Drosophila eiger Mutants Are Sensitive
to Extracellular Pathogens
David S. Schneider*, Janelle S. Ayres, Stephanie M. Brandt, Alexandre Costa, Marc S. Dionne¤, Michael D. Gordon,
Eric M. Mabery, Madeleine G. Moule, Linh N. Pham, Mimi M. Shirasu-Hiza
Department of Microbiology and Immunology, Stanford University, Stanford, California, United States of America
We showed previously that eiger, the Drosophila tumor necrosis factor homolog, contributes to the pathology induced
by infection with Salmonella typhimurium. We were curious whether eiger is always detrimental in the context of
infection or if it plays a role in fighting some types of microbes. We challenged wild-type and eiger mutant flies with a
collection of facultative intracellular and extracellular pathogens, including a fungus and Gram-positive and Gram-
negative bacteria. The response of eiger mutants divided these microbes into two groups: eiger mutants are
immunocompromised with respect to extracellular pathogens but show no change or reduced sensitivity to facultative
intracellular pathogens. Hence, eiger helps fight infections but also can cause pathology. We propose that eiger
activates the cellular immune response of the fly to aid clearance of extracellular pathogens. Intracellular pathogens,
which can already defeat professional phagocytes, are unaffected by eiger.
Citation: Schneider DS, Ayres JS, Brandt SM, Costa A, Dionne MS, et al. (2007) Drosophila eiger mutants are sensitive to extracellular pathogens. PLoS Pathog 3(3): e41. doi:10.
The fruit fly has four main immune mechanisms to fight
circulating microbes. These mechanisms include secreted
antimicrobial peptides (AMPs), melanization, clotting, and
phagocytic hemocytes [1?4]. The most deeply studied of these
mechanisms, AMP secretion, is controlled by the Toll and imd
pathways, which regulate the transcription of AMP genes in
the fat body. Signaling through the Toll and imd pathways is
activated by microbial elicitors; for example, the receptor
peptidoglycan receptor protein LC, one receptor that
triggers imd signaling, is activated by diaminopimelic acid–
containing peptidoglycan [5,6]. This material is found on
most Gram-negative bacteria as well as some Gram-positive
Mutations affecting the Toll and imd pathways severely
immunocompromise flies. Although Toll and imd mutant flies
get sick and die from infections, this does not resemble many
infectious processes in healthy humans. For example, an imd
mutation essentially turns flies into a passive culture medium
for Escherichia coli; the bacteria grow 1,000-fold in 24 h [7,8].
Immunocompromised infected flies likely die from the
enormous load of bacteria that can reach levels of more than
1% of the mass of the fly. Death from massive numbers of
microorganisms can happen in humans, particularly in
immunocompromised patients; however, the infectious
agents that are responsible for the greatest mortality in
humans—Mycobacterium tuberculosis, HIV, Plasmodium falcipa-
rum, and diarrhea-inducing microbes—do not work in this
manner. Instead, relatively small numbers of these infectious
agents cause various pathologies that lead to death. To study
microbial pathogenesis in the fly, it is necessary to follow
microbes that cause disease in wild-type flies.
Pathogens are different from nonpathogenic bacteria and
simple molecular elicitors of innate immunity, and thus the
results we see from experiments with pathogens will be
different than those observed for simple elicitors like E. coli
and Micrococcus luteus. For one thing, pathogens can override
the strong immune defenses of the fly; this causes disease. In
flies, as in humans, there is more than one type of disease that
results from infection. Pathogens have been observed to kill
the fly in at least four different ways: first, overactivation of
the Toll or imd pathways can be pathogenic [9,10]; second,
Vibrio cholera fed to flies kills the fly through the secretion of
toxins that presumably cause physiological changes to the gut
; third, M. marinum causes a wasting disease in flies ;
and fourth, Salmonella typhimurium secretes effectors through
its type III secretory system that increase the pathogenicity of
the microbe. The fly gene eiger, the fly’s sole tumor necrosis
factor homolog, is implicated in causing pathology during
this infection because S. typhimurium–infected eiger mutant
flies live longer than infected wild-type flies . It appears
that there are many physiological routes that can lead to
death following infection.
We have begun to try to understand these physiological
routes to death by analyzing the role played by eiger in a
variety of infections. The eiger mutation divided our group of
pathogens into two unanticipated groups. The first group of
microbes kills eiger mutants more rapidly than wild-type flies;
this group includes a fungus as well as both Gram-positive
and Gram-negative bacterial species. The second group of
microbes kills eiger mutants at the same rate or more slowly
Editor: Frederick M. Ausubel, Massachusetts General Hospital and Harvard Medical
School, United States of America
Received October 23, 2006; Accepted February 6, 2007; Published March 23, 2007
Copyright: ? 2007 Schneider 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.
Abbreviations: AMP, antimicrobial peptide; FITC, fluorescein isothiocyanate; RT,
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
¤ Current address: Departments of Craniofacial Development and Microbiology,
King’s College London, London, United Kingdom
PLoS Pathogens | www.plospathogens.orgMarch 2007 | Volume 3 | Issue 3 | e41 0001
than wild-type flies; this group includes a Gram-positive and a
Gram-negative bacterial species and a mycobacterium.
These results cut across the previous descriptions of
Drosophila immunity, which groups microbes according to
their gross physical characteristics [2,3]. The most obvious
way to explain the grouping of these pathogens in eiger
mutants is not by grouping microbes according to their
Gram-staining properties or kingdom, but rather by their
pathogenesis mechanisms; the first group of microbes
consists of extracellular pathogens while the second group
contains microbes that can grow within professional phag-
ocytes. eiger appears to play a role in innate immunity in
fighting extracellular pathogens but plays a role in driving
pathogenesis when fighting some intracellular pathogens.
This study demonstrates that the examination of real
pathogens and the use of outputs other than AMP tran-
scription leads to the discovery of unanticipated immune
pathways and reveals new complexities in the Drosophila
To determine the role eiger plays in fighting microbial
pathogens, we tested a collection of Gram-positive, Gram-
negative, and fungal pathogens that could cause either
intracellular or extracellular infections (Table 1). Our goal
was to test a broad group of microbes that used different
virulence mechanisms and were recognized by different
innate immunity pathways.
We tested a heterozygous combination of two eiger null
alleles (w1118; egr1/egr3) and compared this to an isogenic w1118
parental strain (Figure 1). eiger mutants died much faster than
wild-type flies when challenged with Beauveria bassiana,
Burkholderia cepacia, Enterococcus faecalis, Staphylococcus aureus,
and Streptococcus pneumoniae (Figure 1). The members of this
group of microorganisms are very different from each other
and include Gram-positive and Gram-negative bacteria and a
fungus. The smallest effect on survival was a 50% reduction in
the mean time of death for B. bassiana infections. The effect
was strongest with eiger mutants infected with S. aureus and
E. faecalis; whereas the chosen doses were nonpathogenic to
wild-type flies (i.e., they died at the same rate as media-
injected controls), eiger mutants died within 2 d. The one
common characteristic of this group is that these microbes
are all expected to produce extracellular infections.
eiger mutant flies did not die faster when infected with the
three facultative intracellular pathogens we tested (Figure 1).
We reported previously that eiger mutants live longer than
wild-type flies when infected with S. typhimurium. This result
led us to suggest that eiger activity could be deleterious for the
fly and was a cause of pathology. We found that M. marinum–
infected eiger mutants also live longer than infected wild-type
flies. Finally, eiger mutants and wild-type flies survive exactly
the same amount of time when infected with Listeria
We tested E. coli as a nonpathogenic control (Figures 1 and
S1). We define a pathogen as a microbe that increases the
death rate of infected flies as compared to a control fly
injected with medium. This microbe was chosen because it
played a major role in characterizing the immune response of
the fly. E. coli is normally a good inducer of innate immune
responses when injected into the fly but will only cause
disease in flies missing the imd pathway. E. coli injection does
not kill eiger mutants or wild-type flies. This suggests either
that eiger does not play a role in fighting a nonpathogenic
infection or that eiger’s role is redundant and therefore
undetectable by this assay.
We next decided to determine whether eiger mutant flies
were killed faster by extracellular pathogens because of
increased bacterial growth or increased pathogenesis by
Table 1. Microbial Strain List
MicrobeStrain Class Infection Toll Mutant Phenotypeimd Mutant Phenotype Reference
The designation ‘‘sensitive’’ indicates that the indicated mutant fly dies faster than a parental control when infected with the listed microbe; ‘‘not sensitive’’ means that there is no change
with regard to the parental phenotype, but because most of these microbes are pathogens, the flies ultimately die from the infection.
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Microbial Pathogenesis in Drosophila
We show that the gene eiger, which is the sole tumor necrosis factor
homolog in the fruit fly, can play opposing roles in the fly’s response
to infections. Sometimes eiger contributes to the disease induced by
an infection, while at other times it is required to fight an infection.
Commonly, the fly’s immune response is described as dividing
microbes into two groups with Gram-positive bacteria and fungi
lying in one group and Gram-negative bacteria lying in the other.
Pathogenic bacteria can also be divided into two groups based on
their behavior in eiger mutant flies, but these two groups differ from
past descriptions. eiger tends to be required for the innate immune
response against extracellular pathogens but tends to cause
pathology during an infection with an intracellular pathogen. We
suggest that eiger is required for innate immune responses that are
effective at fighting extracellular pathogens but are wasteful or
simply ineffective when fighting intracellular pathogens. We show
here that the fly immune response is more complex than previously
recognized and suggest new directions for studying pathogenesis in
addition to innate immunity in the fly.
monitoring bacterial proliferation in infected flies (Figure 2).
As before, bacteria were injected into age-matched male wild-
type parental or isogenic transheterozygous eiger mutant flies.
Flies were collected and homogenized following bacterial
challenge to measure bacterial loads.
Of the four bacterial species that showed increased
virulence in eiger mutants, two—B. cepacia and S. pneumo-
niae—had statistically significant increased growth rates in
eiger mutants. There was no clear effect on E. faecalis growth
rates in eiger versus wild-type flies. S. aureus showed a large
variation in bacterial numbers toward the end of the
infection that suggested a trend toward increased growth;
for example, the highest levels of bacteria found in eiger
mutants were 10,000 higher than those seen in wild-type flies.
However, because the variation was so great in eiger mutants,
the difference between wild-type and eiger mutants was not
statistically significant. We suggest that S. aureus might not
cause synchronous infections and thus that some flies
succumb to infection rapidly and have high numbers of
bacteria while others take longer to die and maintain lower
levels of bacteria. This would result in a huge range of
bacterial titers in a group of injected flies. E. coli were cleared
at comparable rates in eiger and wild-type flies. B. bassiana was
not tested because we do not have a good quantitative
method of measuring fungal growth. In summary, these
bacterial growth measurements show that eiger mutants are
unable to limit the growth of two and possibly three of the
four extracellular pathogens tested. This experiment does not
rule out the possibility that eiger mutants suffer greater
pathological effects of infection but does suggest that eiger in
a wild-type fly plays a role in reducing the numbers of
Figure 1. Survival of eiger Infected Flies
Week-old male flies were infected with pathogens and survival was monitored daily. (A) S. typhimurium; (B) M. marinum, (C) L. monocytogenes, (D) E. coli,
(E) S. aureus, (F) E. faecalis, (G) B. bassiana, (H) S. pneumoniae, (I) B. cepacia. Circles indicate medium-injected eiger; diamonds, microbe-injected eiger;
squares, microbe-injected parental; triangles, medium-injected parental. Medium injection is indicated by a dotted line, while microbe injection is
indicated by a solid line. Statistical significance was determined using log-rank analysis. The infected eiger and wild-type curves in (A), (B), and (E–I) are
significantly different with p , 0.0001 as determined by log-rank analysis.
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Microbial Pathogenesis in Drosophila
The proliferation of facultative intracellular pathogens did
not increase in eiger mutants. As we published previously,
S. typhimurium levels remained constant in an eiger homozy-
gote as compared to an isogenic wild-type fly .
L. monocytogenes numbers decreased significantly in eiger
mutants with respect to wild-type flies even though both
wild-type and mutant flies died at the same rate. Our
interpretation is that eiger is somehow helpful for the growth
of Listeria. We did not measure the growth rate of M. marinum.
There is no simple interpretation, using Toll or imd
signaling, that can explain the difference in sensitivity of
eiger mutants to the tested pathogens. If the eiger mutation
resulted in decreased Toll signaling, then we would expect
Listeria to kill the flies rapidly as has been reported .
Likewise, a reduction of imd activity would immunocompro-
mise the flies to S. typhimurium. imd mutants are very sensitive
to S. typhimurium and will die within 24 h when infected with
as little as a single colony-forming unit . Furthermore, if
imd signaling was affected, we would expect flies to become
sensitive to E. coli, which they do not . We are forced to
conclude that the strong effects of eiger on the pathogenesis
caused by extracellular microbes are not caused by a
reduction in signaling through the Toll and imd pathways.
To test the hypothesis that Toll and imd signaling are not
grossly reduced by a mutation in eiger, we measured the
induction of AMP transcription in eiger and parent strains
(Figure 3). Flies were challenged with S. typhimurium, B. cepacia,
L. monocytogenes, and S. aureus as well as an LB control, and
these conditions were compared to expression levels seen in
uninfected flies. We chose this subset of bacteria because it
included both Gram-positive and Gram-negative examples of
intracellular and extracellular pathogens. Toll signaling was
monitored by following drosomycin gene induction using
quantitative reverse transcription (RT)-PCR, although we
note that there are no perfect Toll responsive genes and
drosomycin is responsive to both Toll and imd signaling . To
measure the output of the imd pathway, we monitored the
expression of the diptericin gene. No statistically significant
changes in drosomycin transcription were seen between
parental and eiger mutant flies. Some conditions caused a
relatively small, but statistically significant increase in
diptericin transcription in eiger mutants. We do not understand
Figure 2. Bacterial Growth in eiger Mutants
Week-old male flies were infected with pathogens, and flies were collected at 0, 2, 6, 24, and 48 h, if there were survivors. Live flies were homogenized
and plated: (A) S. typhimurium; (B) L. monocytogenes; (C) E. coli; (D) S. aureus; (E) E. faecalis; (F) S. pneumoniae; (G) B. cepacia. Data are plotted as box plots
with whiskers. White bars indicate the parental w1118line; gray bars, w1118; eiger1/eiger3mutants. Statistical significance was calculated using two-tailed
PLoS Pathogens | www.plospathogens.orgMarch 2007 | Volume 3 | Issue 3 | e410004
Microbial Pathogenesis in Drosophila
how the slightly higher levels of AMP transcription found in
eiger mutants might affect the eiger phenotype. The important
point is that eiger mutants did not have lower levels of AMP
gene expression than did wild-type flies, demonstrating that
the eiger phenotype is not caused by reduction in Toll or imd
As Toll and imd signaling, and thus the majority of the
antimicrobial peptide response in the fly , does not seem
to be responsible for changes in eiger mutants, we next probed
the cellular immune response (Figure 4). To determine
whether eiger mutations might affect hemocyte function, we
monitored phagocytosis in eiger mutants. Fluorescein iso-
thiocyanate (FITC)-labeled S. aureus were injected into the
hemocoel of wild-type or eiger mutant flies and the injected
flies were given 1 h to phagocytose the particles. S. aureus was
chosen because FITC-labeled dead S. aureus are commercially
available and we predicted that eiger alters the ability of
hemocytes to fight this pathogen. Trypan blue was injected
into the hemocoel of the flies following the 1-h incubation
period. This dye quenches the fluorescence of extracellular
FITC but allows phagocytosed particles to fluoresce brightly
. The flies were examined around the dorsal anterior
abdomen because hemocytes tend to gather in this area.
Changes in phagocytosis can be seen by changes in the
numbers of phagocytosing cells or by the fluorescence
intensity. Both of these characteristics appear altered in eiger
mutants compared to parental flies, suggesting that eiger
mutant hemocytes were either reduced in number or had
reduced phagocytic activity, or both.
We note that uninfected eiger mutants live longer than wild-
type flies. This raises the possibility that the increased survival
of eiger mutant flies after infection with M. marinum or
S. typhimurium is simply a consequence of general increased
longevity. We argue that this possibility is unlikely, because it
assumes that the cause of death due to old age is the same as
the cause of death by infection; this is not observed in other
animals and it is not supported by examination of published
microarray studies of flies dying from infection or old age
[12,17,18]. Instead, we argue that eiger is a driver of patho-
genesis. If eiger is indeed required to fight infections but can
also cause pathology, then the phenotype we observed for a
given infection is likely the sum of the positive and negative
effects of eiger. Regardless of the interpretation of the effects
of eiger on S. typhimurium and M. marinum, the important result
remains that eiger mutations divide the fly’s response to
pathogens into two groups.
Why does eiger affect different microbes in different ways?
We argue that E. coli is removed from flies so rapidly and via
so many mechanisms that the effects of eiger cannot be
measured easily because of redundancy. In contrast, during
an infection with a real pathogen, it may be easier to measure
changes in immunity because the fly is fighting hard for
survival and its immune mechanisms are not acting in a
redundant fashion. Extracellular pathogens are clearly fought
by the fly using eiger-dependent mechanisms because the loss
of eiger results in a deeply sensitive phenotype. It is intriguing
that flies lacking eiger are no worse at fighting intracellular
pathogens. This suggests that these intracellular pathogens
are normally immune to the effects of wild-type eiger.
Intracellular pathogens like M. marinum, L. monocytogenes, and
S. typhimurium use different virulence mechanisms for grow-
ing inside cells, but the common thread is that they can
survive in professional phagocytes. We suggest that eiger
function somehow alters hemocytes to increase their potency
against microbes. The reduced ability of eiger mutant
hemocytes to phagocytose S. aureus compared to wild-type
hemocytes supports this hypothesis. We predict that this
change in potency is effective against pathogens that grow
extracellularly but not against pathogens that have already
developed methods of defeating phagocytes.
We propose that where eiger signaling does not help fight
infection, eiger can cause pathology. This eiger-induced
pathology may be linked to the immune function of eiger—
for example, the induced immune response may be energeti-
cally wasteful or directly toxic. Alternatively, eiger-induced
pathology may be separable from the immune function—for
example, eiger could cause something like muscle wasting in
Figure 3. Antimicrobial Gene Expression Differences between eiger
Mutants and Wild-Type Controls
Flies were injected with an LB control, B. cepacia, S. typhimurium, L.
monocytogenes, or S. aureus or left uninjected. RNA was harvested after a
6-h incubation at 29 8C, and quantitative RT-PCR was used to assay
drosomycin (A) and diptericin (B) gene expression levels relative to a
ribosomal protein 15a control. White bars indicate the w1118parental
control; grey bars, w1118; egr1/egr3One asterisk indicates p , 0.01; two
asterisks, p , 0.001 using Tukey’s multiple-comparison test following
one-way ANOVA. Unmarked parental/mutant pairs do not differ in a
statistically significant manner. No differences were seen with drosmycin
expression between mutant and parental lines, whereas eiger mutant
flies transcribed 2- to 3-fold more diptericin (during a 150- to 1,100-fold
induction compared with uninjected flies) than did their parents, when
challenged with LB, B. cepacia, or S. typhimurium.
Figure 4. Phagocytosis in eiger Mutants
Week-old male flies were injected with FITC-labeled dead S. aureus and
allowed to phagocytose the particles and then were injected with Trypan
blue to quench extracellular fluorescence. The dorsal abdomen of the fly
was photographed under epifluoresence optics. Anterior is on the left.
(A) Wild-type fly (w1118); (B) eiger mutant fly (w1118; egr1/egr3). Bright spots
indicate hemocytes that have phagocytosed the S. aureus. The pictures
are representative of the 15 flies examined for the experiment.
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Microbial Pathogenesis in Drosophila
the fly, as has been suggested for tumor necrosis factor in
Genetic screens that monitored AMP synthesis have been
productive and filled in the Toll and imd pathways but did not
reveal eiger signaling [20,21]. As we have shown, the role eiger
plays in innate immunity cannot be measured using a
nonpathogenic microbe like E. coli. This study demonstrates
that there are important immune mechanisms at work in the
fly that are difficult to see using simple endpoints like
antimicrobial gene expression; however, studies with mi-
crobes that can cause disease in wild-type flies—real
pathogens—can reveal these physiologies.
Materials and Methods
Fly strains. The wild-type parental strain used in all experiments is
white1118. The eiger alleles white1118;egr1and white1118;egr3were kept as
homozygous stocks and crossed to make heterozygous flies as
required . We used this heterozygous combination to reduce the
probability that there were other mutations on the chromosome that
affected pathogenesis. To control for variation in the flies, we
infected only 5- to 7-d-old male flies.
Bacterial strains. Strains used are listed in Table 1.
Pathogen culture conditions. B. bassiana cultures were grown on
malt agar at 29 8C for 2 wk or until a sufficient density was reached,
and the cultures were allowed to sporulate. Anesthetized flies were
shaken on the plates for 30 s to coat flies with spores. Flies were
transferred to fresh vials and incubated at 29 8C for the duration of
the survival experiment.
S. pneumoniae cultures were grown standing at 37 8C 5% CO2in
brain heart infusion medium (BHI) to an OD600of 0.15, and aliquots
were frozen at ?80 8C in 10% glycerol. For infection, an aliquot of
S. pneumoniae was thawed, diluted 1:3 in fresh BHI, and allowed to
adjust at 37 8C 5% CO2for 1.5 h. E. coli, S. typhimurium, E. faecalis, and
S. aureus cultures were grown overnight in Luria Bertani (LB) medium
at 37 8C. L. monocytogenes was grown standing overnight in BHI
B. cepacia, L. monocytogenes, S. pneumoniae, and S. typhimurium were grown
standing, while E. coli, E. faecalis, and S. aureus were shaken. M. marinum
was cultured standing at 29 8C in Middlebrook 7H9 broth
supplemented with Middlebrook oleic acid–albumin–dextrose–cata-
lase enrichment and 0.2% Tween. Then 50 nl of bacteria was injected
at the following optical densities (OD600): B. cepacia, 0.0001–0.001;
E. coli, 0.1; E. faecalis, 0.5; L. monocytogenes, 0.01; M. marinum, 0.05;
S. aureus, 0.001; S. pneumoniae, 0.05; and S. typhimurium, 0.1.
Injection. Five- to 7-d posteclosion male flies were used for
injection. The flies were raised at 25 8C, 65% humidity, on yeasted
dextrose food. Flies were anesthetized with CO2. Injections were
carried out with a pulled glass capillary needle. A picospritzer (Parker
Hannifin, http://www.parker.com) was used to inject 50 nl of liquid
into each fly with needles that were individually calibrated by
measuring the size of the expelled drop under oil. Reproducibility
was measured by determining the number of bacteria injected at time
zero and can be seen in Figure 2. Injected flies were incubated 20 flies
to a vial and placed at 29 8C, 65% humidity with the exception of
B. cepacia and M. marinum. B. cepacia infections were performed at 18
8C in the dark, and humidity was not controlled in this experiment.
This temperature was used because B. cepacia is so pathogenic that it is
difficult to obtain sufficient survival at higher temperatures to
observe changes in death rates. M. marinum infections were carried
out at 25 8C, 65% humidity, in the dark.
Survival curves. Parental flies (w1118) and w1118; eiger1/eiger3mutants
were injected with the microbe of choice or medium as a control.
Sixty flies were assayed for each survival curve, and they were placed
in three vials of 20 flies each. Death was recorded daily. Data were not
censored. Survival curves are plotted as Kaplan-Meier plots, and
statistical significance is tested using log-rank analysis using Prism
software (http://www.prism-software.com). Kaplan-Meier plots are
shown in Figure 1, and survival curves showing the variance in the
data are included in Figure S1. All experiments were performed at
least three times and yielded similar results.
CFU determination. Following challenge with microbes, six
individual flies were collected at each time point. These flies were
homogenized, diluted serially, and plated onto appropriate media
(blood agar for S. pneumoniae, LB for all others). E. faecalis CFUs were
determined by testing three groups of six flies for each time point.
The data are plotted as boxes with whiskers. The median is indicated
with a bold line. The boxes indicate the extent of the third and first
quartiles, while the whiskers show the complete range of the data.
Statistical significance was determined using nonparametric two-
tailed t-tests. All experiments were performed at least three times and
yielded similar results.
Antimicrobial peptide gene expression. Flies were injected with 50
nl of the indicated microbes or controls. Following injection, the flies
were placed in vials containing yeasted dextrose food and incubated
at 29 8C for 6 h. Groups of five flies were homogenized in TriZOL and
stored at ?70 8C until processed. RNA was isolated using a standard
TriZOL preparation, and the samples were treated with DNase
(Promega, http://www.promega.com). Quantitative RT-PCR was per-
formed as described  previously using a Bio-Rad icycler (http://
www.bio-rad.com) and the following primer sets: drosomycin 59-
gracttgttcgccctcttcg, drosomycin 39-cttgcacacacgacgacag, drosomycin
Taqman probe tccggaagatacaagggtccctgtg, diptericin 59-accgcagtacc-
cactcaatc, diptericin 39-cccaagtgctgtccatatcc, and diptericin Taqman
probe cagtccagggtcaccagaaggtgtg. The data shown in Figure 4 include
six biological replicates of each treatment condition, and each data
point was calculated as the mean of two technical replicates. This
experiment was repeated once using a set of three biological
replicates with similar results. The data are plotted as boxes with
whiskers. The median is indicated with a bold line. The boxes indicate
the extent of the third and first quartiles, while the whiskers show the
complete range of the data.
Phagocytosis assays. Flies were injected with 50 nl of 1 mg/ml
FITC-labeled S. aureus (Molecular Probes, http://probes.invitrogen.
com) in water. The flies were allowed to phagocytose the particles for
1 h and then were injected in the thorax with approximately 250 ll of
4% Trypan blue: this quenches the fluorescence of extracellular
bacteria but permits the phagocytosed particles to fluoresce. The
wings of the flies were removed with iris scissors, and the flies were
pinned with a minuten pin and photographed using epifluorescent
illumination with a Leica MZ3 microscope fitted with an ORCA
camera. Images were captured with Openlab (Improvision) software
(http://www.improvision.com). The exposure was set such that the
brightest images had a small number of saturating pixels. The
experiment was repeated three times with at least five flies for each
Figure S1. Variance in Survival of eiger-Infected Flies
Week-old male flies were infected with pathogens and survival was
monitored daily. (A) S. typhimurium; (B) M. marinum, (C) L. mono-
cytogenes, (D) E. coli, (E) S. aureus, (F) E. faecalis, (G) B. bassiana, (H)
S. pneumoniae, and (I) B. cepacia. Circles indicate medium-injected eiger;
diamonds, microbe-injected eiger; squares, microbe-injected parental;
triangles, medium-injected parental. The mean is plotted, and error
bars show the standard deviation from three groups of 20 injected
Found at doi:10.1371/journal.ppat.0030041.sg001 (1.4 MB AI).
We thank E. J. Baron, S. Falkow, N. Silverman, and J. Theriot for
microbial strains. We thank M. Miura for the eiger mutants and the
Author contributions. JSA, SMB, AC, MSD, MDG, EMM, MGM,
LNP, and MMSH contributed equally to this paper. DSS, JSA, SMB,
AC, MSD, MDG, EMM, MGM, LNP, and MMSH conceived and
designed the experiments, performed the experiments, and wrote the
paper. DSS, MSD, MDG, EMM, MGM, LNP, and MMSH analyzed the
data. JSA, SMB, and AC contributed reagents/materials/analysis tools.
Funding. This work was supported by National Institutes of Health
grants AI053080 and AI055651.
Competing interests. The authors have declared that no competing
PLoS Pathogens | www.plospathogens.org March 2007 | Volume 3 | Issue 3 | e410006
Microbial Pathogenesis in Drosophila
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1.Bidla G, Lindgren M, Theopold U, Dushay MS (2005) Hemolymph
coagulation and phenoloxidase in Drosophila larvae. Dev Comp Immunol
2.Brennan CA, Anderson KV (2004) Drosophila: The genetics of innate
immune recognition and response. Annu Rev Immunol 22: 457–483.
3.Hoffmann JA (2003) The immune response of Drosophila. Nature 426: 33–38.
4. Meister M, Lagueux M (2003) Drosophila blood cells. Cell Microbiol 5: 573–
5. Stenbak CR, Ryu JH, Leulier F, Pili-Floury S, Parquet C, et al. (2004)
Peptidoglycan molecular requirements allowing detection by the Drosophila
immune deficiency pathway. J Immunol 173: 7339–7348.
6.Leulier F, Parquet C, Pili-Floury S, Ryu JH, Caroff M, et al. (2003) The
Drosophila immune system detects bacteria through specific peptidoglycan
recognition. Nat Immunol 4: 478–484.
7.Lemaitre B, Kromer-Metzger E, Michaut L, Nicolas E, Meister M, et al.
(1995) A recessive mutation, immune deficiency (imd), defines two distinct
control pathways in the Drosophila host defense. Proc Natl Acad Sci U S A
8.Lemaitre B, Nicolas E, Michaut L, Reichhart J, Hoffmann J (1996) The
dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the
potent antifungal response in Drosophila adults. Cell 86: 973–983.
9. Bischoff V, Vignal C, Duvic B, Boneca IG, Hoffmann JA, et al. (2006)
Downregulation of the Drosophila immune response by peptidoglycan-
recognition proteins SC1 and SC2. PLoS Pathog 2: e14. doi:10.1371/journal.
10. Gordon MD, Dionne MS, Schneider DS, Nusse R (2005) WntD is a feedback
inhibitor of Dorsal/NF-kappaB in Drosophila development and immunity.
Nature 437: 746–749.
11. Watnick TJ, Jin Y, Matunis E, Kernan MJ, Montell C (2003) A flagellar
polycystin-2 homolog required for male fertility in Drosophila. Curr Biol 13:
12. Dionne MS, Pham LN, Shirazu-Hiza MM, Schneider DS (2006) Akt and
FOXO dysregulation contribute to infection-induced wasting in Drosophila.
Curr Biol 16: 1977–1985.
13. Brandt SM, Dionne MS, Khush RS, Pham LN, Vigdal TJ, et al. (2004)
Secreted bacterial effectors and host-produced eiger/TNF drive death in a
Salmonella-infected fruit fly. PLoS Biol 2: e418. doi:10.1371/journal.pbio.
14. Mansfield BE, Dionne MS, Schneider DS, Freitag NE (2003) Exploration of
host-pathogen interactions using Listeria monocytogenes and Drosophila
melanogaster. Cell Microbiol 5: 901–911.
15. De Gregorio E, Spellman PT, Tzou P, Rubin GM, Lemaitre B (2002) The
Toll and Imd pathways are the major regulators of the immune response in
Drosophila. EMBO J 21: 2568–2579.
16. Elrod-Erickson M, Mishra S, Schneider D (2000) Interactions between the
cellular and humoral immune responses in Drosophila. Curr Biol 10: 781–
17. Landis GN, Abdueva D, Skvortsov D, Yang J, Rabin BE, et al. (2004) Similar
gene expression patterns characterize aging and oxidative stress in
Drosophila melanogaster. Proc Natl Acad Sci U S A 101: 7663–7668.
18. Zou S, Meadows S, Sharp L, Jan LY, Jan YN (2000) Genome-wide study of
aging and oxidative stress response in Drosophila melanogaster. Proc Natl
Acad Sci U S A 97: 13726–13731.
19. Jackman RW, Kandarian SC (2004) The molecular basis of skeletal muscle
atrophy. Am J Physiol Cell Physiol 287: C834–C843.
20. Foley E, O’Farrell PH (2004) Functional dissection of an innate immune
response by a genome-wide RNAi screen. PLoS Biol 2: e203. doi:10.1371/
21. Wu LP, Choe KM, Lu Y, Anderson KV (2001) Drosophila immunity: Genes
on the third chromosome required for the response to bacterial infection.
Genetics 159: 189–199.
22. Igaki T, Kanda H, Yamamoto-Goto Y, Kanuka H, Kuranaga E, et al. (2002)
Eiger, a TNF superfamily ligand that triggers the Drosophila JNK pathway.
EMBO J 21: 3009–3018.
23. Schneider D, Shahabuddin M (2000) Malaria parasite development in a
Drosophila model. Science 288: 2376–2379.
24. Pili-Floury S, Leulier F, Takahashi K, Saigo K, Samain E, et al. (2004) In vivo
RNA interference analysis reveals an unexpected role for GNBP1 in the
defense against Gram-positive bacterial infection in Drosophila adults. J Biol
Chem 279: 12848–12853.
25. Dionne MS, Ghori N, Schneider DS (2003) Drosophila melanogaster is a
genetically tractable model host for Mycobacterium marinum. Infect Immun
PLoS Pathogens | www.plospathogens.orgMarch 2007 | Volume 3 | Issue 3 | e41 0007
Microbial Pathogenesis in Drosophila