A high throughput model of Cryptococcus Infection and Cell Biology In Vivo for identifying host and pathogen disease determinants and new therapeutics

I am interested in how our immune system is and is not able to remove micro-organisms that can cause disease. An example of where the immune system is not able to work properly is when a person is infected with HIV (the virus that causes AIDS). Other examples include cancer, during and after organ transplantation and during treatment for rheumatoid arthritis. When the immune system is not working properly we may become ill with a micro-organism that normally would not cause a significant problem. One such micro-organism is the fungus Cryptococcus.
Infections with Cryptococcus are very unusual in those with a normal immune system but in individuals with, for example, severe HIV infection, Cryptococcus is much more common and very serious. The AIDS epidemic has resulted in a large group of people who are susceptible to Cryptococcus and it has been estimated that there are as many as 1 million cases and 600, 000 deaths due to Cryptococcus each year. Fungi that cause disease in humans are uncommon and those that cause serious disease are very rare. Fungi that cause serious disease in humans are difficult to treat because the drugs we have are often quite harmful to us as well.
As well as the Cryptococcus that causes serious disease in those with immune systems that are not working, there is a second type that has been found to cause disease in people who have apparently normal immune systems. This second type is mainly found around the equator but there have been increasing numbers outside this area.
My research is trying to understand these different areas of why Cryptococcus causes disease so that we can find better treatments. The questions I am asking in my research are: What happens to the immune system that means we are seriously infected with Cryptococcus? What makes infection with some types of Cryptococcus more severe (virulent)? Are there any new drugs that we can use to improve the way we treat Cryptococcus?
I will explore these questions using a model of Cryptococcus infection I have developed in the zebrafish. The zebrafish has a similar immune system to our own and I can observe the immune cells of living animals and see what happens during the infection. I am also going to use this model to test possible new drugs. My hope is that through this research we will have a greater understanding of how and why Cryptococcus causes disease and that this will lead to new treatments.

Cryptococcus is a fatal fungal pathogens of humans causing death through meningitis. As a major pathogen of the immunocompromised, especially AIDS patients Cryptococcus causes an estimated six hundred thousand deaths per year. By evading or manipulating phagocytes, particularly macrophages, Cryptococcus is able to cross the immune barriers that normally prevent fatal disseminated disease. Current studies focus on in vitro analysis in isolated cells, such as macrophages or in vivo studies in rodent hosts. However, such approaches are unable to capture the complex cellular events that define how this fatal disease progresses or is stopped. Therefore I have developed a new integrated model of cryptococcosis in zebrafish to simultaneously study host and pathogen factors that determine disease progression and outcome in vivo. The objectives of this model are to identify the underlying causes of cryptococcosis, molecular targets for therapeutic intervention and new therapeutics through my chemical screen. Phase 1a) I will produce quantitative data on disease progression including spatial and temporal growth kinetics. b) I will analyse the innate response to Cryptococcus infection through both imaging of macrophages and neutrophils and measuring expression and activity of molecular markers of innate immunity c) I will study the cell biology of the Cryptococcus:phagocyte interaction in vivo, including the function of the vomocytosis of Cryptococcus from macrophages. Phase 2. I will identify genetic basis of pathogen virulence by examining 4 different groups (directed mutants, 2 hyper-virulent outbreaks and clinical isolates from HIV patients) in my model. Phase 3. I will determine host immune requirements by a) depleting neutrophils and macrophages b) and inhibiting effectors of the innate immune system through knock-down and mutagenesis. Phase 4. I will perform an automated chemical screen to identify new drugs for the reduction of Cryptococcus burden during infection.

Clinical - 1. Cryptococcosis clinical management (5-20 years). My research will either directly identify new therapeutics or generate new understanding of etiology that leads to development of new therapeutics or stimulates clinical studies and trials that will result in improvement in patient outcomes. 2. Clinician research aims (1-5 years). Communication and discussion of my research will influence the research direction of clinician scientists.
NC3Rs - 1. Refinement and reduction in my adult zebrafish use (2-5 years in the first instance). 2. New virulence model of cryptococcosis (2-10 years) I will actively pursue the adoption of my zebrafish embryo model, where relevant, in replacing rodent use with Cryptococcus community. 3. Wider adoption of zebrafish for infection and immunity research (5-10 years). My fellowship will help expand the profile of the zebrafish as versatile model for infection and immunity.
Economy - 1. Chemical screen (5-20 years) My chemical screen has the potential to generate new drugs treatment of cryptococcosis and new reagents for the research community. 2. Zebrafish transgenics (3-10 years) these may have applications in future drug discovery and as reporter lines. 3. Maintaining excellence in medical research (5-20 years). 4. Providing skilled individuals for the UK work force (5-20 years)
Public engagement and outreach - 1. Infection page on CDBG 'fish for science' website (1-10years) 2. Primary school activities (1-5 years) key stage 2 activities based around measuring and microorganisms 3. A-level talks (1-5 years) Supplement to teaching on immunity looking at host pathogen interactions.
Training and career development - 1. Research group career development (1-5 years) ensuring the career development of those in my research group at all levels. 2. Applied microscopy course (1-10 years) training users of the light microscopy facility understanding and problem solving in light microscopy experiments