Date of Degree
PhD (Doctor of Philosophy)
Bradley D. Jones
Francisella tularensis is a highly virulent bacterial pathogen with an extremely low infectious dose (~10 CFU) and high rates of mortality if left untreated (30-60%). F. tularensis has an extensive history as a bioweapon, and there is no vaccine currently licensed. For these reasons the CDC considers F. tularensis a Tier 1 Select Agent. The unlicensed F. tularensis subsp. holarctica Live Vaccine Strain (LVS) provides moderate protection against virulent strains; however, we have discovered that various “wild type” lab stocks differ in their virulence and ability to confer immunity. Genome sequencing of high virulence (RML, LD50 ~200 CFU) and low virulence (ATCC, LD50 ~9,000 CFU) strains has identified nine differences, of which four are non-synonymous substitutions. One such mutation occurs in the ferrous iron uptake gene feoB in RML. While iron is required for cellular function, ferrous iron (Fe2+) can participate in the Fenton reaction with H2O2, leading to inactivation of essential iron-sulfur cluster enzymes. Part of the innate immune response involves mitochondria-derived reactive oxygen species in the cytosol. Fully virulent strains of F. tularensis are known to be highly resistant to such host defenses, and have low levels of intracellular iron. Accordingly, the RML strain was highly resistant to exogenous H2O2in vitro relative to the ATCC strain. An iron-responsive lacZ reporter had ~2-fold higher induction in the RML strain relative to ATCC during iron limitation. Overexpression of the functional feoB allele, but not the RML allele, leads to significantly increased sensitivity to H2O2 and increased killing by primary macrophages stimulated with the cytokine interferon-gamma. Given the connection of iron and H2O2 toxicity, I revisited a previously published transposon screen to determine if any of the mutants identified had a role in iron homeostasis and oxidative stress resistance. One such gene was annotated as bacterioferritin (bfr), which in other bacteria forms a hollow, spherical multimer that oxidizes Fe2+ to Fe3+ and stores the oxidized form in the interior of the sphere. The Δbfr mutant was ~10-fold more sensitive to H2O2 and was attenuated nearly 8-fold in murine intranasal infection in terms of LD50 relative to the parental RML strain. Importantly, the Δbfr mutant allowed us to test the hypothesis that H2O2 resistance is critical for the RML LVS to stimulate productive immunity. At six weeks post-infection, mice previously infected with either RML or the Δbfr mutant were challenged with an infection of 25 CFU of the fully virulent F. tularensis Schu S4 strain. All mice immunized with RML survived this challenge, while all mice immunized with Δbfr succumbed; only displaying a slight increase in time to death. These results are consistent with the hypothesis that the H2O2 resistance of RML LVS mediates increased fitness in a host.
Many organisms on Earth require oxygen and iron to support their growth and metabolism, including many bacteria that can cause disease. One such bacterium, Francisella tularensis, can cause severe pneumonia that may lead to death. The current vaccine against infection comes from a live but weakened version of this bacterium, though it is not very effective. One of the goals of this thesis was to examine the DNA from different lots of this vaccine strain to see if there were genetic differences between lots that were more or less effective at providing immunity. This analysis found that the effective vaccines had a genetic mutation that made these particular strains of the weakened bacteria less able to gather iron from their environment. Certain forms of iron can be highly reactive with oxygen, which can be seen as rust on metal. This form of iron is important for certain metabolic reactions in most living things, but too much of it can be toxic to life. Iron can react with hydrogen peroxide, a common antiseptic. Hydrogen peroxide is also made by the cells of the immune system to kill invading pathogens. My research found that the more effective vaccine strains of Francisella tularensis were resistant to the toxic effects of hydrogen peroxide because they had less iron. I showed that genetically manipulating this strain to make it have more iron made it sensitive to hydrogen peroxide and made it a less effective vaccine.
Copyright 2016 Joshua Robert Fletcher