Gloria Rudenko's Lab Molecular mechanisms mediating immune evasion in the African trypanosome Trypanosoma brucei Dr. Gloria Rudenko Wellcome Senior Fellow in the Basic Biomedical Sciences Reader in Molecular Microbiology Peter Medawar Building for Pathogen Research University of Oxford

Our group is located in the Peter Medawar Building, but we have academic affiliations with the Department of Biochemistry.

Introduction

African trypanosomes

The African trypanosome Trypanosoma brucei causes African Sleeping Sickness in humans, which is endemic to subSaharan Africa and is spread by tsetse flies.  Trypanosomiasis is invariably fatal in humans if left untreated.  Trypanosomes are flagellated unicellular eukaryotes with a genome size about three-fold that of bakers yeast.  Trypanosomes are easily cultured in the laboratory both in vitro and in vivo, and are straightforward to genetically modify. In addition, RNA interference (RNAi) is a highly effective means of inducibly interfering with gene function. Trypanosomes provide a very manipulable experimental system for investigating immune evasion and pathogen-host adaptation.

Bloodstream form T. brucei among red blood cells

Antigenic variation in African trypanosomes

Trypanosomes are unusual parasites in that they multiply extracellularly within the blood of the mammalian host  where they remain fully exposed to continuous immune attack.  Their success as a pathogen is a consequence of their highly sophisticated strategy of antigenic variation of a Variant Surface Glycoprotein (VSG) coat.  As trypanosomes multiply in the bloodstream, the host eventually mounts an antibody response against trypanosomes expressing a given VSG.  However, as trypanosomes can switch to new VSG coat variants not recognised by host antibodies, these can evade antibody mediated lysis and form the next wave of infection.  As trypanosomes have up to a thousand antigenically distinct VSGs, a chronic infection can be mounted lasting for years.  The bloodstream form trypanosome is therefore continuously balancing on the brink of destruction.  It is only through its ability to change its surface coat that it can survive.

Successive waves of VSG variants during a chronic infection

Trypanosomes multiply in the blood of the host until an antibody response results in lysis of recognised variants.  Switched variants have a selective growth advantage until a host antibody response is mounted against these too.

Switching Variant Surface Glycoprotein gene expression

The active VSG gene is located in one of about 20 VSG expression sites located at telomeres (chromosome ends).  VSG expression sites are polycistronic (multiple gene containing) transcription units.  In addition to the telomeric VSG gene, there are a number of families of expression site associated genes, most of unknown function.  It is thought that these expression site associated genes could play a role in adaptation of the trypanosome to life in different species of mammalian host.

VSG expression sites are telomeric transcription units

Schematic of a typical Variant Surface Glycoprotein (VSG) gene expression site transcription unit.  Expression sites are located at telomeres (telomere repeats indicated with triangles).  The promoter is indicated with a flag, and transcription with an arrow.  The coloured boxes indicate different families of expression site associated genes.

Switching the active VSG can involve one of three predominant mechanisms.  Individual trypanosomes have hundreds (up to 1000) silent VSG genes in tandem arrays.  DNA rearrangements can move a previously silent VSG gene into the active VSG expression site transcription unit.  The most important DNA rearrangement mechanism during a chronic infection is duplicative gene conversion.  This results in a previously silent VSG gene being copied into the active VSG expression site, replacing the old copy.  Alternatively, a reciprocal exchange can occur between two telomeres.  Lastly, a VSG switch can be mediated by transcriptional control.  In this case the trypanosome silences one VSG expression site, and activates a new one.

Research

Molecular mechanisms mediating immune evasion in African trypanosomes

Our main aims are to dissect the molecular mechanisms mediating trypanosome immune evasion, both from host antibodies and from complement.  In addition, we would like to understand how the mutually exclusive transcription of VSG expression sites is controlled.

VSG is monitored in the cell cycle

First of all, we are investigating the role of the protective VSG coat in bloodstream form trypanosomes.  The VSG coat is composed of a dense layer of VSG molecules that cover the entire bloodstream form trypanosome shielding invariant surface molecules like receptors.  This VSG coat can be considered analogous to a fur coat, with the individual VSG molecules attached to the cell surface analogous to hairs.  We attempted to make "naked" bloodstream form trypanosomes, by blocking VSG synthesis using tetracycline inducible VSG RNA interference (RNAi).  Surprisingly, there was no significant depletion of VSG within the coat.  Instead, blocking VSG synthesis triggers a rapid and specific cell-cycle arrest.

Induction of VSG RNAi results in a growth arrest before cell division

Microscopic analysis of T. brucei cell cycle progression during the course of tetracycline inducible VSG RNAi (indicated in hours).  Cells have one nucleus (N) and kinetoplast (K).  As the cells undergo S phase, first the kinetoplast divides and then the nucleus undergoes mitosis. From Sheader et al. (2005) PNAS  102: 8716-8721.

Using scanning electron microscopy in collaboration with the Gull laboratory, normal cells immediately before cell division were compared with those arrested before cell division after the induction of VSG RNAi.  Cells that have arrested after the induction of VSG RNAi are shorter and broader than normal precytokinesis cells, although they have the same volume.  This indicates that the cells have changed their shape in the face of a restriction on the protective VSG coat, possibly as a means of minimising their surface area to volume ratio.  Secondly, the cells appear to have arrested at a very precise stage of the cell cycle with parallel flagellar pockets (invagination where the flagellum exits the cell).

Cells stalled after VSG RNAi arrest at a very precise cell cycle stage
	0h VSG RNAi
	24h VSG RNAi
Scanning electron microscopy of a normal bloodstream form trypanosome immediately before cell division (above).  Below is a trypanosome that has stalled immediately before cell division after the induction of tetracycline inducible VSG RNAi. Scanning electron microscopy performed by Sue Vaughan and Keith Gull (Dunn School of Pathology, Oxford). From Sheader et al. (2005) PNAS  102: 8716-8721.

All of these results are compatible with VSG being monitored during the cell cycle of African trypanosomes.  We propose that we have discovered a novel checkpoint that is triggered in the absence of VSG synthesis.  This results in a rapid and specific cell cycle block preventing what would otherwise be disastrous dilution of the protective VSG coat.  We are presently investigating the mechanism behind this cell cycle arrest.

Antigenic variation studied using VSG RNAi in the absence of immune selection

After performing VSG RNAi, cells undergo a cell cycle arrest and eventually die.  However, eventually trypanosomes emerge which escape this arrest.  Analysis of these revertant cells shows that most have switched to a new VSG coat.  As different VSGs typically have very different amino acid sequences, the switched trypanosomes can escape the VSG RNAi operating against the old variant.

Induction of VSG221 RNAi selects for switches to new VSG variants

Western blot analysis of protein lysates from T. brucei where VSG221 RNAi was induced for increasing lengths of time.  The VSG221 expressing population disappears, and is replaced by variants which have switched to different VSGs, including VSG1.8.  BIP is used as a loading control. Modified from Aitcheson et al. (2005) Mol Micro 57: 16-8-1622.

This allowed us to develop a method using VSG RNAi rather than immune selection to select for VSG switch variants.  This allows a very rapid and efficient means for generation of many hundreds of VSG switch variants entirely in vitro.  Various phenotyping and genotyping screens allow us to categorise the different VSG switch variants according to the mechanism that must have been used to mediate the switch.  We can identify the new VSGs that were activated by sequencing the VSG cDNAs amplified by RT-PCR.  This provides an extremely powerful means for studying VSG switching in a very experimentally amenable fashion. 

Reproducible preferential hierarchy of VSG activation after selection using VSG RNAi

Characterisation of VSG switch variants generated in different VSG RNAi experiments.  The newly activated VSG was identified by sequencing VSG cDNA.  The percentage of switch variants expressing a given VSG is indicated with bars.  We found a reproducible preferential hierarchy in which VSGs were activated. From Aitcheson et al. (2005) Mol Micro 57: 16-8-1622.

Transcriptional control of VSG expression sites

Last, we are trying to understand how VSG expression sites are turned on and off in a mutually exclusive fashion in bloodstream form T. brucei. We are attempting to identify genes involved in VSG expression site silencing and control. In addition, we would like to understand how all of the VSG expression sites in insect form T. brucei are downregulated. We have discovered the first gene shown to play a role in downregulation of VSG expression sites in T. brucei (TbISWI). TbISWI is a trypanosome member of the ISWI family of chromatin remodeling proteins. TbISWI plays a role in expression site silencing in both bloodstream and insect form trypanosomes.

Blocking synthesis of TbISWI using RNAi leads to derepression of silent VSG expression sites as monitored using flow cytometry of a GFP gene inserted into a silent VSG expression site. At the bottom are FACs traces of GFP derepression measured in the FITC channel after the induction of TbISWI RNAi for 80 hours. From Hughes et al. (2007) EMBO J 26: 2400-2410.

TbISWI-GFP fusion protein located in the nucleus of trypanosomes at different stages in the cell cycle. The number of nuclei (N) or kinetoplasts (K) are indicated. From Hughes et al. (2007) EMBO J 26: 2400-2410.

We would now like to determine which genomic sequences TbISWI binds, and if TbISWI is involved in silencing other areas of the trypanosome genome in addition to the silent VSG expression sites. In addition, we would like to determine the protein partners of TbISWI in order to dissect how the transcriptional silencing mediated by TbISWI operates.

Future plans

We would like to dissect the molecular machinery providing the link between VSG synthesis and progression through the trypanosome cell cycle.  Does the cell cycle arrest after a block in VSG synthesis indeed play a protective role for the trypanosome?  We would like to better understand how the dense VSG coat protects the trypanosome from complement mediated lysis.  We have developed a very amenable experimental system for studying VSG switching completely in vitro.  What are the molecular mechanisms behind the preferential hierarchies of VSG switching?  Lastly, we are very interested in VSG expression site control.  VSG expression sites can be silenced in both bloodstream and insect form life-cycle stages.  How are the VSG expression sites silenced in insect form trypanosomes where VSG expression is unnecessary?  In contrast in bloodstream form trypanosomes, how does the counting machinery behind the mutually exclusive transcription of one VSG expression site operate?  Hopefully, the answers to these questions will give us a better handle on this slippery pathogen.

Recent Publications

1. Young R, Taylor JE, Kurioka A, Becker M, Louis EJ, and Rudenko G. (2008) Isolation and analysis of the genetic diversity of repertoires of VSG expression site containing telomeres from Trypanosoma brucei gambiense, T. b. brucei and T. equiperdum. BMC Genomics 9: 385.
2. Hughes K., Wand M, Foulston L, Young R, Harley K, Terry S, Ersfeld K and Rudenko G (2007) A novel ISWI is involved in VSG expression site downregulation in African trypanosomes. EMBO J 26: 2400-2410.
3. Rudenko G. and Taylor J.E. (2006) Switching trypanosome coats: what's in the wardrobe? Trends in Genetics 22: 614-620.
4. Rudenko G. (2005) Maintaining the protective variant surface glycoprotein coat of African trypanosomes. Biochem Soc Trans. 33: 981-2.
5. Aitcheson, N., Talbot, S., Shapiro, J., Hughes, K., Adkin, C., Butt, T., Sheader, K. and Rudenko G. (2005) VSG switching in Trypanosoma brucei: antigenic variation analysed using RNAi rather than an immune system. Molecular Microbiology 57: 1608-1622.
6. Sheader, K., Vaughan, S., Minchin, J., Hughes, K., Gull, K. and Rudenko G. (2005) Variant Surface Glycoprotein RNAi triggers a precytokinesis cell cycle arrest in African trypanosomes. Proceedings of the National Academy of Sciences USA 102: 8716-8721.
   (Covered in Editors' choice: Science (2005) 308: 1843).
7. Becker M., Aitcheson N., Byles E., Wickstead, B., Louis E. and Rudenko G. (2004) Isolation of the repertoire of VSG expression site containing telomeres of Trypanosoma brucei 427 using Transformation Associated Recombination in yeast. Genome Research 14: 2319-2329.
8. Sheader K., te Vruchte, D. and Rudenko G. (2004) Bloodstream form specific upregulation of VSG expression sites and procyclin in Trypanosoma brucei after inhibition of DNA synthesis or DNA damage. Journal of Biological Chemistry 279: 13363-13374.
9. te Vruchte D., Aitcheson N. and Rudenko G. (2003) Downregulation of Trypanosoma brucei VSG expression site promoters on circular Bacterial Artificial Chromosomes. Molecular and Biochemical Parasitology 128: 123-133.

Contact details

Dr. Gloria Rudenko
The Peter Medawar Building for Pathogen Research
University of Oxford
South Parks Road
Oxford OX1 3SY
United Kingdom

Tel: +44 1865 281 548
FAX: +44 1865 281 894
E-mail: gloria.rudenko@medawar.ox.ac.uk

If you are interested in our research, and would like to inquire about a position in our laboratory, please send me an E-mail with your CV. I can let you know if vacancies (post-doc or research assistant) are currently available, or will be available within the immediate future. In addition, if you would like to inquire about a PhD position or other research project within the lab please E-mail for more details.

Lab members

Contact

Lab office Tel: +44 1865 281 540

Viola Denninger viola.denninger@bioch.ox.ac.uk	Post-doc Wellcome Trust MSc. University of Tuebingen, Tuebingen, Germany PhD University of Tuebingen, Tuebingen, Germany
Megan Lindsay megan.lindsay@bioch.ox.ac.uk	Post-doc Wellcome Trust BSc. Loyola College in Maryland, Baltimore, MD, USA PhD Johns Hopkins University, Baltimore, MD, USA
Tara Stanne tara.stanne@bioch.ox.ac.uk	Post-doc Wellcome Trust BSc. Mount Allison University, New Brunswick, Canada PhD University of Göteborg, Göteborg, Sweden
Manish Kushwaha manish.kushwaha@medawar.ox.ac.uk	PhD student Inlaks Foundation, Clarendon award BSc. University of Delhi, New Delhi, India MSc. Jawaharlal Nehru University, New Delhi, India
Mani Narayanan mani.narayanan@queens.ox.ac.uk	PhD student Clarendon award, Queens College BSc. University of Delhi, Delhi, India MSc. University of Oxford, Oxford, England
Nadina Vasileva nadina.vasileva@bioch.ox.ac.uk	PhD student Krebs Memorial Scholar - Biochemical Society, BBSRC BSc. (Hons) University of Pretoria, Pretoria, South Africa
Alexander Fullbrook alexander.fullbrook@bioch.ox.ac.uk	Post-graduate research assistant Wellcome Trust BSc. University of Edinburgh, Edinburgh, Scotland, UK

Links

• University of Oxford
• Department of Biochemistry
• The Wellcome Trust
• WHO trypanosomiasis page
• Sanger Centre- Trypanosome genomes
• Trypanosome telomeres