The cell cycle of Entamoeba histolytica

Entamoeba histolytica, is a microaerophilic protist, which causes amoebic dysentery in humans. This unicellular organism proliferates in the human intestine as the motile trophozoite and survives the hostile environment outside the human host as the dormant quadri-nucleate cyst. Lack of organelles – such as mitochondria and Golgi bodies – and an unequal mode of cell division, led to the popular belief, that this organism preceded other eukaryotes during evolution. However, data from several laboratories have shown that, contrary to this belief, E. histolytica is remarkable in its divergence from other eukaryotes. This uniqueness is witnessed in many aspects of its biochemical pathways, cellular biology and genetic diversity. In this context, I have analysed the cell division cycle of this organism and compared it to that of other eukaryotes. Studies on E. histolytica, suggest that in its proliferative phase, this organism may accumulate polyploid cells. Thus 'checkpoints' regulating alternation of genome duplication and cell division appear to be absent in this unicellular protist. Sequence homologs of several cell cycle regulating proteins have been identified in amoeba, but their structural divergence suggests that they may not have equivalent function in this organism. The regulation of cell proliferation in E. histolytica, may be ideally suited to survival of a parasite in a complex host. Analysis of these molecular details may offer solutions for eradicating the pathogen by hitherto unknown methods.     

New observations of the nuclear division in Entamoeba histolytica. The chromosomes

The morphologic organization of the nucleus and DNA during the nuclear division of Entamoeba histolytica was examined. The DNA of dividing amebic trophozoites was visualized with the fluorescent probe, Hoechst 33258 for light microscopy, and a DNA-specific antibody and phosphotungstic acid for electron microscopy. These techniques demonstrated features of the dividing amebic nuclei and the presence of spherical DNA-containing bodies corresponding to the condensed chromosomes. Based on light microscopy observations the number of chromosomes in E histolytica is five. Microtubules (MT) radiating from the microtubule organizing center (MTOC) were observed attached to the putative chromosomes.     

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.     

Hydrogen peroxide-induced apoptosis-like cell death in Entamoeba histolytica

The microaerophilic intestinal parasitic protozoan Entamoeba histolytica has been previously shown to be highly susceptible to oxidative stress induced by hydrogen peroxide. However the mechanism of cell death was not investigated. Studies presented in this paper demonstrate several morphological features in the parasite when exposed to H₂O₂ which are identical to metazoan apoptotic phenotype indicating a possible apoptosis-like cell death exhibited by E. histolytica in response to H₂O₂ treatment. Trophozoite cell shrinkage, DNA fragmentation, phosphatidyl serine externalization and increased endogenous reactive oxygen species level have been observed in the protozoan parasite when exposed to 2.0 mM H₂O₂ for different time periods. Although the parasite genome is completely devoid of any of the homologues of mammalian caspases it still codes for a huge number of cysteine proteases which may take over the apoptotic function of the caspases. But the present study indicates the existence of a cysteine protease independent programmed cell death in the parasite since E-64 the specific cysteine protease inhibitor could not rescue the cells from H₂O₂ induced apoptosis-like cell death.     

Entamoeba histolytica: collagenolytic activity and virulence

Several axenic strains of pathogenic and nonpathogenic Entamoeba histolytica were tested for their capacity to digest native radioactive type I collagen gels and to produce liver abscesses when injected into the liver of newborn hamsters. The results demonstrate that the pathogenic strains of amebas (HM1:IMSS, HM3:IMSS, HM38:IMSS and HK9) have a collagenolytic activity that closely correlates with their in vivo capacity to produce liver lesions. The nonpathogenic isolate (Laredo) did not show collagenolytic activity and failed to produce lesions in the liver of newborn hamsters. The results also demonstrate that type I collagen obtained from rodents and cats is degraded less by amebic collagenase than is bovine collagen, which is similar to human collagen. These findings suggest that species susceptibility to invasive infection may depend, among other factors, on the characteristics of the extracellular components of host tissues.     

Entamoeba histolytica: transferrin binding proteins

Entamoeba histolytica trophozoites depend on iron for their growth; thus, they must use some host iron-containing molecules to fulfill this requirement. In this work we report that amoebas are able to utilize human holo-Tf as iron source and to recognize it through transferrin binding proteins. By use of an anti-human transferrin antiserum in an immunoblotting assay, two main polypeptides with apparent molecular masses of 70 and 140 kDa were found in total extract of trophozoites cultured in vitro. However, when a monoclonal anti-human transferrin receptor antibody was used, only one band with molecular mass of 140 kDa was observed. Both the human transferrin and the monoclonal antibody recognized a protein on the amoebic surface, demonstrated by confocal microscopy. Furthermore, the complex transferrin-transferrin binding protein was internalized by an endocytic process and probably dissociated inside the cell. This mechanism could be one manner in which E. histolytica acquires iron from the human host transferrin.     

Introns of Entamoeba histolytica and Entamoeba dispar.

The genome of Entamoeba histolytica is considered to possess very few intervening sequences (introns), as only 5 intron-containing genes from this protozoan parasite have been reported so far. However, while sequencing a number of genomic contigs as well as three independent genes coding for ribosomal protein L27a, we have identified 9 additional intron-containing genes of E. histolytica and the closely related species Entamoeba dispar, indicating that introns are more common in these organisms than previously suggested. The various amoeba introns are relatively short comprising between 46 and 115 nucleotides only and have a higher AT-content compared to the corresponding exon sequences. In contrast to higher eukaryotes, amoeba introns do not contain a well-conserved branch point consensus, and have extended donor and acceptor splice sites of the sequences G     

Chemotaxis by Entamoeba histolytica

A micropore membrane procedure to assay taxis by Entamoeba histolytica is described and the results of studies of responses to a variety of soluble substances, bacteria, an rat colon washings using this procedure are reported. Trophozoites migrated in blind well chambers through 8-micron pore size polycarbonate membranes but not nitrocellulose membranes up to 12 micron pore size. Amoebae were attracted toward fresh axenic culture medium (TYI-S), an enzymatic hydrolysate of casein (Trypticase), and a partially purified preparation of N-acetylneuraminic acid from egg mucin, but not purified N-acetylneuraminate or a variety of other low molecular weight metabolites. The response was verified as chemotaxis by checkerboard analysis. Amoebae migrated most dramatically toward suspensions of all of seven bacterial species tested, including motile and non-motile, gram-negative and gram-positive rods and cocci. This response was diminished when the bacteria concentration gradient was eliminated. The response to bacteria culture filtrates was less than 10% of that to bacterial suspensions. A response to clarified washings from the rat colon was detected; this was diminished but not eliminated by filter sterilization of the washings. We concluded that some soluble molecules, possibly of intermediate molecular size, whole bacteria, and both soluble and particulate components of the rat colon provide tactic stimuli for E. histolytica. Scanning electron micrographs of trophozoites migrating towards attractants through membranes showed narrow, extended pseudopodia entering the membrane pores, and enlarging spheres exiting as the cells proceeded through.     

Interdependence of cell cycle events inSchizosaccharomyces pombe. Terminal phenotypes of cell division cycle mutants arrested during dna synthesis and nuclear division

Summary Temperature-sensitive cell division cycle (cdc) mutants of the fission yeastSchizosaccharomyces pombe, previously characterized as defective in nuclear division were examined by thin section electron microscopy. All of the mutants failed to enter mitosis, rather they accumulated at one of four distinct terminal phenotypes. Class one were arrested with a nucleus rectangular in cross-section and a laterally situated spindle pole body (SPB). The second group had spherical or rectangular nuclei with a single SPB. The sole member of the third group wascdc 27. K 3, which had a spherical crenated nucleus with a single SPB from which microtubules emerged and extended into the cytoplasm. Allelic variants ofcdc 25 comprised the fourth group all of which displayed aberrant nuclear morphologies. Utilizing this ultrastructural data together with a knowledge of the transition points of these mutants a model for the interdependence of certain cell cycle event is proposed in which the initiation of DNA synthesis is uncoupled from the replication and separation of the SPB. This paper also provides new information on SPB structure inS. pombe. This is discussed in connection with the transient assembly of both spindle and cytoplasmic microtubules.     

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.     

Reducing agents and Entamoeba histolytica

Entamoeba histolytica remains an important but enigmatic parasite. It displays both non-pathogenic and invasive pathogenic types, which can be distinguished clinically and by isoenzyme markers. Yet as debated in Parasitology Today last year(1), the relationship between these two forms remains unclear. Bacterial associates and reducing agents are known to play on important role in the culture of E. histolytica, and possibly in its differentiation and invasive mechanisms. This article briefly reviews available information on the role o f reducing agents, and explores the possibility that bacteria may play a role in reduction o f toxic oxygen product - thereby promoting the virulence of E. histolytica. The review is not definitive, but should help to stimulate further research in this neglected area.     

The nuclear division and parasexual cycle inPenicillium

The vegetative nuclear division inPenicillium differs from classical mitosis, and a model for the division process is presented. In early divisional stages the interconnected chromosomes are arranged in a ring which breaks, giving rise to a linear configuration which divides by longitudinal splitting. The break may occur with equal probability between each of the chromosomes. At the end of the division process the daughter nuclei regain ring structure. One of the chromosomes is believed to represent a separation center for all the chromosomes. In diploid strains the two haploid genomes show a close somatic association and the nuclear configurations occurring during the divisional cycle are identical to those in the haploids. The double break of the ring structures in diploids will give recombinant nuclei in certain cases. The model explains the available data of the parasexual cycle, and both diploidization and haploidization are believed to represent singlestep processes.     

Entamoeba histolytica: Collagenolytic Activity and Virulence1

Several axenic strains of pathogenic and nonpathogenic Entamoeba histolytica were tested for their capacity to digest native radioactive type I collagen gels and to produce liver abscesses when injected into the liver of newborn hamsters. The results demonstrate that the pathogenic strains of amebas (HM1:IMSS, HM3:IMSS, HM38:IMSS, and HK9) have a collagenolytic activity that closely correlates with their in vivo capacity to produce liver lesions. The nonpathogenic isolate (Laredo) did not show collagenolytic activity and failed to produce lesions in the liver of newborn hamsters. The results also demonstrate that type I collagen obtained from rodents and cats is degraded less by amebic collagenase than is bovine collagen, which is similar to human collagen. These findings suggest that species susceptibility to invasive infection may depend, among other factors, on the characteristics of the extracellular components of host tissues.