African Trypanosomes & Sleeping Sickness II. Sleeping Sickness and Trypanosomes I zLife cycle and biology of trypanosomes zSleeping sickness, differences.

African Trypanosomes & Sleeping Sickness II

Sleeping Sickness and Trypanosomes I

Life cycle and biology of trypanosomesSleeping sickness, differences between

gambiense and rhodesienseNagana, kachexia and TNFDrugs used to treat trypanomiasisTse tse flies, fly control

Trypanosomes II

Why is African trypanosomiasis such a deadly disease?

Important pathways discovered along the way to understand this problem: trans splicing and GPI anchors

The nuts & bolts of trypanosme gene expression control

Why is trypanosomiasis so deadly?

Infection is chronic and ultimately fatal if left untreated.

How can the slender trypomastigote form of the parasite survive in the human bloodstream?

Why is trypanosomiasis so deadly?

Trypanosomes are highly susceptible to antibodies and complement

They live fully exposed to antibodies in the blood stream

They induce a very strong antibody response

Still they manage to thrive in the same host for a year or longer, until the host dies

Why is trypanosomiasis so deadly?

The number of parasites found in the blood of humans and animals infected with trypanosomes is not constant, but shows characteristic waves of parasitemia

The window of time between parasitemia peaks is about 5-7 days

Why is trypanosomiasisso deadly?

Infection is characterized by periodic waves of parasitemia

Each wave represents a single antigenically distinct clone or serotype

Antibodies produced in the first week against clone A will not react with clone B

The changing display of different antigens is called antigenic variation

Antigenic variation is an important form of immune evasion

Antigenic variation

The entire population of trypanosomes within an infected animal seems antigenically uniform

But at a very low frequency divergent (so called switched) serotypes are encountered

Antigenic variation

Trypanosomes are covered with a dense surface coat

Variant specific antisera strongly react with this surface coat

Surface coats from different clones are antigenically distinct

Antigenic variation

Trypsin treatment completely removes the surface coat from Trypanosomes (trypsin is a protease, an enzyme that specifically digests proteins)

This treatment also abolishes antibody binding

This suggests that the antigenic determinant on the surface is a protein

Antigenic variation

The surface coat is made up almost entirely by a single protein the Variant Surface Glycoprotein or VSG

This protein is highly immunogenic and distinguishes the clones in successive parasitemia peaks

VSGs from different parasitemia peaks differ in their amino acid sequence

Lessons learned along the way: the GPI anchor

When genes for T. brucei VSGs were sequenced they were shown to encode a c-terminal hydrophopic peptide that could anchor the protein

However when the proteins were sequenced this part was absent -- how is this soluble protein kept in the membrane?

VSG is anchored into the membrane via a glycolipid anchor (glycosyl-phosphatidylinositol or GPI)

Lessons learned along the way: the GPI anchor

Initially thought to be specific for trypanosomes GPI anchors have been shown to be present in all eukaryotic organisms

The GPI anchor is synthesized as a precursor glycolipid in the endoplasmic reticulum by sequential addition of sugar molecules to a phospholipid

The mature precursor contains a terminal ethanolamine phosphate which can form a peptide bond with the c-terminal carboxyl group of the protein

Antigenic variation

GPI anchors allow very dense packing of molecules on the surface of the parasite

VSGs forms a dense coat on the surface of the trypanosome

This coat is equivalent of the coat form by lipophosphoglycan in Leishmania

Antigenic variation

All VSGs are 65 kDA glycoproteins, and are present on the surface as dimers

The outer domain is highly variable and the only conservation detected is the position of cysteines

Other (non-variant) proteins like transferrin receptor or hexose transporter are hidden in the this surface coat

Antigenic variation

6-10% of the total genome of African trypanosomes is coding for VSGs (more than 1000 genes)

Only one is expressed at a given time the other 999 genes are shut down and completely silent (allelic exclusion)

At a low frequency a switch to a different gene occurs, if the host develops antibodies against the previous VSG the new clone is strongly selected

What is the advantage of expressing a single VSG? How is expression controlled? What mechanisms can you think of by which a cell

could control gene expression and protein abundance?

Antigenic variation

mRNA derived from only a single VSG gene can be detected at one time

VSG expression is controlled at the level of transcription initiation

Regulation of promoter activity is used to control gene expression in many organisms

Transcription in trypanosomes is polycistronic

But, only very few promoters have been identified in trypanosomes and they did not seem to control the expression

Also surprisingly transcription in trypanosomes was found to be polycistronic

Polycistronic means that a number of genes are transcribed at the same time into one long messenger RNA

In bacteria this message is translated into protein, in trypanosomes further processing is needed and this processing might confer additional level of control

Transcription in trypanosomes is polycistronic

Individual mature mRNAs are derived from large polycistronic transcripts by a process called trans-splicing

In this process mRNAs for individual genes are cut out of the polycistronic transcript and a short RNA transcribed from a different locus (the splice leader) is attached to it 5’ end

Initially this was thought to be the key to regulation – but it is not.

Antigenic variation

If it is not the promoter or the processing maybe it is the exact location in the genome that predisposes a specific VSG for expression

Where are active and inactive genes in the genome?

How could a location based system switch?

VSGs are expressed from telomeric polycistronic expression sites

Transcription in trypanosome is polycistronic as we have seen

Active VSG genes are allways at the “ends” of chromosomes (telomeres)

Genes are read in (20) expression sites like CDs in CD players but only one CD player appears to be playing at a time

How do you get a new CD in and how are the CD players controlled

Several mechanisms for switching have been discovered

The most common mechanism of VSG switching requires physical transposition of a new VSG gene into the active expression site

Antigenic variation

There are several mechanisms but the most common mechanism of VSG switching requires physical transposition of a new VSG gene into the active expression site

Transposition of VSG genes occurs by intra- or intermolecular recombination

This explains switching but not really why one gene is active and all the others are silent

Antigenic variation

Regulation could be achieved by modification of chromatin (by sticking on a read me or do not read me label)

Indeed active and inactive sites differ in the amount of a special modified base called J (-glucosyl-hydroxy-methyluracil)

But is this the chicken or the egg? Recent work from Dr. Sabatini’s lab here at UGA shows that J is likely not

controlling expression but is important for switching & recombination

For the next experiment we need a mushroom

http://www.mushroomexpert.com

Amantia bisporingea, the Destroying Angel

VSG is transcribed by Pol I -amanitin is a specific and highly

potent RNA polymerase inhibitor Cells have specialized RNA

polymerases to transcribe different genes

In most cells mRNA which encodes proteins is transcribed by the RNA polymerase Pol2 (this enzyme can be inhibited by the toxin amanitin)

Ribosomal RNA is generally transcribed by Pol1 (which is resistant to the toxin)

VSG transcription is insensitive to -amanitin suggesting it is transcribed by the highly processive Pol I (however all other mRNAs for proteins seem to be made using Pol II as everywhere else)

How could this help to explain allelic exclusion?

Drug

rRNA

tubulin

VSG

African trypansome cellular architecture

Nucleoulus

Nucleus

Kinetoplast

How is a single expression site activated?

Location, location, location

PolI is found in two spots in blood stream forms: the nucleolus (where rRNA is made) and a second locus outside of the nucleolus

insect mammal

Pol I

DNA

Nature 414:759-63

How is a single expression site activated?

The additional spot of Pol I is not the nucleolus (Fib in red is a nucleolus marker)

Nature 414:759-63

How is a single expression site activated?

active VSG

inactive VSG

Active, but not inactive VSG expression sites colocalize with the extranuclear Pol I spot. GFP in green shows the position of the respective VSG gene in the nucleus

Nature 414:759-63

Antigenic variation

Only a single VSG gene out of ~1000 is expressedExpression occurs out of teleomeric expression

sites (the tape recorder)To switch genes on they are transposed into an

active expression site by several mechanismsExpression seems to be controlled by physical

association of the expression site with a single POL1 transcription particle per nucleus

There are 1000 CDs, 20 CD players but only one is plugged in