Lecture 10 African Sleeping Sickness and Antigenic Variation
Antigenic Variation
The entire trypanosome population seems antigenically uniform but at a very low frequency divergent (so called switched) serotypes are encountered
The switch to a new serotype is not recognized by the host antibody population
“Switchers” survive & proliferate leading to a new wave of parasitemia
Serotype switching continues Antigenic Variation
1 Antigenic Variation
T. brucei is covered with a dense surface coat
Variant specific antisera strongly react with surface coat
Surface coats from different clones are antigenically distinct
Antigenic Variation
No protease treatment
Trypsin (or other protease) treatment completely removes the surface coat from T. brucei
This treatment also abolishes antibody binding
This suggested that the antigenic determinant on the surface is made of protein
+ protease treatment
2 Surface coat consists of a single glycoprotein
65 kDa glycoprotein
C-terminus anchored in the membrane (GPI-anchor)
Only epitopes in the N-terminal 1/3 are exposed
Constant and variable regions
VSG forms dimers
VSGs from different clonal variants have same molecular weight, but different amino acid composition
Different VSG share only 16% amino acid similarity, but yet adopt a nearly identical tertiary structure!
Variant Surface Glycoprotein
• Single VSG type uniformly covers surface of parasite (107 copies) • VSG forms 12-15 nm electron dense surface coat • VSG dimers form a densly packed surface coat
3 Variant Surface Glycoprotein
Variable region
Constant region
Different VSG share only 16% similarity, but yet adopt a nearly identical tertiary structure!
T. brucei life cycle
non-dividing fuel=? Dividing form mVSG coat fuel=glucose mito=? VSG coat mito=“off”
Dividing form fuel=amino acids non-dividing Procyclin coat fuel=glucose mito=“on” VSG coat mito=“low”
4 T. brucei has ~ 1000 different VSG genes
Great variability of chromosome size among isolates
11 diploid megabase chromosomes, intermediate size, and about 100 minichromosomes - all classes contain VSG genes
6-10% of the total DNA codes for VSGs (~1000 genes)
Only a single VSG is expressed at a time!
At a low frequency a switch to a different gene occurs, the host developed antibodies against the previous VSG so Genome organization the new clonal cell line is strongly selected. 11 Megabase chromosomes (1-6 Mbp) 1-7 Intermediate chromosomes (200-700 kbp) ~100 Minichromosomes (50-150 kbp)
VSG Antigenic Variation
Immune VSG destruction switch by host Proliferation
What is the advantage to expressing a single VSG?
What mechanisms can you think of that could control gene expression and protein abundance?
How is VSG expression controlled?
5 Genomic Location of VSGs
The VSG Expression Site
Long polycistronic transcript Approximately 20-40 Bloodstream expression sites (BES) in the genome Active VSG genes are always at the “ends” of the chromosomes (telomeres)
6 VSG in Minichromosomes
VSG genes at minichromosome telmomers
Switching via telomere conversion or reciprocal telomere exchange
Mechanisms of Switching
7 Creation of Mosaic VSGs
VSG switching
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
8 Expression Sites
Regulation could be achieved by modification of chromatin
JJJJJ JJJJ active VSG
JJJJJ J J J J JJJJ JJJJ JJJJ X inactive VSG
The hyper-modified Base J
β-glucosyl-hydroxy-methyluracil a T variant
Base J But is J a chicken or an egg?
Expression Site Body (ESB)
How is a single expression site activated? LOCATION!
Differential localization of RNA polymerase I
rRNA transcription in other eukaryotes by RNA Pol I
usually RNA Pol II transcribes proteins coding sequences
Localizes to nucleolus in PF and BSF
Procyclic Bloodstream Extranulcleolar in BSF
9 Expression Site Body (ESB)
Procyclic
Bloodstream
Red: anti-fibrillarin - nucleolus marker Green: anti-RNA Pol I
The additional spot of RNA Pol I localization is NOT the nucleolus
Expression Site Body (ESB)
Active 221ES
Inactive 121ES
Active, not inactive VSG expression sites co-localize with the extranuclear Pol I spot.
GFP shows the position of the respective VSG genes in the nucleus
10 Transcriptional analysis of expression sites
Transcription of ES sites during development
Initiation occurs in several sites, but is abortive
Only in an active ES site is RNA elongation productive
Hypothesis: there is a limited supply of factors (transcription) connecting Pol I polymerase to elongation/processing machinery
Hypothesis: these factors are located in the ESB
Antigenic Variation Key Points
General features of Antigenic Variation (non-viral) Requires a family of variant sruface antigen genes Requires a mechanism to express only one gene at a time Requires a mechanism to switch genes
Trypanosomes - ~2000 VGS genes (variant surface glycoprotein)
Expression occurs out of telomeric expression sites (ES) (tapes/tape recorder or CDs/ CD player)
Expression seems promoter independent
To switch genes on, they are transposed into an active expression site by several mechanisms
Expression seems to be controlled by a physical association of ES with a single RNA Pol I transcription particle (location) per nucleus
11 Subnuclear location of Pol I
Navarro Model
12 Taking a Tryp(anosome) Across the Blood-Brain Barrier - Part 1 Review by Masocha et al 2007 Phys & Behav 92:110-114
Endothelium
Major structural elements
Data acquired from animal models - experimental infections with T. brucei brucei Laminin α5
Taking a Tryp(anosome) Across the Blood-Brain Barrier - Part 1 Masocha et al 2004 J. Clin Invest 114:689-694
Endothelial Laminin membrane Trypanosome
Parenchymal membrane
Laminin α4 Laminin α5
13 In vitro BBB Model
Taking a Tryp(anosome) Across the Blood-Brain Barrier - Part 2 Grab et al 2004 J. Parasitol 90:970-979.
Data acquired from in vitro BBB tissue culture models: T. brucei gambiensi
Laminin α4 Laminin α5
14 Taking a Tryp(anosome) Across the Blood-Brain Barrier - Part 2
Grab et al 2004 J. Parasitol 90:970-979.
15