Defense Date

2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Microbiology & Immunology

First Advisor

Guy Cabral

Second Advisor

Francine Marciano-Cabral

Third Advisor

Kathleen McCoy

Fourth Advisor

Michael McVoy

Fifth Advisor

Sandra Welch

Abstract

Acanthamoeba culbertsoni is an opportunistic free-living amoeba that is causative of granulomatous amoebic encephalitis (GAE), a chronic and often fatal central nervous system (CNS) disease that is most prevalent in immune compromised individuals. One hallmark of this disease is the formation of granulomas within the CNS, which are commonly absent in immune compromised individuals. Granulomas are usually composed of amoebae, microglia (CNS macrophages), macrophages, T cells, B cells, and neutrophils. Previous studies have demonstrated that microglia respond to Acanthamoeba by producing pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF)-α, interleukin (IL)-1α, and IL-1β. In addition, activated microglia and macrophages have been demonstrated to be cytolytic (i.e., amoebicidal) to Acanthamoeba. Furthermore, previous studies also indicated that Acanthamoeba secrete a myriad of factors including proteases. The role of these proteases during GAE has not been fully elucidated; however, it is thought that these factors may aid in the chronic persistence of Acanthamoeba within the CNS by modulating the host immune response. Using two-dimensional (iso-dalt) gel electrophoresis, we demonstrated that A. culbertsoni secrete factors that degrade culture medium proteins. Initial gelatin zymography studies demonstrated that propagation of A. culbertsoni in medium with high iron content leads to augmentation of protease activity. Gelatin zymography in concert with protease inhibitors demonstrated that A. culbertsoni secrete proteases predominantly of the serine protease class. Using an in vitro co-culture model, we demonstrated that co-culture of A. culbertsoni with mouse microglial-like cells (BV-2 cells) results in the augmentation of A. culbertsoni serine protease activity and stimulation of pro-inflammatory cytokine and chemokine protein expression by microglial-like cells. However, the A. culbertsoni-elicited proteases were shown to degrade microglial-like cell elicited cytokines and chemokines. Collectively, our results suggest that A. culbertsoni- secreted serine proteases may play a role in A. culbertsoni CNS immune evasion by increasing A. culbertsoni CNS dissemination via the diminution of granuloma formation and by dampening microglial-dependent cytokine response.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

May 2009

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