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Tomography of flagellar pocket Lacomble et al., J Cell Sci. 122:1081-90.

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Presentation on theme: "Tomography of flagellar pocket Lacomble et al., J Cell Sci. 122:1081-90."— Presentation transcript:

1 Tomography of flagellar pocket Lacomble et al., J Cell Sci. 122:1081-90.

2 Sleeping Sickness and Trypanosomes

3 Trypanosomias zTrypanosomes were first described in frogs 1855 zGriffith Evans identifies T. evansi as agent of surra (a horse and camel disease) in 1880 zDavid Bruce identifies T. brucei as cause of Nagana and demonstrates transmission by Tse-tse flies David Bruce, 1855-1931

4 Trypanosome biology

5 The kinetoplast consists of a complex network of concatenated DNA cirlces Mensa-Wilmot lecture will tell you what the circles are for

6 Trypanosome biology

7 Tse tse flies zParasites are taken up with the blood meal (stumpy forms are cell cycle arrested and ‘ready to go’ for the next host zTransformation into procyclic trypomastigotes in the midgut zMigration into the ectoperitrophic space where parasites replicate zPassage into salivary glands, differentiation into epimastiogotes which attach to the epithelium and massively replicate zTransformation into infectious metacyclic trypomastigotes zAgain these are cell cycle arrested ‘sleepers’

8 Trypanosmes have sex and likely it happens in the salivary gland zGenetic exchange occurs (e.g. double drug resistance occurs after coinfection of fly with single resistant parents) zGenetic exchange/sex is likely not obligatory to complete fly development and population genetics suggest “modest” sex zNobody has seen it, yet it likely involves fusion and meiosis (progeny appears largely diploid but there are also polyploids) zMost likely this exchange/fusion occurs among detached salivary gland epimastigotes

9 Trypanosmes have sex and likely it happens in the salivary gland GFP & RFP only parents early on in salivary gland Red, green & yellow progeny Gibson et al., Parasites & Vectors 2008, 1:4

10 Insect stages and blood stream forms are very different zDifferent stages express different sets of surface proteins zInsect forms have large mitochondria with many cristae zInsect stages have an aerobic metabolism and a full respiratory chain zBlood stream forms only engage in glycolysis and excrete pyruvate and glycerol zNote that transmission stages do not replicate and are arrested in development (ladies in waiting)

11 Several species of trypanosomes cause disease in domestic animals and man zT. brucei rhodesiense & gambiense cause sleeping sickness zT. brucei brucei, T. congolense and T. vivax cause Nagana in cattle zT. equiperdum causes sexually transmitted disease in horses and camels (interestingly, T. equiperdum is a recent ‘petite’ mutant of T. brucei (loss of mitochondrial genome or kDNA) Lai et al. 2008, zLoss of oxidative phosphorylation locks parasite into BS form – or the other way around, leaving Africa and tse- tse transmission makes the mitochondrion dispensable z(There are trypanosomes infecting many species of animals and even plants and every single deer in the State of Georgia)

12 Sleeping sickness in man

13 zTrypanosomes multiply in the tissue around the initial bite site zThis often results in a characteristic local inflamation the trypansomal chancre zFrom there they enter the blood and lymphatic system

14 Sleeping sickness in man zEnlargement of the lymphatic glands (especially in the posterior triangle of the neck) can be an early sign of the diseasese (Winterbottom sign, not as common in rhodesiense infection). zAspiration of swollen gland often reveals parasites.

15 Sleeping sickness in man zOnce parasites enter blood stream fever sets in (low and irregular in gambiense and high and periodic in rhodesiense zGeneral toxic symptoms include headache, facial oedema, nausea and vomiting,back and bone pain zSymptoms at this stage are rather mild in gambiense but can be servere in rhodesiense with often fatal outcome

16 Sleeping sickness in man zThe second stadium of trypansomiasis is characterized by progressive anemia and kachexia.  Both features are primarily due to extremely high serum levels of TNF   TNF  was isolated both as factor with tumor necrotic effect as well as kachexin inducing wasting in nagana

17 Sleeping sickness in man zIn later stages of infection parasites pass the blood brain barrier and infect the CNS zPresence of parasites leads to meningo- encephalitis with progressive neurological involvement, which ultimately ends in coma (sleeping sickness) zUntreated trypanosomiasis is always fatal

18 Sleeping sickness in man zThe progressive encephalitis can cause severe dementia with sometimes aggressive behavior zDisease progression especially CNS invasion is much faster in rhodesiense zGambiense can take a year or two rhodesiense usually passes the blood brain barrier within a month

19 Nagana is the major impediment to cattle production in Africa zAlmost the entire area of subsaharan Africa which is suitable for cattle is Tsetse infested zHigh losses due to anemia and cachexia especially in productive breeds

20 Wild animals are important reservoirs for human and cattle trypanosomiasis

21 Why is trypanosomiasis so deadly? zTrypanosomes are highly susceptible to antibodies and complement zThey live fully exposed to antibodies in the blood stream zThey induce a very strong antibody response zStill they manage to thrive in the same host for a year or longer

22 Why is trypanosomiasis so deadly? zInfection is characterized by periodic waves of parasitemia

23 Why is trypanosomiasis so deadly? zInfection is characterized by periodic waves of parasitemia zEach wave represents a single antigenically distinct clone or serotype

24 Antigenic variation zThe entire trypanosome population seems antigenically uniform but at a very low frequency divergent (so called switched) serotypes are encountered

25 Antigenic variation zTrypanosomes are covered with a dense surface coat zVariant specific antisera strongly react with this surface coat zSurface coats from different clones are antigenically distinct

26 The surface coat consists of a single 65 kDa glycoprotein zA single protein can be labeled on the surface of trypanosomes zUpon parasite lysis this protein becomes soluble and can be purified to homogeneity fairly easily. George Cross

27 Different antigenic variants have different surface glycoproteins zVSGs from different clonal isolates have the same molecular weight but vastly different amino acid compositions zVaccination with a given VSG protects against challenge with the homologous isolate but not against another variant.

28 VSGs share a common structure zAll VSGs are 65 kDA glycoproteins zMost contain classical N-linked glycans and all are anchored via a GPI glycolipid (cross reacting determinant) zTwo domains can be cleaved by trypsin zThe outer domain is highly variable and the only conservation detected is the position of cysteines zVSG forms dimers

29 Antigenic variation zVSG dimers form a densly packed surface coat zOther (non-variant) proteins like transferrin receptor or hexose transporter are hidden in this coat

30 Trypansomes harbor ~1000 different VSG genes zThe genomic organization of trypanosomes is quite complex with 20 chromosomes and 100 mini chromosomes zGreat variability of chromosome size between isolates z6-10% of the total DNA is coding for VSGs (~1000 genes) zOnly one is expressed z3 very peculiar details emerged from studying the mRNA of VSG: all trypanosome mRNAs seem to have the same 5’end, and the VSG mRNA encodes a hydrophobic c- terminus absent from the mature protein sequence, VSG message is transcribed by Pol I

31 Antigenic variation zmRNA derived from only a single VSG gene can be detected at one time zVSG expression is controlled at the level of transcription initiation zRegulation of promoter activity is used to control gene expression in many organisms

32 Transcription in trypanosomes is polycistronic zBut, only very few promoters have been identified in trypanosomes and they did not seem to regulate the expression of VSG zAlso surprisingly transcription in trypanosomes was found to be polycistronic zPolycistronic means that a number of genes are transcribed at the same time into one long messenger RNA zIn bacteria this message is translated into protein, in trypanosomes further processing is needed

33 Transcription is polycistronic zThe 39 first (5’) base pairs of all trypanosme mRNAs are identical, this sequence is not found in the genomic locus of these genes zIndividual mature mRNAs are derived from large polycistronic transcripts and short SL-RNAs by trans- splicing (details in Mensa- Wilmot lecture) zThis might help control – but was shown not to be the key to antigenic variation

34 Location in the genome?

35 VSGs are expressed from telomeric polycistronic expression sites zActive VSG genes are typically at the “ends” of chromosomes (telomeres) zThey are found in “expression sites” zGenes are read in (~20) expression sites like tapes in a tape recorder but only one recorder is playing at a time zHow do you get a new tape in and how are the recorders controlled e.g. switched on and off?

36 Several mechanisms for switching have been described

37 Antigenic variation zTransposition of VSG genes occurs by intra- or intermolecular recombination zThis explains switching but not really why one gene is active and all the others are silent

38 Antigenic variation zRegulation could be achieved by modification of chromatin  Indeed active and inactive sites differ in the amount of a special modified base called J (  -glucosyl-hydroxy-methyluracil - a T variant) and there are newly discovered differences in histone methylation and acetylation patterns (Bob Sabatini will go over this in detail on Monday)

39 For the next experiment we need a mushroom Amantia bisporingea, the Destroying Angel

40 VSG is transcribed by Pol I   -amanitin is a specific and highly potent RNA polymerase inhibitor zCells 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) zRibosomal 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) zHow could this help to explain allelic exclusion? Drug rRNA tubulin VSG

41 African trypansome cellular architecture Nucleoulus Nucleus Kinetoplast

42 How is a single expression site activated? zLocation, location, location zPolI antibody detects two spots in blood stream forms: the nucleolus (where rRNA is made) and a second locus outside of the nucleolus Navarro M, Gull K. Nature 414:759-63

43 How is a single expression site activated? zThe additional spot of PolI is not the nucleolus Navarro M, Gull K. Nature 414:759-63

44 How is a single expression site activated? zThe extranuclear PolI structure is transcriptionally active control  -amanitin Navarro M, Gull K. Nature 414:759-63

45 How is a single expression site activated? active VSG inactive VSG zLabeling of the expression sites using GFP-Lac zActive, not inactive VSG expression sites colocalize with the extranuclelarPolI spot Navarro M, Gull K. Nature 414:759-63

46 Antigenic variation zOnly a single VSG gene out of ~1000 is expressed zExpression occurs out of teleomeric expression sites (the tape recorder) zTo switch genes on they are transposed into an active expression site by several mechanisms zExpression seems promoter independent zInactive DNA is modified zExpression seems to be controlled by physical association of ES with a single POL1 transcription particle per nucleus

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