John Samuelson

Early lateral transfer of genes encoding malic enzyme, acetyl‐CoA synthetase and alcohol dehydrogenases from anaerobic prokaryotes to Entamoeba histolytica

The fermentation enzymes, which enable the microaerophilic protist Entamoeba histolytica to parasitize the colonic lumen and tissue abscesses, closely resemble homologues in anaerobic prokaryotes. Here, genes encoding malic enzyme and acetyl‐CoA synthetase (nucleoside diphosphate forming) were cloned from E. histolytica, and their evolutionary origins, as well as those encoding two alcohol dehydrogenases (ADHE and ADH1), were inferred by means of phylogenetic reconstruction. The E. histolytica malic enzyme, which decarboxylates malate to pyruvate, closely resembles that of the archaeon Archaeoglobus fulgidus, strongly suggesting a common origin. The E. histolytica acetyl‐CoA synthetase, which converts acetyl‐CoA to acetate with the production of ATP, appeared to be closely related to the Plasmodium falciparum enzyme, but it was no more closely related to the Giardia lamblia acetyl‐CoA synthetase than to those of archaea. Phylogenetic analyses suggested that the adh1 and adhe genes of E. histolytica and Gram‐positive eubacteria share a common ancestor. Lateral transfer of genes encoding these fermentation enzymes from archaea or eubacteria to E. histolytica probably occurred early, because the sequences of the amoebic enzymes show considerable divergence from those of prokaryotes, and the amoebic genes encoding these enzymes are in the AT‐rich codon usage of the parasite.

Cloning and expression of chitinases of Entamoebae

Entamoeba histolytica (Eh) and Entamoeba dispar (Ed) are protozoan parasites that infect hundreds of millions of persons. In the colonic lumen, amebae form chitin-walled cysts, the infectious stage of the parasite. Entamoeba invadens (Ei), which infects reptiles and is a model for amebic encystation, produces chitin synthase and chitinase during encystation. Ei cysts formation is blocked by the chitinase-inhibitor allosamidin. Here molecular cloning techniques were used to identify homologous genes of Eh, Ed, and Ei that encode chitinases (EC 3.2.1.14). The Eh gene (Eh cht1) predicts a 507-amino acid (aa) enzyme, which has 93 and 74% positional identities with Ed and Ei chitinases, respectively. The Entamoeba chitinases have signal sequences, followed by acidic and hydrophilic sequences composed of multiple tandemly arranged 7-aa repeats (Eh and Ed) or repeats varying in length (Ei). The aa compositions of the chitinase repeats are similar to those of the repeats of the Eh and Ed Ser-rich proteins. The COOH-terminus of each chitinase has a catalytic domain, which resembles those of Brugia malayi (33% positional identity) and Manduca sexta (29%). Recombinant entamoeba chitinases are precipitated by chitin and show chitinase activity with chitooligosacharide substrates. Consistent with previous biochemical data, chitinase mRNAs are absent in Ei trophozoites and accumulate to maximal levels in Ei encysting for 48 h.

Primary sequences of two P-glycoprotein genes of Entamoeba histolytica

Two P-glycoprotein genes (EhPgp1 and EhPgp2) from the protozoan parasite Entamoeba histolytica were sequenced from a genomic library made with the DNA of an emetine-resistant ameba mutant, which overexpresses mRNAs homologous to segments of the human mdr1 (P-glycoprotein) gene. The open reading frames for EhPgp1 and EhPgp2 were 1302 and 1310 amino acids long, respectively, and showed a 67% positional identity with each other and 41% and 40% positional identities, respectively, with human mdr1 gene. Within each ameba P-glycoprotein were the ATP-binding sites found twice in eukaryotic P-glycoproteins and once in prokaryotic transport proteins. Hydropathy plots of the ameba P-glycoproteins were nearly superimposable on that of the human mdr 1, showing 2 homologous halves, each containing an ATP-binding site and 6 hydrophobic transmembrane domains that form the putative channel. A phylogenetic tree showed that the Entamoeba P-glycoproteins are more related to the human and mouse P-glycoproteins than to the Plasmodium and Leishmania P-glycoproteins. Also identified in the E. histolytica genomic library were 2 P-glycoprotein pseudogenes, each with a frame shift and stop codons in identical places within the amino ATP-binding site. In conclusion, the 2 E. histolytica P-glycoproteins encoded by the EhPgp1 and EhPgp2 genes are similar in structure to the mammalian P-glycoproteins and so may be involved in energy-dependent drug efflux by this human parasite.

Emetine-resistant mutants of Entamoeba histolytica overexpress mRNAs for multidrug resistance.

Although drug therapy is critical for control of amoebiasis, little is known about mechanisms of drug resistance by E. histolytica parasites. Here we tested the hypothesis that multidrug resistant (mdr) amoeba mutants, similar to mdr tumor cells, are drug resistant based upon overexpression of a P-glycoprotein pump that effluxes drugs from the cells. Using primers to conserved regions of the human P-glycoprotein and the polymerase chain reaction (PCR), we identified multiple 344 base par segments of amoeba DNA similar to the mammalian P glycoprotein. The amino acid sequences of amoeba mdr-like PCR products were from 53 to 97 identical with each other, 55 to identical to human mdr1 sequences, and 41-44% identical with P. falciparum mdr-like sequences. On northern blots, the mdr-like PCR products identified amoeba mRNAs 4.5-5 kilobases long, similar to the 5 kilobase mRNAs reported for the mammalian mdr gene. These mRNAs were increased at least seven times in emetine resistant mutant clone C2 amoebae versus wild-type clone A parasites. Further, the expression of the mdr-like mRNAs was increased three to four times when clone C2 mutants were grown under drug pressure versus the same parasites grown without emetine. In contrast, the number of genomic copies of the mdr-like DNA segments was not increased in the mutant clone C2 versus the wild-type clone A amoebae, and no rearrangements of the mdr-like DNA segments by the mutant were identified on Southern blots. In conclusion there appears to be a family of mdr-like genes in E. histolytica, which may be involved in drug resistance by the parasite.

Cloning of the Eh cdc2 gene from Entamoeba histolytica encoding a protein kinase p34cdc2 homologue

To understand better growth regulation in the protozoan parasite, Entamoeba histolytica (Eh), a homologue of the cdc2 gene encoding the yeast cyclin-dependent protein kinase, p34cdc2, has been cloned and sequenced. This gene, called Eh cdc2, contains a 79-bp intron located in the same place as the second of four introns in the Schizosaccharomyces pombe cdc2 gene. The sequence of an Eh cdc2 cDNA confirms the conserved eukaryotic splice donor (GT) and acceptor (AG) sites and shows that Eh is able to splice mRNAs. The spliced Eh cdc2 open reading frame is 291 amino acids (aa) long, encoding an M(r) 33,806 protein. The primary sequence of Eh cdc2 is most like those of cdc2 homologues Eg1 of Xenopus laevis and CDK2 of man (52% aa identity with each) and codes for (i) the serine (Ser), threonine (Thr), and tyrosine residues phosphorylated in p34cdc2 proteins, (ii) 32 of 33 aa conserved in other Ser/Thr protein kinases, and (iii) the sequence PVTSVRE instead of PSTAIRE found in most p34cdc2 proteins. This is the first cell-division-cycle regulatory protein homologue, as well as the first intron identified from Eh.

Hatching chemokinesis and transformation of miracidia of schistosoma mansoni

Hatching, chemokinesis, and transformation of miracidia of Schistosoma mansoni were examined with a light microscope equipped with a video recording system. Saline, linearly and reversibly, inhibited miracidial hatching and swimming. Both hatching and swimming were inhibited at 4 C and 12 C and accelerated at 34 C relative to rates at 22 C. Hatching was an explosive event that began with ciliary beating when the egg was placed in artificial pond water (APW) and culminated in the parasites escape from the shell in 100 to 300 msec. Broken egg shells had sharp, complementary edges. Neither miracidia nor eggs swelled prior to hatching. Accumulation of miracidia in a spot of snail conditioned water (SCW) occurred rapidly due to a 60-75% decrease in the exit rate from the spot, rather than by an increase in the entry rate. The turning rate in SCW increased tenfold and the time spent in the spot was 6 times that of controls. Eserine sulfate inhibited miracidial turning and accumulation in SCW. Parasites accumulated in a spot of serotonin by increasing their rate of turning. Miracidia transformed to sporocysts in either complex media containing serum, RPMI-1640, Hanks salts or phosphate buffered saline, but not in amino acids or vitamins. Transformation was inhibited when miracidia were incubated with serotonin or when miracidia had not been exposed previously to APW.

LINEs and SINE-like elements of the protist Entamoeba histolytica

A survey of whole genome shotgun sequences of the protozoan parasite Entamoeba histolytica revealed three families of non-long terminal repeat (LTR) retrotransposons or long interspersed elements (LINEs) (called EhLINEs in this report). The 4.8 kb EhLINEs each had a single open reading frame with a putative nucleic acid binding motif (CCHC) and restriction enzyme-like endonuclease domain located downstream of the reverse transcriptase (RT) domain. Phylogenetic analysis of the RT domain placed the EhLINEs in the R4 clade of non-LTR elements, a mixed clade of non-LTR elements that includes members from nematodes, insects, and vertebrates. EhLINE1 (which was previously identified as HMc and EhRLE) shared a common 3 end with a highly transcribed 0.55 kb short interspersed element (SINE)-like element previously identified as IE or ehapt2 and called EhLSINE1 in this report. Similarly, EhLINE2 shared a common 3 end with a highly transcribed 0.65 kb SINE-like element called EhLSINE2 in this report. The shared 3 end sequences of the EhLINEs and EhLSINEs suggested that EhLINEs are involved in the retrotransposition of the EhLSINEs. EhLSINEs were flanked by target site duplications and contained conserved 5 sequences, which likely regulate their transcription. The EhLSINEs are the first protist SINE-like elements identified that share a common 3 sequence with LINEs, and the first SINE-like elements that have been associated with the R4 clade of non-LTR elements.

Molecular cloning of a rho family gene of Entamoeba histolytica

An Entamoeba histolytica gene (Eh rho1) was cloned that encodes a putative low-molecular-mass GTP-binding protein, most similar to the ras homologue rho. The Eh rho1 open reading frame was 208 amino acids long and encoded a 23-kDa protein similar to Saccharomyces cerevisiae RHO1-RHO4 and CDC42 and human rhoA, rac1, and G25K gene products. This similarity was greatest at the NH2 terminus of Eh rho1 where two GTP-binding sites and a possible effector site were conserved. A cysteine residue at the COOH terminus of Eh rho1 was followed by eight hydrophobic amino acids rather than the three hydrophobic amino acids present in other ras family proteins.

HETEROGENEITY OF ENTAMOEBA HISTOLYTICA RAC GENES ENCODING P21RAC HOMOLOGUES

Entamoeba histolytica (Eh), the parasite that causes amebic dysentery, is the only protozoan that phagocytoses bacteria, epithelial cells and red blood cells. Numerous low-molecular weight GTP-binding proteins, called p21rac, are implicated in signal transduction and actin polymerization during phagocytosis by macrophages and Dictyostelium discoideum (Dd). Here, molecular cloning techniques were used to obtain four Eh rac genes that encoded putative p21rac, as well as segments of two Eh rac pseudogenes. The predicted Eh p21rac, which share 55-81% amino acid (aa) identities with each other, include one that closely resembles the p21rac1 of man, Dd, Drosophila melanogaster and Caenorhabditis elegans; two that resemble the p21racC of Dd; and one that is unique. An alignment of the Eh rac ORF with other rac family proteins reveals multiple aa that distinguish p21rac1, p21racC and p21cdc42. We conclude that the Eh genes encoding amebic p21rac, which are the first identified from a protozoan parasite, are numerous and heterogeneous.

Entamoeba histolytica: Physiology of multidrug resistance

Cross-resistance to unrelated drugs has been previously observed in multidrug-resistant carcinoma cells and the goal of this work was to determine whether a similar mechanism existed in Entamoeba histolytica. An emetine and a colchicine-resistant clone, C2(90) (IC50 = 62 microM, and 1.5 mM, respectively), and the parental clone, A (IC50 = 5 microM and 1 mM, respectively), were analyzed for resistance to other drugs and for the effect of verapamil. Both clones, C2(90) and A, exhibited similar resistance to both daunomycin (IC50 = 50 microM) and actinomycin D (IC50 = 13 nM). In the presence of verapamil, the IC50 for emetine was reduced to 0.5 microM, while the IC50 for colchicine was reduced to 0.3 mM. These results demonstrate that verapamil reverses both emetine and colchicine resistance in the mutant C2(90). In uptake experiments with [3H]emetine, drug accumulation was lower in resistant trophozoites. However, in the presence of verapamil, drug accumulation was increased in clone C2(90) to a level close to that of the parental strain, clone A. These results are consistent with observations made using malaria and multidrug-resistant tumor cells and suggest that a P-glycoprotein-like molecule may play a role in drug resistance in E. histolytica.