Mosaic representation of the cytoskeleton of a Trypanosoma brucei insect-form cell. Bar: 1 µm.
Cell Biology of Parasitic ProtozoaWe are very interested in the cell biology of parasites for two reasons: firstly because of the importance of understanding specific parasite biology to disease prevention; and secondly because many parasites can be used as ‘simple’ (and manipulable) organisms to investigate cell biology of eukaryotes more generally. Of particular interest to us are cellular and molecular aspects of sleeping sickness.
African trypanosomiasisSleeping sickness is a disease of Equatorial and Sub-equatorial Africa, in which it affects half million people per year (TDR). The disease is marked by progressive lethargy, recurrent fever and headaches. If untreated, it terminates in somnolence, coma and death. Treatment, however, is as old as World War II, the time when drugs effective against syphilis were discovered to also eradicate the sleeping sickness etiological agent from the body. These drugs are crude poisons and, among the only 10% affected people that receive any treatment intervention, 1:20 patients die because of its toxicity. This is why we are interested in African trypanosomes – if we wish to help in combating sleeping sickness, we must understand its causative agent, Trypanosoma brucei. It is in the biological differences found between the parasite and the host that clues for new drug development are most likely to be found.
Antigenic variation enables the parasite to evade the host adaptive immune system. Bar: 10 µm.
The flagellar pocket of African trypanosomesAfter being transmitted by the bite of a tsetse fly, African trypanosomes are able to survive extracellularly in the human bloodstream while being fully exposed to the immune system. They accomplish this feat through the expression of a series of immunologically-distinct cell surface coats. Each coat is produced from a single type of variant surface glycoprotein (VSG). Periodic switching of the single expressed VSG gene from a vast silent library enables the parasite to avoid clearance by the hosts adaptive immune response, hence prolonging infection and increasing the chances of transmission.
For this strategy of immune evasion, trypanosomes must maintain the surface coat free of many invariant surface receptors that could otherwise elicit an immune response from which the organism could not escape. Crucially, this must be done while maintaining the ability to perform the essential biological functions of nutrient uptake and secretion through the surface. This is achieved through specialisation of the plasma membrane at the base of the flagellum to create a protected invagination – the flagellar pocket – in which the invariant receptors for endocytosis are concentrated. This relatively small region of membrane is the sole site for all endo- and exocytosis performed by the parasite.
At the posterior end of the parasite the surface membrane invaginates around the flagellum to form the flagellar pocket (FP). A collar closes the pocket making it a protected compartment. CCP, clathrin-coated pit; BB, basal body; K, kinetoplast. Bar: 250 nm.
Discovering proteins at the the host-parasite interfaceBecause of its major role in pathogenicity, it’s inhibitory that very few flagellar pocket proteins have been identified to date, severely limiting functional studies, and hampering the development of potential treatments. Our most recent research, in Cambridge and Nottingham, has used an integrated biochemical, proteomic and bioinformatic strategy to identify surface components of the human parasite Trypanosoma brucei. This ‘surfeome’ contains previously known flagellar pocket proteins as well as multiple novel components, and is significantly enriched in proteins that are essential for parasite survival. Validation by cellular localisation has shown that the majority of surfeome constituents are bona fide surface-associated proteins and, as expected, the majority present at the flagellar pocket. Moreover, our work represents the largest systematic analysis of trypanosome surface molecules to date and provides further evidence that the cell surface is compartmentalised into three distinct domains with free diffusion of molecules in each, but selective, asymmetric traffic between.
Definition and validation of a bloodstream-form Trypanosoma brucei cell surface proteome. 25 ESPs (‘enriched in surface proteome’) were localised by GFP-tagging (right panel; the flagellar pocket is indicated by yellow arrowhead).
3D tomographic model of the African trypanosome flagellar pocket (seen in white at the centre of the image). Other organelles and structures include the microtubules of the flagellum (orange), pellicule (light blue) and rootlet (dark blue), the nuclear envelope (pink), Golgi apparatus (yellow) and endoplasmic reticulum (green).
Endocytosis and pathogenicityDuring my postdoc in Oxford, I studied how the flagellar pocket is organized and compartmentalized to perform its critical functions. Some of this research investigated the cytoskeletal boundaries that demarcate the flagellar pocket membrane domain. One question raised by this was how macromolecules that are essential to parasite growth cross these barriers and gain access to the pocket lumen, such that they can be internalised by the cell. The answer to this question came when we slowed down the trypanosome endocytic capacity by placing the parasite at 0℃. Under these cold conditions, trypanosomes continue to swim, and macromolecules are able to enter the pocket, but internalization is blocked. When this is done, markers of both receptor-mediated and fluid-phase endocytosis given to cold trypanosomes are concentrated in a continuous 'channel' connecting the outside of the cell to the flagellar pocket interior. Importantly, this channel traverses those boundaries that protect the pocket environment, and we believe that this represents the major route of transport into the organelle - and hence into the cell.
Cytoarchitecture critical for parasitismThe architectural arrangement of the cytoskeleton and other organelles is highly polarised around the flagellar pocket. The inheritance of this architectural arrangement through the trypanosome cell cycle was also investigated while in Oxford, because the duplication timings and rotational movements that direct the correct inheritance of a functional flagellar pocket to the next generation of daughter cells are likely to be critical to disease progression.
A 3D tomographic reconstruction (far left) and segmentation models of the Trypanosoma brucei cell interior. Electron tomography has revealed a cytoarchitecture that is critical for the success of this parasite. Bar at top left: 250 nm.