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Entamoeba histolytica

By Levi Clancy for Student Reader on

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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
IngestionCysts are ingested.
TransformCysts differentiate into trophozoites in stomach.
DivisionTrophozoite multiplies in small intestine.
InvasionTrophozoites 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 lectinAdheres mucin/cells, serum resistance.
Fibronectin/collagenAdheres extracellular matrix receptors.
Cysteine proteinasesInvasion through the extracellular matrix.
AmoebaporeLysis of target cells.
PhospholipasesLysis of target cells.
CytoskeletonAdhesion 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.

What Attaches?
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:

StarveCells were starved for methionine.
LabelLabelled methionine was added to label all proteins.
LyseCells were lysed and run through a Gal/GalNAc column.
WashColumn was washed until no radiolabel came off.
EluteGal 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:
Ameobapore: Lysis of Target Cells

Ameobapore proteins are a family of 5kDa (77 amino acid) proteins whose three members have ~50% similarity to each other. Amoebapores form β-barrel pores in membranes that allow the pathogen to lyse the host cells after binding via GalNAc lectins. One amoebapore is specifici to eukaryotic membranes and another to prokaryotic membranes. Multiple types of ameobapores mediate lysis of different cell types (i.e. host cells that are attacked versus bacteria cells that are engulfed for nourishment). Note that the membrane lipids of Entamoeba histolytica are impervious to ameobapore activity.

To study amoebapores, comparisons were made between Entamoeba histolytica and E. dispar. These two species are extremely similar except that Entamoeba dispar is a harmless commensal. These two species may be distinguished via PCR, morphology and lytic activity. RNA silencing can mimic the effect of E. dispar. AmoebaporeA is specific to eukaryotic membranes. It was reduced via RNA silencing to undetectable levels. AmoebaporeA- trophozoites were introduced into SCID mice and caused significantly smaller amebic liver abscesses compared to the parental strain.

Cysteine Proteinases: Invasion Through Extracellular Matrix to Reach Cells

At least six cysteine proteases are expressed in Entamoeba histolytica and none are expressed in E. dispar -- this indicates they are likely virulence factors. Some cysteine proteases are secreted and can disrupt a host cell monolayer; CP5 causes host cells to roll up into little balls and detach from the dish. Comparing the genome sequences of CP5 in E. histolytica and a homologous gene in E. dispar, it seems that E. dispar has accumulated enough mutations in this gene to have a non-functional version.

AdhesionGalNAc Lectin adheres to host cells.
ApoptosisGalNAc Lectin induces apoptosis of host cells.
VirulenceParasite virulence proportional to amount of lectin.
UptakeMediates uptake of gut bacteria for feeding.
EncystationGalNAc Lectin binding induces encystation in gut.
AntibodiesInducing antibody response → acquired immunity.