The World Health Organization estimates that over seven billion individuals succumb to infectious disease annually and that they are the number one cause of death in the young. Fundamentally, each of these cases stems from an inability of the individual's innate immune system to eliminate infection coupled with the infectious agent capitalizing on the lag period of several days required for the adaptive immune system to prime against the specific pathogen. Despite dramatic advances in the understanding of the innate and adaptive immune systems, the host-pathogen interface is a relatively unexplored frontier. To date, some of the best studied bacterial killing mechanisms at this interface include reactive oxygen species (ROS), nitric oxide (NO), and hydrolases. Notwithstanding this body of knowledge, there is relatively little knowledge of exactly how these components interact with one another to contribute to bacterial killing. A potential explanation for how these effector proteins interact at the host-pathogen interface was recently discovered, with the characterization of macrophage-expressed Mpeg1. It was revealed that this mRNA encodes a protein domain, MACPF, which predicts a pore-forming, perforin-like molecule. Two already described pore-forming molecules of immune defense are the membrane attack complex of complement (MAC) that kills extracellular bacteria, and Perforin-1 of cytotoxic lymphocytes that kills virus-infected and cancer cells. Preliminary studies with Mpeg1 demonstrated that Mpeg-1 cDNA encodes a membrane- associated pore-forming protein, designated Perforin-2 (P2). These studies further characterized P2 as being constitutively expressed in all hematopoietic cells, and inducible in all cells. Furthermore, P2 seems to be a vital component as knockdown of P2 with siRNA blocks intracellular killing of pathogenic bacteria. My goal is to determine the mechanisms of P2 activation, culminating in polymerization and intracellular bacterial killing. I hypothesize that the cytoplasmic domain of P2 is the master regulator of activation and polymerization, that the cytoplasmic tail contains several key domains that undergo post-translational modification to become activated, and that the cytoplasmic tail will interact with several key adaptor proteins to regulate polymerization as well as localization within cells. The objectives of this application are to (i) identify the key cytoplasmic domains and amino acid residues necessary for polymerization to occur utilizing a functional intracellular bacterial killig assay;and (ii) characterize the P2-cytoplasmic domain interacting proteins that promote polymerization of P2 utilizing loss of function studies and transmission electron microscopy. A compelling aspect of this work is the potential to describe the host-pathogen interface with the addition of P2 as a common mediator in the previously described bacterial killing paradigm. By unraveling the molecular mechanisms triggering P2 polymerization and activation of bacterial attack, the proposed studies may provide new targets for drug interventions in the treatment of infections.

Public Health Relevance

Infectious diseases are the second leading cause of death worldwide, claiming approximately 16.2 percent of individuals who die each year, with a disproportionate number of deaths occurring in children under five years old. The dramatic impact of infectious disease on worldwide morbidity and mortality is highlighted in the top ten causes of death from the World Health Organization in 2008, with four of the top ten causes of death stemming from infectious disease. As each of these diseases arise from an inability of the host immune system to eliminate infection, the elucidation of the mechanism of Perforin-2 mediated killing will enable a better understanding of the host-pathogen interface and the respective processes that each infection utilizes to guarantee success.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1)
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Adger-Johnson, Diane S
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University of Miami School of Medicine
Schools of Medicine
Coral Gables
United States
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McCormack, Ryan M; Szymanski, Eva P; Hsu, Amy P et al. (2017) MPEG1/perforin-2 mutations in human pulmonary nontuberculous mycobacterial infections. JCI Insight 2:
McCormack, Ryan; Bahnan, Wael; Shrestha, Niraj et al. (2016) Perforin-2 Protects Host Cells and Mice by Restricting the Vacuole to Cytosol Transitioning of a Bacterial Pathogen. Infect Immun 84:1083-1091
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McCormack, Ryan M; de Armas, Lesley R; Shiratsuchi, Motoaki et al. (2015) Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria. Elife 4:
McCormack, Ryan; Podack, Eckhard R (2015) Perforin-2/Mpeg1 and other pore-forming proteins throughout evolution. J Leukoc Biol 98:761-8
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McCormack, Ryan; de Armas, Lesley; Shiratsuchi, Motoaki et al. (2013) Killing machines: three pore-forming proteins of the immune system. Immunol Res 57:268-78