Identification of Unknown Antioxidant Defense Mechanisms of Francisella tularensis
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Issue Date
2014
Authors
Rane, Harshita Mangesh '14
Degree
MS in Pharmaceutical Sciences
Advisor
Malik, Meenakshi
Committee Members
Yager, Eric
Stein, Markus
Feleder, Carlos
Stein, Markus
Feleder, Carlos
Journal Title
Journal ISSN
Volume Title
Abstract
Francisella tularensis (F. tularensis), an intracellular gram negative bacterium, is the causative agent of tularemia. The pneumonic form of this disease is fatal and may cause up to 30-60% mortality in untreated cases. However, its main significance arises due to the fact that it is considered as a deadly bioterrorism agent. The Centers for Disease Control and Prevention (CDC) has classified F. tularensis as a Category A select agent due to its ease of aerosolization, retention of infectivity in small droplets, and the ability of as few as ten aerosolized bacteria to cause fatal disease in humans.
The F. tularensis antioxidant enzymes, the superoxide dismutase B and C (SodB and SodC) and the catalase (KatG) of F. tularensis have been identified as important virulence factors. Previous published work has shown that these enzymes are required for resistance to oxidative stress, intramacrophage survival and virulence in mice. However, work done in our laboratory suggests that there are additional unknown antioxidant mechanisms which act independently or in concert with the known classical antioxidants to render robust oxidant resistance to F. tularensis. By screening a transposon insertion library of F. tularensis LVS in the presence of H2O2, we have identified four oxidant sensitive mutants in aromatic amino acid transporter (FTL_0283, FTL_0283-34), putative EmrA1 secretion protein (FTL_0687), SIS domain protein (FTL_0864), and Transposase (FTL_0042). Our results demonstrate the emrA1 mutant is highly sensitive for H2O2 as well as super oxide generating compounds paraquat and pyrogallol. Additional studies conducted showed that EmrA1 contributes to oxidative resistance by affecting the secretion of F. tularensis antioxidant enzymes SodB and KatG.
Upon characterizing this mutant further, we found that the emrA1 mutant exhibits diminished intramacrophage growth that can be restored to wild type F. tularensis LVS levels by either transcomplementation, inhibition of reactive oxygen species (ROS) generation or infection in NADPH oxidase deficient (gp91Phox-/-) macrophages. The emrA1 mutant is attenuated for virulence, which is restored by infection in gp91Phox-/- thus mimicking in vitro results.
To conclude, our results demonstrate that antioxidant defenses of F. tularensis are important virulence factors and that EmrA1 is a key component of this important virulence machinery.
The F. tularensis antioxidant enzymes, the superoxide dismutase B and C (SodB and SodC) and the catalase (KatG) of F. tularensis have been identified as important virulence factors. Previous published work has shown that these enzymes are required for resistance to oxidative stress, intramacrophage survival and virulence in mice. However, work done in our laboratory suggests that there are additional unknown antioxidant mechanisms which act independently or in concert with the known classical antioxidants to render robust oxidant resistance to F. tularensis. By screening a transposon insertion library of F. tularensis LVS in the presence of H2O2, we have identified four oxidant sensitive mutants in aromatic amino acid transporter (FTL_0283, FTL_0283-34), putative EmrA1 secretion protein (FTL_0687), SIS domain protein (FTL_0864), and Transposase (FTL_0042). Our results demonstrate the emrA1 mutant is highly sensitive for H2O2 as well as super oxide generating compounds paraquat and pyrogallol. Additional studies conducted showed that EmrA1 contributes to oxidative resistance by affecting the secretion of F. tularensis antioxidant enzymes SodB and KatG.
Upon characterizing this mutant further, we found that the emrA1 mutant exhibits diminished intramacrophage growth that can be restored to wild type F. tularensis LVS levels by either transcomplementation, inhibition of reactive oxygen species (ROS) generation or infection in NADPH oxidase deficient (gp91Phox-/-) macrophages. The emrA1 mutant is attenuated for virulence, which is restored by infection in gp91Phox-/- thus mimicking in vitro results.
To conclude, our results demonstrate that antioxidant defenses of F. tularensis are important virulence factors and that EmrA1 is a key component of this important virulence machinery.
Citation
Rane HM. Identification of unknown antioxidant defense mechanisms of Francisella Tularensis. Ann Arbor (MI): Proquest LLC; 2014. 84 p.
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