Molecular and phylogenetic analysis of PCR-amplified cyclin-dependent kinase (CDK) family sequences from representatives of the earliest available lineages of eukaryotes

Cyclin-dependent kinase (CDK) and cell division control (CDC2) sequences are strongly conserved among eukaryotes and may complement the use of other sequence families in eukaryotic phylogenetic inference. We synthesized degenerate PCR primers to amplify the catalytic region of CDK homologs in representatives of the earliest available lineages of eukaryotes. CDK family sequence-based, maximum-likelihood distance measurements with neighbor joining, and Fitch-Margoliash least-squares analyses produced unrooted dendrograms that included protists, yeasts, and higher eukaryotes. Bootstrap confidence estimates supported CDK-based phylogenetic groupings among the protists, fungi, and vertebrates although resolution within these groups was often insignificant. However, Trichomonas vaginalis and Giardia lamblia exhibited two of the most divergent CDK-like sequences consistent with rRNA-phylogenetic inference of early divergence of these eukaryotic lineages. In the evolution from unicellular to multicellular organisms, a constellation of amino acid residues aligning with the human, CDK N-terminal sheet may have undergone abrupt replacement.Abbreviations CDC cell division control - CDK cyclin-dependent kinase family - PCR polymerase chain reaction Correspondence to: D.E. Riley     

Eukaryotic checkpoints are absent in the cell division cycle ofEntamoeba histolytica

Fidelity in transmission of genetic characters is ensured by the faithful duplication of the genome, followed by equal segregation of the genetic material in the progeny. Thus, alternation of DNA duplication (S-phase) and chromosome segregation during the M-phase are hallmarks of most well studied eukaryotes. Several rounds of genome reduplication before chromosome segregation upsets this cycle and leads to polyploidy. Polyploidy is often witnessed in cells prior to differentiation, in embryonic cells or in diseases such as cancer. Studies on the protozoan parasite,Entamoeba histolytica suggest that in its proliferative phase, this organism may accumulate polyploid cells. It has also been shown that although this organism contains sequence homologs of genes which are known to control the cell cycle of most eukaryotes, these genes may be structurally altered and their equivalent function yet to be demonstrated in amoeba. The available information suggests that surveillance mechanisms or ‘checkpoints’ which are known to regulate the eukaryotic cell cycle may be absent or altered inE. histolytica.     

Genome Re-duplication and Irregular Segregation Occur During the Cell Cycle of Entamoeba histolytica

Heterogeneity of genome content is commonly observed in axenic cultures of Entamoeba histolytica. Cells with multiple nuclei and nuclei with heterogenous genome contents suggest that regulatory mechanisms that ensure alternation of DNA synthesis and mitosis are absent in this organism. Therefore, several endo-reduplicative cycles may occur without mitosis. The data also shows that unlike other endo-reduplicating organisms, E.histolytica does not undergo a precise number of endo-reduplicative cycles. We propose that irregular endo-reduplication and genome partitioning lead to heterogeneity in the genome content of E.histolytica trophozoites in their proliferative phase. The goal of future studies should be aimed at understanding the mechanisms that are involved in (a) accumulation of multiple genome contents in a single nucleus; (b) genome segregation in nuclei that contain multiple genome contents and (c) maintenance of genome fidelity in E. histolytica.     

Galactose/N-acetylgalactosamine lectin: The coordinator of host cell killing

Entamoeba histolytica is an enteric parasite that can kill host cells via a contact-dependent mechanism. This killing involves the amoebic surface protein referred to as the Gal/GalNAc lectin. The Gal/GalNAc lectin binds galactose and N-acetylgalactosamine allowing the adherence of amoebas to host cells. Involvement of the lectin in the pathogenesis ofE. histolytica infection will be reviewed in this paper. The lectin has been shown to have very specific and substantial effects on adherence, cytotoxicity, and encystation. There is also possible involvement of the lectin in phagocytosis and caspase activation in host cells.     

Acetylation is the most abundant actin modification in Entamoeba histolytica and modifications of actin's amino‐terminal domain change cytoskeleton activities

Actin is one of the most conserved, abundant, and ubiquitous proteins in all eukaryotes characterised to date. Posttranslation modifications of actin modify the organisation of the actin‐rich cytoskeleton. In particular, chemical modifications of actin's amino‐terminal region determine how filamentous actin is organised into scaffolds. After assuming that protein modifications account for the multiple functional activities exerted by the single actin in Entamoeba histolytica, we profiled posttranslational modifications of this protein. Acetylation (on 21 different amino acids) was the most abundant modification, followed by phosphorylation. Furthermore, the glycine residue at Position 2 in E. histolytica's actin (Gly2, not found in most other eukaryotic actins) was found to be acetylated. The impact of Gly2 on the amoeba's life cycle and pathogenicity was then assessed in mutagenesis experiments. We found that Gly2 was necessary for cell morphology and division, parasite–host cell adhesion, and host invasion in an in vitro model of amoebic human infection.     

Novel regulatory roles of PtdIns(4,5)P2 generating enzyme EhPIPKI in actin dynamics and phagocytosis of Entamoeba histolytica

Motility and phagocytosis are the two important processes that are intricately linked to survival and virulence potential of the protist parasite Entamoeba histolytica. These processes primarily rely on actin‐dependent pathways, and regulation of these pathways is critical for understanding the pathology of E. histolytica. Generally, phosphoinositides dynamics have not been explored in amoebic actin dynamics and particularly during phagocytosis in E. histolytica. We have explored the roles of PtdIns(4,5)P₂ as well as the enzyme that produces this metabolite, EhPIPKI during phagocytosis. Immunofluorescence and live cell images showed enrichment of EhPIPKI in different stages of phagocytosis from initiation till the cups progressed towards closure. However, the enzyme was absent after phagosomes are pinched off from the membrane. Overexpression of a dominant negative mutant revealed a reduction in the formation of phagocytic cups and inhibition in the rate of engulfment of erythrocytes. Moreover, EhPIPKI binds directly to F and G‐actin unlike PIPKs from other organisms. PtdIns(4,5)P₂, the product of the enzyme, also followed a similar distribution pattern during phagocytosis as determined by a GFP‐tagged PH‐domain from PLCδ, which specifically binds PtdIns(4,5)P₂ in trophozoites. In summary, EhPIPKI regulates initiation of phagocytosis by regulating actin dynamics.     

Extracellular vesicles released by anaerobic protozoan parasites: Current situation

Extracellular vesicles (EVs) have emerged as a ubiquitous mechanism for transferring information between cells and organisms across all three kingdoms of life. Parasitic unicellular eukaryotes use EVs as vehicles for intercellular communication and host manipulation. Pathogenic protozoans are able to modulate the immune system of the host and establish infection by transferring a wide range of molecules contained in different types of EVs. In addition to effects on the host, EVs are able to transfer virulence factors, drug‐resistance genes and differentiation factors between parasites. In this review we cover the current knowledge on EVs from anaerobic or microaerophilic extracellular protozoan parasites, including Trichomonas vaginalis, Tritrichomonas foetus, Giardia intestinalis and Entamoeba histolytica, with a focus on their potential role in the process of infection. The role of EVs in host: parasite communication adds a new level of complexity to our understanding of parasite biology, and may be a key to understand the complexity behind their mechanism of pathogenesis.     

Histone Synthesis inTrypanosoma cruzi

Trypanosoma cruziis an ancient, parasitic eukaryote which does not undergo chromatin condensation during cell division. This behavior may be explained if one considers the strong amino acid sequence divergence ofTrypanosomahistones compared to higher eukaryotes. In the latter organisms histone synthesis is coupled to DNA replication. Considering the nonconserved amino acid sequence ofT. cruzihistones, as well as the absence of chromatin condensation in this organism, we have studied histone synthesis in relation to DNA replication in this parasite. We have found that core histones and a fraction of histone H1 are synthesized concomitantly to DNA replication. However, another fraction of histone H1 is constitutively synthesized.     

Amoebic PI3K and PKC Is Required for Jurkat T Cell Death Induced by Entamoeba histolytica

The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.     

Cloning and characterization of an ftsZ homologue from a bacterial symbiont of Drosophila melanogaster

A 1194 by open reading frame that codes for a 398 amino acid peptide was cloned from a gt11 library of Drosophila melanogaster genomic DNA. The predicted peptide sequence is very similar to three previously characterized protein sequences that are encoded by the ftsZ genes in Escherichia coli, Bacillus subtilis and Rhizobium meliloti. The FtsZ protein has a major role in the initiation of cell division in prokaryotic cells. Using a tetracycline treatment that eradicates bacterial parasites from insects, the ftsZ homologue has been found to be derived from a bacterium that lives within the strain. However, polymerase chain reaction (PCR) amplification of the gene from treated embryos suggests that it is not derived from a gut bacterium. Nevertheless, by amplifying and characterizing part of the 16S rRNA from this bacterium we have been able to demonstrate that it is a member of the genus Wolbachia, a parasitic organism that infects, and disturbs the sexual cycle of various strains of Drosophila simulans. We suggest that this ftsZ homologue is implicated in the cell division of Wolbachia, an organism that fails to grow outside the host organism. Sequence and alignment analysis of this ftsZ homologue show the presence of a potential GTP-binding motif indicating that it may function as a GTPase. The consequences of this function particularly with respect to its role in cell division are discussed.     

Close sequence identity between ribosomal DNA episomes of the non-pathogenicEntamoeba dispar and pathogenicEntamoeba histolytica

Entamoeba dispar andEntamoeba histolytica are now recognized as two distinct species-the former being nonpathogenic to humans. We had earlier studied the organization of ribosomal RNA genes inE. histolytica. Here we report the analysis of ribosomal RNA genes inE. dispar. The rRNA genes ofE. dispar, like their counterpart inE. histolytica are located on a circular rDNA molecule. From restriction map analysis, the size ofE. dispar rDNA circle was estimated to be 24·4 kb. The size was also confirmed by linearizing the circle withBsaHI, and by limited DNAseI digestion. The restriction map of theE. dispar rDNA circle showed close similarity to EhR1, the rDNA circle ofE. histolytica strain HM-1:IMSS which has two rDNA units per circle. The various families of short tandem repeats found in the upstream and downstream intergenic spacers (IGS) of EhR1 were also present inE. dispar. Partial sequencing of the cloned fragments ofE. dispar rDNA and comparison with EhR1 revealed only 2·6% to 3·8% sequence divergence in the IGS. The region Tr and the adjoiningPvuI repeats in the IGS of EhR1, which are missing in thoseE. histolytica strains that have one rDNA unit per circle, were present in theE. dispar rDNA circle. Such close similarity in the overall organization and sequence of the IGS of rDNAs of two different species is uncommon. In fact the spacer sequences were only slightly more divergent than the 18S rRNA gene sequence which differs by 1·6% in the two species. The most divergent sequence betweenE. histolytica andE. dispar was the internal transcribed spacer, ITS2. Therefore, it was concluded that probes derived from the ITS1 and ITS 2 sequences would be more reliable and reproducible than probes from the IGS regions used earlier for identifying these species.     

Two types of ftsZ genes isolated from the unicellular primitive red alga Galdieria sulphuraria.

FtsZ plays a crucial role in bacterial cell division, and may be involved in plastid division in eukaryotes. To investigate the evolution of the dividing apparatus from prokaryotes to eukaryotes, the ftsZ genes were isolated from the unicellular primitive red alga Galdieria sulphuraria. Two ftsZ genes (GsftsZ1 and GsftsZ2) were isolated. This suggests that duplication and divergence of the ftsZ gene occurred in an early stage of plant evolution. A comparison of the FtsZs of G. sulphuraria and other organisms shows that FtsZ is highly and universally conserved among prokaryotes, primitive eukaryotic algae, and higher plants. The GsftsZ2 gene seems to contain an intron. Southern hybridization analysis of the G. sulphuraria chromosomes separated by CHEF revealed that each ftsZ gene and its flanking region may be duplicated.     

Entamoeba histolytica Induces Cell Death of HT29 Colonic Epithelial Cells via NOX1-Derived ROS

Entamoeba histolytica, which causes amoebic colitis and occasionally liver abscess in humans, is able to induce host cell death. However, signaling mechanisms of colon cell death induced by E. histolytica are not fully elucidated. In this study, we investigated the signaling role of NOX in cell death of HT29 colonic epithelial cells induced by E. histolytica. Incubation of HT29 cells with amoebic trophozoites resulted in DNA fragmentation that is a hallmark of apoptotic cell death. In addition, E. histolytica generate intracellular reactive oxygen species (ROS) in a contact-dependent manner. Inhibition of intracellular ROS level with treatment with DPI, an inhibitor of NADPH oxidases (NOXs), decreased Entamoeba-induced ROS generation and cell death in HT29 cells. However, pan-caspase inhibitor did not affect E. histolytica-induced HT29 cell death. In HT29 cells, catalytic subunit NOX1 and regulatory subunit Rac1 for NOX1 activation were highly expressed. We next investigated whether NADPH oxidase 1 (NOX1)-derived ROS is closely associated with HT29 cell death induced by E. histolytica. Suppression of Rac1 by siRNA significantly inhibited Entamoeba-induced cell death. Moreover, knockdown of NOX1 by siRNA, effectively inhibited E. histolytica-triggered DNA fragmentation in HT29 cells. These results suggest that NOX1-derived ROS is required for apoptotic cell death in HT29 colon epithelial cells induced by E. histolytica.     

Biochemistry of Entamoeba: A review

Entamoeba histolytica, the protozoan parasite causing amoebiasis, is carried by approximately 10% of the world's population although only a minority of carriers of the organism present active clinical symptoms. Although Entamoeba are classified as eukaryotes, the biochemistry of these organisms has a number of unusual facets which are reminiscent of prokaryotes. It has indeed been suggested that these cells represent early evolutionary forms that have been successful in surviving unchanged in the protected environment in which they reproduce (the intestine). Glycolysis in Entamoeba lacks several of the typical eukaryotic glycolytic enzymes. The pentose phosphate shunt enzymes are missing completely. Another unusual feature of the glycolytic path is the utilization of PP_i instead of ATP in a number of enzyme reactions e.g. phosphofructokinase. Entamoeba is the only eukaryote in which one of these PP_i utilizing enzymes, phosphoenolpyruvate carboxytransferase, has been found. The respiratory pathway is also an unusual one. Entamoeba are classified as anaerobes but the cells do have an affinity for oxygen. The oxygen is reduced to water at the end of a respiratory chain which is not well understood but which operates withough cytochromes or mitochondria. The nucleic acid, protein and lipid metabolic pathways have not been well studied and interest has mainly focused on the proteolytic processes of the amoeba which have been implicated in the pathogenic, histolytic behaviour of the parasite. Despite these efforts the mechanism of attack of the parasite and the stimuli that cause it to invade the host are not yet clear. This understandably is the goal of much of the present studies concerning E. histolytica but the organism also deserves study in its own right as an example of an organism that has an unusual biochemistry and may represent an early stage in the evolution of eukaryotic cells.     

Molecular Changes in Entamoeba histolytica in Response to Bacteria1

Entamoeba histolytica, the protozoan parasite, is the causative agent of amoebiasis. The degree of virulence, as inferred from invasiveness, of potentially pathogenic strains may be regulated by both host and parasite factors that determine the gut environment. One such factor that plays an important role is the bacterial flora in the gut. Previous studies have clearly shown that bacterial flora is an important determinant of virulence in E. histolytica. However, the exact nature of changes induced in E. histolytica in response to bacteria and their role in virulence is not clear. In this study the levels of a number of molecules potentially important in virulence mechanisms were determined in E. histolytica cells grown with and without normal human bacterial flora, using enzyme-linked immunosorbent assay. Significant changes were observed only after the E. histolytica cells had been adapted to grow with bacterial flora for a number of generations, and not in short term culture.     

Differentiation of Entamoeba histolytica: a possible role for enolase

The study of the encystation process of Entamoeba histolytica has been hampered by the lack of experimental means of inducing mature cysts in vitro. Previously we have found that cytoplasmic vesicles similar to the encystation vesicles of Entamoeba invadens are present in E. histolytica trophozoites only in amebas recovered from experimental amebic liver abscesses. Here we report that a monoclonal antibody (B4F2) that recognizes the cyst wall of E. invadens also identifies a 48 kDa protein in vesicles of E. histolytica trophozoites recovered from hepatic lesions. This protein is less expressed in trophozoites continuously cultured in axenical conditions. As previously reported for E. invadens, the B4F2 specific antigen was identified as enolase in liver-recovered E. histolytica, by two-dimensional electrophoresis, Western blot and mass spectrometry. In addition, the E. histolytica enolase mRNA was detected by RT PCR. The antigen was localized by immunoelectron microscopy in cytoplasmic vesicles of liver-recovered amebas. The B4F2 antibody also recognized the wall of mature E. histolytica cysts obtained from human samples. These results suggest that the enolase-containing vesicles are produced by E. histolytica amebas, when placed in the unfavorable liver environment that could be interpreted as an attempt to initiate the encystation process.     

Preliminary evidence on existence of transplasma membrane electron transport in Entamoeba histolytica trophozoites: a key mechanism for maintaining optimal redox balance

Entamoeba histolytica, an amitochondriate parasitic protist, was demonstrated to be capable of reducing the oxidized form of α-lipoic acid, a non permeable electron acceptor outside the plasma membrane. This transmembrane reduction of non permeable electron acceptors with redox potentials ranging from −290 mV to +360 mV takes place at neutral pH. The transmembrane reduction of non permeable electron acceptors was not inhibited by mitochondrial electron transport inhibitors such as antimycin A, rotenone, cyanide and azide. However, a clear inhibition with complex III inhibitor, 2-(n-heptyl)-4-hydroxyquinoline-N-oxide; modifiers of sulphydryl groups and inhibitors of glycolysis was revealed. The iron-sulphur centre inhibitor thenoyltrifluoroacetone failed to inhibit the reduction of non permeable electron acceptors whereas capsaicin, an inhibitor of energy coupling NADH oxidase, showed substantial inhibition. p-trifluromethoxychlorophenylhydrazone, a protonophore uncoupler, resulted in the stimulation of α-lipoic acid reduction but inhibition in oxygen uptake. Mitochondrial electron transport inhibitors substantially inhibited the oxygen uptake in E. histolytica. Transmembrane reduction of α-lipoic acid was strongly stimulated by anaerobiosis and anaerobic stimulation was inhibited by 2-(n-heptyl)-4-hydroxyquinoline-N-oxide. Transmembrane redox system of E. histolytica was also found to be sensitive to UV irradiation. All these findings clearly demonstrate the existence of transplasma membrane electron transport system in E. histolytica and possible involvment of a naphthoquinone coenzyme in transmembrane redox of E. histolytica which is different from that of mammalian host and therefore can provide a novel target for future rational chemotherapeutic drug designing.     

Natural Synchronisation for the Study of Cell Division in the Green Unicellular Alga Ostreococcus tauri

Ostreococcus tauri (Prasinophyceae) is a marine unicellular green alga which diverged early in the green lineage. The interest of O. tauri as a potential model to study plant cell division is based on its key phylogenetic position, its simple binary division, a very simple cellular organisation and now the availability of the full genome sequence. In addition O. tauri has a minimal yet complete set of cell cycle control genes. Here we show that division can be naturally synchronised by light/dark cycles and that organelles divide before the nucleus. This natural synchronisation, although being only partial, enables the study of the expression of CDKs throughout the cell cycle. The expression patterns of OtCDKA and OtCDKB were determined both at the mRNA and protein levels. The single OtCDKA gene is constantly expressed throughout the cell cycle, whereas OtCDKB is highly regulated and expressed only in S/G2/M phases. More surprisingly, OtCDKA is not phosphorylated at the tyrosine residue, in contrast to OtCDKB which is strongly phosphorylated during cell division. OtCDKA kinase activity appears before the S phase, indicating a possible role of this protein in the G1/S transition. OtCDKB kinase activity occurs later than OtCDKA, and its tyrosine phosphorylation is correlated to G2/M, suggesting a possible control of the mitotic activity. To our knowledge this is the first organism in the green lineage which showed CDKB tyrosine phosphorylation during cell cycle progression.