PhD: IICB/Calcutta University/ 1986
Cell cycle of Entamoeba histolytica
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
have used the following approaches to study the amoeba cell cycle:
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.
of DNA content and cell cycle phases of Entamoeba by flow cytometry:
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.
Inhibition of gene expression by double strand RNA interference in
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.
Genome analysis of E.histolytica
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.
Genomic analysis of S/MAR sequences in E.histolytica
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.
Medicinal plant extracts showing anti-amoebic activity
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.