Date of Award

Fall 12-2018

Level of Access

Open-Access Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

Advisor

Robert Wheeler

Second Committee Member

Julie Gosse

Third Committee Member

Clarissa Henry

Additional Committee Members

Roger Sher

John Singer

Abstract

Innate immunity has developed elegant processes for the detection and clearance of invasive fungal pathogens. Disseminated candidiasis is of significant concern for those with suppressed immune systems or indwelling medical equipment, and mortality in these groups approaches 70%. Poor patient outcomes have spurred the need to understand how this non-motile pathogen spreads in the host. Technical limitations have previously hindered our ability to visualize the role of innate immunity and host tissue barriers in the spread of C. albicans in vivo. Using the zebrafish model to overcome these limitations, we have examined three potential host-mediated mechanisms of dissemination: movement inside phagocytes (“Trojan Horse”), inflammation, and endothelial barrier disruption.

Both neutrophils and macrophages respond to yeast-locked and wild type C. albicans in our zebrafish infection model, and time lapse imaging has revealed that macrophages may support yeast spread in a “Trojan Horse” mechanism. Similarly, inflammation is associated with the influx of immune cells to the site of infection which often precedes dissemination events. Loss of immune cell response or inflammation does not alter dissemination dynamics, however, so other mechanisms of yeast spread are also involved. Time lapse imaging also demonstrated the role of the endothelium on direct uptake of yeast into the bloodstream, suggesting the endothelium aids in yeast escape. When blood flow is blocked, yeast continue to cross the endothelium, but dissemination level is reduced. With loss of both phagocytes and blood flow, yeast disseminate but the distance traveled may be reduced. Together, our data suggests a two-step process: (1) yeast are brought across the endothelium with the assistance of phagocytes or direct uptake of host tissue, and then (2) utilize blood flow or phagocytes to travel to distant sites.

This suggests the host can contribute to the spread of yeast throughout the body. Surprisingly, innate immune cells do not always contain or kill the pathogen, instead allowing it grow if released in other tissues. This work will help to identify specific host and pathogen molecules which initiate cues for yeast uptake and spread, pinpointing areas for therapeutic targeting.

Movie 2.1.avi (1041654 kB)
Movie 2.1 Yolk injection

Movie 2.2.avi (30477 kB)
Movie 2.2 Phagocytes leave the infection site

Movie 2.3.avi (170840 kB)
Movie 2.3 Macrophages carry and release yeast

Movie 2.4.avi (8253 kB)
Movie 2.4 Non-lytic exocytosis of yeast

Movie 2.5.avi (1760540 kB)
Movie 2.5 Blood flow blockade in zebrafish

Move 2.6.avi (325 kB)
Movie 2.6 Endothelial cell interaction

Available for download on Monday, August 31, 2020

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