Publication Date

2013-05-08

Availability

Embargoed

Embargo Period

2015-05-08

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Microbiology and Immunology (Medicine)

Date of Defense

2013-04-04

First Committee Member

Eckhard R. Podack

Second Committee Member

Kenneth A. Fields

Third Committee Member

Mathias Lichtenheld

Fourth Committee Member

Gregory V. Plano

Fifth Committee Member

Amjad Farooq

Sixth Committee Member

Olivera Stojadinovic

Abstract

Pore-forming proteins such as the membrane attack complex (MAC) of the complement system and Perforin of cytotoxic lymphocytes are potent host defense effectors employed by the immune system. These proteins share a structurally conserved domain, MACPF that is responsible for ring formation and membrane insertion of these toxic molecules. The objective of the current study was to identify the function of a third MACPF-containing pore-former of the immune system, Macrophage expressed gene 1 (Mpeg1). Mpeg1 encodes a highly conserved, membrane-bound protein which we have designated Perforin-2 (P-2). P-2 is induced upon monocyte maturation, is constitutively expressed by macrophages and upregulated by lipopolysaccharides (LPS) and interferon-gamma (IFN-gamma). Evidence is presented herein that macrophages employ P-2 to mediate intracellular killing of bacteria. Gene silencing was performed using P-2-specific siRNA which led to uncontrolled Salmonella typhimurium growth. To further characterize the protein, a P-2 fusion protein with a fluorescent tag was transfected into macrophages and analyzed for localization and function. P-2 in macrophages resides in the endoplasmic reticulum, Golgi network and recycling endosomes where it would be available for trafficking to phagocytosed microbes. Combined inhibition of the established bactericidal mechanisms, reactive oxygen species and nitric oxide, with P-2 resulted in bacterial survival equal to that of P-2 inhibition alone suggesting that oxidative pathways of toxicity are downstream of P-2 action. Finally, site-directed mutagenesis of a conserved tyrosine in the cytoplasmic domain of P-2 rendered the protein non-functional in killing against Mycobacterium smegmatis suggesting that phosphorylation of this residue is important for activation of P-2. Therefore, P-2 represents a novel, highly conserved cytotoxic effector molecule expressed by macrophages with important implications for the control of microbial infection and host defense.

Keywords

innate; bacteria; macrophages; pore-forming; MACPF

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