What we do:
Our lab studies the molecular basis of pathogenesis of two medically important parasites, Toxoplasma gondii and Entamoeba histolytica. The work is aimed at understanding the virulence determinant that each parasite uses in causing disease, specifically how T. gondii evades the human immune response by converting to a dormant bradyzoite stage and how E. histolytica causes invasive colonic and hepatic disease. In the recent past our efforts have been split evenly in studying both parasites, but we are now switching exclusively to the investigation of Entamoeba biology.
The work on E. histolytica is focused at identifying unique virulence mechanisms that the parasite has developed for causing invasive disease using a combination of genetic and genomics based approaches.
Entamoeba histolytica is a protozoan parasite that causes colonic and liver disease. Approximately 50 million people have invasive disease annually resulting in 100,000 deaths per year making it the second most common cause of parasitic death in humans. The disease is predominantly seen in developing countries where it seriously affects the young and the old, malnourished individuals and pregnant women. The high prevalence of infection is due to fecal contamination of food and water supply, factors that cannot be immediately remedied due to limited financial resources in these countries. Since these socio-economic factors are difficult to correct in the short term, strategies and methods for disease eradication and prevention must be developed. To proceed in this direction it is critical that we understand the molecular mechanisms and virulence determinants necessary for the parasite to invade and cause disease in its human host. There are no significant environmental or animal reservoirs of the parasite making the possibility of a vaccine strain and disease eradication a feasible goal.
cycle and disease manifestations
During its life cycle the parasite converts between two stages: a trophozoite which invades the host epithelial cells causing colonic and hepatic disease and a cyst stage which transmits disease by surviving
in challenging environments. Infection begins by adherence of the trophozoite in the colon to colonic mucins and colonic epithelial cells and can lead to either asymptomatic colonization (90% of all infections), amebic colitis (in 10% of all infected individuals) or extraintestinal disseminated disease (most commonly liver abscess; occurring in 10% of people with invasive diarrhea). In the process of causing invasive diarrhea in the large intestine the trophozoites interact with enteric bacteria, attach to and invade the colonic epithelium, adapt to changing oxygen tensions, and ingest erythrocytes. The molecular mechanisms that the parasite utilizes during these events are poorly understood.
A closely related species: Entamoeba dispar
An interesting aspect of amebic biology that may give insights into amebic virulence is the recent reclassification of Entamoeba histolytica into:
(a) Entamoeba histolytica (formerly named the pathogenic zymodemes
of E. histolytica) and
(b) Entamoeba dispar (formerly named the non-pathogenic zymodemes of E. histolytica).
E. dispar and E. histolytica are morphologically identical and phylogenetically closely related (~98% identity of rRNA sequences). Both species have a similar host range but have vastly different properties with regard to pathogenicity in vivo. Both E. histolytica and E. dispar are able to colonize humans but only E. histolytica is able to cause invasive disease (colitis and extraintestinal manifestations). Aggressive behavior such as tissue destruction and erythrophagocytosis is not seen with E. dispar in vivo. In fact E. dispar has never been documented to cause invasive disease in humans and a recent panel of experts concluded that colonization with the parasite does not necessitate treatment.
Identification of the genetic differences between the parasites has been an active area of study since classification of the virulence determinant(s) that contribute to species-specific pathogenetic potential will greatly advance our understanding of the disease process. Studies of other E. histolytica virulence determinants have shown quantitatively variable expression between the two species. Genes of the three amebapore isoforms (A, B, C) are present in both species. However, E. dispar has comparatively reduced protein levels of amebapore A and B and almost absent levels of amebapore C the isoform which has been shown to have the highest cytolytic activity. To date only four genes have been described which are present in E. histolytica but absent or significantly degenerate in E. dispar (EhCP1, EhCP5, EHAPT2 and an Arg-rich protein “Ariel1”). Overexpression of EhCP1 and EhCP5 in E. dispar has been attempted but unsuccessful thereby making elusive the genetic proof of these molecules in species-specific virulence. The molecular basis of virulence of E. histolytica may thus be more fully characterized by understanding the factors that make it pathogenetically distinct from E. dispar.
available to study ameba biology
Many molecular and genetic tools are available for the study of ameba biology. These include manipulation of the parasite using a variety of methodologies including transient and stable transfection, regulated gene expression, antisense and dominant-negative technologies. The ability to integrate DNA into the genome has been elusive to date thereby not allowing studies with targeted disruption of genes. However the redundancy of the gene families and the possible multi-ploidy of the parasite make this approach, even if technically feasible, less attractive. Animal models have been developed for amebic colitis and amebic liver abscess allowing the in vivo analysis of amebic virulence. The genome sequencing effort (see below) is also providing many new insights into parasite biology. Although in vitro encystation has not yet been accomplished in E. histolytica, it is readily possible in Entamoeba invadens, a reptilian ameba, and provides us with an opportunity to study the complete life cycle of the parasite.
The genome of E. histolytica is currently being sequenced by a collaborative effort between The Institute for Genomic Research (TIGR) (http://www.tigr.org/tdb/e2k1/eha1/) and the Sanger Center (http://www.sanger.ac.uk/). The genome data was recently published (Nature. 2005 Feb. 24;433(7028):865-8).
What we do
Our goal is to understand the genetic basis of virulence in Entameoba. Towards that end we are interested in identifying genes that distinguish virulent from nonvirulent parasites and genes that enable the parasite to cause invasive disease. Genes identified in this manner are then genetically and biochemically characterized and their roles in amebic pathogenesis investigated.
Currently our focus is to use a genomics based approach to investigate the molecular basis of amebic pathogenesis. We have developed an 11,000 clone DNA microarray for E. histolytica (based on sequenced and annotated clones from the TIGR/SANGER genome sequencing efforts) and are utilizing it to identify genetic loci associated with virulence. Microarray technology can be used to answer a multitude of questions including population genetics, genetic diversity, regulation of gene expression and transcriptional response to a given stimulus. Additionally we are developing genetic tools for gene regulation in Entamoeba.
For a review on microarray technology and its applications, please see Nature Genetics supplement, December 2002.
The current projects in the lab can be categorized into three sections:
a) Extent of divergence between pathogenic E. histolytica and nonpathogenic E. dispar.
b) Can we correlate a genotype with virulence?
a) What amebic genes are important in causing invasive disease?
b) Identify gene expression differences between “virulent” and “nonvirulent” strains