NON-INVASIVE IMAGING OF HOST RESPONSE IN A ZEBRAFISH MODEL OF CANDIDEMIA

 NON-INVASIVE IMAGING OF HOST RESPONSE IN

A ZEBRAFISH MODEL OF CANDIDEMIA

By Kimberly M. Brothers

Thesis Advisor:  Dr. Robert T. Wheeler

A Lay Abstract of the Thesis Presented

 in Partial Fulfillment of the

Requirements for the Degree of

Doctor of Philosophy

(in Biomedical Sciences)

August, 2012

Human innate immunity is the first line of defense that prevents microbial infection and plays a critical role in controlling opportunistic infections.  Candida albicans is a ubiquitous fungus that normally associates asymptomatically with the gastrointestinal tract and skin of approximately 50% of humans, but causes invasive infections in immunocompromised patients.  This fungus is responsible for 80% of hospital-acquired bloodstream fungal infections and approximately one-third of these C. albicans infections kill the patient.  C. albicans can switch in vitro between budding yeast and filamentous forms to evade phagocytosis by macrophages and neutrophils, to puncture macrophage membranes, to escape detection, and to penetrate epithelial cells. Although it is not known how C. albicans interacts with immune cells in human infection, mouse infection studies suggest the ability to switch from one form to another is required for virulence.  A key question is how these shape changes occur during active infection, and unfortunately it is not possible to image fungi and immune cells non-invasively during infection of a mouse. To overcome this difficulty, we have developed a transparent zebrafish model of bloodstream C. albicans infection to determine how fungi and immune cells interact during infection. We show that infection of zebrafish larvae with as few as 10 yeast cells causes lethal disseminated disease that shares important traits with disseminated disease in mammals: dimorphic fungal growth, dependence on hyphal growth for virulence, and dependence on the phagocyte oxidase for immunity.  Using fluorescently marked immune cells and fungi, we find that within macrophages, phagocytosed yeast can remain viable and even divide without germinating.  In a similar fashion, although neutrophils kill yeast, the vast majority of Candida cells within neutrophils are viable.  We visualized the cellular impact of loss of phagocyte oxidase activity on both host and pathogen, finding that it mediates the vast majority of oxidative stress in fungi while limiting both fungal proliferation and filamentous growth. This model holds additional promise in the long-term for high-throughput screening of candidate virulence factors and novel anti-fungal drugs.