Anuradha Lohia
Professor, Biochemistry
PhD: IICB/Calcutta University/ 1986

Cell cycle of Entamoeba histolytica

 Our research is focused on the regulation of cell cycle progression and differentiation of the human pathogen, Entamoeba histolytica. E.histolytica is a protozoan parasite, which infects and causes morbidity to at least 500 million people worldwide. This organism is extremely difficult to grow in the laboratory and standard microbiological methods cannot be used for the study of this protozoan. Similarly, classical methods of genetics cannot be used to obtain mutants or to identify the functions of genes as is routinely used for most microbes. Inspite of these drawbacks, our results support the growing notion that the unique biology of this parasite has probably evolved in response to its survival strategies as a parasite  

 We have used the following approaches to study the amoeba cell cycle:

a) Cloning of homologs of eukaryotic genes regulating the cell division cycle from E.histolytica.

 Since cell cycle regulating genes from Entamoeba could not be isolated by functional complementation in yeast mutants, we designed degenerate oligonucleotide primers derived from the conserved domains of eukaryotic cell cycle genes. Using these primers with E.histolytica genomic DNA, we amplified homologs of several of these genes by PCR. These genes were cloned and sequenced to identify their homology to the eukaryotic genes. With this approach we characterized Entamoeba homologs of cdc2, rho, ras and rap, mos family kinase (mfk),  rac, gamma tubulin,  minichromosome maintenance (MCM) genes, p21 activated kinase, INO1 and diaphanous.

The sequencing of the cDNA and the genomic clones of the Eh cdc2 gene revealed that the ORF of the gene was interrupted by a 79 bp intron. This was the first demonstration of the presence of an intron in E.histolytica. Before this, it was largely believed that E.histolytica genes did not contain introns. In yet another study, we demonstrated that the Eh MCM3 and Eh PAK genes were transcribed from a common genomic sequence such that the 3’ end of Eh PAK transcript overlapped with the 5’ end of Eh MCM3 gene. This unusual genome organization showed no intergenic regulatory sequences, such as a promoter, between the two genes suggesting that gene expression of Eh MCM3 was regulated by novel means.

We have raised polyclonal antisera against each of the proteins after overexpressing these genes as fusion proteins in E. coli. These antisera were used to study expression and subcellular localization of the proteins by flow cytometry and confocal microscopy. Subcellular localization of gamma tubulin by immuno-fluorescence showed that, unlike other eukaryotes, E.histolytica trophozoites had several extranuclear MTOCs in each cell.

 A majority of eukaryotes have developed an intricate machinery for precisely replicating their genome only once per cell cycle. “Checkpoints” or the surveillance action of proteins, ensuring the successful completion of one event before another can begin, control the progression of the cell division cycle in these organisms. Therefore, DNA re-duplication is not permitted until mitosis has been completed. This paradigm appears to be altered in the regulation of the cell division cycle of Entamoeba histolytica.   

Our group has shown that asynchronously growing amoeba cells contain heterogeneous populations of varying DNA contents, ranging from 1n to 8n per cell. Thus completion of DNA replication is not tightly coupled to cell division and polyploid cells are commonly observed. It was demonstrated that polyploidy was an intrinsic property of growing cells irrespective of the age of the culture. Thus, DNA reduplication occurred at least 3-4 times before cell division occurs in E.histolytica. Using a laser cytometer, we have demonstrated that individual nuclei of E.histolytica trophozoites may contain from 1n to 20n genome contents. 

Since differentiation of E.histolytica trophozoites to the infectious cyst form is not possible in axenic culture, the model reptilian species E.invadens was used to study changes in DNA content and cell cycle during conversion of trophozoites to cysts. We have shown that during vegetative growth DNA synthesis continues beyond 4n genome content whereas during differentiation, DNA synthesis arrests at 4n genome content. Since the amoeba cell cycle lacks checkpoints to ensure cell division after genome duplication, the cell cycle proteins of E.histolytica may not be subject to the same regulation as their eukaryotic counterparts. It is possible that other, yet unknown, regulatory proteins ensure a broad fidelity in the maintenance of genome content in this organism. The repertoire of cell cycle genes which we have cloned can be exploited for the study of the basic biology of this organism and as potential candidates for drug targeting.

 c) Inhibition of gene expression by double strand RNA interference in Entamoeba histolytica  

 We have developed a method to obtain stable transformants expressing dsRNA to down regulate expression of specific genes. This was done by cloning a short fragment of DNA from the gene of interest in a head to head orientation with an intervening non-specific stuffer fragment, first in the E.coli vector pBS. Subsequently the construct was subcloned downstream of the actin promoter in the amoeba expression vector pJST4. Using this strategy we have been able to downregulate expression of several cell cycle genes.

 d) Genome analysis of E.histolytica

 The genome sequence of this parasite was completed at TIGR and Sanger Centre. Our laboratory was designated to analyse and annotate sequence homologs of eukaryotic cell cycle genes from the amoeba genome. Our results show that a large number of sequence homologs or paralogs of eukaryotic cell cycle genes could not be identified in E.histolytica. This discovery has led us to look for novel proteins that may have replaced eukaryotic genes as well as to identify unique processes for cell cycle control in this parasite.

e) Genomic analysis of S/MAR sequences in E.histolytica

 We have shown that S/MAR sequences such as Eh MRS2 are substrates for Eh Methyl transferase. We are now studying the role of S/MAR sequences in regulation of gene expression by epigenetic mechanisms in E.histolytica.

f) Medicinal plant extracts showing anti-amoebic activity

 We have purified crude extracts of several medicinal plants known to be used for diarrhoea and dysentery in ayurvedic literature. From our studies we have found that these extracts cause cytotoxicity and cell death in E.histolytica.  We are in the process of purifying these compounds and elucidating their structure.