Publication Date

2017-05-05

Availability

Embargoed

Embargo Period

2019-05-05

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Microbiology and Immunology (Medicine)

Date of Defense

2016-06-20

First Committee Member

Thomas R. Malek

Second Committee Member

Enrique A. Mesri

Third Committee Member

Gregory V. Plano

Fourth Committee Member

Michael A. Kolber

Fifth Committee Member

Nirupa Chaudhari

Sixth Committee Member

Noula D. Shembade

Seventh Committee Member

Gabriel Nunez

Abstract

The World Health Organization estimates that more than seven million individuals succumb to infectious disease annually and are the leading cause of mortality in the young. At a fundamental level each of these cases stems from a combination of two factors—the inability of the innate immune system to eliminate the offending pathogen and the ability of the infectious agent to capitalize on this delay before the adaptive immune response takes effect. A potential explanation for how the effectors of this response interact at the host-pathogen interface was recently discovered with the characterization of macrophage-expressed Mpeg1—hereafter referred to as Perforin-2. It was predicted that Perforin-2 may be the third defined pore-forming protein critical to immune response, with the primary goal of current studies to understand the role of Perforin-2 in host innate immunity. It was found that Perforin-2 is expressed constitutively in phagocytes and inducibly in parenchymal cells, and that the protein prevents proliferation of bacterial pathogens in vitro. Owing to the ability of Perforin-2 to be induced in all cells, certain bacterial pathogens are able to disrupt the induction of Perforin-2 transcription in host cells. Perforin-2 is a transmembrane protein of cytosolic vesicles –derived from multiple organelles-that translocate and fuse with bacterium containing vesicles. It was found that cullin-RING E3 ubiquitin ligase (CRL) complex containing cullin-1 and bTRCP monoubiquitinate Perforin-2 in response to pathogen associated molecular patterns. This ubiquitination triggers a redistribution of Perforin-2 and is essential for bactericidal activity. Unsurprisingly, pathogens like Yersinia pseudotuberculosis and enteropathogenic Escherichia coli disarm this defense mechanism by injecting cell cycle inhibiting factors (Cifs) into mammalian cells to deamidate the ubiquitin-like protein NEDD8. Because CRL activity is dependent on NEDD8, Cif effectively blocks ubiquitin-dependent trafficking of Perforin-2 and thus, its bactericidal activity. Following trafficking, Perforin-2 polymerizes and forms large 100 Å pores in the bacterial surface with cleavage products present in bacteria. Likely owing to this pore-formation, Perforin-2 is also required for bactericidal activity of known anti-bacterial mediators including reactive oxygen and nitrogen species. In vivo significance of Perforin-2 was confirmed with knockout mice that are unable to control bacterial infection and have a significantly worse prognosis than wild-type animals after infection with Salmonella enterica serovar typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Chlamydia. Furthermore, these studies have identified human Perforin-2 clinical mutations contributing to decreased innate immune defense as well as identified Perforin-2 as a key molecule in host inflammation.

Keywords

Perforin-2; MACPF; human immunology; pathogenic bacteria; infectious disease

Available for download on Sunday, May 05, 2019

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