Insights into the opportunistic fungal pathogen Cryptococcus and neutrophilic inflammation using zebrafish models

The innate immunity provides the first line of defence against infection and inflammation. Zebrafish are a proven model for understanding the in vivo biology of infection and immunity. Here I describe how I have developed and used zebrafish models to understand three different aspects of infection and immunity: 1) The development and use of a zebrafish model of the human fungal infection Cryptococcus neoformans 2) Understanding the virulence of the hypervirulent Cryptococcus gattii and 3) The mechanisms of action of the immunosuppressive drug mycophenolate mofetil (MMF). I have established an innate in vivo model for macrophage response to Cryptococcus by injecting cryptococci into zebrafish embryos. I have developed a high-content imaging method in a zebrafish model of cryptococcosis. This approach enabled me the discovery that while macrophages are critical for control of C. neoformans, a failure of macrophage response is not the limiting defect in fatal infections. I found that phagocytosis is inhibited early in infection and that increases in cryptococcal number are driven by intracellular proliferation. Moreover, macrophages favourably phagocytose cryptococci with smaller polysaccharide capsules and that capsule size is greatly increased over twenty-four hours of infection, a change that is sufficient to severely limit further phagocytosis. I then used the zebrafish model of cryptococcosis to determine the virulence of C. gattii. I have identified a mutant in the hyper virulent strain R265 that is attenuated in vivo. The attenuation of the mutant, R265 GFP14 was further confirmed in a mouse model of infection. I analysed the interaction of macrophages and R265 GFP cryptococci in zebrafish and found that the transgenic R265 GFP was rapidly cleared. Whole genome sequences revealed that R265 GFP14 has 32 kb deletion in chromosome 1, resulting in the loss of six genes. R265 wild-type and R265 GFP14 were characterised for carbon sources utilisation. Finally, following up on colleagues’ use of my zebrafish model of cryptococcosis, I investigated the action of MMF on neutrophilic inflammation. I showed that MMF treatment resulted in neutrophil cell death by apoptosis in vivo, thereby reducing neutrophilic inflammation. Thus in this thesis, I demonstrate how I combined the study of infection and immunity to better understand diseases that cause the biggest disease burden in humans. I pioneered novel approaches to studying cryptococcosis using an experimental zebrafish model, which demonstrated for the first time how the early interactions with macrophages determined the outcome of infection. I subsequently used my model to study the virulence of an emerging pathogen, Vancouver strain R265 of C. gattii, identifying a genome region that may be important for virulence. Finally, from my cryptococcosis model a new mechanism for the immunosuppressant mycophenolate mofetil was identified in macrophages. Using my expertise in neutrophilic inflammation I was able to show that there was a second mechanism in neutrophils and this may explain the usefulness of this drug in treating chronic inflammation.