Cryptococcal meningitis is an often fatal disease which rose to prominence with the advent of the HIV epidemic. Currently, nearly one million HIV patients are affected per year, two thirds of whom do not survive. It is known from animal models that cryptococcal spores and yeast initially establish infections in the lung, but subsequently disseminate to the brain to cause meningoencephalitis. There is evidence for multiple intracellular and extracellular pathways by which this occurs, but to date it has not been possible to visualize the full process.
We have developed a model using larval zebrafish which allows direct visualization of pathogenesis from the initial encounter with phagocytes to CNS-specific spread. Purified spores expressing nuclear eGFP (enhanced green fluorescent protein) are microinjected into the caudal vein or pericardium of 48 hour zebrafish larvae. Individual larvae can be observed repeatedly over several days via video microscopy. Germinating yeast continue to express eGFP in their nuclei, and can be tracked and quantified. Using transgenic zebrafish with fluorescently labelled macrophages, we are able to monitor the cellular events in high resolution over time.
As seen in other animal models, spores are readily phagocytosed by macrophages, regardless of injection site. Many are able to survive and germinate, as indicated by changes in morphology.
Germinated yeast can then escape from the phagocytes and disseminate to the CNS via the blood stream.
After close interactions with the vasculature, they are found in the perivascular spaces of the brain, once again surrounded by macrophages. This localization is strikingly similar to that seen in human pathology.
Observations with this model provide for the first time a picture of cryptococcal infection from phagocytosis of spores all the way to dissemination to the brain. Germination into a form capable of specific dissemination to the CNS takes place inside macrophages. Once the germinated yeast reach the bloodstream they interact with endothelial cells and colonize the brain. With the available host and cryptococcal mutant strains and other molecular tools, this model promises to be very useful for probing the molecular mechanisms of cryptococcal meningitis.
J. M. Davis,
C. Hull, None
A. Huttenlocher, None