Glycogen phosphorylase sequences from the amitochondriate protists, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica and Giardia intestinalis

Glycogen phosphorylase genes or messages from four amitochondriate eukaryotes, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica (two genes) and Giardia intestinalis, have been isolated and sequenced. The sequences of the amitochondriate protist enzymes appear to share a most recent common ancestor. The clade containing these sequences is closest to that of another protist, the slime mold (Dictyostelium discoideum), and is more closely related to fungal and plant phosphorylases than to mammalian and eubacterial homologs. Structure-based amino acid alignment shows conservation of the residues and domains involved in catalysis and allosteric regulation by glucose 6-phosphate but high divergence at domains involved in phosphorylation-dependent regulation and AMP binding in fungi and animals. Protist phosphorylases, as their prokaryotic and plant counterparts, are probably not regulated by phosphorylation.     

Presidential address: rediscovering parasites using molecular tools--towards revising the taxonomy of Echinococcus, Giardia and Cryptosporidium

Molecular biology has provided parasitologists with a fantastic variety of techniques that have had a major impact on research into parasites and parasitism. Molecular tools have revealed the extent and nature of genetic diversity in parasites and this information has made a significant contribution to studies on the population genetics and evolutionary biology of parasites. Similarly, epidemiology has benefited enormously from the application of molecular tools in terms of studying parasite life cycles and transmission, and in the development of specific and sensitive methods for diagnosis and surveillance. However, the theme I wish to develop in this paper is concerned with the contribution molecular tools have made to parasite taxonomy and systematics, and in particular, the fact that in many cases molecular tools are validating the proposals made many years ago by taxonomists and biologists which were discounted or not fully accepted at the time. To do this I have chosen four examples (Echinococcus, Entamoeba, Giardia, Cryptosporidium) where recent research involving molecular characterisation has confirmed observations made many years ago and has resulted in a need to revise the taxonomy of different groups of parasites.     

In vitro effect of nitazoxanide against Entamoeba histolytica,Giardia intestinalis and Trichomonas vaginalis trophozoites

Nitazoxanide, a 5-nitrothiazolyl derivative, is effective in the treatment of a broad range of parasitic infections. In vitro, it is active against several protozoa, including Cryptosporidium parvum, Blastocystis hominis, and Giardia intestinalis. The objective of this study was to determine the in vitro effect of nitazoxanide on the growth and morphology of three anaerobic protozoa (Entamoeba histolytica, Giardia intestinalis, and Trichomonas vaginalis) and to compare these effects with those of metronidazole and albendazole. A subculture method was used to determine the concentrations required to inhibit growth by 50% or 90% (IC50 and IC90,). Nitazoxanide exhibited IC50, and IC90 values of 0.017 and 0.776 microg/ml respectively, against E. histolytica, 0.004 and 0.067 microg/ml against G. intestinalis, and 0.034 and 2.04 6 microg/ml against T. vaginalis. Based on the IC90 values, nitazoxanide was more toxic than metronidazole and albendazole against E. histolytica; albendazole and nitazoxanide were more toxic than metronidazole against G. intestinalis; and metronidazole was the most toxic drug against T. vaginalis. The effects of nitazoxanide on trophozoite ultrastructure of all three parasites included cell swelling and distorted cell shape, a redistribution of vacuoles, plasma membrane damage, and the formation of extensive empty areas in the cytoplasm of the protozoa.     

Lipid metabolism in mucous-dwelling amitochondriate protozoa

Entamoeba, Giardia, and trichomonads are the prominent members of a group known as 'mucosal parasites'. While Entamoeba and Giardia trophozoites colonise the small intestine, trichomonads inhabit the genitourinary tracts of humans and animals. These protozoa lack mitochondria, well-developed Golgi complexes, and other organelles typical of higher eukaryotes. Nonetheless, they have developed unique metabolic pathways that allow them to survive and multiply in the small intestine and reproductive tracts by scavenging nutrients from the host. Various investigators have shown that these protozoa are unable to synthesise the majority of their own lipids and cholesterol de novo; rather, they depend mostly on supplies from outside sources. Therefore, questions of how they transport and utilise exogenous lipids for metabolic purposes are extremely important. There is evidence suggesting that these parasites can take up the lipids and cholesterol they need from lipoprotein particles present in the host and/or in the growth medium. Studies also support the idea that individual lipid and fatty acid molecules can be transported without the help of lipoproteins. Exogenous phospholipids have been shown to undergo fatty acid remodelling (by deacylation/reacylation reactions), which allows these protozoa to alter lipids, bypassing the synthesis of entirely new phospholipid molecules. In addition, many of these amitochondriates are, however, capable of elongating/desaturating long-chain fatty acids, and assembling novel glycophospholipid molecules. In this review, progress in various aspects of lipid research on these organisms is discussed. Attempts are also made to identify steps of lipid metabolic pathways that can be used to develop chemotherapeutic agents against these and other mucosal parasites.     

Lycorine induces cell death in the amitochondriate parasite, Trichomonas vaginalis, via an alternative non-apoptotic death pathway

In this study, the mechanism of action of the pro-apoptotic alkaloid lycorine on an amitochondriate cell, the parasite Trichomonas vaginalis, was investigated. The cytotoxicity of lycorine against T. vaginalis was studied from 2.5 to 1000 μM and several important ultrastructural alterations were observed by electron microscopy. Lycorine arrested the T. vaginalis cell cycle, although no hallmarks of apoptosis, such as apoptotic bodies, were observed. Consequently, the underlying mechanism of action fails to completely fulfill the criteria for apoptosis. However, some similarities to paraptotic cell death were observed.     

YI-S, a Casein-free Medium for Axenic Cultivation of Entamoeba histolytica, Related Entamoeba, Giardia intestinalis and Trichomonas vaginalis

ABSTRACT. Pancreatic digests of casein are major ingredients of media used in the axenic cultivation of lumen-dwelling parasitic protozoa, especially Entamoeba, Giardia , and trichomonads. The digest used almost exclusively in the development of these media, Medo-Peptone (Trypticase® BBL), has not been available since 1981. Moreover, none of dozens of similar type digests tested since then in our laboratory has proved equal to Medo-Peptone, and in the last two years it has become increasingly difficult to obtain new batches which will support even modest growth of Entamoeba histolytica . In response to this problem we have developed a casein-free medium, YI-S, consisting of a nutrient broth, vitamin mixture and serum. We recommend it as a replacement for the casein-dependent medium TYI-S-33, currently the most widely used for axenic culture of Entamoeba histolytica and other lumen-dwellers.     

Entamoeba histolytica trophozoites transfer lipophosphopeptidoglycans to enteric cell layers

Transfer of antigens frequently follows adhesion of protozoan parasites to host cells. We were interested in such transfer from the Entamoeba surface to enterocytes following adhesion of trophozoites. Therefore, cocultures of enterocytes in vitro and ex vivo with Entamoeba histolytica (strain HM-1:IMSS) or Entamoeba dispar (strain SAW760) trophozoites were processed for immunocytochemistry. The EH5 monoclonal antibody against amoebic proteophosphoglycans marked a dotted pattern on the apical side of enterocytes in in vitro cocultures with HM-1:IMSS and SAW760 trophozoites. Basolateral staining was present in cocultures following dysfunction of tight junctions, or when trophozoites made direct contact with the basolateral side of enterocytes in in vitro and ex vivo cocultures. Based on the molecular mass in Western blot, the transferred proteophosphoglycan was identified as a lipophosphopeptidoglycan. In conclusion, trophozoites transfer LPPG to the apical side of enterocytes following adhesion and prior to dysfunction of tight junctions.     

Mitochondrial-type hsp70 genes of the amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and two microsporidians

Genes encoding putative mitochondrial-type heat shock protein 70 (mit-hsp70) were isolated and sequenced from amitochondriate protists, Giardia intestinalis, Entamoeba histolytica, and two microsporidians, Encephalitozoon hellem and Glugea plecoglossi. The deduced mit-hsp70 sequences were analyzed by sequence alignments and phylogenetic reconstructions. The mit-hsp70 sequence of these four amitochondriate protists were divergent from other mit-hsp70 sequences of mitochondriate eukaryotes. However, all of these sequences were clearly located within a eukaryotic mitochondrial clade in the tree including various type hsp70 sequences, supporting the emerging notion that none of these amitochondriate lineages are primitively amitochodrial, but lost their mitochondria secondarily in their evolutionary past.     

Cloning and partial characterization of Entamoeba histolytica PTPases

Reversible protein tyrosine phosphorylation is an essential signal transduction mechanism that regulates cell growth, differentiation, mobility, metabolism, and survival. Two genes coding for protein tyrosine phophatases, designed EhPTPA and EhPTPB, were cloned from Entamoeba histolytica. EhPTPA and EhPTPB proteins showed amino acid sequence identity of 37%, both EhPTPases showed similarity with Dictyostelium discoideum and vertebrate trasmembranal PTPases. mRNA levels of EhPTPA gene are up-regulated in trophozoites recovered after 96h of liver abscess development in the hamster model. EhPTPA protein expressed as a glutathione S-transferase fusion protein (GST::EhPTPA) showed enzymatic activity with p-nitrophenylphosphate as a substrate and was inhibited by PTPase inhibitors vanadate and molybdate. GST::EhPTPA protein selectively dephosphorylates a 130kDa phosphotyrosine-containing protein in trophozoite cell lysates. EhPTPA gene codifies for a 43kDa native protein. Up-regulation of EhPTPA expression suggests that EhPTPA may play an important role in the adaptive response of trophozoites during amoebic liver abscess development.     

Inhibition of gene expression with double strand RNA interference in Entamoeba histolytica

In order to inhibit gene expression in Entamoeba histolytica, we have developed a method based on expressing double strand RNA interference constructs in stable transformants. The 5' end of Eh Dia was cloned head to head with an intervening non-specific stuffer fragment in the E. histolytica expression vector pJST4. This construct was transformed in E. histolytica HM1:IMSS trophozoites and stable transformants were selected with 20microg/ml G418. Our results show that expression of Eh Dia was completely inhibited in these transformants. These stable transformants could be maintained indefinitely without expression of Eh Dia. This method therefore provides an effective tool to study the phenotypic changes, which occur due to inhibition of gene expression in the absence of mutants and other microbiological manipulations in this protozoan parasite.     

Electron microscopy and cytochemistry analysis of the endocytic pathway of pathogenic protozoa

Endocytosis is essential for eukaryotic cell survival and has been well characterized in mammal and yeast cells. Among protozoa it is also important for evading from host immune defenses and to support intense proliferation characteristic of some life cycle stages. Here we focused on the contribution of morphological and cytochemical studies to the understanding of endocytosis in Trichomonas, Giardia, Entamoeba, Plasmodium, and trypanosomatids, mainly Trypanosoma cruzi, and also Trypanosoma brucei and Leishmania.     

Mitochondrial type iron-sulfur cluster assembly in the amitochondriate eukaryotes Trichomonas vaginalis and Giardia intestinalis, as indicated by the phylogeny of IscS

Pyridoxal-5'-phosphate-dependent cysteine desulfurase (IscS) is an essential enzyme in the assembly of FeS clusters in bacteria as well as in the mitochondria of eukaryotes. Although FeS proteins are particularly important for the energy metabolism of amitochondrial anaerobic eukaryotes, there is no information about FeS cluster formation in these organisms. We identified and sequenced two IscS homologs of Trichomonas vaginalis (TviscS-1 and TviscS-2) and one of Giardia intestinalis (GiiscS). TviscS-1, TviscS-2, and GiiscS possess the typical conserved regions implicated in cysteine desulfurase activity. N-termini of TviscS-1 and TviscS-2 possess eight amino acid extensions, which resemble the N-terminal presequences that target proteins to hydrogenosomes in trichomonads. No presequence was evident in GiiscS from Giardia, an organism that apparently lacks hydrogenosmes or mitochondria. Phylogenetic analysis showed a close relationship among all eukaryotic IscS genes including those of amitochondriates. IscS of proteobacteria formed a sister group to the eukaryotic clade, suggesting that isc-related genes were present in the proteobacterial endosymbiotic ancestor of mitochondria and hydrogenosomes. NifS genes of nitrogen-fixing bacteria, which are IscS homologs required for specific formation of FeS clusters in nitrogenase, formed a more distant group. The phylogeny indicates the presence of a common mechanism for FeS cluster formation in mitochondriates as well as in amitochondriate eukaryotes. Furthermore, the analyses support a common origin of Trichomonas hydrogenosomes and mitochondria, as well as secondary loss of mitochondrion/hydrogenosome-like organelles in Giardia.     

A phagocytosis mutant of Entamoeba histolytica is less virulent due to deficient proteinase expression and release

Cysteine proteinases are key virulence factors of Entamoeba histolytica that are released during the process of invasion. We used a chemical mutant of E. histolytica strain HM-1:IMSS, clone L6, which is deficient in virulence, phagocytosis, and cysteine proteinase activity to help define the mechanisms of cysteine proteinase release. All cysteine proteinase genes of wild type HM-1 were present in the L6 mutant genome, but three of the major expressed proteinases, ehcp1, ehcp2, and ehcp5 were both transcribed, translated, and released at lower levels in L6. We hypothesized that a central protein such as the calcium binding protein 1, EhCaBP1, which is required for both phagocytosis and exocytosis might be deficient in this mutant. We found that both mRNA and proteinase levels of EhCaBP1 were decreased in L6. These findings provide an important link between phagocytosis, passive release of multiple cysteine proteinases, and attenuated virulence of this E. histolytica mutant.     

Entamoeba histolytica mitosomes: organelles in search of a function

It has been more than eight years since the discovery of mitosomes (mitochondrial remnant organelles) in the intestinal human pathogen Entamoeba histolytica. Despite detailed knowledge about the biochemistry of this parasite and the completion of the E. histolytica genome sequencing project no physiological function has yet been unequivocally assigned to these organelles. Entamoeba mitosomes seem to be the most degenerate of all endosymbiosis-derived organelles studied to date. They do not appear to participate in energy metabolism and may have dispensed completely with the proteins required for iron-sulphur cluster biosynthesis. However, the large number of mitosomes found in E. histolytica trophozoites hints at a significant biological role for these organelles in their natural environment. Identifying the protein complement of mitosomes will provide answers as to their biological significance and the reason(s) for their retention in this parasite.     

Giardia lamblia: identification and characterization of Rab and GDI proteins in a genome survey of the ER to Golgi endomembrane system

To investigate the complexity of the endomembrane transport system in the early diverging eukaryote, Giardia lamblia, we characterized homologues of the GTP-binding proteins, Rab1 and Rab2, involved in regulating vesicular trafficking between the endoplasmic reticulum and Golgi in higher eukaryotes, and GDI, which plays a key role in the cycling of Rab proteins. G. lamblia Rab1, 2.1, and GDI sequences largely resemble yeast and mammalian homologues, are transcribed as 0.66-, 0.62-, and 1.4-kb messages, respectively, and are expressed during growth and encystation. Western analyses detected an abundant Rab/GDI complex at approximately 80 kDa, and free GDI (60 kDa) in both trophozoites and encysting cells. Immunoelectron microscopy with antibody to Rab1 localized Rab with ER, encystation secretory vesicles, and lysosome-like peripheral vesicles. GDI associated with these structures, and with small vesicles found throughout the cytoplasm, consistent with GDI's key role in Rab cycling between organelles within the cell.     

Loss of dsRNA-based gene silencing in Entamoeba histolytica: implications for approaches to genetic analysis

The ability to regulate gene expression in the protozoan parasite Entamoeba histolytica is critical in determining gene function. We previously published that expression of dsRNA specific to E. histolytica serine threonine isoleucine rich protein (EhSTIRP) resulted in reduction of gene expression [MacFarlane, R.C., Singh, U., 2007. Identification of an Entamoeba histolytica serine, threonine, isoleucine, rich protein with roles in adhesion and cytotoxicity. Eukaryotic Cell 6, 2139-2146]. However, after approximately one year of continuous drug selection, the expression of EhSTIRP reverted to wild-type levels. We confirmed that the parasites (i) contained the appropriate dsRNA plasmid, (ii) were not contaminated with other plasmids, (iii) the drug selectable marker was functional, and (iv) sequenced the dsRNA portion of the construct. This work suggests that in E. histolytica long term cultivation of parasites expressing dsRNA can lead to the loss of dsRNA based silencing through the selection of “RNAi” negative parasites. Thus, users of the dsRNA silencing approach should proceed with caution and regularly confirm gene down regulation. The development and use of constructs for inducible expression of dsRNA may help alleviate this potential problem.     

Entamoeba histolytica: fibrilar aggregates in dividing trophozoites

Entamoeba histolytica trophozoite cytokinesis is dependent upon cytoskeletal elements such as filamentous actin and myosin. Here we present confocal and transmission electron microscopy studies of this process. A sequence in the formation of the contractile ring was shown with rhodamine-phalloidine staining. Ultrastructural analysis revealed the presence of fibrilar aggregates in the cytoplasm of dividing trophozoites. Among them two filaments of different diameter were identified. These aggregates presented repeating assemblies of thin and thick filaments that in cross section revealed a muscle-like appearance. Our results suggest that these aggregates constitute the contractile ring responsible for the separation of daughter cells.     

Photocatalytic disinfection of Giardia intestinalis and Acanthamoeba castellani cysts in water

In this study, disinfection of water containing Giardia intestinalis and Acanthamoeba castellani cysts with TiO2 and modified catalyst silver loaded TiO2 (Ag-TiO2) was investigated. Destruction of the parasites was evaluated after UV illumination of the suspension consisting 5 x 10(8)-13.5 x 10(8)cysts/mL in the presence of 2g/L neat or modified TiO2 at neutral pH. In the initial stage, the solid photocatalyst particles penetrated the cyst wall and then oxidant species produced by TiO2/UV destroyed both cell wall and intracellular structure. In the case of G. intestinalis inactivation (disinfection) performance of TiO2/UV system reached 52.5% only after 25 min illumination and total parasite disinfection was achieved after 30 min illumination. However, silver loaded TiO2 seemed to be more effective as this loading provided better catalytic action as well as additional antimicrobial properties. Cell viability tests showed that parasite cysts, their walls in particular, were irreversibly damaged and cysts did not re-grow. Nevertheless the studied system seemed to be ineffective for the inactivation of A. castellani. Inactivation percentages of TiO2/UV and Ag-TiO2/UV systems were far lower than that of UV alone, being 50.1% and 46.1%, respectively.