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ABSTRACT: Accumulation of multiple copies of the genome in a single nucleus and several nuclei in a single cell has previously been noted in Entamoeba histolytica, contributing to the genetic heterogeneity of this unicellular eukaryote. In this study, we demonstrate that this genetic heterogeneity is an inherent feature of the cell cycle of this organism. Chromosome segregation occurs on a variety of novel microtubular assemblies including multi-polar spindles. Cytokinesis in E. histolytica is completed by the mechanical severing of a thin cytoplasmic bridge, either independently or with the help of neighboring cells. Importantly, cytokinesis is uncoupled from the nuclear division cycle, both temporally and spatially, leading to the formation of unequal daughter cells. Sorting of euploid and polyploid cells showed that each of these sub-populations acquired heterogeneous DNA content upon further growth. Our study conclusively demonstrates that genetic heterogeneity originates from the unique mode of cell division events in this protist.
PLoS Neglected Tropical Diseases 02/2009; 3(4):e409. · 4.69 Impact Factor
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ABSTRACT: The formin family of proteins mediates dynamic changes in actin assembly in eukaryotes, and therefore it is important to understand the function of these proteins in Entamoeba histolytica, where actin forms the major cytoskeletal network. In this study we have identified the formin homologs encoded in the E. histolytica genome based on sequence analysis. Using multiple tools, we have analyzed the primary sequences of the eight E. histolytica formins and discovered three subsets: (i) E. histolytica formin-1 to -3 (Ehformin-1 to -3), (ii) Ehformin-4, and (iii) Ehformin-5 to -8. Two of these subsets (Ehformin-1 to -3 and Ehformin-4) showed significant sequence differences from their closest homologs, while Ehformin-5 to -8 were unique among all known formins. Since Ehformin-1 to -3 showed important sequence differences from Diaphanous-related formins (DRFs), we have studied the functions of Ehformin-1 and -2 in E. histolytica transformants. Like other DRFs, Ehformin-1 and -2 associated with F-actin in response to serum factors, in pseudopodia, in pinocytic and phagocytic vesicles, and at cell division sites. Ehformin-1 and -2 also localized with the microtubular assembly in the nucleus, indicating their involvement in genome segregation. While increased expression of Ehformin-1 and -2 did not affect phagocytosis or motility, it clearly showed an increase in the number of binucleated cells, the number of nuclei in multinucleated cells, and the average DNA content of each nucleus, suggesting that these proteins regulate both mitosis and cytokinesis in E. histolytica.
Infection and immunity 07/2008; 76(6):2368-78. · 4.21 Impact Factor
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ABSTRACT: 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.
Bioscience Reports 01/2008; 27(6):373-84. · 2.38 Impact Factor
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ABSTRACT: Earlier studies have established two unusual features in the cell division cycle of Entamoeba histolytica. First, microtubules form a radial assembly instead of a bipolar mitotic spindle, and second, the genome content of E. histolytica cells varied from 1x to 6x or more. In this study, Eh Klp5 was identified as a divergent member of the BimC kinesin family that is known to regulate formation and stabilization of the mitotic spindle in other eukaryotes. In contrast to earlier studies, we show here that bipolar microtubular spindles were formed in E. histolytica but were visible only in 8-12% of the cells after treatment with taxol. The number of bipolar spindles was significantly increased in Eh Klp5 stable transformants (20-25%) whereas Eh Klp5 double-stranded RNA (dsRNA) transformants did not show any spindles (< 1%). The genome content of Eh Klp5 stable transformants was regulated between 1x and 2x unlike control cells. Binucleated cells accumulated in Eh Klp5 dsRNA transformants and after inhibition of Eh Klp5 with small molecule inhibitors in control cells, suggesting that cytokinesis was delayed in the absence of Eh Klp5. Taken together, our results indicate that Eh Klp5 regulates microtubular assembly, genome content and cell division in E. histolytica. Additionally, Eh Klp5 showed alterations in its drug-binding site compared with its human homologue, Hs Eg5 and this was reflected in its reduced sensitivity to Eg5 inhibitors - monastrol and HR22C16 analogues.
Cellular Microbiology 02/2007; 9(2):316-28. · 5.46 Impact Factor
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ABSTRACT: Clostridial glucosylating cytotoxins inactivate mammalian Rho GTPases by mono-O glucosylation of a conserved threonine residue located in the switch 1 region of the target protein. Here we report that EhRho1, a RhoA-like GTPase from the protozoan parasite Entamoeba histolytica, is glucosylated by clostridial cytotoxins. Recombinant glutathione S-transferase-EhRho1 and EhRho1 from cell lysate of Entamoeba histolytica were glucosylated by Clostridium difficile toxin B and Clostridium novyi alpha-toxin. In contrast, Clostridium difficile toxin A, which shares the same mammalian protein substrates with toxin B, did not modify EhRho1. Change of threonine 52 of EhRho1 to alanine prevented glucosylation by toxin B from Clostridium difficile and by alpha-toxin from Clostridium novyi, which suggests that the equivalent threonine residues are glucosylated in mammalian and Entamoeba Rho GTPases. Lethal toxin from Clostridium sordellii did not glucosylate EhRho1 but labeled several other substrate proteins in lysates from Entamoeba histolytica in the presence of UDP-[14C]glucose.
Applied and Environmental Microbiology 01/2007; 72(12):7842-8. · 3.83 Impact Factor
