Coming soon.
Since VSG (variant surface glycoprotein) was first discovered in the 1970’s, many researchers have believed it to be the only antigen on the cell surface of infective-form Trypanosoma brucei. With approximately 10 million copies of a single VSG type enshrouding the parasite, this thick surface coat enables African trypanosomes to survive extracellularly in the human bloodstream while being fully exposed to the innate immune system. Whilst periodic switching of the expressed VSG gene from a vast silent library enables the parasite to avoid clearance by the host’s adaptive immune response, hence prolonging infection and increasing the chances of transmission.
What led you to study cell surface proteins?
This strategy of immune evasion must be sustained whilst the parasite performs all of the cell surface functions that are essential for survival, such as nutrient uptake, secretion and cell signalling. The issue is that we don’t know the identity of most of the proteins carrying out those biological processes. By the mid 2000’s, they were poorly characterized, but included a transferrin receptor, a hexose transporter, a repertoire of adenylate cyclases and two families of type I transmembrane domain proteins.
This had been something that piqued my interest early in my postdoc (Oxford, 2005-2009), when I was using 4D electron tomography to reconstruct the membrane and cytoskeleton architecture and cell polarity that are critical for parasitism. It just seemed to me to be important to determine if the surface protein discovery had been completed. Because for an extracellular parasite that has invested in the strategy of antigenic variation, knowing the invariant antigens seemed like a worthwhile (but probably very difficult) goal.
It was in 2007, I think, while reading a paper in a pub that, on a whim, I thought of an idea for how to identify the proteins on the surface of the parasite. When I moved to Cambridge to start my independent career, I put that idea into practice. It involved the chemical modification of live cells with an amine-reactive derivative of fluorescein. That was pretty novel at the time: cell surface proteomics had frequently used the biotin-avidin based system to isolate plasma membrane proteins. But the specificity of this approach is often compromised by high background and, in the case of trypanosomes, made worse by the fact that the parasite biotinylates its own endogenous proteins.
I picked fluorescein for several reasons: firstly because it’s cell-impermeable, ensuring that only surface membrane proteins from intact cells would be labelled by covalent modification of accessible lysine residues. Secondly because I knew from my PhD work that very good anti-fluorescein antibodies are commercially available, and antigen-antibody columns can be washed to high stringency. Fluorescein also has an advantage that it can be followed visually or by fluorimetry during preparations. So, following chemical labelling, labelled trypanosomes were solubilized and fluoresceinated surface proteins purified by affinity chromatography. The purification method was further optimized by a VSG depletion step to increase sensitivity of detection of less abundant surface proteins, and on-column enzymatic removal of N-glycans to improve mass spectrometry identification of glycosylated surface proteins.
It paid off. Using sensitive, semi-quantitative mass spec, bioinformatics and cell biology, we validated a cell surface protein atlas (we also call it ‘surfeome’) that contains 175 components. Since then, cell surface biology has been the focus of my research, as I’m keen to explore the biological functions and possible applications of novel surface receptors and transporters.
Why is the discovery of surface proteins important?
Cell surface proteins are major targets of biomedical research because of their exposure to the extracellular environment and, as such, accessibility for pharmacological intervention. The surfeome defined 175 surface-exposed proteins, 110 of which are invariant (encoded by a single-copy gene) and mostly parasite specific (not found in the host). This demonstrate its potential as a source of drugable targets, opening the door to new strategies for disease treatment and control.
What are the main areas of focus for your group?
What intrigues me now are the biological function of the novel receptors, transporters and channels we discovered, and relevant mechanisms, such as their role in scavenging macromolecules from the host blood, communicating between individuals in the parasite population, or modulating infection and immune response.
We are also interested in basic biology of membrane proteins, particularly with regards to protein sorting and membrane barriers. The surfeome atlas supports a model whereby trypanosome surface organization is determined by control of access to any of three membrane domains on the plasma membrane. Diffusion within each domain is essentially free for most surface proteins. However, selective diffusion barriers exist between these domains, and our work has so far indicated that these barriers must be asymmetric. This model raises major questions with regards to the mechanisms underlying protein sorting and retention in these cells, and elucidating such mechanisms in any eukaryote remains a formidable challenge.
What are the potential biomedical applications of your findings?
Over 1 billion people in the world today suffer from one or more neglected tropical disease (NTD). These diseases are “neglected” primarily because they attract disproportionately low R&D investment (there is little commercial incentive to develop drugs for a patient population that can’t afford them). As such, there has been a big push toward drug repurposing. Antibody-drug conjugates (ADCs), for example, are effective ways to repurpose cancer immunotherapeutics, and rely on knowledge of validated, surface-exposed receptors. Colleagues in Bristol and Cambridge are working with chemical manufacturers to develop antibodies against such receptors as delivery routes for ADCs which kill trypanosomes. Our cell surface protein atlas offers many novel targets for this approach.
Equally, vaccinology pipelines in collaborators’ labs can test 10s of vaccine candidates in a relatively rapid manner. What is required to make effectively use of these pipelines or the development of new diagnostics are sets of validated, surface-exposed, parasite-specific invariant antigens. The infective-form surfeome has been priming these studies, which could realistically have a meaningful impact on disease control.
Ultimately, I hope our research will make an impact both on the scientific community and on society.
Coming soon.
As parasites cross new borders, and livestock diseases become endemic in new areas, the need for disease models is on the rise. For animal African trypanosomiasis, most of our knowledge is centred on one parasite species Trypanosoma brucei which does not capture all biological phenomena relevant to disease. The spread of animal African trypanosomiasis, and research efforts to control it, are particularly driving the demand for new experimental systems.
Researchers in the lab have led responses to this demand. Initially focusing on T. congolense, the major agent of animal African trypanosomiasis, we have harnessed the advancements in genetic technologies and genome sequencing to develop a model and accompanying tools that allow direct and regulatable transgenesis of multiple types in the pathogenic stage of this species. We have also built a system to allow inducible RNA-interference (RNAi), demonstrating knockdown of multiple essential and non-essential genes; and describe a stable cell line which enables transfection efficiencies compatible with the production of genome-scale high-complexity libraries.
Advancing therapeutic discoveries. Importantly, we show that genetically-modified forms of T. congolense are still infectious and can be used in animal models of disease. We have created stable high-bioluminescence lines that enable monitoring the course of infections by in vivo imaging. These tools have significantly increased the efficiency of on-going vaccinology and drug development studies in collaborators’ labs.
Accelerating research. The work was worth it, and changed T. congolense from a technically challenging organism to a routine model for functional genetics (read more in Awuah-Mensah et al. 2021 PLoS Pathogens). One of the best rewards we get is the very early adoption of our model in fundamental biology, drug evaluation, vaccinology and disease pathology studies.
Aiming for higher ambition. We have now turned our attention to the second most important livestock disease agent, T. vivax, for which growth of infective forms outside of animals doesn’t exist (let alone the means to genetically modify them). We have partnered with colleagues in Edinburgh and Glasgow to define an optimal medium composition to the particular nutritional requirements of the infective form of T. vivax, in a manner similar to our recent achievements in T. congolense. Our goal is to develop sustained long-term culturing of the parasite blood stage in vitro.
We will also use our expertise in engineering specially designed tools to genetically modify infective-form T. vivax, allowing us and others to begin to address some of the fundamental questions about the biology of this significant parasite.
Fighting livestock disease to ensure food security in some of the poorest communities in the world highlights the importance of collaborative work. One element of this project has been the establishment of a formal collaboration with the UK-based product development company GALVmed, in hope to speed up drug discovery against this devastating disease.