Date of Award

1-1-2015

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Nanoscale Science and Engineering

Program

Nanoscale Sciences

Content Description

1 online resource (xiii, 148 pages) : color illustrations.

Dissertation/Thesis Chair

J. Andres Melendez

Committee Members

Sara Brenner, Nathaniel Cady, Shekhar Bakshi, Nadine Hempel

Keywords

calcium, catalase, Francisella, hydrogen peroxide, TRPM2, Francisella tularensis, TRP channels, Calcium channels, Cellular signal transduction

Subject Categories

Immunology of Infectious Disease | Microbiology | Molecular Biology

Abstract

As an innate defense mechanism, macrophages produce reactive species that weaken pathogens and serve as secondary messengers to modify signaling responses involved in immune function. The gram-negative bacterium F. tularensis utilizes its antioxidant armature to limit the host immune response but the mechanism behind this suppression has not been defined. Here we establish that F. tularensis limits Ca2+ entry thereby limiting actin reorganization and IL-6 production in a redox-dependent fashion. Wild-type (LVS) or catalase deficient F. tularensis (∆katG) show distinct profiles in their H2O2 scavenging capacity, 1 pM/sec and 0.015 pM/sec, respectively. Murine alveolar macrophages infected with ∆katG display distinct intracellular Ca2+ profiles and fail to respond to a secondary calcium influx stimulated by H2O2. Additionally, ΔkatG infected macrophages displayed limited Ca2+ influx in response to the Ca2+, ionophore ionomycin. Basal increases in cytosolic Ca2+ as well as insensitivity to H2O2-mediated Ca2+ entry in response ΔkatG infection are reversed by the calcium release modulators, 2-Aminoethyl diphenylborinate (2APB) or SKF-96365. 2APB but not SKF abrogated ∆katG-dependent increases in macrophage actin remodeling and IL-6 secretion. 2APB is a potent inhibitor of Ca2+ entry through the transient receptor potential melastatin 2 (TRPM2) channel. Increases in basal Ca2+, actin polymerization and IL-6 production are reversed in TRPM2-null macrophages infected with ∆katG. Together our findings provide compelling evidence that F. tularensis catalase restricts ROS to temper calcium signaling and limit host immune function.

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