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.