Research
Pathogen cell surface
In mammalian hosts including man, the entire extracellular surface of the cell is covered by a molecular monolayer of a single protein, the variant specific glycoprotein (VSG). The VSG monolayer functions at two levels, first by undergoing antigenic variation it permits a trypanosome population to persist in the bloodstream. Second, it forms a physical barrier to protect underlying invariant cell surface proteins, such as the ISGs, from the host immune system. A large number of VSGs with very different primary sequences are able to perform this function as the tertiary structure is highly conserved.The VSG N-terminal domain has an elongated tertiary structure, largely defined by two long alpha helices, with the long axis perpendicular to the cell surface. We have recently solved the structure of further C-terminal domains and a model of the C-terminal domain of VSG ILTat1.24 (surface) and the glycosylphosphatidylinositol oligosaccharide (sticks) arising from Nicola Jones’ work is below.
Are all VSGs equivalent or are some more favoured than others and if so why? We are currently sequencing VSG cDNAs from field isolates to determine whether new VSGs are present or whether the same VSGs start recurring. This approach is helped by using genetically isolated trypanosomes such as T. evansi from Malaysia.
Invariant cell surface proteins and receptors
A range of invariant surface proteins, including ISG75 and ISG65 have the same alpha helices as the VSG N-terminal domain, this infers that they also have a similar elongated structure. These observations pose two questions that we are currently trying to answer.
What is the function of the ISGs? This is being addressed through the generation of tetracycline-inducible RNA interference lines that are able to ablate ISG expression and through the production of null mutants.
How are ISGs integrated into the VSG monolayer? We are aiming to produce a dynamic model of the VSG monolayer using the known dimensions of the VSG and the most accurate values for the density of VSG dimers on the cell surface that will allow testable predictions such as the degree of penetration by molecules of a known size. Currently, only the structure of the VSG is known and we are aiming to solve the structure of one of the ISG family members and recombinant ISG64 in sufficient quantities for crystalisation trials.
Most isolates of Trypanosoma brucei are unable to infect humans as they are lysed by a factor present in the high density lipoprotein. However, in T. b. rhodesiese the expression of SRA is sufficient to confer resistance to human serum. SRA is also related to VSGs but contains a large internal deletion of ~120 amino acids. We have modelled the structure of SRA using the VSG N-terminal domain structure. This has been highly successful in suggesting future experimental approaches, below is a comparison of models of a VSG and SRA. We are currently aiming to solve the structure of SRA binding its ligand human apolipoprotein L-I.
The regulation of genes transcribed by RNA polymerase II in trypanosomes
Transcription of protein coding genes in kinetoplastid protozoa is unusual as arrays of genes are transcribed polycistronically and individual mRNAs processed from the precursor RNA by trans splicing of a capped mini-exon onto the 5' end and cutting and polyadenylation at the 3' end. The transcription of most genes is constitutive and therefore mRNA levels are determined post-transcriptionally.
We have been investigating the regulation of mRNA levels using two developmentally regulated genes: GPI-PLC (which encodes the glycosyphosphatidylinositol-specific phospholipase C) and ISG65 (which encodes invariant surface glycoprotein 65). The reason for using developmentally regulated genes as a model is that they provide a ready assay for genes that are necessary for mRNA turnover. Both mRNAs are not normally present in insect stage (procyclic) trypanosomes and any disruption of pathways required for the rapid turnover of these mRNAs results in their appearance in procyclic cells. What is the basis for the developmental regulation of mRNA levels? The approach we are using is to make mutated versions of the genes that have lost developmental regulation.
Regulation of the overall rate of gene expression
Heat shock has been used to investigate the response of mRNA metabolism to growth arrest and stress. Heat shock causes a rapid and large decrease in polysome abundance, with a consequent reduction in translation and a rapid decrease in total mRNA levels. Both polysome disassembly and loss of mRNA are selective; most mRNAs decrease in level, but ~300 are unaffected or actually increase. The decrease in mRNA levels is probably caused by a combination of reduced transcription and maturation combined with accelerated turnover. Heat shock provides an excellent system for studying changes in mRNA on growth arrest and on return to proliferation, the changes are rapid, reversible and selective.
In procyclic trypanosomes, the heat shock response is initiated by temperature of ~41oC; within 15 minutes there is a large decrease in the number of polysomes per cell and a selective reduction in the translation of most but not all mRNAs. The reduction of translation is not dependent on phosphorylation of eIF2A on T169 and the mechanism of down regulating initiation of translation during heat shock remains unclear. Within 30 minutes, heat shock stress granules appear in the cytoplasm at the cell periphery, the number of P-bodies increases and the XRNA-containing focus at the posterior pole of the cell increases in size. The XRNA-containing focus at the posterior pole is novel and no equivalent structure has been reported previously. At the same time the total mRNA in the cell decreases so by 1 hours the amount per cell has been halved. The loss is selective, some mRNAs are not affected. The decrease in mRNA levels is probably caused by a combination of reduced transcription and maturation combined with normal turnover and, in some cases, accelerated turnover. The appearance of heat shock stress granules and increased numbers of P-bodies are co-incident with the alterations in mRNA metabolism and it can be speculated that they operate by providing different compartments for mRNA within the cell each of which can act as a gateway for a particular fate. The behaviour of a particular mRNA will be determined by how it partitions between the different compartments. The mechanism that regulates this partitioning is central to the understanding how mRNA levels are regulated and, along with the regulation of eIF2A activity, is the main focus of our research.
Publications
Key publications:
Recently
Peacock L, Ferris V, Sharma R, Sunter J, Bailey M, Carrington M, Gibson W. (2011) Proc Natl Acad Sci U S A. 108:3671-6. Identification of the meiotic life cycle stage of Trypanosoma brucei in the tsetse fly.
Kramer S. Carrington M (2011). Trends in Parasitology 27:23-30. Trans-acting proteins regulating mRNA maturation, stability and translation in trypanosomatids.
Schwede A, Jones N, Engstler M, Carrington M (2011). Mol. Biochem. Parasitol. 175:201-4. The C-terminal domain of the Trypanosoma brucei VSG is not accessible to antibodies
Kramer S, Queiroz R, Ellis L, Hoheisel JD, Clayton C, Carrington M (2010) J. Cell Sci. 123, 699-711. The RNA helicase DHH1 is central to the correct expression of many developmentally regulated mRNAs in trypanosomes.
Schwede A, Carrington M (2010). Parasitology 137: 2029-2039. Bloodstream form trypanosome plasma membrane proteins: antigenic variation and invariant antigens.
Kramer S, Kimblin N, Carrington M (2010) BMC Genomics May 5;11:283. Genome-wide in silico screen for CCCH-type zinc finger proteins of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major.
Sharma R, Gluenz E, Peacock L, Gibson W, Gull K, Carrington M (2009). Trends in Parasitology 25, 517-524.The heart of darkness – Trypanosoma brucei in the tsetse fly.
Field M, Carrington M (2009). Nature Rev. Microbiol. 7:775-786. The trypanosome flagellar pocket.
Thomson R, Molina-Portela P, Mott H, Carrington M Raper J. (2009). Proc. Natl. Acad. Sci. 106, 19509-19514. Gene therapy with baboon trypanosome lytic factor eliminates both animal and human infective African trypanosomes.
Kramer S, Queiroz R, Ellis L, Hoheisel JD, Clayton C, Carrington M (2009) J. Cell Sci. 123,699-711. The RNA helicase DHH1 is central to the correct expression of many developmentally regulated mRNAs in trypanosomes.
