The Apical Complex: a Targeted Investigation of the Molecular Functions of this Structure Essential to Apicomplexan Parasite Invasion and Replication.

Apicomplexans are a large group of single-celled pathogens that cause significant disease in humans. The most notorious member of this group causes malaria, a devastating disease in many developing regions of the world and responsible for over 500 million cases, and 0.6 million deaths per year. Other apicomplexan species cause widespread human diseases: cryptosporidiosis, a leading cause of fatal infant diarrhoea; and toxoplasmosis, which infects approximately 30% of all humans. Further, several apicomplexans infected domesticated animals causing significant loss to human food production.

All apicomplexans cause disease by growing and dividing within the cells of their human (or animal) hosts. They enter their host's cells by non-destructively forming a temporary pore in the host cell wall, through which they slide in and then reseal. By keeping the host cell alive it provides a constant source of food and nutrients to the parasite. Only once the parasites have divided to many times the original number do they burst open and kill the human cell, and then actively seek out and invade new host cells.

The key structure used by the pathogen during invasion of the host's cells is a structure called the apical complex, and this is a shared feature of all apicomplexan pathogens. The apical complex can be observed using microscopes as an internally reinforced point at the tip of the cell that acts as a cellular nozzle or syringe. At its apex is a small opening, through which it first releases sticky molecules onto the pathogens surface that help it adhere to its host's cells. Subsequently, it uses this opening to inject invasion factors directly into its host that create the invasion pore through which it enters the host. Further pathogen molecules are then released from the apical complex that enable it to feed on its host. Thus, the apical complex is at the heart of these coordinated events of host cell invasion, and is therefore an essential structure for disease formation.

Despite the importance of the apical complex, this structure remains poorly understood. Few of the molecular components are known, and this limits understanding of its architecture, assembled, and how it provides the general functions that it does. This study will use multiple approaches to identify and catalogue the molecular components of the apical complex. It will employ the apicomplexan Toxoplasma as the best developed experimental system for apicomplexans. A detailed model of the architecture of the apical complex will be generated, and using genetic tagging of individual structural components, their assembly and behaviour will be characterised in live cells, including during the invasion events. The function of the individual components, and of the apical complex as a whole, will then be dissected by selectively removing one component at a time at a genetic level. This will provide a mechanistic understanding of how the apical complex achieves the deadly tasks of host cell penetration and exploitation.

An integrated understanding of the apical complex organisation and function will provide clearer insights into how apicomplexan organisms have achieved such success as pathogens. Moreover, understanding their mechanisms of pathogenesis provides the best opportunity for strategically designing disease treatment strategies.

This project will use the genetically amenable Toxoplasma gondii as a model to study the architecture and function of the apical complex. T. gondii can be readily genetically modified by targeted recombination, allowing gene-tagging with reporters, inducible protein knockdowns by promoter swaps, and gene knockouts. Detailed phenotypic investigations of cell morphology, ultrastructure, growth, invasion and replication will be undertaken to characterise the behaviour and functions of individual apical complex structures. These procedures are all established in the Waller laboratory.

Identification of novel apical complex proteins will use three independent approaches. 1) Comparative bioinformatics using known peripheral cytoskeletal proteins from related ciliates. 2) Proximity-dependant biotinylation, using either promiscuous biotin ligase (BirA*) or modified peroxidases (APEX) fused to known apical complex proteins, to tag and identify further new components of this structure. 3) Immuno pulldowns of interaction partners of known apical complex proteins. Protein locations will be validated, and a detailed structural and behavioural model of the apical complex will be made using reporter-tagged proteins and live cell imaging, immuno-fluorescence (including with 3D-SIM super-resolution microscopy) and electron microscopy.

Functional dissection of apical complex proteins will be undertaken by inducible knockdown (tetracycline-responsive promoter replacement and controllable degradation domains), and gene knockouts using modified versions of the Cre/Lox recombinase system, and CRISPR gene excision. The effects of selective protein depletion will be measured and characterised by high-resolution microscopy and sensitive assays for parasite growth, replication and invasion processes (e.g. invasion efficiency, cell motility, regulated microneme and rhoptry protein secretion). These approaches will allow thorough interrogation of apical complex assembly and function.

This project will have broad and substantial academic impact, both in the UK and worldwide. In the long term it could contribute to health and economic improvements in developing regions of the world through aiding the development of better disease treatment strategies. Impact will also occur through training of postdoctoral and laboratory staff, postgraduate and undergraduate students, and direct engagement with the public.

The Academic Community:
This project will provide direct insight to how the apical complex provides an effective means for parasites to invade and proliferate within their host's cells, and a framework for how this structure has evolved and allowed apicomplexan parasites to be so successful. These findings will be of great interest to both the academic community studying invasion processes in apicomplexans, but will also impact broader research groups interested in host-parasite interactions and the evolution of cellular diversity. These findings will be widely disseminated via publications in open access journals, presentations at national and international conferences and invited seminars, and via my laboratory's website.

Translational work:
This work has the potential to contribute to ongoing development of intervention strategies for apicomplexan diseases. For instance, vaccine development to protect against malaria has focused on parasite molecules presented on the parasite surface during the invasion stages when the parasites are extracellular and exposed directly to the host immune system. The controlled secretion of these surface molecules occurs via the apical complex. Knowledge of apical secretion that this project will generate will contribute to understanding the presentation of these vaccine candidates. Apicomplexan parasites have proved exceptionally difficult for the development of effective vaccines, or stable reliable chemotherapeutics. New insights into these basic processes are required to direct efforts to develop and refine intervention strategies. I expect that this work will ultimately contribute to improved treatments, lessening the disease burden and consequent economic burdens borne disproportionately by the developing world.

Training:
The postdoctoral research fellow and research technician will be trained in parasite biology including advanced molecular-genetic manipulation of cells. Further, they will be trained in data analysis and rigorous scientific method, communication and presentation skills. The postdoctoral fellow will also be trained in project design and management. These skills will be highly transferable to other aspects of biomedical research either within academia or industry. Both staff will be mentored in career progression and opportunities throughout the three year project and beyond, and their training will provide ongoing impact to medical science and technology in the future.

Education:
The Waller lab provides ongoing opportunities for school and undergraduate research placements, and continuously hosts such students. Parasite-host interactions are often unexpected facets of biology to many students who typically think about organisms in isolation. This project will continue to provide original research projects to students in their formative years as potential scientists, and as ambassadors for medical parasitology amongst their peers, families and social networks.

Outreach:
Through my ongoing engagement activities with the wider public, including with school students and teachers, at university open days and through the media, I will make the relevance and outcomes of this project, and medical parasitology in general, accessible to a wide audience. The public are typically fascinated and intrigued by parasite-human interactions, and my experience is that this engagement has a profound impact on their appreciation of this area of medical science. This promotes future generations to participate in and support scientific research