CS-specific CD8+ T cells in the spleen was relatively short-lived, decreasing to half of its original value by 4 wk and to one-third at 8 wk after AdPyCS inoculation. The decrease in splenic CS-specific CD4+ T cells was even more rapid. Most importantly, a single dose of inoculation of AdPyCS into mice rendered them highly resistant to sporozoite challenge, resulting in a 93% inhibition of liver stage development of the parasites. This protective effect was primarily mediated by CD8+ T cells, as shown by depletion of this T cell population, while depletion of the CD4+ T cell population had only a minor effect on anti-plasmodial activity. Moreover, the inoculation of mice with AdPyCS induces sterile immunity in a significant proportion of mice, preventing the occurrence of parasitemia
— id: 8435, year: 1997, vol: 158, page: 1268, stat: Journal Article,

Directed mutagenesis of the Trypanosoma cruzi trans-sialidase enzyme identifies two domains involved in its sialyltransferase activity
Smith LE; Eichinger D
1997 Apr;7(3):445-451, Glycobiology
Of the increasing number of sialidases found to be made by microorganisms, the trypanosome trans-sialidase is unique in its added ability to efficiently carry out a sialyltransferase reaction using preformed glycoconjugates. The enzyme is predicted to have a multidomain structure, with one domain containing sequence and expected structural features found in bacterial sialidases. The trans-sialidase is very similar in overall sequence to another trypanosome enzyme that has only sialidase activity. Hybrid expression constructs containing pieces of these trypanosome trans-sialidase and sialidase genes were used to determine which regions of trans-sialidase are required for sialyltransferase activity. Two domains were found to influence the enzymatic activity: the N-terminal catalytic domain, and a downstream domain that resembles an Fn3-like module
— id: 56509, year: 1997, vol: 7, page: 445, stat: Journal Article,

Isolation and expression of an open reading frame encoding sialidase from Trypanosoma rangeli
Smith LE; Uemura H; Eichinger D
1996 Jul;79(1):21-33, Molecular & biochemical parasitology
Several protozoan parasites of human have been found to express enzymes capable of releasing terminal sialic acid residues from host glycans. These include enzymes similar in activity to bacterial and viral sialidases, as well as a novel type of enzyme, trans-sialidase, which can transfer sialic acid from one carbohydrate chain to another. Here we report the isolation of a gene and a gene fragment from the kinetoplastid Trypanosoma rangeli which encode products related in sequence to the trans-sialidase enzyme of T. cruzi. The gene fragment ORF is nearly identical to that of the complete gene, which encodes an enzymatically inactive protein. When the ORF of the gene fragment is fused to fragments from related genes, it encodes a product with sialidase activity. Both predicted T. rangeli protein products also have other potential structural features found in bacterial sialidases and in members of a previously described Trypanosoma trans-sialidase superfamily
— id: 56892, year: 1996, vol: 79, page: 21, stat: Journal Article,

Trans-sialidase genes expressed in mammalian forms of Trypanosoma cruzi evolved from ancestor genes expressed in insect forms of the parasite
Briones, M R; Egima, C M; Eichinger, D; Schenkman, S
1995 Aug;41(2):120-131, Journal of molecular evolution
The trans-sialidase of Trypanosoma cruzi mammalian forms transfers sialic acids from host's cell-surface glycoconjugates to acceptor molecules on parasite cell surface. To investigate the mechanism by which the mammalian stages of Trypanosoma cruzi have acquired their trans-sialidase, we compared the nucleotide and predicted amino acid sequences of trans-sialidase genes expressed in different developmental stages and strains of Trypanosoma cruzi with the sialidase gene of Trypanosoma rangeli and the sialidase genes of the prokaryotic genera Clostridium, Salmonella, and Actinomyces. The trans-sialidase gene products of Trypanosoma cruzi have a significant degree of structural and biochemical similarity to the sialidases found in bacteria and viruses, which would hint that horizontal gene transfer occurred in Trypanosoma cruzi trans-sialidase evolutionary history. The comparison of inferred gene trees with species trees suggests that the genes encoding the T. cruzi trans-sialidase of mammalian forms might be derived from genes expressed in the insect forms of the genus Trypanosoma. The branching order of trees inferred from T. cruzi trans-sialidase sequences, the sialidase from Trypanosoma rangeli, and bacterial sialidases parallels the expected branching order of the species and suggests that the divergence times of these sequences are remarkably long. Therefore, a 'vertical' inheritance from a hypothetical eukaryotic trans-sialidase gene expressed in insect forms of trypanosomes is more likely to have occurred than the horizontal gene transfer from bacteria, and thus explains the presence of this enzyme in the mammalian infective forms of Trypanosoma cruzi
— id: 78795, year: 1995, vol: 41, page: 120, stat: Journal Article,

Identification of a developmentally regulated transcript expressed during encystation of Entamoeba invadens
Sanchez L; Enea V; Eichinger D
1994 Sep;67(1):125-135, Molecular & biochemical parasitology
Differentiation of trophozoites into cysts in Entamoeba species has been described morphologically and to a lesser extent biochemically, but studies of stage specific gene expression have not been reported. At present Entamoeba invadens is the only species that can be induced to differentiate in axenic culture and is a useful model system for the human parasite Entamoeba histolytica. Using this model system, we performed cDNA-mRNA hybridization experiments to compare the RNA populations from trophozoites and from parasites at different stages of cyst formation. We detected the accumulation of a population of stage specific transcripts between 8 and 22 h after parasites are placed in induction medium. To identify genes involved in the trophozoite-cyst transformation we carried out a differential screening of a cDNA library. This yielded a clone that represents a stage specific gene whose transcripts are barely detectable in vegetatively grown trophozoites and maturing cysts, but are readily detected at the onset of cyst formation. Other features of the gene and its predicted protein product(s) are described
— id: 56703, year: 1994, vol: 67, page: 125, stat: Journal Article,

Increased levels of polyadenylated histone H2B mRNA accumulate during Entamoeba invadens cyst formation
Sanchez LB; Enea V; Eichinger D
1994 Sep;67(1):137-146, Molecular & biochemical parasitology
We have isolated cDNA clones of a member of the histone H2B protein family by differential screening of an Entamoeba invadens cDNA library with cDNA probes from vegetatively growing or encysting parasites. The cDNA clones reveal two polyadenylation sites, 26 nucleotides and 31 nucleotides downstream from the stop codon. RNA species recognized by E. invadens histone H2B clones are found at increased levels during cyst formation. Histone H2B RNA could be detected in both the poly(A)+ and poly(A)- RNA fractions, with stage-specific differences in the steady state levels of the two RNAs: trophozoites contain predominantly the poly(A)- RNA, while encysting parasites express predominantly the poly(A)+ RNA. Southern blot analysis suggests that both forms are transcribed from a single copy gene
— id: 56704, year: 1994, vol: 67, page: 137, stat: Journal Article,

Structural and functional properties of Trypanosoma trans-sialidase
Schenkman S; Eichinger D; Pereira ME; Nussenzweig V
1994 ;48:499-523, Annual review of microbiology
Sialic acids and sialidases play important roles in cellular interactions and modulate the recognition of pathogenic microbes by mammalian host cells. Protozoan parasites of the genus Trypanosoma express a unique sialic acid-metabolizing enzyme. This enzyme, named trans-sialidase (TS), catalyzes the transfer of sialic acids from host glycoconjugates to acceptor molecules of the parasite plasma membrane. In African trypanosomes, the agents of sleeping sickness, TS is found only in forms developing within the insect vector, and the enzyme sialylates the major surface protein. In Trypanosoma cruzi, the causative agent of Chagas' disease in Central and South America, TS is expressed both in the insect and mammalian forms of the parasite. The T. cruzi enzyme has been biochemically characterized, and the gene encoding the enzyme has been cloned. The enzyme sialylates abundant mucin-like molecules present on the surface of the parasite. Several lines of evidence suggest that TS and sialic acid acceptors on the surface of T. cruzi participate in host-parasite interactions and mediate the initial stages of the trypanosomes' invasion of host cells
— id: 56524, year: 1994, vol: 48, page: 499, stat: Journal Article,

A proteolytic fragment of Trypanosoma cruzi trans-sialidase lacking the carboxyl-terminal domain is active, monomeric, and generates antibodies that inhibit enzymatic activity
Schenkman, S; Chaves, L B; Pontes de Carvalho, L C; Eichinger, D
1994 Mar 18;269(11):7970-7975, Journal of biological chemistry
trans-Sialidase isolated from trypomastigote forms of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is multimeric and heterogeneous in size. We show here that limited proteolysis of tans-sialidase with papain yields a single monomeric polypeptide chain of 70 kDa that conserves full enzymatic activity on soluble and membrane-bound substrates. The papain fragment lacks most of the 12-amino acid repeats of the carboxyl-terminal domain that comprises about 50% of the native trans-sialidase. When injected into rabbits, the papain-generated fragment induces antibodies that inhibit trans-sialidase activity and trypomastigote sialylation. The repeats are also not required for the stability of the enzyme or for the correct folding during the biosynthesis in Escherichia coli, but seem essential for trans-sialidase oligomerization. We conclude that trans-sialidase is composed of two structurally and functionally independent domains
— id: 78796, year: 1994, vol: 269, page: 7970, stat: Journal Article,

Demonstration of heat-shock protein 70 in the sporozoite stage of malaria parasites
Tsuji M; Mattei D; Nussenzweig RS; Eichinger D; Zavala F
1994 ;80(1):16-21, Parasitology research
Three monoclonal antibodies generated by immunization of mice with Plasmodium berghei-infected red blood cells were found to react with the 75-kDa heat-shock protein (HSP70) present in liver stages and erythrocytic forms of the parasites. These antibodies were shown to react with a recombinant protein encoding the carboxyl terminal half of PfHSP70 (aa 365-681). Differently from earlier results, we clearly demonstrated that HSP70 was also expressed in the sporozoite stage, using these monoclonal antibodies in an immunofluorescence and Western immunoblot assay. These monoclonal antibodies react not only with sporozoites of P. berghei, the parasites originally used for the immunization, but also with sporozoites of several other rodent and human plasmodial species. Passive transfer of these monoclonal antibodies into naive mice, simultaneously injected with sporozoites, failed to neutralize the infectivity of P. berghei sporozoites and to inhibit the development of liver stages of P. yoelii
— id: 13020, year: 1994, vol: 80, page: 16, stat: Journal Article,

Trypanosoma cruzi trans-sialidase and cell invasion
Schenkman, S; Eichinger, D
1993 Jun;9(6):218-222, Parasitology today
Trypanosoma cruzi does not synthesize sialic acid but does contain a trans-sialidase, an enzyme capable of transferring sialic acid between host glycoconjugates and the parasite. Sialic acids are negatively charged carbohydrates attached to the terminal non-reducing end of glycoproteins and glycolipids, and their presence can dramatically influence many cell-surface recognition processes. Since sialic acids have been implicated in several ligand-receptor interactions, including the interaction of pathogenic viruses, bacteria and protozoans with their hosts, the expression of trans-sialidase and the acquisition of sialic acid by T. cruzi may be relevant to the interaction of the parasite with the host, and consequently may influence the pathobiology of Chagas disease. In this review, Sergio Schenkman and Daniel Eichinger discuss recent data about the structure and function of T. cruzi trans-sialidase
— id: 78797, year: 1993, vol: 9, page: 218, stat: Journal Article,

Only some members of a gene family in Trypanosoma cruzi encode proteins that express both trans-sialidase and neuraminidase activities
Uemura H; Schenkman S; Nussenzweig V; Eichinger D
1992 Nov;11(11):3837-3844, EMBO journal
Trypomastigotes, the blood stage form of the human parasite Trypanosoma cruzi, contain an enzyme on their surface, trans-sialidase, which catalyses the transfer of sialic acid from host glycoconjugates to acceptors on its own cell surface. At least a subset of the sialic acid-bearing acceptor molecules are involved in parasite invasion of host cells, an essential step in the life cycle of the parasite. Another trypomastigote surface enzyme that affects host cell invasion is neuraminidase and recent evidence suggests that both trans-sialidase and neuraminidase activities may be expressed by the same proteins on the parasite surface. We describe here the isolation and expression of several members of a trans-sialidase--neuraminidase gene family from T.cruzi. One of the isolated genes does indeed encode a protein with both trans-sialidase and neuraminidase activities, while other members of the gene family encode closely related proteins that express neither enzymatic activity. Chimeric protein constructs combining different portions of active and inactive genes identified a region of the gene necessary for enzymatic activity. Sequence analysis of this portion of the gene revealed a limited number of amino acid differences between the predicted active and inactive gene products
— id: 61957, year: 1992, vol: 11, page: 3837, stat: Journal Article,

Circumsporozoite protein of Plasmodium berghei: gene cloning and identification of the immunodominant epitopes
Eichinger DJ; Arnot DE; Tam JP; Nussenzweig V; Enea V
1986 Nov;6(11):3965-3972, Molecular & cellular biology
The gene encoding the circumsporozoite (CS) protein of the rodent malaria parasite Plasmodium berghei was cloned and characterized. A cDNA library made from P. berghei sporozoite RNA was screened with a monoclonal antibody for expression of CS protein epitopes. The resulting cDNA clone was used to isolate the CS protein gene from a lambda library containing parasite blood-stage DNA. The CS protein gene contains a central region encoding two types of tandemly repeated amino acid units, flanked by nonrepeated regions encoding amino- and carboxy-terminal signal and anchorlike sequences, respectively. One of the central repeated amino acid unit types contains the immunodominant epitopes
— id: 61980, year: 1986, vol: 6, page: 3965, stat: Journal Article,

The murine sex-limited protein (Slp): reassessment of its sex limitation
Ferreira A; Eichinger D; Nussenzweig V
1982 Oct;129(4):1506-1508, Journal of immunology
Previous studies in which an alloantiserum was used to measure Slp levels indicated that in certain inbred strains of mice (Slpa), this protein was sex-limited, that is, present only in males. We raised several monoclonal antibodies directed against different epitopes of Slp and used them to develop a sensitive two-site immunoradiometric assay. Using this assay we detected Slp in serum of all Slpa females previously thought to be phenotypically negative. The levels of Slp in these females are about 0.2 to 4% of that of the males of the same inbred strain. The molecule found in serum of females was isolated by affinity chromatography and was found to have the characteristic three-chain structure of male Slp. These findings establish that the presence of Slp in Slpa females is the rule rather than the exception. Quantitative differences similar in magnitude to those found between males and females were also detected among Slpa males; i.e., Sd males have about 100 times more Slp than Sp males. The mechanisms by which androgens determine the extensive quantitative male-female differences, and by which the S region determines large variations among Slpa males, are unknown
— id: 62014, year: 1982, vol: 129, page: 1506, stat: Journal Article,

A SIMPLIFIED TWO-DIMENSIONAL ELECTROPHORETIC TECHNIQUE
FERREIRA, A; EICHINGER, D
1981 ;43(3):291-299, Journal of immunological methods
— id: 40324, year: 1981, vol: 43, page: 291, stat: Journal Article,