By Levi Clancy for Student Reader on
Entamoeba histolytica is the third parasitic killer worldwide. Despite this, it infects many more than it kills.
It has a worldwide distribution and lacks a vector, instead transmitting as cysts via fecal-oral route. Poor sanitation leads to contaminated water and a high incidence of this disease. It is diagnosed via microscopy, ELISA and/or PCR. Entamoeba histolytica is a huge single-celled and single-nucleus amitochondriate that colonizes the large intestines and lyses tissues (Giardia lamblia colonizes the small intestines and has two nuclei). It engulfs bacteria and red blood cells for nourishment, and moves using pseudopodia. In place of mitochondria, E. histolytica has intracytoplasmic vacuoles, and also crystalline bodies containing ribonucleoprotein helices.
Entamoeba histolytica secretes proteinases that dissolve host tissues, kills host cells on contact, and engulfs red blood cells. E. histolytica trophozoites also invade the intestinal mucosa, lysing host cells and causing ulcers (amoebic colitis). Amoebas can breach the mucosal barrier and travel through the portal circulation to the liver, where they cause abscesses that are 100% fatal if untreated. Amoebic liver abscesses grow inexorably and, at one time, were almost always fatal, but now even large abscesses can be cured by one dose of antibiotic. A variety of virulence factor are currently being investigated to better deﬁne pathogenic mechanisms. Entamoeba histolytica (pathogen) is a distinct species from Entamoeba dispar (a harmless commensal).
Life Cycle of Entamoeba histolytica
|Ingestion||Cysts are ingested.|
|Transform||Cysts differentiate into trophozoites in stomach.|
|Division||Trophozoite multiplies in small intestine.|
|Invasion||Trophozoites invade the colon.|
|Some trophozoites encyst in the colon and pass in the feces to continue the cycle. In a small percentage of infections, the trophozoite also invades other organs, such as the liver and brain, and causes abscesses.|
Factors Implicated in Pathogenesis
|GalNAc lectin||Adheres mucin/cells, serum resistance.|
|Fibronectin/collagen||Adheres extracellular matrix receptors.|
|Cysteine proteinases||Invasion through the extracellular matrix.|
|Amoebapore||Lysis of target cells.|
|Phospholipases||Lysis of target cells.|
|Cytoskeleton||Adhesion plates, endocytosis, motility.|
GalNAc Lectin: Adherence to Mucin/Cells
What's the deal?
Killing of human cells requires adherence of parasite to target cells. Entamoeba histolytica has a lectin protein that binds carbohydrates on the host cell, specifically galactose and N-acetyl-D-galactosamine. Hence, the Entamoeba histolytica lectin is known as GalNAc Lectin.
Addition of 50mM galactose or N-acetyl-D-galactosamine to host cells in vitro blocked the adherence of the parasite to the host cells. This assay indicated that sugar was involved in adherence. However, it was unclear whether the sugar moieties were on the parasite or host cells.
But Which Has the Sugar?
Host cells were found that had a glycosylation enzyme mutation such that terminal Gal/GalNAc sugars were added to host surface proteins. This strain was resistant to killing by E. histolytica, indicating that the sugars required for parasite-host attachment are on the host.
What's the Parasite Lectin?
Affinity chromatography identified the protein for adherence:
|Starve||Cells were starved for methionine.|
|Label||Labelled methionine was added to label all proteins.|
|Lyse||Cells were lysed and run through a Gal/GalNAc column.|
|Wash||Column was washed until no radiolabel came off.|
|Elute||Gal was added in excess to elute bound proteins.|
|Two proteins were found, which turned out to be the 170 kDa heavy and 35kDa light chains, normally connected as a heterodimer via a disulfide bond. The heavy subunit contains a carbohydrate recognition domain that binds host cell sugars. The C-terminal region is involved in intracellular signaling. Each subunit is encoded by a separate gene, of which there are multiple different copies.|
Many Roles of GalNAc Lectin?
From here, mass spectrometry identified the protein sequence and then PCR was used to amplify and clone the gene. With the gene in hand, it can be used to induce overexpression or to knock out the gene in the parasite.
The following functions of GalNAc Lectin were identified:
|Adhesion||GalNAc Lectin adheres to host cells.|
|Apoptosis||GalNAc Lectin induces apoptosis of host cells.|
|Virulence||Parasite virulence proportional to amount of lectin.|
|Uptake||Mediates uptake of gut bacteria for feeding.|
|Encystation||GalNAc Lectin binding induces encystation in gut.|
|Antibodies||Inducing antibody response → acquired immunity.|