Reciprocal Analysis of Francisella novicida Infections of a Drosophila melanogaster Model Reveal Host-Pathogen Conflicts Mediated by Reactive Oxygen and imd-Regulated Innate Immune Response

Abstract

The survival of a bacterial pathogen within a host depends upon its ability to outmaneuver the host immune response. Thus, mutant pathogens provide a useful tool for dissecting host-pathogen relationships, as the strategies the microbe has evolved to counteract immunity reveal a host's immune mechanisms. In this study, we examined the pathogen Francisella novicida and identified new bacterial virulence factors that interact with different parts of the Drosophila melanogaster innate immune system. We performed a genome-wide screen to identify F. novicida genes required for growth and survival within the fly and identified a set of 149 negatively selected mutants. Among these, we identified a class of genes including the transcription factor oxyR, and the DNA repair proteins uvrB, recB, and ruvC that help F. novicida resist oxidative stress. We determined that these bacterial genes are virulence factors that allow F. novicida to counteract the fly melanization immune response. We then performed a second in vivo screen to identify an additional subset of bacterial genes that interact specifically with the imd signaling pathway. Most of these mutants have decreased resistance to the antimicrobial peptide polymyxin B. Characterization of a mutation in the putative transglutaminase FTN_0869 produced a curious result that could not easily be explained using known Drosophila immune responses. By using an unbiased genetic screen, these studies provide a new view of the Drosophila immune response from the perspective of a pathogen. We show that two branches of the fly's immunity are important for fighting F. novicida infections in a model host: melanization and an imd-regulated immune response, and identify bacterial genes that specifically counteract these host responses. Our work suggests that there may be more to learn about the fly immune system, as not all of the phenotypes we observe can be readily explained by its interactions with known immune responses.

Full text

Reciprocal Analysis of
Francisella novicida
Infections of a
Drosophila melanogaster
Model Reveal Host-Pathogen
Conflicts Mediated by Reactive Oxygen and imd-
Regulated Innate Immune Response
Madeleine G. Moule, Denise M. Monack, David S. Schneider*
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America
Abstract
The survival of a bacterial pathogen within a host depends upon its ability to outmaneuver the host immune response.
Thus, mutant pathogens provide a useful tool for dissecting host-pathogen relationships, as the strategies the microbe has
evolved to counteract immunity reveal a host’s immune mechanisms. In this study, we examined the pathogen Francisella
novicida and identified new bacterial virulence factors that interact with different parts of the Drosophila melanogaster
innate immune system. We performed a genome-wide screen to identify F. novicida genes required for growth and survival
within the fly and identified a set of 149 negatively selected mutants. Among these, we identified a class of genes including
the transcription factor oxyR, and the DNA repair proteins uvrB, recB, and ruvC that help F. novicida resist oxidative stress.
We determined that these bacterial genes are virulence factors that allow F. novicida to counteract the fly melanization
immune response. We then performed a second in vivo screen to identify an additional subset of bacterial genes that
interact specifically with the imd signaling pathway. Most of these mutants have decreased resistance to the antimicrobial
peptide polymyxin B. Characterization of a mutation in the putative transglutaminase FTN_0869 produced a curious result
that could not easily be explained using known Drosophila immune responses. By using an unbiased genetic screen, these
studies provide a new view of the Drosophila immune response from the perspective of a pathogen. We show that two
branches of the fly’s immunity are important for fighting F. novicida infections in a model host: melanization and an imd-
regulated immune response, and identify bacterial genes that specifically counteract these host responses. Our work
suggests that there may be more to learn about the fly immune system, as not all of the phenotypes we observe can be
readily explained by its interactions with known immune responses.
Citation: Moule MG, Monack DM, Schneider DS (2010) Reciprocal Analysis of Francisella novicida Infections of a Drosophila melanogaster Model Reveal Host-
Pathogen Conflicts Mediated by Reactive Oxygen and imd-Regulated Innate Immune Response. PLoS Pathog 6(8): e1001065. doi:10.1371/journal.ppat.1001065
Editor: Neal Silverman, University of Massachusetts Worcester, United States of America
Received March 9, 2010; Accepted July 26, 2010; Published August 26, 2010
Copyright: ß2010 Moule et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by RO1AI060164. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: dschneider@stanford.edu
Introduction
The outcome of any infection, whether it be clearance of the
infecting pathogen, establishment of a persistent infection, or even
death of the host is determined by contributions from both the host
and the microbe [1]. To infect a susceptible host microbes express
virulence factors, genes that allow the pathogen to invade, colonize,
and survive within the host and cause essential pathology. In
response, the host initiates an immune response that attempts to
clear the pathogen and increase tolerance to the ensuing infection
[2]. Consequently, in addition to genes that allow the bacteria to
invade host cells and obtain nutrients from its host, a subset of the
virulence factors expressed by the microbe must address the need of
the bacteria to counteract the host immune response. Exploring this
complex interplay between host and pathogen can help us to
understand bacterial pathogenesis and define the contributions of
the host immune system to bacterial virulence.
One way to explore the host-pathogen relationship is to apply
model systems that allow us to dissect the genetics of both sides of
the equation simultaneously in vivo [3]. In this study, we examine
the host-pathogen interactions of Francisella novicida with an insect
host, Drosophila melanogaster, and identify aspects of fly immunity
that are most important for fighting F. novicida infection as well as
the bacterial virulence factors that interact with each of these
specific immune responses. Drosophila is used as a model of innate
immunity because its simplicity and the ease at which it can be
used for both forward and reverse genetics has allowed for the
identification and characterization of aspects of the innate immune
response that are conserved across evolution [4–6]. The fly
immune response has three effector arms: an inducible antimi-
crobial peptide (AMP) response, a reactive oxygen response
mediated by the activation of the enzyme phenoloxidase and the
deposition of the pigment melanin, and a cellular immune
response in which foreign invaders are phagocytosed by Drosophila
hemocytes [7]. The humoral AMP response has been studied
extensively and shown to be regulated by two pattern recognition
pathways, Toll and imd which have been well-characterized and
described, but the regulatory mechanisms of the melanization and
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cellular immune responses have only recently become the focus of
increased interest and have not yet been fully elucidated [8].
Previous studies with pathogenic bacteria in the fly have shown
that virulence factors that function in the vertebrate hosts of these
pathogens are often required for the microbe to survive in the fly
[6]. Recently, this has been shown to be true for the live vaccine
strain (LVS) of the virulent pathogen Francisella tularensis, a Gram-
negative facultative intracellular bacterium that is the causative
agent of tularemia [9]. F. tularensis can infect a wide range of hosts
that includes humans, but is more commonly associated with
rabbits and small rodents. Unlike many of the pathogens used in
previous fly studies, F. tularensis also has a documented arthropod
vector phase in its natural life-cycle [10,11]. While many
arthropod-vectored pathogens can only be transmitted by a single
specific species, F. tularensis is able to infect arthropods ranging
from ticks to multiple species of mosquito to biting flies such as
deerflies [12–14]. This makes the Drosophila model system
particularly useful for studying both general F. tularensis host-
pathogen interactions and insect-specific factors.
To date, the fly has primarily been used to dissect the function
of known bacterial virulence factors or to demonstrate conserva-
tion between fly and vertebrate defenses. Less has been done to use
forward genetic approaches in the microbe to identify virulence
factors de novo. As immunologists we tend concentrate on known
signaling pathways that have proven simple to study, are of interest
to those working in vertebrates because they are conserved, and
those that fit our idea of what the fly’s immune response should be.
In other words, experiments are driven by the interests of the
scientists and not the pathogens. We took a more ecology-based
approach and determined, from scratch, what this fly pathogen
needs to kill the fly. The advantage of the fly is that it is
inexpensive, rapid to use and has extensive genetic tools. The fly
could be a useful tool for the identification of new virulence factors
rather than a system used to study known factors.
To identify new virulence factors and examine their interactions
with the fly immune system, we used the Francisella novicida strain
U112 to infect flies and performed a genome-wide screen to
identify factors required for growth and/or survival within the fly.
Many of the genes that we identified are required for resistance to
the Drosophila innate immune response, particularly to the
oxidative stress produced by melanization. This is interesting in
particular, because until recently, this pathway had been discarded
as having no relevance to microbial infections in the fly [15–17].
Our work demonstrates that bacterial mutants can be used as
probes of the host immune system to identify what aspects of
innate immunity are most important in determining the outcome
of an infection.
To identify additional interactions between the host immune
system and bacterial virulence factors, we performed a second
genetic screen in which we compared the ability of F. novicida
mutants to grow in wild type flies to flies with an immunity defect
known to affect fly survival in F. tularensis infections. These flies
lack a functional imd signaling pathway, and we anticipated that
this would reveal bacterial mechanisms necessary to circumvent
the imd-regulated immune response. The imd pathway is
primarily described as inducing antimicrobial peptides. Although
we identified bacterial genes required to resist antimicrobial
peptide killing in vitro, we were particularly intrigued to find a
subset of genes that when mutated did not appear to change F.
novicida sensitivity to the antimicrobial peptide we tested yet
showed an altered phenotype in imd mutants. This suggests the
possibility that there are previously undescribed immune mech-
anisms that are regulated by the imd pathway.
Results
Characterization of Francisella tularensis ssp. novicida
infections of Drosophila melanogaster
We infected flies with F. novicida strain U112, a wild type strain
that causes virulent infections in its natural mouse and rabbit hosts
but is not pathogenic to humans. Previous work using the Live
Vaccine Strain (LVS) of F. tularensis demonstrated that F. tularensis
grows to high bacterial levels within flies and causes a lethal
infection [9]. Infections of the fly with the U112 strain were
consistent with this result, although we found the U112 strain to be
slightly more virulent in Drosophila than the LVS strain, killing the fly
with a median time to death (MTD) of 5 days post-infection with
10
3
CFU (Figure 1A and Figure S1). As few as 5 CFU of F. novicida
U112 were sufficient to kill the fly, and bacterial growth within the
fly was exponential approaching 5610
7
CFU per fly before they
succumbed to the infection. Regardless of the initial dose, F. novicida
infections quickly reached the same high levels of bacteria; colony
counts in flies receiving a low dose caught up to the 10
8
fold higher
dose within two days (Figure 1B and Figure S2).
In mammalian infections, F. tularensis is a facultative intracellular
parasite that primarily grows within macrophages [18]. However,
due to the extremely high bacterial levels observed within the fly
we speculated that a large proportion of the bacteria were growing
extracellularly. To test this idea, we performed gentamycin chase
assays, infecting flies with F. novicida and subsequently injecting
them with the non-cell permeable antibiotic gentamycin at various
timepoints post infection. This assay kills off extracellular bacteria
while leaving bacteria growing within cells intact and allowing us
to determine whether the bacteria are growing intracellularly or
extracellularly By 24 hours post infection, approximately 1610
4
CFU per fly were found to be localized intracellularly. However,
the average total population of bacteria in infected flies was at least
1610
5
CFU per fly, demonstrating that a significant population of
bacteria was located extracellularly. Over the course of the
infection the total bacteria population increased to 1610
7
CFU/
fly, while the intracellular population remained steady, indica-
ting that the extracellular population was responsible for the
Author Summary
To infect a host and survive attacks from the host immune
system, bacteria express genes that allow them to
counteract immune responses. By identifying these genes
we can learn how hosts fight infections and how bacteria
resist immune attacks. We identified Francisella novicida
genes that interact with the fruit fly immune system by
performing a genetic screen of bacterial mutants. We
identified genes that when mutated cause the bacteria to
grow poorly within the fly. Many of these genes were
shown to help the bacteria survive oxidative stress,
providing resistance to an immune response known as
melanization. We then identified bacterial genes that
interact with another branch of the immune system, the
imd pathway, by performing a second screen in imd
mutant flies. We identified bacterial mutants that cannot
grow in wild-type flies but are rescued in imd mutants,
indicating an interaction with this pathway. We followed
up one example from this screen and found that mutants
in the gene FTN_0869 grow normally inside cells, but
cannot grow extracellularly. We found that this was due to
being unable to resist previously unexplored aspects of
the imd-regulated immune response that help fight off F.
novicida infections.
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exponential increase in bacteria seen during fly infections
(Figure 1C). This is roughly similar to what has been observed
using the LVS strain, although the absolute numbers differ slightly
possibly due to the differences in virulence between the two
bacterial strains, differences in the host strains or environmental
conditions [9].
Identification of F. novicida virulence factors with a
Transposon Site Hybridization (TraSH) screen
Having demonstrated that the fly can support F. novicida growth
we applied this model to the identification of bacterial genes that
were important for establishing infection within the fly. Previous
work has shown that many F. tularensis virulence factors that are
required for growth in mammalian models are also essential in
insect infections, including the Francisella Pathogenicity Island
(FPI) genes iglB, iglC, and iglD and the transcription factor mglA
[9,19]. To expand upon this work, we sought to identify additional
bacterial virulence factors and provide an opportunity to discover
new biology using a forward genetic approach.
Using a transposon insertion library of F. novicida mutants we
performed an in vivo screen for mutants with altered growth rates
compared to wild-type bacteria using a Transposon Site
Hybridization (TraSH) assay. Briefly, flies were infected with the
pooled library and the infection was allowed to proceed for two
days, at which point the bacterial populations in each fly were
harvested. Genomic DNA was then purified from this population
of bacteria and from the original input library that had not been
subjected to the stresses found within the fly. RNA was amplified
from the site of each transposon insertion and the two populations
of RNAs were compared by microarray analysis. We identified
mutants representing 149 F. novicida genes that were negatively
selected with a false discovery rate (FDR) of 5%, indicating that
these genes were essential for bacterial growth and survival within
the fly (Table S1). 41 of these genes had previously been identified
as negatively selected in a similar TraSH analysis performed with
the same mutant library in an in vivo mouse model; this list includes
the known virulence genes iglC, iglD, pdpA, and pdpB, and mglA
[20]. In addition, 11 genes from our screen overlapped with data
from a negative selection screen performed by Kraemer et al. using
an inhalation model to observe respiratory infections in the mouse,
and an additional 8 overlapped with a signature-tagged mutagen-
esis screen performed by Su et al. that also used an intranasal route
of inoculation [21,22]. The overlap between our TraSH assay and
additional Francisella genome-wide screens is shown in Table 1 and
Figure 2. Interestingly, no genes were identified in all four unique
screens, although 7 genes were identified in our fly TraSH and at
least two other screens. These genes were the hypothetical proteins
FTN_1682 and FTN_1016, the RNA methyltransferase yibK, the
ABC transporter yjjK, the amino acid antiporter FTN_0848, iglC
and iglD. The degree of overlap between our fly screen and similar
mouse screens both supports the hypothesis that our screen in
Drosophila can be used to identify virulence factors that are
conserved between insect and mammalian infections, and also
presents the possibility of identifying virulence factors unique to
the arthropod vector stage of the F. novicida life cycle.
Confirmation of negatively selected mutants
To confirm the results of the TraSH screen, we tested 65 of the
negatively selected mutants individually by competition assay,
focusing on mutants that had particularly large decreases in
abundance and/or showed homology to bacterial genes that we
predicted could play a role in immune evasion or modulation.
Transposon insertion mutants of each gene containing a kanamycin
resistance cassette were tested to determine their ability to grow in
Figure 1.
Francisella novicida
is capable of infecting Drosophila. Wild-type flies were injected with F. tularensis novicida U112 and survival and
growth were monitored over the course of the infection. (A) Survival of wild-type flies following injection of 10
3
CFUs of F. novicida. Median-time-to
death (MTD) is approximately five days post infection when incubated at 29uC. Log-rank analysis of the Kaplan-Meyer survival curves showed
statistical significance with a P value of ,0.0001. Figure S1 provides variance data for these and other survival curves. (B) Growth of F. novicida in
wild-type flies. Injection of a range of initial doses between 5 and 5*10
4
CFUs per fly results in bacterial growth to up to approximately 5*10
7
CFU per
fly within 4–5 days post-infection at 29uC. (C) Intracellular and extracellular populations of bacteria within the fly following infection with 5*10
3
CFU/
fly, as determined by survival of bacteria within cells following injection of the non-cell-permeable antibiotic gentamycin. Horizontal lines indicate
mean CFU/fly. (D) GFP expressing F.novicida within a larval hemocyte.
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Table 1. Overlap between Drosophila TraSH screen and screens for Francisella virulence factors in a mouse model.
U112 loc Schu4 loci Gene Product Gene [20] [21] [22]
FTN_1683 FTT0028c drug:H+antiporter-1 (DHA1) family protein x
FTN_1682 FTT0029c conserved hypothetical protein xx
FTN_1658 FTT0052 Histidyl-tRNA synthetase hisS x
FTN_1657 FTT0053 major facilitator superfamily (MFS) transport protein x
FTN_1654 FTT0056c major facilitator superfamily (MFS) transport protein x
FTN_1617 FTT0094c sensor histidine kinase qsec x
FTN_1582 FFF0134 hypothetical membrane protein x
FTN_0210 FTT0295 hypothetical protein x
FTN_0217 FTT0303c L-lactate dehydrogenase lldD x
FTN_0493 FTT0397 5-methylthioadenosine\S-adenosylhomocysteine nucleosidase mtn x
FTN_0494 FTT0398c hypothetical membrane protein x
FTN_0495 FTT0399c BNR/Asp-box repeat protein x
FTN_0554 FTT0463 tRNA/rRNA methyltransferase yibK x x
FTN_0546 FTT0455c dolichyl-phosphate-mannose-protein mannosyltransferase family protein x
FTN_0599 FTT0509c conserved hypothetical protein x
FTN_1066 FTT0615c metal ion transporter protein x
FTN_1038 FTT0645c conserved hypothetical membrane protein x
FTN_1016 FTT0667 hypothetical protein xx
FTN_1220 FTT0790 bacterial sugar transferase family protein x
FTN_1214 FTT0797 glycosyl transferase, family 2 x
FTN_1196 FTT0810c conserved hypothetical UPF0133 protein ybaB x
FTN_0344 FTT0829c Aspartate:alanine antiporter x
FTN_0416 FTT0891 lipid A 1-phosphatase lpxE x
FTN_0429 FTT0903 hypothetical protein x
FTN_0824 FTT0947c major facilitator superfamily (MFS) transport protein, pseudogene x
FTN_0840 FTT0961 modulator of drug activity B mdaB x
FTN_0848 FTT0968 amino acid antiporter xx
FTN_1243 FTT1224c DNA repair protein recO recO x
FTN_0891 FTT1013 holliday junction DNA helicase, subunit B ruvB x
FTN_1257 FTT1239 membrane protein of unknown function x
FTN_1276 FTT1257 membrane fusion protein x
FTN_0666 FTT1312c excinuclease ABC, subunit A uvrA x
FTN_0664 FTT1314c Type IV pili fiber building block protein x
FTN_1310 FTT1345 conserved hypothetical protein; conserved hypothetical protein pdpB; pdpB1 x
FTN_1319 FTT1354 conserved hypothetical protein; conserved hypothetical protein pdpC x
FTN_1321 FTT1356c intracellular growth locus, subunit D; subunit D iglD; iglD1 x x
FTN_1322 FTT1357c intracellular growth locus, subunit C; subunit C1 iglC; iglC1 x x
FTN_1357 FTT1394c ATP-dependent exoDNAse (exonuclease V) beta subunit recB x
FTN_1417 FTT1447c Phosphomannomutase manB x
FTN_1501 FTT1490 monovalent cation:proton antiporter-1 x
FTN_1439 FTT1531 3-ketoacyl-CoA thiolase fadA x
FTN_1513 FTT1503 site-specific recombinase xerC x
FTN_0337 FTT1600c fumarate hydratase, class I fumA x
FTN_0036 FTT1647c diyroorotate dehydrogenase pyrD x
FTN_0035 FTT1648c Orotidine 5-phosphate decarboxylase pyrF x
FTN_1715 FTT1736c two component sensor protein kdpD x
FTN_1753 FTT1759c Oxidase-like protein, pseudogene x
FTN_1745 FTT1767c phosphoribosylglycinamide formyltransferase 2 purT x
FTN_1762 FTT1782c ABC transporter, ATP-binding protein yjjK x x
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competition with wild-type F. novicida. Each mutant was mixed with
wild-type bacteria and injected into wild-type Drosophila at a 1:1
ratio. Infected flies were incubated for 48 hours, at which point the
bacteria in each fly were plated and the ratio of mutant to wild-type
bacterial was determined. A competitive index of 1 represents an
equal ratio of mutant to wild-type bacteria, while competitive
indexes of less than one indicate that the mutant is attenuated.
Mutants that were determined to be statistically significantly less
than 1 by one sample t-test in a minimum of three repetitions were
considered attenuated and are listed in Figure 2.
Mutants that were confirmed as negatively selected included
kdpA, kdpC, kdpD, and kdpE, components of 2-component
regulatory system that responds to turgor pressure, a number of
genes known to be regulated by the virulence factor mglA, members
of the Major Facilitator Superfamily (MFS) thought to be involved
in substrate transport and drug resistance, multiple genes know to be
involved in DNA repair, and a number of hypothetical proteins.
(Figure 3A and data not shown) 56 of the mutants tested showed
attenuated phenotypes by competition assay, with competitive
indexes ranging from 0.6-0.007. The results of the competition
Figure 2. Summary of results of negatively selected mutant phenotypes. Mutants with confirmed attenuated phenotypes by competitive
index are categorized by their sensitivity to oxidative stress and polymyxin and phenotype in imd mutant flies. To be considered attenuated, each
mutant listed in this table was determined to have competitive indexes that were statistically significantly less than 1 by one sample t-tests with a
maximum p value of ,0.05. +indicates increased sensitivity, 2indicates decreased sensitivity, and 0 indicates no change. N/A indicates that the
assay was not applicable to that mutant, and N/D indicates test not done. In the ‘‘imd rescue’’ column, +indicated that the phenotype is rescued in
imd mutants, 2indicates no rescue. The ‘‘screens in vertebrate models’’ column indicates which mutants were identified in screens for F.novicida
mutants previously.
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assays indicated that the microarray data produced by the TraSH
assays is useful for predicting negatively selected mutants but was
somewhat non-quantitative; the degree of attenuation as measured
by microarray analysis did not always correlate with the strength of
the phenotype observed by competition assay.
F. novicida mutants demonstrating increased sensitivity
to oxidative stress are attenuated in Drosophila
One set of negatively selected mutants stood out as particularly
interesting because they indicated a bacterial requirement for
resistance to oxidative stress within the fly. These mutants included
the LysR family transcriptional regulator oxyR. The homologue of
this gene in E. coli has been shown to sense hydrogen peroxide and
induce the transcription of downstream genes that provide
protection against oxidative stress [23]. We also identified a
number of genes that are essential for repairing damage to DNA
such as that caused by reactive oxygen, including uvrA, uvrB,
recB, ssb, mutM, and ruvC [24] (Figure 3B). This result is
consistent with a screen for attenuated F. novicida U112 transposon
mutants using an inhalation method of inoculation, which
identified the DNA repair proteins recO and recA [21].
The fly’s melanization immune response produces reactive
oxygen as an effector and thus we hypothesized that these bacterial
genes were involved in helping F. novicida resist melanization
Figure 3. A negative selection screen of
F. novicida
mutants identifies bacterial genes important for bacterial growth and survival
within the fly. Transposon Site Hybridization (TraSH) experiments were used to identify bacterial mutants that failed to replicate within Drosophila
at a rate similar to wild-type bacteria. (A) Candidate mutants were tested individually using competition assays in which each mutant was injected
into flies at a 1:1 ratio with wild type bacteria. Following 2 days of infection, the bacteria from each fly was plated and a competitive index was
determined using the ratio of mutant bacteria to wild type bacteria and comparing that to the input ratio. One sample t-tests showed that all
mutants shown had competitive indexes significantly different from 1. The P values for each mutant are mglA,0.0001, pdpB,0.0001, kdpD,0.0001,
FTN_0494,0.001, FTN_1657,0.0001, FTN_1654,0.0001, FTN_1099 = 0.007, pilA,0.0001, pilC = 0.0035, FTN_1452,0.0001, fumA = 0.0047,
FTN_1719 = 0.0003, FTN_1276,0.0001, FTN_0921 = 0.0001, and talA = 0.0016. Two genes that were identified by TraSH but not confirmed as
statistically significant, FTN_0346 and FTN_0392 are shown on the far right. Horizontal lines indicate the geometric mean. (B) Mutants of interest
identified in the TraSH analysis include bacteria that are impaired in their ability to resist oxidative stress damage, including the transcriptional
regulator oxyR and multiple DNA repair pathway genes. One sample t-tests showed that all mutants shown had competitive indexes significantly
different from 1. The P values for each mutant are oxyR,0.0001, uvrA,0.0001, uvrB = 0.0004, recB,0.0001, ssb,0.0001, and ruvC,0.0001. Horizontal
lines indicate the geometric mean. (C) Disk diffusion assay comparing oxyR to U112 wild-type bacteria demonstrated increased susceptibility to
reactive oxygen produced by hydrogen peroxide (D) DNA damage repair mutants are also sensitive to oxidative stress as measured by disk diffusion
assay with hydrogen peroxide. Error bars represent standard error. All of the mutants are statistically different than U112 as measured by two-tailedt-
tests with P values of oxyR,0.0001, uvrA = 0.0008, uvrB = 0.0002, recB = 0.0008, mutM = 0.0008, ssb = 0.0006, and ruvC = 0.0005 (E) oxyR and uvrB
mutants are rescued in CG3066 mutant flies which are unable to produce a melanization response. Both rescues are statistically significant, with P
values in a 2-tailed t-test of 0.0165 and 0.0002 respectively. Horizontal lines indicate mean values.
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[25,26]. To test this idea, we first performed in vitro disc diffusion
assays to determine the sensitivity of each mutant to hydrogen
peroxide (H
2
O
2
) and paraquat [27]. The oxyR mutants were
extremely sensitive to both H
2
O
2
and paraquat (Figure 3C (H
2
O
2
)
and data not shown (paraquat)). In addition, all of the DNA
damage repair mutants showed a significant degree of sensitivity to
both reactive oxygen-producing agents (Figure 3D). Taken
together, these data suggest that we identified a class of F. novicida
genes that are essential for wild-type growth and survival within
the fly, genes which help the bacteria to resist oxidative stress.
Interestingly, 4 DNA repair genes, uvrA, recB, recO, and uvB,
were identified in one screen of Francisella mutants in mice,
suggesting that reactive oxygen species are a threat to the bacteria
in mammalian infections as well.
To determine whether melanization is a critical factor limiting
the growth of F. novicida, we performed competition assays using
the oxyR mutant in CG3066 mutant flies. These flies do not
induce melanization upon infection [17]. We found that the
growth defect of oxyR with respect to wild type bacteria was
rescued in non-melanizing CG3066 flies (Figure 3E). This supports
the idea that melanization is the reactive oxygen producing
immune response for limiting the growth of F. novicida in the fly.
We therefore took our collection of negatively selected mutants
and tested them for sensitivity to reactive oxygen. We found 25
mutants with increased sensitivity to oxidative stress and 2 with
decreased sensitivity (Figure 2). Thus we were able to assign
functions to these genes based on their behavior in a fly
pathogenesis screen. This group of mutants makes up 45 percent
of the mutants with attenuated growth phenotypes in the fly,
demonstrating that oxidative stress mediated immunity is an
important aspect of the fly’s defenses against this pathogen and
that an important class of F. novicida virulence factors in fly
infections are genes that help the bacteria to counteract the effects
of reactive oxygen.
Identification of F. novicida genes involved in bacterial
resistance to imd-regulated innate immune responses
Having demonstrated that the TraSH method was useful for
identifying genes required for growth in the fly and that many of
these mutants were involved in counteracting the oxidative stress
response of the fly, we decided to expand our study to examine an
additional immune pathway and look for similar interactions. We
chose to focus on one of the most intensely studied aspects of the
fly innate immune response, NF-kB signaling pathways. Drosophila
has two well-characterized NF-kB pathways (Toll and imd) that
are responsible for sensing the presence of microbes and inducing
an immune response [7]. Previous work by others demonstrated
that the imd pathway is an important component of the Drosophila
innate immune response to the LVS strain of F. tularensis, while the
Toll pathway is not [9]. To confirm this for F. novicida U112 strain,
we infected flies with null mutations in Toll and imd pathway
genes. Two separate alleles of imd, imd
1
and the null allele
imd
10191
, as well as mutants lacking the NF-kB homologue Relish
showed significantly increased sensitivity to infections with the
U112 strain; in contrast, mutants in the Toll pathway components
Dif
1
and dMyD88
C03881
showed no significant difference com-
Figure 4. Negative selection screens in Drosophila immunity mutants identify
F. novicida
mutants that help the bacteria resist the
imd-regulated host innate immune response. Survival of Toll and imd pathway mutants infected with F. novicida at 29uC. (A) Two null alleles of
imd, imd
10191
and imd
1
backcrossed to OR backgrounds were tested, and both are significantly different from OR flies with log-rank test P values of
.0.0001. (B)The Toll pathway is represented by loss of function alleles of two Toll pathway members, Dif and MyD88. Neither are statistically different
from wild-type with log-rank test P values of 0.0866 and 0.0582 respectively. (C) Confirmation of mutants identified in the TraSH analysis as
attenuated in wild-type flies and rescued in imd mutant flies. All rescues are statistically significant as measured by two-tailed t-tests, with P values of
pmrA = 0.0006, FTN_0869= 0.0001, FTN_0889= 0.0113, udp= 0.0004, glpD,0.0001, nadC = 0.0465, and FTN_0649 = 0.0030. Horizontal lines indicate
the geometric means of the samples. (D) Sensitivity of imd rescue mutants to Polymyxin B as measured by disk diffusion assay. Error bars represent
standard error. pmrA, FTN_0889, udp, glpD and FTN_0649 are statistically significantly different from U112 with 2-tailed t-test P values of 0.0299,
0.0041, 0.0495, 0.0065 and 0.0447 respectively. FTN_0869 and nadC are not significantly different than wild-type U112, with P values of 0.1404 and
0.8130.
doi:10.1371/journal.ppat.1001065.g004
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pared to wild type (Figure 4A (imd alleles only, relish data not
shown) and Figure S1). Therefore, we focused on F. novicida genes
required to resist the fly’s imd mediated response.
To identify such genes, we repeated our TraSH analysis, this
time infecting imd mutant flies and compared the results to those
found in wild-type flies. We identified 36 genes that appeared to be
negatively selected in wild type flies and at least partially rescued in
imd mutant flies (Table S2) Subsequent confirmation of these
results by competition assay using transposon insertion mutants
revealed a subset of 7 mutants that showed reproducible large
rescue phenotypes in imd flies (Figure 4C). These genes were the
orphan response regulator pmrA, the gene FTN_0889 which is a
helix-turn-helix protein and putative transcriptional regulator,
glpD which is an anaerobic glycerol-3-phosphate dehydrogenase,
the nicotinate-nucleotide pyrophosphorylase nadC, a uridine
phosphorylase udp, FTN_0649, a FAD-dependent 4Fe-4S ferro-
doxin, and FTN_0869, a hypothetical protein that encodes a
putative transglutaminase that is regulated by the virulence factor
mglA [28].
Since the imd pathway has been well-characterized as being
responsible for inducing antimicrobial peptide (AMP) mRNA
levels in response to F. novicida and other bacterial pathogens, the
simplest explanation for the rescue of these bacterial mutants in
flies lacking an intact imd pathway is that they have increased
sensitivity to AMPs. This idea is further supported by identification
of pmrA in our rescue screen, as pmrA has previously been shown
to be sensitive to the antimicrobial peptide polymyxin B in vitro
[29]. Therefore, we wished to determine whether the other F.
novicida genes identified in our rescue screen are also sensitive to
AMPs. There are 7 families of AMPs in Drosophila, and more
than 2 dozen individual AMPs can be expressed during an
infection, producing a complex bacteriocidal cocktail. Among the
characterized AMPS, four families have been implicated in killing
Gram-negative microbes, attacin, cecropin, diptericin, and
drosocin. The first three families contain cation rich peptides
while drosocin is described as proline rich [7]. It is currently
impossible to recreate in vitro, the array of AMPs brought to bear
on an infecting microbe in vivo. We therefore tried testing
individual AMPs for their effects on F. novicida mutants.
Unfortunately, few of these Drosophila AMPs are available
commercially. We tested a commercial preparation of cecropin
and did not detect activity against F.novicida on plates (data not
shown). We turned to the cationic antimicrobial peptide
polymyxin B, which has been used to model AMP sensitivity in
F. tularensis in multiple studies [29,30].
Of the seven genes we confirmed to be rescued in imd mutant
flies, we found that five of these genes, pmrA, FTN_0889, glpD,
udp, and FTN_0649 were indeed more sensitive to polymyxin B in
vitro. Suprisingly, mutants in the genes FTN_0869 and nadC did
not show any phenotypes in these assays, suggesting that the imd
rescue phenotype of these mutants may not be due AMP
sensitivity, or at least not to cationic AMP sensitivity (Figure 4D).
To determine how common this phenotype was, we expanded our
analysis to include the entire set of confirmed attenuated mutants
described in Figure 2. We found that twelve of the fourteen F.
novicida mutants that were rescued in imd mutant flies on arrays
showed altered sensitivity to polymyxin B, whereas this was the
case with just five of the thirty eight mutants not rescued in an imd
mutant (Figure 2). These five mutants were likely exceptions as
they also had defects in reactive oxygen sensitivity and in the
absence of an imd mediated response would still be sensitive to a
melanization response. In our entire set of attenuated mutants,
only nadC and FTN_0869 mutants demonstrated the unique
phenotype of rescue in an imd mutant fly without showing any
increased sensitivity to AMPs, so we chose to focus on one of these
genes, the putative transglutaminase FTN_0869 for further
analysis.
FTN_0869 deletion mutants are attenuated in wild-type
flies due to clearance of extracellular bacteria by an imd-
dependent immune response
The mutation in the gene FTN_0869 was intriguing as it clearly
grows better in imd mutants as compared to wild type flies yet the
mutant does not demonstrate altered sensitivity to the antimicro-
bial peptide we tested. The fly produces dozens of AMPs at once
and not all of them work by the same mechanism, therefore it is
impossible and illogical to eliminate the possibility that a single
untested AMP or combination of imd induced AMPs might be
responsible for killing F. novicida. Regardless, the resistance of
FTN_0869 mutants to an AMP raises the question that the imd
pathway might be generating an immune response that was not
AMP mediated. In addition, the fact that this gene is regulated by
the virulence factor mglA which regulates the F. tularensis
pathogenicity island and many other important virulence factors
suggested that it could be particularly important to F. tularensis
pathogenesis. To determine the extent of the attenuation of
FTN_0869 mutants, we examined the growth and survival of these
bacteria in individual infections.
We observed that with a starting dose of 5610
3
bacteria the
FTN_0869 mutant took significantly longer to kill wild-type flies
than did wild-type F. novicida (Figure 5A). This phenotype was
completely rescued in imd mutant flies, with both FTN_0869
mutants and wild-type bacteria killing the fly with a mean time to
death of 7 days, consistent with the sensitivity phenotype observed
for imd flies (Figure 5B and Figure S1). In wild-type Drosophila, the
FTN_0869 mutant did not develop the high bacterial loads found
in wild type flies; wild type F. novicida can reach titers of 5610
7
CFU per fly within 4 days while the FTN_0869 mutant did not
grow higher than 5610
5
CFU/fly (Figure 5C). Again, this
phenotype was abrogated in imd mutant flies, in which both wild
type bacteria and FTN_0869 mutants were able to grow to similar
high titers. (Figure 5D) This suggested that an imd-regulated
immune response was preventing the FTN_0869 mutants from
growing as well as wild-type U112 bacteria in the fly. We infer that
the decreased bacterial population was responsible for the
decreased virulence observed in terms of fly survival.
The attenuated phenotype of mglA mutants in mouse cells is
due to the inability of these mutants to survive and replicate
intracellularly [31]. Since FTN_0869 is regulated by mglA, we
sought to determine whether the same was true for this mutant.
We performed gentamycin chase assays on wild-type U112, mglA
mutants, and FTN_0869 mutants. As expected, the mglA mutants
showed no bacterial growth within the fly but rather were partially
cleared very quickly following injection into the fly, and were
completely unable to establish an intracellular population
(Figure 6A). This suggests that the small intracellular population
may be important, if not essential, for the establishment of a
successful infection. In contrast, the FTN_0869 mutants had a
robust albeit slightly reduced intracellular population as compared
to wild type bacteria, but demonstrated a unique phenotype with
little to no extracellular bacteria present in wild-type flies
(Figure 6B). By testing sensitivity to gentamycin in vitro, we were
able to show that this was due to lack of extracellular bacteria
rather than an increased sensitivity of the FTN_0869 mutant to
gentamycin (Figure S3). Again, loss of the imd pathway in the host
animal eliminated this effect; the extracellular population of
FTN_0869 mutant bacteria grew to similar levels as wild-type
bacteria in imd mutant flies (Figure 6C).
Probing Innate Immunity Using Francisella
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This result suggested that the extracellular population of
bacteria was unable to persist in the extracellular space of infected
flies due to an immune mechanism that is controlled by the imd
signaling pathway. Therefore, we were interested in investigating
this mutant further to determine what effector arm of the innate
immune system was responsible for the clearance of extracellular
bacteria. We hypothesized that this clearance could be due to
either an increased activation of the imd pathway by the
FTN_0869 mutants, AMP activity that we were unable to test in
vitro, or a novel component of the imd-regulated immune response.
To determine if the imd pathway is induced more intensely by the
FTN_0869 mutant, to rule out the possibility that this gene is able
to downregulate the imd immune response, we measured the
induction of imd-regulated AMPS as a readout for imd pathway
activation. We used quantitative real-time RT-PCR to monitor
the levels of Diptericin, Drosocin, Drosomycin, Attacin, Cecropin
and Metchnikowin at 1,2,5 and 24 hours post-infection. We found
that only Metchnikowin, Cecropin, and Diptericin were strongly
induced in response to F. novicida infections and that the transcript
levels of each of these highly-induced AMPs peaked at 24 hours
post-infection (Figure 7A and data not shown). All of these AMPs
were induced to similar levels during infections with either the
wild-type or FTN-0869 mutant bacteria, with no statistically
significant difference between induction by wild-type or mutant
bacteria at any timepoint. This confirms that the imd pathway is
indeed activated by F. novicida and that the gene FTN_0869 does
not have an effect on the induction of the imd pathway.
Clearance of extracellular FTN_0869 mutants is not
dependent on antimicrobial peptides or melanization
We wished to probe the role of AMPs in clearing F. novicida
further. There are more than 30 antimicrobial peptides in the fly
and purified Drosophila AMPs are not readily available and the
AMPs are always expressed together during an immune response;
as described above, this makes it difficult to directly test the role of
AMP activity on F. novicida growth in the fly. We therefore tried an
indirect approach to test their importance. Recent work in the
beetle Tenebrio molitor demonstrated that the majority of bacteria
injected into the insect is cleared in less than an hour post-
infection, much faster than antimicrobial peptides can be
upregulated, transcribed, and synthesized [32]. Using this larger
insect model, Haine et al. were able to conclusively demonstrate
that insect antimicrobial peptide activity is induced slowly, and
thus is not responsible for the bulk of the bacterial clearance. The
analysis of antimicrobial peptide induction in the fly relies on the
analysis of mRNA transcript levels, which are less accurate
kinetically than a direct measurement of antimicrobial activity but
nevertheless suggest that a slow induction with transcript levels
only rising hours after infection and peaking at 6–24 hours post-
infection for various AMPs [33]. To determine if the kinetics of
Figure 5.
F. novicida
deletion mutants of a putative transglutaminase are severely attenuated in virulence and growth. (A) Survival of
wild-type and FTN_0869 mutant bacteria in wild-type flies at 25uC. FTN_0869 mutants demonstrate significantly lower survival compared to wild-type
F. novicida with a P value by log-rank analysis of ,0.0001. The MTD for U112 is 9 days at 25uC, while the MTD for FTN_0869 is 12 days post infection,
with a P value by log-rank analysis of ,0.0001. (B) Survival of wild-type and FTN_0869 mutant bacteria in imd mutant flies at 25uC. The FTN_0869
phenotype is now partially rescued as the FTN_0869 mutant and wild-type Francisella die with MTDs of 7 and 8 days respectively at 25uC, a 4-fold
decrease in the spread between mutant and wild-type survival. (C) Total wild type and FTN_0869 mutant growth in wild type OR flies, showing a
growth defect of FTN_0869 mutants. At each timepoint, U112 and the FTN_0869 mutants are significantly different with P values from a 2-tailed t-test
of ,0.0001. At 24 hours post-infection, U112 has 8.5-fold more CFU/fly than the FTN_0869 mutant. At 48 hours, U112 has 8.9-fold more bacteria, and
at 72 hours, U112 infected flies have a full 45-fold more bacteria than the FTN_0869 mutant, with a difference between mutant and wild-type of
5.9*10
5
at 24 hours, 1.7*10
6
at 48 hours, and 1.1*10
7
at 48 hours. Horizontal lines represent the mean CFU/fly at each timepoint. Error bars represent
standard error. (D) Growth of wild-type and FTN_0869 mutant bacteria in imd mutant flies, showing rescue of the growth defect. The difference
between the mean number of CFUs of wild-type and FTN_0869 has decreased at every timepoint, with only a 2.9-fold increase in wild-type bacteria
compared to FTN_0869 bacteria at 24 hours, 6-fold more bacteria in U112 infected flies at 48 hours, and only 4-fold more U112 bacteria than
FTN_0869 bacteria at 72 hours. Horizontal lines represent the mean CFU/fly at each timepoint. Error bars represent standard error.
doi:10.1371/journal.ppat.1001065.g005
Probing Innate Immunity Using Francisella
PLoS Pathogens | www.plospathogens.org 9 August 2010 | Volume 6 | Issue 8 | e1001065

extracellular bacterial clearance coincide with AMP induction, we
performed gentamycin chase assays at early timepoints post
infection. As early as 1 hour post-infection much of the FTN_0869
mutant population had already been cleared from the fly; in
contrast the extracellular population of wild-type bacteria did not
substantially decrease (Figure 7B). By two hours post-infection, the
timepoint at which both wild-type and mutant bacteria had begun
to enter cells, the wild-type bacteria now had both intracellular
and extracellular populations, while in FTN_0869 mutant
infections only the intracellular bacteria had survived clearance.
By five hours post infection the extracellular population of U112
wild-type bacteria had begun to increase while the titer of
FTN_0869 mutants did not and only the intracellular population
remained. This supported the notion that imd-induced AMPs
were not responsible for the clearance of extracellular FTN_0869
mutant bacteria, as the bulk of this clearance occurred within an
hour post-infection before AMP activity would be upregulated.
We next sought to determine if one of the other effector arms of
the fly innate immune system could be causing this phenotype. We
first examined the effects of reactive oxygen species on the
FTN_0869 mutants. Unlike many of the genes we isolated from
our TraSH screen, the FTN_0869 mutants did not show increased
sensitivity to reactive oxygen species produced by H
2
O
2
or
paraquat in vitro as measured by disk diffusion assay (Figure 2). We
next examined the effects of melanization in vivo by infecting
CG3066 mutant flies with wild type and FTN_0869 mutants.
Unlike the oxyR mutants, the FTN_0869 mutants were just as
attenuated in CG3066 mutants as they are in the wild-type control
(Figure 7C) suggesting that these mutants do not have a defect in
resisting reactive oxygen stress and that melanization is not
A.
B.
C.
CFU/fly
7000
6000
5000
4000
3000
2000
1000
000.5 125
Total
Intracellular
Total
Intracellular
CFU/fly
2000
1000
0
00.5 12
5
U112 mglA
Time (Hours) Time (Hours)
0
10
102
103
104
105
106
108
CFU/fly
107
024 48
Total
Intracellular
0
10
102
103
104
105
106
108
CFU/fly
107
024 48
Total
Intracellular
U112
U112
FTN_0869
FTN_0869
Time (Hours)
Time (Hours)
Time (Hours) Time (Hours)
0
10
102
103
104
105
106
108
CFU/fly
107
0
10
102
103
104
105
106
108
CFU/fly
107
Total
Intracellular
Total
Intracellular
024 48 024 48
Figure 6.
F. novicida
mglA mutants are unable to survive intracellularly, while FTN_0869 mutants are unable to survive
extracellularly. (A) OR flies were treated with gentamycin at 0, 0.5, 1, 2, and 5 hours post infection after incubation at 29uC. Total CFUs per fly and
intracellular CFUs as determined following gentamycin treatment for U112 and mglA mutant bacteria. U112 infected flies are represented on the left
in red, mglA infection on the right in green. By 30 minutes post infection 1.5% of U122 CFUs are intracellular, at 1 hour post infection this has
increased to 2.3%, by 2 hours 9.6% of the bacteria are intracellular, and by 5 hours, 43.4% of U112 is intracellular. In contrast, none of the mglA
mutant bacteria is intracellular is intracellular at any timepoint. Horizontal lines represent the mean of all of the data points. (B) Total CFUs in OR wild-
type flies and intracellular CFUs and 0, 24, and 48 hours for U112 and FTN_0869 mutant bacteria. U112 infections are graphed on the left in red,
FTN_0869 mutant infection on the right in blue. At 24 hours post infection, 8.2% of the U112 CFUs are intracellular, and by 48 hours only 2% is
intracellular because the extracellular population is increasing while the intracellular population remains steady. In contrast, at 24 hours post
infection 54% of FTN_0869 mutant bacteria is intracellular and 30% is still intracellular at 48 hours, due to the extracellular population failing to
increase. Horizontal lines represent the mean. (C) Total CFUs in imd mutant flies and intracellular CFUs and 0, 24, and 48 hours for U112 and
FTN_0869 mutant bacteria. U112 infections are graphed on the left in red, FTN_0869 mutant infection on the right in blue. At 24 hours post-infection
the percentages of intracellular U112 bacteria in imd flies is similar to those seen in wild-type flies, with 4.6% and 0.4% intracellular at 24 and 48 hours
respectively. However, in imd flies the extracellular population of FTN_0868 mutant bacteria is rescued such that only only 15% of the CFUs are
intracellular at 24 hours and only 2% is intracellular at 48 hours post-infection. Horizontal lines represent the mean.
doi:10.1371/journal.ppat.1001065.g006
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responsible for the FTN_0869 imd rescue phenotype. We
concluded that the imd rescue phenotype of FTN_0869 mutants
not likely due to cationic antimicrobial peptides or melanization
and rather suggested a third category of F. novicida interactions
with the fly immune system as shown in Figure 2.
Discussion
Our goal was to dissect the host-pathogen interactions between
Francisella and D. melanogaster. To identify components of this
complex system we used a combination of three genetic techniques
that enabled us to determine the contributions of both host and
microbe to the virulence of the infection. First, we identified
bacterial virulence factors necessary to infect the fly using a library
of F. novicida mutants. Second, we used fly immunity mutants to
confirm which host immune pathways were essential for fighting F.
novicida infections. Finally, we combined these two techniques to
identify subsets of bacterial virulence factors that allow the bacteria
to counter-respond to specific immune attacks and evade immune
clearance. This paper identifies genes from both the pathogen and
the host that are components of each of these aspects of the host-
pathogen relationship.
To identify bacterial virulence factors, we performed an in vivo
screen that identified 149 bacteria genes that are important for
growth and survival within the fly. 41 of the 149 genes had
previously been identified in a similar screen performed with the
same bacterial library in the mouse indicating that many bacterial
virulence factors are conserved between host species [20–22].
Genes that overlap between the Drosophila and mouse screens
include known virulence factors such as mglA, iglC, and iglD, a
number of various transporters, and some of the DNA repair genes
we identified as helping F. novicida to survive oxidative stress. The
remaining genes are unique to our screen performed in the fly
model. These genes could either represent F. novicida genes that
play a role specific to arthropod vectors, demonstrate a stronger
phenotype in insects than in mammals, or were not identified in
previous screens for experimental reasons.
We note that of the 26 F.novicida mutants identified as being
sensitive to reactive oxygen, 7 (27%) had been previously identified
as being important for virulence in vertebrates. In contrast, of the
16 mutants we found to be polymixin sensitive, only 1 (7%) was
identified previously as being important for virulence in verte-
brates. The numbers in this study are small enough that
differences in representation could be due to chance and therefore
future work with more pathogens will be required to confirm the
trends seen here; that said, analysis of interactions with the fly’s
reactive oxygen based immune response seems to be useful
predictor of genes that will be of interest to those studying
Figure 7. The clearance of extracellular FTN_0869 mutant is not due to altered imd pathway activation, antimicrobial peptide
induction, or the
Drosophila
melanization response. (A) Antimicrobial peptide RNA levels for Cecropin, Diptericin, and Metchnikowan as
determined by quantitative RT-PCR. Error bars represent standard error. Antimicrobial peptide induction is not significantly different between wild-
type and FTN_0869 mutant F. novicida infections for any of the AMPs tested at any timepoint. (B) Gentamycin chase experiments for early time
points, before the induction of antimicrobial peptides. The kinetics of the clearance of extracellular FTN_0869 mutant bacteria are too rapid to be
attributed to antimicrobial peptide induction. At one hour post infection, clearance of FTN_0869 mutants has already begun. While only 2.8% of U112
CFUs are intracellular at 1 hour, 29% of FTN_0869 mutant bacteria is already intracellular presumably because the total number of CFUs present in
the fly has reduced from 10
4
per fly to 2*10
3
per fly. By two and five hours post-infection at 29uC wild-type bacteria have both extracellular and
intracellular populations and have begun to replicate. At 2 hours post infection, the total CFU’s of U112 per fly has doubled from 1*10
4
to 2*10
4
with
14% of the CFUs intracellular. At 5 hours post-infection, the mean total CFUs per fly is 4*10
4
with 45% intracellular at 5 hours. In contrast, at 2 hours
post infection, 27.6% of the FTN_0869 mutant bacteria are intracellular and the total CFUs per fly remains steady at 10
3
/fly. At 5 hours post infection
the bacterial levels have increased slightly to 3*10
3
/fly but the intracellular population has increased to 80% of the total CFUs/fly. Horizontal lines
indicate the mean. (C) Competitive indexes of FTN_0869 mutants in wild-type w
1118
flies and in non-melanizing CG3066 mutant flies two days post-
infection at 29uC. There is no statistically significant difference between the competitive indices in wild-type and non-melanizing flies, with a P value
of 0.601 by 2-tailed t-test, showing that the FTN_0869 mutants are not rescued in Drosophila melanization mutants. Horizontal lines represent the
geometric mean of each data set.
doi:10.1371/journal.ppat.1001065.g007
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vertebrates. In contrast, analysis of the AMP and imd sensitive
mutants is not as robust a tool for identifying mutations that will
are relevant in vertebrates.
Secondary screens of these mutants revealed important patterns
that shed light onto what particular stresses F. novicida encounters
within the fly. 25 of the 56 mutants that we confirmed to have
reduced competitive indexes compared to wild-type F. novicida
were also hyper sensitive to oxidative stress in vitro. This indicates
that preventing or repairing damage caused by reactive oxygen
species is an important survival strategy for F. novicida in insect
infections. Of particular interest among the genes that were
sensitive to oxidative stress was the gene oxyR, which has
homology to an E. coli transcriptional regulator that senses and
responds to the presence of hydrogen peroxide by inducing the
transcription of catalases and other genes that can counteract
oxidative stress
23
. In addition, our screen identified multiple genes
in DNA damage repair pathways that are also sensitive to
oxidative stress [24,25]. We expect that these genes are required to
repair damage caused by reactive oxygen species to DNA as has
been suggested by Kraemer et al [21].
Of the three effector arms that have been characterized in the
immune response occurring within the fly’s body cavity, the major
producer of oxidative stress is the melanization response [15,17,26].
Therefore, we speculated that the large number of negatively
selected bacterial mutants with oxidative stress sensitivity pheno-
types suggested that the melanization response plays a large role in
the fly’s immune response to F. novicida. To test this hypothesis, we
performed competition assays with the oxyR mutants in fly mutants
that lack a melanization response. As expected, the attenuation of
these mutants was rescued in flies that do not melanize and
therefore would be expected to not produce toxic oxygen species.
This demonstrated that melanization is an essential component of
the fly immune response against F. novicida and demonstrated that
we could use our characterizations of bacterial genes to learn about
the fly immune system and understand the host-pathogen
relationship. We note that reactive oxygen is a well-established
immune effector in the Drosophila gut. Perhaps most microbes
encountering the fly will face this immune barrier before
encountering internal immune defenses. Thus protection against
reactive oxygen is doubly important for fly pathogens [34].
Because microbes must withstand the host immune system to
mount a successful infection, we were able to exploit the inherent
ability of bacteria to function as metaphorical immunologists to
identify which aspects of fly immunity were important to F. novicida
infections. We next sought to determine if this system could be
used in the reverse direction by manipulating the fly immune
system to identify which bacterial virulence factors were
responsible for interacting with one specific aspect of innate
immunity. We did this by performing a second round of our in vivo
screen for bacterial mutants in an immunocompromised fly. We
focused on the imd-regulated humoral immune response, which
had previously been identified as important for fighting F. tularensis
infections [9]. We confirmed that the imd pathway, but not the
Toll pathway, was essential in combating F. novicida infections, and
performed our TraSH assay in imd mutant flies. We identified a
subset of bacterial virulence factors that were important for
infections of wild-type flies but not imd flies; this imd-regulated
immune response has been primarily characterized for its role in
the induction of antimicrobial peptides and therefore we tested
these mutants for their sensitivity to a cationic, membrane active
antimicrobial peptide, polymyxin B [29,30]. As expected, twelve of
the fourteen mutants were sensitive to polymyxin killing in vitro,
providing another example of how resistance to host immune
responses is an important component of bacterial virulence.
We identified 2 bacterial mutants that were not sensitive to
polymyxin in vitro despite being rescued in imd mutants flies. This
phenotype was unexpected as the majority of the literature
suggests imd signaling drives antimicrobial peptide production and
this is its most important job. We propose three explanations for
this phenotype. First, the rescue phenotype could be due to specific
sensitivity to additional antimicrobial peptides that were not tested
in vitro; the bacteria show no sensitivity to polymyxin but could be
sensitive to one of the 30 or more AMPs synthesized by flies.
Second, the rescue phenotype could be due to the bacterial gene
being an inhibitor of the imd pathway; in this case the bacteria
would have wild type sensitivity to AMPs but would encounter
increased concentrations of them in the fly because the bacteria
could not inhibit AMP production. Finally, the rescue phenotype
of these bacterial mutants could be due to an aspect of imd-
regulated immunity that has not been previously described.
To differentiate between these possibilities, we chose one imd-
rescue mutant, the putative transglutaminase FTN_0869 to
characterize further in terms of its interactions with the fly
immune system. We chose this gene because it had a strongly
attenuated phenotype in wild-type flies that was significantly
rescued in imd mutants and because it has previously been shown
to be regulated by the virulence factor mglA, which is essential for
F. novicida intracellular growth
28
. More recently, the homologue of
this gene in the extremely virulent Type A F. tularensis ssp. tularensis
strain Shu4 was identified in a transcriptional analysis of genes that
are upregulated inside mouse bone marrow-derived macrophages
(BMMs) [35]. Interestingly, FTN_0869 deletion mutants in the less
virulent U112 strain are unable to replicate in BMMs, but mutants
of the homologue of this gene, FTT0989 in the SCHU4 strain did
not demonstrate any intracellular replication defect [28,35].
Further characterization of the FTN_0869 mutants showed that
these mutants are attenuated for both lethality to the fly and
bacterial growth in an imd-dependent manner. However, unlike
its transcriptional regulator mglA, the FTN_0869 mutant is
capable of intracellular growth within flies, but is incapable of
surviving in the extracellular space. This phenotype is consistent
with what is observed in mouse bone marrow-derived macro-
phages with the virulent Shu4 strain, but not with the phenotype
of FTN_0869 deletion mutants in the U112 strain. The reason for
this difference is unclear, but it is interesting to note that the ability
of the putative transglutaminase deletion mutants to grow
intracellularly correlates with its virulence in mammalian and
insect hosts.
We found that the phenotype of FTN_0869 deletion mutants in
flies is imd-dependent, and used this phenotype to investigate the
role of the imd pathway in clearing the extracellular bacteria. With
this mutant, we were able to show that the imd rescue phenotype
of this particular mutant was not due to modulation of the imd
pathway because AMP genes downstream of imd are induced to
similar amounts in infections with wild-type and FTN_0869
bacteria. By examining the kinetics of the clearance of extracel-
lular bacteria, we were able to limit the possibility that other imd-
induced antimicrobial peptides that we did not test in vitro were
causing the attenuation of the FTN_0869 mutant. Using non-
melanizing mutants, we were able to rule out melanization as the
cause of this phenotype, leaving us with the possibility that imd
could either be regulating the cellular immune response or an
uncharacterized effector arm of fly immunity. Thus the
FTN_0869 phenotype suggested a third category of host-pathogen
interactions between F. novicida and the Drosophila innate immune
system. Future work with this mutant and other imd-rescue
mutants identified in our screen could provide further insight into
the biology of the imd-regulated fly immune response.
Probing Innate Immunity Using Francisella
PLoS Pathogens | www.plospathogens.org 12 August 2010 | Volume 6 | Issue 8 | e1001065

In summary, reciprocal studies of a pathogen, F. novicida and a
host, D. melanogaster, allowed us to identify genes in the pathogen
required to counteract, evade, or resist host immune responses and
allow bacterial growth and survival. These studies identified two
branches of host immunity that are important for fighting F.
novicida infections, melanization and imd-regulated immune
responses and helped us to understand how the bacteria resists
these responses. By identifying the mechanism of one or two
bacterial mutants based on their sequence or interaction with fly
mutants we developed assays to identify the mechanism of mutants
with unknown function. Our work with one of these mutants,
FTN_0869, taught us that there is likely more to learn about the
fly immune system as there are classes of F. novicida mutants that
cannot immediately be explained by their interactions with the
melanization response or AMPs. Our screen allowed us to pose
directed questions and focus our investigations on particular
aspects of the host immune system and the microbial strategies to
evade this immune response, helping us to identify and
characterize components of the host-pathogen relationship.
Materials and Methods
Drosophila strains
All experiments were performed in wild-type Oregon Red (OR)
flies unless otherwise noted. The imd mutant fly line imd
10191
is a
null allele with a 26-nucleotide deletion at amino acid 179 that
results in a frameshift mutation and has been backcrossed onto an
OR background. The Toll pathway alleles tested in this study are
Dif
1
which is a complete loss of function mutant and MyD88
C03881
The CG3066
KG02818
Sp7 mutant flies are PiggyBack insertion
mutants on a w
1118
background (Bloomington stock num-
ber 13494), and w
1118
flies are used as the wild-type control for
these experiments. All experiments were performed on 5–7 day
old age-matched male flies that were maintained on dextrose
medium at 25uC and 65% humidity in a 12:12h light dark cycle.
Bacterial cultures
Francisella novicida strain U112 was used for all experiments
described in this paper. Bacterial stocks were grown in Tryptic Soy
Broth (TSB) supplemented with 0.2% L-cysteine and cultured
overnight under aerobic conditions at 37uC. Cultures were grown
to an OD
600
of 1.5–2 and diluted in PBS to OD
600
0.005–0.01 for
fly infections.
Fly infections
Flies were anaesthetized with CO
2
and injected with 50
nL
of
bacteria using a glass needle and a Picospritzer III injector system
(Parker Hannifin). Each fly was injected in the ventrolateral
surface of the fly abdomen and placed into fresh vials with no
more than 20 flies per vial to prevent crowding. Following
infection, the flies were incubated at either 25uCor29uC as noted.
Each survival curve was performed using 3 replicates of 20 flies
each for a total of 60 flies per condition and each experiment was
performed a minimum of three times. The number of dead flies
was monitored daily and Kaplan-Meier survival curves were
generated using GraphPad Prism software, and statistical analysis
was performed using log-rank analysis.
Determination of bacterial CFUs and gentamycin-chase
assays
Individual infected flies were homogenized in 100mL of PBS,
serially diluted, and plated onto Mueller-Hinton (MH) agar plates
supplemented with 0.025% ferric pyrophosphate (Sigma), 0.1%
glucose, 0.025% calf serum (GIBCO), and 0.02% Iso-VitaleX
(Benton Dickinson). Plates were incubated overnight and colonies
were counted to determine the number of bacterial colony forming
units (CFUs) per fly. Statistical significance was determined using
unpaired two-tailed t-tests. Gentamycin chase experiments were
performed as described about except that 50nL of 1mg/mL of
gentamycin was injected into each fly 3 hours prior to plating [36].
TraSH experiments
Three sets of 30 flies were injected with 50nL of the trash
library. Each fly received approximately 2*10
5
CFUs of bacteria,
representing approximately 2-fold coverage of the library. The
infection was allowed to proceed for two days at 29uC, at which
point each fly was homogenized and plated onto MH agar. Plates
were incubated at 37uC overnight, and the bacteria were collected
and pooled and DNA was collected by phenol-chloroform
extraction. Each pool was divided in half and digested with either
BfaI or RsaI (NEB) and re-pooled to be used as a template for in
vitro transcription with a MegaScript T7 Kit (Ambion). The RNA
was then purified and used for reverse transcription using a
SuperScript III First Strand Synthesis Kit (Invitrogen) and random
hexamer primers. The resulting cDNA was labeled with amino-
allyl dUTP using Klenow (exo-) enzyme (NEB). The input pool
was then labelled with Cy5 and the day 2 pools with Cy3 and
hybridized to Francisella microarrays as has been previously
described [28]. Data was normalized using the Stanford Micro-
array Database according to the median log
2
Cy5/Cy3 and
filtered using a Cy3 net median intensity of 150 and a regression
correlation of .0.6. The dataset was then analyzed using SAM
software using a blocked 2-class analysis to identify differences
between the input and wild-type or input and imd mutant samples
with a false discovery rate of 5% [37].
Construction of bacterial mutants
Genes that were selected for further analysis were knocked out
of F. novicida individually to create deletion mutants. Briefly, 500bp
of sequence 59and 39to the gene of interest was amplified from
genomic F. novicida DNA using Phusion DNA Poylmerase (NEB),
and fused onto either side of a kanamycin cassette using a sewing
PCR reaction.
38
The resulting PCR products were then
transformed into chemically competent F. novicida U112 as
described [28] and the mutants were confirmed by PCR.
Competition assays
To confirm the bacterial growth attenuation phenotypes, 50nL
of a 1:1 ratio of mutant and wild-type bacteria at an OD
600
of 0.01
was injected into flies. The infection was allowed to proceed for 2
days at 29uC, following which the flies were homogenized and
plated onto MH agar plates with and without 30 mg/mL of
kanamycin. Since only the mutant bacteria is capable of growing
in kanamycin media, we were able to determine the number of
wild-type and mutant bacterial CFUs for each fly by subtracting
the number of mutant bacterial CFUs from the total CFUs per fly.
A competitive index (CI) was determined using the formula
CI = (mutant CFU day 2/wild-type CFU day 2)/(mutant CFU
input/wild-type CFU input).
Disk diffusion assays
To determine the sensitivity of various F. novicida mutants to
oxidative stress and antimicrobial peptides, disk diffusion assays
were performed using protocols adapted from Mohapatra et al.
and Bakshi et al. [27,29]. Briefly, 50mL of overnight cultures of
bacteria were plated onto MH agar plates to create a lawn of
bacteria. Plates were allowed to dry for 10 minutes, and then 6mm
Probing Innate Immunity Using Francisella
PLoS Pathogens | www.plospathogens.org 13 August 2010 | Volume 6 | Issue 8 | e1001065

Whatman filter paper disks (Fisher Scientific) were placed onto
each plate and inoculated with 10mL of 100mM freshly diluted
hydrogen peroxide (Sigma) or 10mL of a 10 mg/mL stock of
polymyxin B. Plates were incubated overnight and the diameter of
the zone of inhibition was measured for each sample. Three zones
were measured for each mutant and each experiment was
repeated three times.
qRT-PCR of antimicrobial peptides
The fold increase of antimicrobial peptide expression follow-
ing infection by wild-type and FTN_0869 mutant F. novicida was
determined by isolating RNA from infected flies 6 and 24 hours
post-infection by trizol extraction and performing qRT-PCR
analysis using an iScript One-Step RT-PCR kit with SYBR Green
(Bio-Rad) and a Bio-Rad icycler. The following primer sets were
used: cecropin 5950-tcttcgttttcgtcgctctc-39, cecropin 3959-
cttgttgagcgattcccagt-39, drosomycin 5959-gacttgttcgccctcttcg-39,
drosomycin 3959-cttgcacacacgacgacag-39, diptericin 5959-ac-
cgcagtacccactcaatc-39, diptericin 3959-cccaagtgctgtccatatcc-39,
attacin 5959-caatggcagacacaatctgg-39, attacin 3959-attcctgg-
gaagttgctgtg-3, drosocin 5959-ttcaccatcgttttcctgct-39, drosocin 39
59-agcttgagccaggtgatcct-39, metchinkowin 5959-tcttggagcgatttt-
tctgg39, metchnikowin 3959aataaattggacccggtcttg-39, ribosomal
protein 15a 59-tggaccacgaggaggctagg, 39-gttggttgcatcctcggtga.
Supporting Information
Figure S1 Alternate plots of survival curves. A second
representation of each survival curve presented in Figure 1A,
Figure 3A, B, and Figure 4A, B using line graphs showing percent
survival rather than Kaplan-Myer survival curves in order to show
error bars for each timepoint. Error bars represent standard error.
Found at: doi:10.1371/journal.ppat.1001065.s001 (1.15 MB EPS)
Figure S2 Dose dependency of F. novicida infections of the fly.
Doses ranging from 5–50,000 CFU/fly kill the fly with MTDs of
4–6 days post infection. The doses presented in this figure
correspond with the doses plotted by CFU in Figure 1B.
Found at: doi:10.1371/journal.ppat.1001065.s002 (0.54 MB EPS)
Figure S3 F. novicida gentimicin sensitivity. Sensitivity of wild-
type U112 bacteria and FTN_0869 mutant bacteria to the
antibiotic gentamycin as measured by growth in culture overnight
at 37uC.
Found at: doi:10.1371/journal.ppat.1001065.s003 (0.74 MB EPS)
Table S1 Negatively selected mutants identified by TraSH assay
in OR flies
Found at: doi:10.1371/journal.ppat.1001065.s004 (0.04 MB
XLS)
Table S2 Bacterial mutants that are attenuated in wild-type flies
and rescued in imd mutant flies by in a TraSH assay
Found at: doi:10.1371/journal.ppat.1001065.s005 (0.02 MB XLS)
Acknowledgments
We thank Dave Weiss for providing the bacterial mutant library used in
these experiments, and Annie Brotcke for providing the FTN_0869
deletion mutant. We thank Stephen Popper for invaluable assistance with
the microarray SAM analysis, and Moria Chambers for assistance with the
fly experiments. Finally, we thank Peter Sarnow, David Relman, Mary
Beth Mudgett and all the members of the Schneider and Monack lab for
helpful discussions and feedback on this project.
Author Contributions
Conceived and designed the experiments: MGM DMM DSS. Performed
the experiments: MGM. Analyzed the data: MGM DMM DSS.
Contributed reagents/materials/analysis tools: MGM. Wrote the paper:
MGM DMM DSS.
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