Molecular and cellular dissection of kinesin motors in Apicomplexa to reveal roles in parasite proliferation

The purpose of this research is to discover how replication of intracellular parasites is driven at a molecular and cellular level. We will focus our studies on Apicomplexan parasites because they are pathogens that cause diseases that are medically and economically important. These include Babesia, Eimeria and Neospora - which affect domesticated livestock causing huge economic losses - and Plasmodia, which causes malaria in a variety of vertebrates including man and kills 584,000 humans worldwide. The life cycles of these parasites are complex and alternate between sexual and asexual replicative stages in distinct hosts. Their replicative and proliferative mechanisms are just beginning to be understood. By understanding how these parasites replicate, we hope to first, provide general insight into the mechanisms and evolution of cell replication. Secondly, we hope to uncover unique features of parasite specific replication because this knowledge promises to help in the development of novel anti-parasite drugs.

In the same way as our bodies have a skeleton that provides us with support and strength, the cells of parasites have a skeleton - called the cytoskeleton - which also provides support and structure. The cytoskeleton is involved in many important aspects of the parasite life cycle, including cellular transport, architecture and replication. Studying the cytoskeleton is important both so we can understand how normal cells work and how the parasite cytoskeleton differs from the host cells that they infect. This knowledge can be used to specifically target the cytoskeleton of pathogenic organisms with drugs that kill these organisms and prevent disease.

In particular, this project will focus on a part of the cytoskeleton called microtubules. These are long cylindrical structures that act like tracks along which molecular transport motors carry cellular cargo. The motors that we will study are called kinesins and there are many different types, all of which move along microtubules. In this project, we will be investigating kinesins thought to be important for cell replication. We want to know both how parasite kinesins use cellular fuel to move along microtubules during replication and in what cellular context they perform these roles.

The work by the Birkbeck research team will involve studying the three-dimensional structure of the cytoskeleton, because knowing what the cytoskeleton looks like will contribute to our understanding of how it works in the parasites themselves. We will use a very powerful microscope - an electron microscope - to take pictures of individual cytoskeleton molecules and then use computational analysis to combine these pictures and calculate their three-dimensional shape. A very important aspect of the proposed project is that we will also study the function of kinesins in malaria parasites themselves, in collaboration with experts in parasite cell biology at the University of Nottingham. Unlike many other such disease-causing parasites, our collaborators at Nottingham work on a species of malaria parasite (called P. berghei) that can be studied at all stages of its complex life cycle that are involved in the pathogenesis and transmission of the infection. Along with the ease with which this parasite can be genetically manipulated, this means that the contributions of different kinesins to parasite cell replication can be tested systematically.

Initial analysis suggests that the kinesins from parasites such as malaria are different compared to kinesins from other organisms, including livestock and humans. This means that we might be able to find drugs that can block parasite kinesins - and thereby parasite replication - and not human kinesins. Such drugs could be very promising for development as anti-parasite treatments. Studying the structure and function of the parasite kinesins will allow us to investigate this idea.

The goal of this project is elucidation of the mechanisms of the microtubule-based replicative machinery in apicomplexan parasites. Apicomplexa are evolutionarily diverse protozoan parasites, many of which are important pathogens affecting humans and livestock. This includes Babesia, Eimeria and Neospora, which affect domesticated livestock causing huge economic losses. The parasite life cycles are complex and alternate between sexual and asexual replicative stages in distinct hosts. Their replicative mechanisms are just beginning to be understood.
Kinesins are a large superfamily of ATP-driven, microtubule-dependent motors. They perform many essential functions in eukaryotic replication but their roles in Apicomplexa are poorly understood. The proposed work will study the molecular and cellular basis of kinesin function in apicomplexan replication in vitro and in vivo. We will reconstitute putative mitotic kinesin-microtubule complexes from purified components and biophysically characterize their interactions. The structures of microtubule-bound parasite kinesins will be studied using near-atomic (<5Å) resolution cryo-electron microscopy to 1) visualise parasite-specific features of the kinesin-microtubule interface, and 2) reveal nucleotide-dependent conformational changes that drive force generation. P. berghei is a genetically tractable model organism and an excellent system in which to study sexual and sporogonic stages in the mosquito as well as the asexual stages in vivo. This allows the link between cell division roles and post-division morphogenesis to be studied comprehensively. We will perform a systematic functional screen of all kinesins to study how their activities contribute to replication throughout the parasite life cycle. In vivo studies will also enable us to look for regulatory mechanisms and interaction partners for these motors during different replicative phases and thereby provide insight into a poorly understood aspect of parasite biology.

Who will benefit from this research?
- Pharmaceutical and veterinary medicine companies
- Livestock farmers
- The wider public
- UK economy
- Women in science

How will they benefit from this research?

The work described will lead to a greater understanding of essential mechanisms involved in proliferation/replication and cell division in parasites affecting animal and human health. Kinesin motors could be potential targets for novel anti-parasite agents that block parasite replication and our research will aid development of these. Beneficiaries from this aspect of the research would be pharmaceutical and veterinary medicine companies, as greater understanding from academic studies such as ours could lead to more effective generation of improved anti-parasite drugs and, therefore, improved sales. We would aim to engage these beneficiaries as soon as possible. This work would also ultimately lead to benefits for livestock farmers whose output could be increased by control of livestock health. In the future, novel pharmaceuticals would also benefit human health.

Science and technology is a key facet of global economy, and we will liase with UCL Business (UCLB), who work with Birkbeck researchers on technology development and intellectual property matters, and Nottingham University's IP Commercialisation Office, to maximise the impact of our discoveries. This will ultimately have benefits for the economic competitiveness of the UK. It is essential to retain talented young researchers in the UK, and the proposed research programme will provide an attractive research opportunity for excellent young scientists looking for multi-disciplinary and collaborative areas of discovery. In addition, transferable skills - such as time- and project management, presentation and collaboration - that can be applied in all employment sectors - will be acquired, particularly through transferable skills training within the Institute of Structural and Molecular Biology and at Nottingham University.

We will aim to make our research available not only to the academic community, but also to the general public. The PIs have proven track-records of public communication of science: Prof Moores was the 2006 winner of the prestigious DeMontfort medal for science communication (SET for Britain), has attended a BBSRC Media Training Day, and has presented her research to a general audience for example as part of Birkbeck Science Week 2012. Prof Tewari liaises regularly with the Press and Media office at Nottingham University to communicate her research findings to the wider community. She has presented her research on the malaria parasite at the Community Day of the University of Nottingham, to which the general public is invited, and regularly trains summer students through the Nuffield fellowship and Erasmus student programmes. The appointed PDRAs and PIs will undertake to design lab web-pages which are accessible to the general public and will seek to participate in other public understanding of science activities, for example by inviting sixth-form students to visit the lab and experience the day-to-day life of scientists. During the project period, the PIs will arrange school visits to inspire future scientists, and will continue to be involved in advancing gender equality in science, engineering and technology through involvement with the Athena SWAN programme. Prof Moores is coordinator of her Department's self-assessment team and is actively involved in mentoring and career development activities.