Date of Award

8-2015

Rights

© 2015 Zachary Tranchemontagne

Document Type

Thesis

Degree Name

Master of Science in Biological Sciences

Department

Biological Science

First Advisor

Kristin Burkholder

Second Advisor

Geoffrey Ganter

Third Advisor

Ling Cao

Abstract

Community-acquired Methicillin-resistant Staphylococcus aureus (CA-MRSA) strain USA300 is a major cause of invasive drug-resistant skin and soft tissue infections in humans. Although S. aureus is a well-recognized extracellular pathogen, recent reports that USA300 survives inside host macrophages suggest that the intramacrophage environment may be a niche for persistent infection. Intramacrophage survival requires bacteria to avoid destruction in the phagosome; however, mechanisms by which USA300 evades phagosomal defenses are unclear. We examined the fate of the USA300-containing phagosome in human THP-1 macrophages by evaluating phagosomal acidification and maturation, and by testing the impact of phagosomal conditions on bacterial viability. Utilizing confocal microscopy, we discovered that the USA300-containing phagosome acidified rapidly, and colocalized with the late endosome and lysosome protein LAMP-1. Interestingly, significantly fewer phagosomes containing live USA300 associated with lysosomal hydrolyses cathepsin D and β-glucuronidase than those containing dead bacteria, suggesting that USA300 harbors the ability to perturb lysosomal fusion during macrophage infection. We then examined the impact of phagosomal acidification on USA300 intracellular viability and found that inhibition of acidification significantly impairs USA300 survival, as well as negatively impacts virulence gene regulator agr expression. Together, these results suggest that USA300 survives inside macrophages by altering phagolysosome formation, as well as relying on vacuolar acidification as a trigger for virulence.

Comments

Master's thesis

This digital object has been funded in part with Federal funds from the National Science Foundation, Division of Graduate Education, under Award No. #0841361, "The Interactions of Biology, Chemistry and Physics at the Land-Ocean Interface: A Systemic PARTnership Aimed at Connecting University and School (SPARTACUS)", to the University of New England.

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