Oxidative stress resistance in the Francisella tularensis live vaccine strain is associated with

166 Pages·2017·1.37 MB·English

by  Joshua Robert Fletcher

University of Iowa
Iowa Research Online
Theses and Dissertations
Spring 2016
Oxidative stress resistance in the Francisella tularensis live
vaccine strain is associated with genetic variability in the ferrous
iron uptake gene feoB
Joshua Robert Fletcher
University of Iowa
Follow this and additional works at: https://ir.uiowa.edu/etd
Part of the Genetics Commons
Copyright 2016 Joshua Robert Fletcher
This dissertation is available at Iowa Research Online: https://ir.uiowa.edu/etd/3083
Recommended Citation
Fletcher, Joshua Robert. "Oxidative stress resistance in the Francisella tularensis live vaccine strain is
associated with genetic variability in the ferrous iron uptake gene feoB." PhD (Doctor of Philosophy)
thesis, University of Iowa, 2016.
https://doi.org/10.17077/etd.c5lqs7hk
Follow this and additional works at: https://ir.uiowa.edu/etd
Part of the Genetics Commons
Oxidative stress resistance in the Francisella tularensis Live Vaccine Strain is
associated with genetic variability in the ferrous iron uptake gene feoB

by

Joshua Robert Fletcher

A thesis submitted in partial fulfillment
of the requirements for the Doctor of Philosophy
degree in Genetics in the
Graduate College of
The University of Iowa

May 2016

Thesis Supervisor: Professor Bradley D. Jones
Copyright by
JOSHUA ROBERT FLETCHER
2016
All Rights Reserved
Graduate College
The University of Iowa
Iowa City, Iowa

CERTIFICATE OF APPROVAL

____________________________

PH.D. THESIS

_________________

This is to certify that the Ph.D. thesis of

Joshua Robert Fletcher

has been approved by the Examining Committee for
the thesis requirement for the Doctor of Philosophy degree
in Genetics at the May 2016 graduation.

Thesis Committee: ____________________________________________
Bradley D. Jones, Thesis Supervisor

____________________________________________
Lee-Ann H. Allen

____________________________________________
Craig D. Ellermeier

____________________________________________
Mary E. Wilson

____________________________________________
John R. Kirby
To my family.

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ACKNOWLEDGEMENTS
No scientist’s endeavors occur in a vacuum, unless you happen to be a
computer scientist in the 1940’s or an astronaut. I am neither. I owe much
gratitude to: my friends and colleagues Matt Faron and Jed Rasmussen, two
fellow graduate students in the lab who were soundboards for my ideas more
often than they probably wanted to be, and who offered many a great
conversation about books, science, life, the universe and Rick Astley’s place in it;
everyone in the Francisella group who helped me learn the field and think critically
about the details of experiments; Bram Slutter from the Harty lab for his help and
expertise with T cell biology; our collaborator Katy Bosio from Rocky Mountain
Labs for being generous with data and strains that served as the foundation for
this document; my mentor Brad Jones for giving me space in the lab and the
freedom to explore it; the Genetics program for admitting me and allowing me to
develop as a scientist. Lastly, I owe an enormous debt of gratitude to my friends
and family for supporting me even though I often put science ahead of them.

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ABSTRACT
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
2+
required for cellular function, ferrous iron (Fe ) 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
H2O2 in 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.
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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,
2+ 3+
spherical multimer that oxidizes Fe to Fe 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.

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PUBLIC ABSTRACT
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.

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Table of Contents
LIST OF FIGURES ...............................................................................................ix
TABLE: PRIMER SEQUENCES ........................................................................... 1
CHAPTER I .......................................................................................................... 3
INTRODUCTION .................................................................................................. 3
Francisella tularensis early history .................................................................... 3
Tularemia .......................................................................................................... 6
F. tularensis taxonomy and ecology .................................................................. 8
Francisella tularensis genetics ........................................................................ 10
Bacterial factors that mediate pathogenesis .................................................... 13
Iron uptake in F. tularensis .............................................................................. 17
Intracellular lifecycle ........................................................................................ 20
Immunity to Francisella species ...................................................................... 26
CHAPTER II ....................................................................................................... 30
A VIRULENT BIOVAR OF F. TULARENSIS LVS IS INTRINSICALLY MORE
RESISTANT TO HYDROGEN PEROXIDE ........................................................ 30
Introduction ..................................................................................................... 30
Materials and Methods .................................................................................... 32
Results ............................................................................................................ 36
LVS biovars have similar PiglA-lacZ expression and lack low molecular
weight O-antigen glycosylated proteins........................................................ 36
2+
RML LVS has less intracellular Fe than the ATCC or Iowa LVS ............... 38
FeoB D471Y does not complement E. coli ΔfeoB fhuF::λplac ..................... 42
Resistance to H2O2 correlates with feoB D471Y allele ................................. 43
Resistance to H2O2 also requires Dyp peroxidase ....................................... 45
Neither feoB nor dyp are essential for intracellular growth in A549 or
BMM cells .................................................................................................... 47
Discussion ....................................................................................................... 62
CHAPTER III ...................................................................................................... 67
CHARACTERIZATION OF BACTERIOFERRITIN IN OXIDATIVE STRESS
RESISTANCE ..................................................................................................... 67
Introduction ..................................................................................................... 67
Materials and methods .................................................................................... 69
Results ............................................................................................................ 72
A bacterioferritin mutant has modest reduction in PfslA-lacZ activity ............. 72
Bacterioferritin promoter activity is not significantly induced in iron
limiting media ............................................................................................... 73
Bacterioferritin protects against H2O2 but is not required for intracellular
growth in A549 cells ..................................................................................... 75
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