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Drosophila melanogaster as a model host for studying Pseudomonas

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Article  in  Nature Protocol · February 2009

DOI: 10.1038/nprot.2009.124 · Source: PubMed

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 PROTOCOL

Drosophila melanogaster as a model host for studying

Pseudomonas aeruginosa infection
Yiorgos Apidianakis1,2 & Laurence G Rahme1–3
1Department of Surgery, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, USA. 2Shriners Burns Institute, Boston, Massachusetts, USA.
3Department of Microbiology and Molecular Genetics, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, USA. Correspondence should
be addressed to L.G.R. (rahme@molbio.mgh.harvard.edu).

Published online 13 August 2009; doi:10.1038/nprot.2009.124

Conservation of host signaling pathways and tissue physiology between Drosophila melanogaster and mammals allows for the
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

modeling of human host–pathogen interactions in Drosophila. Here we present the use of genetically tractable Drosophila models of
bacterial pathogenesis to study infection with the human opportunistic pathogen Pseudomonas aeruginosa. We describe and compare
two protocols commonly used to infect Drosophila with P. aeruginosa: needle-pricking and injector-pumping. Each model has relevance
for examining host components and bacterial factors in host defense and virulence. Fly survival and bacterial proliferation within host
flies can be assessed as a measure of host susceptibility and pathogen virulence potential. The profiles of host responses toward
P. aeruginosa virulent and non-virulent strains can be determined, enabling the identification of interaction-specific genes that could
potentially favor or limit the initiation and progression of infection. Both of the protocols presented herein may be adapted for the
inoculation and study of other microbial pathogens. P. aeruginosa cell preparation requires 24 h, fly inoculation 1 h, and fly survival
and bacterial proliferation 1–4 d.

INTRODUCTION
Each partner in a host–pathogen antagonistic interaction uses
specialized strategies—the host to respond to and combat the
pathogen, and the pathogen to circumvent these host defenses.
Indeed, the ultimate success of an infectious agent is often deter-
mined by the interplay between these competing strategies. Host
defenses can be general and directed against many pathogens, or
conversely, they can target-specific threats. Meanwhile, pathogen
mechanisms can exploit normal host processes by interfering with
their regulation or activity. The host pathogen interaction studies
can therefore show the role of a pathogen’s virulence gene(s) and
the importance of host components during infection.
Here we focus on the use of Drosophila melanogaster to model
Pseudomonas aeruginosa pathogenesis. P. aeruginosa is a clinically
important opportunistic human pathogen1 that shows an extra-
ordinarily broad host range, infecting vertebrates, insects, nema-
todes, and plants2–5. The virulence mechanisms used by
P. aeruginosa to infect these phylogenetically diverse hosts are
remarkably well conserved6,7, suggesting that the dissection of
these mechanisms in Drosophila could provide an understanding of
P. aeruginosa virulence mechanisms in mammals. P. aeruginosa
causes clinical infections that range from acute to chronic1,8. This
pathogen has a variety of clinical presentations, including respira-
tory, gastrointestinal, eye, urinary tract, bone and joint infections,
as well as dermatitis and soft tissue infections, with many of these
leading to sepsis1. Hence, particular infection models offer certain
advantages depending on the tissue or organ in question.
Drosophila has emerged as an ideal model organism for examin-
ing the genetic control of immune recognition and response
because of the high degree of conservation between fly and
mammalian innate immune systems9. Furthermore, cellular and
tissue physiology of the striated muscle in the fly thorax and the
intestinal epithelium of this genetically tractable organism are
conserved in mammals. Hence, despite the absence of an adaptive
immune system in Drosophila and important anatomical
differences between mammals and flies, including vasculature
that is absent in flies, and the presence of cuticular structures lining
the fly epidermis, trachea, midgut and hindgut10,11, host–microbe
interaction studies in this organism provide important insights
relevant to mammalian innate immunity as well as epithelial and
muscle homeostasis. Furthermore, it is possible to conduct sys-
tematic large-scale screens for the identification of microbial factors
relevant in pathogenesis in this simple model host. P. aeruginosa
infections can induce broad humoral and cellular physiological
changes9,12. Hence, Drosophila provides the important advantage of
allowing the assessment of P. aeruginosa interactions with epithelial,
muscle, blood and fat-body cells.
Methods used to inoculate Drosophila with pathogens
Here we describe two inoculation methods routinely used in our
laboratory to model P. aeruginosa pathogenesis in adult Drosophila:
(1) the thoracic or abdominal needle pricking3,12–15 method and
(2) the injector pumping12 method. Variations of these Drosophila
infection protocols have been described previously by other
groups6,8,14–22. A third method, the feeding assay, although exten-
sively used in our lab, and published by other research groups8,17,23,
is not presented here.
The selection of the inoculation method to be used should
depend on the biological question at hand (see summary in
Table 1). For instance, the needle pricking and feeding assay
methods are considerably more facile and amenable to large-scale
screening than the injector pumping method. These protocols
could potentially be adapted for the inoculation of a wide spectrum
of microbial pathogens. However, the limitations imposed by the
risks in handling certain pathogens and standardization (e.g.,
medium, temperature and inoculum concentration) have to be
considered. Indeed, all three methods and variations thereof have
been used extensively to infect Drosophila adults with a variety of
pathogens (Table 2). Among all of the different microbial

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 PROTOCOL

TABLE 1 | Inoculation method comparison.

Primary infection Bacterial presence/
Method site infection Reproducibility Pros and cons Bacterial kinetics
Needle Injury site (local) Wound site and blood Good Facile, permits assessment of both Exponential
pricking stream infection local and systemic effects, and
bacterial dissemination potential;
amenable to large-scale screen
Injector Systemic Blood stream infection Excellent Controlled inoculum load; less Exponential
pumping amenable to large-scale screen
Feeding Intestinal (local) Intestinal Fair Feeding solution manipulation, Constant load of 104–105
colonization/infection amenable to large-scale screen; CFUs per fly
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

constant feeding on concentrated

bacteria

pathogens capable of causing fly infections assayed to date, TABLE 2 | Partial list of microbes studied in Drosophila.
Pseudomonas species are some of the most virulent, with a lethal Needle Injector
dose being less than five bacteria per fly when introduced in the pricking pumping Feeding
hemolymph13,24. Gram negative
1. Thoracic or abdominal needle pricking involves the use of a Agrobacterium tumefaciens56 O
tungsten needle, dipped in a bacterial suspension, to inflict a Erwinia carotovora56,57 O O
wound in the fly thoracic cuticular epithelium and underlying Enterobacter cloacae56 O
muscle (Fig. 1a) or in the abdominal epithelium (arrow in Fig. 1b). Escherichia coli21,22,56 O O
We and others have shown that this method introduces bacteria Burkholderia cepacia22 O
locally at the wound site; there the bacteria proliferate and subse- Salmonella typhimurium22 O
quently disseminate throughout the fly body over a period of Mycobacterium marinum22 O
time12,14,15,19. This method creates a wound infection and allows Mycobacterium smegmatis58 O
for the assessment of both local and systemic effects. It has been used Pseudomonas aeruginosa3,8,12,15–17,19,20 O O O
extensively to inoculate a variety of microbes into the fly thorax Pseudomonas entomophila24,59 O O
(Table 2), including the non-pathogenic bacteria Escherichia coli and Serratia marcescens11,55 O O
Micrococcus luteus21, a whole array of bacterial human pathogens25, Vibrio cholerae31,60 O O
as well as fungal human pathogens, such as Cryptococcus neoformans, Neisseria sp.23 O
Candida albicans and Aspergilus fumigatus26–28. Gram positive
To study the wound infections that result from trauma, the Enterococcus faecalis22,32,61 O O O
needle pricking assay with a low dose of B100 bacteria per fly Staphylococcus aureus22,61,62 O O
would be most appropriate. This assay offers several advantages in Micrococcus luteus21,56 O O
the assessment of highly virulent strains, as low numbers of Listeria monocytogenes22 O
bacterial cells, introduced primarily locally, subsequently spread Streptococcus pneumoniae22 O
systemically. Thus, the ability of a bacterial strain to colonize, Bacillus subtilis63 O
invade and evade host immune responses at the site of injury, and Bacillus megaterium63 O
eventually systemically, can be assessed. For example, thoracic Streptococcus pyogenes62 O
needle pricking inoculation enabled us to study host responses at Streptococcus sp.23 O
the site of injury and infection, and permitted the assessment of Propionibacterium acnes23 O
early local host responses during the interaction of P. aeruginosa Actinomyces sp.23 O
with the fly musculature12. However, if abdominal pricking is used, Staphylococcus sp.23 O
the thoracic local muscle homeostatic response to injury and Rothia sp.23 O
infection12 (see below) can not be assessed. Micrococcus roseus64 O
2. Injector pumping produces primarily systemic inoculation, as Fungi
the bacteria are injected directly in the Drosophila hemolymph Candida albicans27,61,65 O
through injector-mediated pumping of bacterial inoculum Cryptococcus neoformans26 O O
(Fig. 1d). This method allows for the assessment of systemic effects, Aspergillus fumigatus66–68 O
as it promptly distributes bacteria throughout the fly body12, Mucor circinelloides68 O
whereas the small diameter pulled-glass capillary tip inflicts only Rhizopus oryzae68 O
a minimal wound (Fig. 1d). In addition, the injector pumping Cunninghamella bertholletiae68 O
method enables inoculation using precise doses of bacteria12,22 and Fusarium moniliforme69 O
viruses29,30. Scedosporium sp.69 O
Injector-mediated pumping and a low dose of B100 bacteria per Metarhizium anisopliae28 O
fly could be used for bacteremia studies. High doses (up to 107 Beauveria bassiana70,71 O

1286 | VOL.4 NO.9 | 2009 | NATURE PROTOCOLS

 PROTOCOL

Figure 1 | Drosophila infection experimental set-

up. (a) For the needle pricking assay, flies and the a Bacterial solution d

le

y
lar
ed
lid of a 1.5-ml tube with the bacterial suspension

pil
Ne

Ca
are placed on a CO2 pad. The flies are pricked (red
circle) with a tungsten needle that has been Brush

dipped into the bacterial solution. (b,c) The Brush

dorsolateral thorax is punctured with a relatively
thick (0.2 mm main body) needle (arrows Infected flies Infected flies
delineate the diameter of the needle).
(d) For the injector pumping assay, flies are
Uninfected flies
placed on the CO2 pad and the pulled-glass Uninfected flies
capillary tip is inserted into the fly thorax (red
circle) introducing a precise volume of bacterial b e
solution in the fly’s body. (e,f) The dorsolateral
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

thorax is punctured with a relatively thin glass

(o0.5 mm tip) capillary tip (arrows and dashed
lines delineate the diameter of the tip). For
abdominal pricking or injector pumping, the
needle or capillary tip is inserted parallel to the
anteroposterior body axis in the dorsolateral
abdominal cuticle, close to the junction
between the thorax and the abdomen
(white arrows in b,e).
c f
bacteria per fly) can be used to mimic late
stages of infection. For example, a dose of
105 bacteria per fly, which is considered to
be a high dose, can facilitate comparative
evaluation of Pseudomonas gene expression
for various bacterial strains that grow dif-
ferentially during infection (see Experi-
mental design). Nevertheless, when high
levels of bacteria are introduced, caution should be taken as this
type of infection quickly bypasses many critical steps of the natural
infection process.
3. Feeding assay infection establishes a population of 104–105
bacteria per fly following continuous feeding on Luria–Bertani
(LB) broth medium containing bacteria. This assay mimics intest-
inal infection and it is used to introduce various microbes into the
fly intestine (Table 2), including Enterococcus faecalis, Serratia
marcescens and Vibrio cholerae11,31,32. The feeding method disse-
minates P. aeruginosa selectively into the gastrointestinal tract, yet
as with the other types of inoculation, the bacteria cause morbidity
and ultimately lethality, usually within 2–15 d (ref. 17). This
method has been described by other groups8,17,23 and thus it will
not be presented here.
Experimental design
Bacterial and fly selection. All three infection models mentioned
above allow the assessment of the contribution of a specific
bacterial factor in virulence and/or the relevance of specific host
immune components in defense against the pathogen of interest.
The use of fly and bacterial mutant lines can reveal signaling
pathways that function to limit or promote the severity of patho-
genesis, both in terms of lethality and the kinetics of bacterial
invasion and proliferation. In such studies, the fly or bacterial
mutant strain ideally should be compared with: (1) the isogenic
parental strain and (2) the genetic rescue of the mutant strain by
replacement of the mutated gene with the corresponding wild-type
gene. Furthermore, phenotypic differences may be observed when
bacterial3,8 or fly strains of different genetic backgrounds are
compared (Fig. 2a,d). Isogenic genetic-background flies should
be used when differences between strains due to infection are slight.
If the isogenic parental fly strain is not available in cases in which
slight differences between strains are observed, then the mutant
flies should be backcrossed for many generations (ideally 6 or
more) to an available wild-type strain to minimize genetic back-
ground variations33. This is accomplished easily when the specific
mutation has a visible phenotype, such as a distinctive eye color,
but may require an elaborate genetic scheme when there is no such
definitive marker34. Alternatively, multiple mutations in the same
gene or signaling pathway can be compared with a wild-type strain
to increase confidence of the results.
As the age of the flies has a significant impact on their
susceptibility to infection (Fig. 2b), it is recommended that
flies of similar age (ideally 5–7 d old) should be used in all
experiments within a study. Depending on the specific fly and
bacterial strain interaction, a wide variety of differences in suscept-
ibility can be observed between males and females. For example, a
pronounced sex difference is evident in Oregon-RS flies inoculated
with P. aeruginosa (Fig. 2c). However, no such sex difference
emerges when other strains (i.e., Canton-S) are used (data not
shown). Although female flies have the advantage of larger size,
which facilitates handling and anatomical evaluation, and of a
more robust humoral response to infection (Y.A. and L.G.R.,
unpublished data and refs. 35,36), male flies are generally preferred
due to the absence of fertilization, which impacts Drosophila
immune physiology37. To minimize potential sex-dependent
effects, flies should be sex-sorted and only one sex should be
used in a given assay.

NATURE PROTOCOLS | VOL.4 NO.9 | 2009 | 1287

 PROTOCOL

Figure 2 | Parameters that affect mortality a b c

and growth rate in the needle pricking assay. 100 100 1-d old 100 Males
Typical results of mortality (a–e) and growth 7-d old
80 80 15-d old 80 Females

Survival (%)
rate (f) assessment in the needle pricking 30-d old
60 60 60
assay. (a) Two wild-type strains of different
40 Oregon-RS 40 40
genetic background differ in resistance to Canton-S
infection. (b) Young flies (1 and 7 d old) 20 20 20

are more resistant to infection than older 0 0 0

flies (15 or 30 d old). (c) Male and female 30 32 34 36 38 40 42 30 35 40 45 50 0 100 200 300 400 500

flies of the Oregon RS genotype show differences d e f

in resistance to infection with the low 100 100 18°C 8
21°C 7
virulence Pseudomonas aeruginosa isolate CF5. 80 80
6

CFUs per fly

25°C

Survival (%)
(d) Three P. aeruginosa isolates differ markedly in 60 60 5
4
their virulence potential. (e,f) Temperature 40
CF5 40 3 25°C
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

has a major function in fly survival rate (e), 20

PA14
20 2 21°C
and growth rate, as shown in flies infected PA2 1
0 0 0
with the highly virulent strain PA14 (f). 0 8 16 24 32 40 48 0 20 40 60 80 100 120 0 6 12 18 24 30 36 42 48
Error bars in all graphs represent s.d. of the mean. Time (h) Time (h) Time (h)
Differences in all graphs were statistically
evaluated and found to be significant (P o0.05). Injury without infection or injection with buffer does not typically cause fly mortality (data not shown).

Assessing pathogenicity. The fly mortality caused by P. aerugi-
nosa correlates with the ability of the bacteria to proliferate3.
Therefore, monitoring percentage survival and bacterial prolifera-
tion over time can provide information about fly resistance to
P. aeruginosa infection. These readouts serve as measures of the
pathogen’s virulence potential and host resistance to specific
bacterial strains. Bacterial titers and survival kinetics of wild-type
and mutant strains should be compared and carried out in
parallel. A power analysis indicates that a sample size of ten flies
per bacterial strain would provide 80% power (b ¼ 0.20) to
detect a 50% difference in survival, assuming a 85–90% mortality
rate in the wild-type strain, based on a two-tailed Fisher’s
exact test for comparing survival proportions (version 5.0, nQuery
Advisor, Statistical Solutions). A statistical analysis of fly survival
kinetics can be carried out using the Kaplan–Meier survival
estimate38, such as in the SPSS software package (version 15,
SPSS). The differences in the Kaplan–Meier fly survival curves
between treatments can be assessed using the log-rank method
(Mantel–Haenszel)38.
An estimation of the bacterial titers present in whole flies
or in specific fly tissues can provide information about the
pathogen’s ability to proliferate within the host or colonize a
specific tissue. To determine whether or not colony forming units
(CFUs) differ significantly in vivo between wild-type and an
isogenic mutant bacterial strain, a standard t-test can be used to
compute the P-values39. The CFU data for each strain tested can be
plotted against time post-infection, including s.d. for all data
points. Alternatively, individual CFU data points per condition
can be plotted for a given time point with the calculated
mean also represented on the graph. In addition, bacterial gene
expression in vivo can be assessed and provide a more direct
evaluation of the expression of specific virulence genes during
infection. However, differences in bacterial gene expression
between P. aeruginosa strains showing differential growth rates
in vivo should be assessed at early time points (up to 12 h
post inoculation), when bacterial numbers are comparable across
subjects. The volume of the primary inoculum in this case
should be high enough to yield sufficient amounts of RNA for
expression studies15.
Host responses to infection can be assessed on a whole genome
level (through microarray analysis, i.e., using Drosophila Affymetrix
Genechips), or on a single gene level, through qRT–PCR (quanti-
tative reverse transcription-PCR), or through reporter gene
fusions13,40–45. Host–pathogen response signatures arising from
whole genome studies can serve as biomarkers of susceptible versus
non-susceptible interactions. A comparison of Drosophila
responses elicited in response to a virulent P. aeruginosa strain,
such as PA14, with those elicited in response to a non-virulent
isogenic mutant strain will show genes whose expression patterns
differ in susceptible versus non-susceptible host–pathogen interac-
tions. More specifically, such comparisons can uncover host genes
that promote or limit the initiation or progression of infection
facilitated by a specific virulence factor or pathway. For statistical
analysis of transcriptome studies, publicly available software can be
used, such as dChip and SAM. Student’s t-test may also be used to
compute P-values of qRT–PCR replicate measurements or in
experiments in which reporter gene expression is measured quan-
titatively. The non-statistical tool GuiGraph can be used to provide
rapid visualization and to aid the interpretation of large gene-
expression data sets13,46,47.

MATERIALS
REAGENTS . 10 mM MgSO4 (sterilize before use)
. Bacterial strains: Wild-type (PA14, PA2 and CF5) and PA14-isogenic mutant . Appropriate adult fly lines, Oregon-RS or Canton-S can be availed from
P. aeruginosa strains can be obtained from the Rahme and Ausubel the Bloomington Stock Center.
Laboratories48, respectively ! CAUTION P. aeruginosa cultures should be . LB broth (Fisher Scientific, cat. no. BP1427) ! CAUTION Infected flies
autoclaved before disposal. should be killed by freezing at
20 1C and disposed of as a biohazard
. LB agar plates (Fisher Scientific, cat. no. BP1425, prepared with antibiotics if material.
used) or Pseudomonas Isolation Agar (Difco, cat. no. BD292710). . Autoclaved water
. Antibiotics (rifampicin 0.1 mg ml
1, if used) . 99% Ethanol

1288 | VOL.4 NO.9 | 2009 | NATURE PROTOCOLS


. Common grade fly food reagents agar, sucrose, yeast and cornmeal
(Genesee Scientific Corporation)
. Vials (Genesee Scientific Corporation, cat. no. 32-116)
. Cotton balls (Genesee Scientific Corporation, cat. no. 51-101)
. Tegosept (Genesee Scientific Corporation, cat. no. 20-258)
. Propionic acid (Sigma, cat. no. p-1386)
. Trizol reagent (Invitrogen, cat. no. 15596-026)
EQUIPMENT
. Centrifugation tubes and grinding pestles (Fisher Scientific, cat. no.
K749521-1590)
. Glass capillaries (3.500 , Drummond Scientific Company, cat. no. 3-000-203-G/X)
. Paintbrush (size 0)
. Tungsten stainless steel needle, with approximate diameters of 0.01 mm at
the tip and 0.2 mm across the main needle body, held in a pin vise
(Ernest F. Fullam, cat. no. 54270)
. Injector (Nanoject II, Drummond Scientific Company, cat. no. 3-000-204)
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols
PROTOCOL
REAGENT SETUP
Fly food Conventional 1% (wt/vol) agar, 0.6% (wt/vol) sucrose, 3% (wt/vol)
yeast, 4.4% (wt/vol) cornmeal supplemented with 0.36% (vol/vol) propionic
acid and 0.11% (wt/vol) Tegosept should be used to rear and maintain flies
before and after infection. Other conventional recipes can be used as well.
EQUIPMENT SETUP
Injection setup For needle pricking or pump injection, flies are kept
anesthetized on a CO2 pad and handled using a paintbrush under a
stereoscope (Fig. 1a,d). During fly inoculation, the bacterial culture is placed in
an inverted 1.5-ml microcentrifuge tube lid proximal to the flies to be
inoculated; for needle pricking the fly is dipped in the bacterial suspension
(Fig. 1a). For injection pumping, the bacterial solution to be injected should
be aspirated in a pulled-glass capillary (Fig. 1d) using an injection device,
such as the Nanoject II apparatus. The glass capillary has a softening
point of 780 1C. Many researchers pull the tips and then break them

off using forceps. The tip size should be approximately 0.03–0.08 mm in

. Stereoscopic microscope ( Stemi 2000-C, Zeiss) equipped with a controllable diameter. Once the tips are pulled, they must be ‘backfilled’ with a non-
CO2 flow pad compressible fluid (e.g., mineral oil or silicone) before attachment
. Fly incubators with high humidity capacity (60–75%), adjustable to the injector. Backfilling can be achieved easily using a 30-guage, 200 needle
temperature, and a 12-h light/12-h dark cycle and a syringe.

PROCEDURE

Fly preparation TIMING 21–24 d
1| Grow appropriate fly strains on conventional fly food (see REAGENT SETUP). The flies should preferentially be 5–7 d old.
Therefore, collect newly eclosed flies, then allow them to age for 5 more days at 25 1C. A wider range of fly ages (e.g., 5–10 d)
can be used if retrieval of an adequate number of flies is a limitation, such as when flies are collected from the progeny of
low-yielding crosses. Regardless, always use control groups that are run in parallel. Run experiments using 10–20 male flies per
group for all assays to achieve statistical significance in the results. If fly mutants are tested, always include a wild-type group
in the experiment in addition to a mock-infected group (see Bacterial and fly selection). Flies can become contaminated with
various microbes, in which case extra measures must be taken for decontamination.
? TROUBLESHOOTING

Bacterial inoculum preparation TIMING 1 d 

2| Streak frozen glycerol stocks onto fresh LB agar plates containing the specific antibiotic selection and incubate overnight
(O/N, B16 h) at 37 1C. If the virulence phenotype of a specific bacterial gene is to be assessed, use only isogenic mutant
strains and always include the parental strain as a control (see Bacterial and fly selection).

3| Inoculate single colonies from the fresh plates into tubes containing 5 ml LB broth and incubate overnight at 37 1C,
shaking at 250 r.p.m. or rotating at 120 r.p.m. to obtain sufficient aeration for good cell growth.

4| The next day, subculture by diluting (1:100–1:200) the O/N culture in 5 ml of LB such that the optical density at
600 nm (OD600 nm) is 0.05 or less. Depending on the strain used, aeration and volume, bacterial doubling times may vary.
The OD600 nm should be equal for all bacterial strains to be compared.

5| Incubate the cultures at 37 1C until they reach an OD600 nm of 3.0, which corresponds to approximately 3–5  109 cells
per ml. A 1:100 dilution of an O/N culture (OD600 nm B4.5) takes B5 h to reach an OD600 nm of 3.0 when rotated at 120 r.p.m.

6| Centrifuge 1 ml of each culture for 2 min at 11,000g. Discard the supernatant using a pipette (1 ml), but carefully retain
the viscous material that includes bacterial polysaccharides on the top of the pellet to improve reproducibility.

7| Wash once using 1 ml of 10 mM MgSO4 to remove traces of LB and centrifuge as in Step 6. Re-suspend the cells in 1 ml of
10 mM MgSO4 and determine the OD600 nm.

8| Prepare an inoculum of OD600 nm ¼ 0.03 by serially diluting the cells in MgSO4. To get a final OD of 0.03 begin by
preparing 1 ml of OD600 nm 3.0 culture (Step 7) and subsequently double dilute OD600 nm 3.0 culture (single dilution being 0.3,
double to 0.03) in 1 ml. This dilution contains an approximate concentration of 3–5  107 cells per ml. Use higher or lower
dilutions to reduce or increase inoculum concentrations, respectively.


Fly inoculation assay TIMING 1 h
9| Choose the most appropriate inoculation mode, such as needle pricking (Fig. 1a) or injector pumping (Fig. 1d), for the
biological question to be addressed (see ‘Methods used to inoculate Drosophila with pathogens’).

NATURE PROTOCOLS | VOL.4 NO.9 | 2009 | 1289

 PROTOCOL


(A) Needle pricking assay TIMING 1 h
(i) Anesthetize the flies with CO2 and place them as a group at the center of the CO2 pad next to the bacterial
solution (100 dilution). Flies should be handled with a paintbrush to minimize the chances of injury (Fig. 1a).
(ii) Sterilize a tungsten needle with ethanol, and shake it to remove any residual ethanol before dipping the tip of the
needle into the bacterial suspension.
m CRITICAL STEP To prevent bacterial sedimentation in the culture, regularly expel and re-aspirate the bacterial
suspension or mix the bacterial suspension in between fly inoculations by stirring the suspension with the needle.
(iii) Prick the fly’s thorax by inserting the needle midway into the thorax, at the mid-point along the anteroposterior axis and
dorsolaterally along the dorsoventral axis of the thorax (Fig. 1b,c). This effectively inoculates B100 bacteria locally in
the fly thorax. An insertion midway into the thorax imposes a thoracic wound, which is desirable in wound infection stu-
dies. For an abdominal inoculation, insert the needle parallel to the anteroposterior body axis in the dorsolateral abdom-
inal cuticle close to the junction between the thorax and the abdomen (white arrow in Fig. 1b). Such positioning will
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

avoid injury of the fly heart. Place the pricked fly aside using the paintbrush, and dip the needle into the bacterial suspen-
sion to inoculate the next fly. Repeat this procedure until all flies in the particular treatment group have been pricked.
This will take B5 min per treatment group when ten flies are used. To cause a lesser injury, insert only the very tip of the
tungsten needle, which is only 0.01 mm in diameter as opposed to 0.2 mm at the main body. When only the very tip is
used, B30 bacteria will be delivered at the site of inoculation.
(iv) Return the pricked flies to the fly-food vial.
m CRITICAL STEP To prevent flies from sticking in the food, leave the vials on their sides until the flies have recovered
from the anesthesia.
m CRITICAL STEP Infected flies incubated at 18, 21, 25 or 29 1C show differential mortality kinetics (Fig. 2e),
as bacteria may proliferate more slowly at lower temperatures and temperature might affect host defense responses49,50.
Generally, 21 1C is an optimal temperature for assessing bacterial mutants that show an intermediate virulence phenotype.
(v) Proceed to Step 10 to assess pathogenicity.

(B) Injector pumping assay TIMING 1 h
(i) Set up the injector and a pulled-glass capillary according to the manufacturer’s instructions (see Drummond Scientific
Company manual; overview in EQUIPMENT SETUP).
(ii) Place a drop of the 100 diluted bacterial culture from Step 8 onto a piece of parafilm (B5 cm  5 cm) under a
stereoscope. Use the pulled-glass capillary attached to the injector to aspirate the bacterial suspension.
m CRITICAL STEP To prevent bacterial sedimentation, see section 8A (ii) above.
(iii) Anesthetize the flies using CO2 and place them as a group at the center of the CO2 pad (Fig. 1d).
(iv) Penetrate the fly cuticle using the capillary tip at the mid-point along the anteroposterior axis and dorsolaterally
along the dorsoventral axis of the thorax (Fig. 1e,f), and pump 9.6 nl of inoculum, corresponding to a dose of B100 bac-
teria per fly. For abdominal inoculation, inject into the dorsolateral abdominal cuticle at the junction between the thorax
and abdomen22 (white arrow in Fig. 1e).
(v) Put the injected fly aside and repeat until all flies in the current treatment group have been injected.
(vi) Return the injected flies to the fly-food vial and measure pathogenicity as described in Step 10.


Pathogenicity assessment TIMING 1–4 d
10| Assess pathogenicity by determining the percentage of surviving flies (option A), the bacterial growth within fly tissues
(option B), or the host responses (option C). Any of these options can be used for each inoculation method, but options
A and B are conventionally used for primary assessments of pathogenicity.

(A) Fly survival TIMING 1–4 d
(i) To determine fly survival, simply count the number of living flies Bevery 6 h post-inoculation until flies begin
dying, which usually occurs after 30 h at 21 1C, and then every 1–3 h once mortality commences. Monitoring should
continue until fly survival ceases to change for a full 24-h period, which usually occurs after 3 d. Determine the
percentage survival as a function of time. For statistical analysis of the fly survival kinetics, see Experimental design:
Assessing Pathogenicity. To confirm whether immobile flies lying on their sides or backs are really dead, tap the vials.
? TROUBLESHOOTING
(ii) Exclude the flies inoculated with a dose of B100 bacteria per fly that die within 6 h of the treatment from the
survival analysis because early death is probably a result of extreme injury or stress, and cannot be
attributed to infection.
m CRITICAL STEP The percentage of excluded flies should not be more than 5% of the total number of flies. If it is
45%, exclude this batch of flies from the analysis because such a high early-mortality rate indicates excessive injury or
stress to the flies.

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 PROTOCOL

(iii) Carry out three or more independent repetitions of each experiment and analyze the results using statistical
methods described in the Experimental design: Assessing Pathogenicity.

(B) Bacterial growth TIMING 1–4 d
(i) Assessment of bacterial growth on a per fly basis is another important measure of infection. Remove ten flies from the
vials at various time points following infection and homogenize each fly individually in 0.1 ml of 10 mM MgSO4 in a
1.5-ml centrifuge tube using a plastic pestle.
m CRITICAL STEP An estimation of CFUs per fly should be conducted immediately following the needle pricking
assay to assess, at time 0, the average number of injected bacteria. The particles, after grinding, should be smaller that
B1/10 of the intact fly volume to ensure efficient bacterial release.
(ii) Plate serial 10 dilutions of the fly extract on LB agar plates supplemented with appropriate antibiotics and count
CFUs after B18 h of incubation at 37 1C. Using the same procedure, also plate the fly extract of uninfected flies on
plates without antibiotics to check for possible fly contamination.
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

? TROUBLESHOOTING
(iii) Carry out three or more independent repetitions of each experiment and analyze the results using statistical
methods described in the Experimental design: Assessing Pathogenicity.

(C) Host responses TIMING 1 h
(i) To study host responses toward P. aeruginosa infection, using microarrays, qRT-PCR or protein analysis methods,
anesthetize flies with CO2, and collect whole flies or the fly parts of interest. Flies can be collected at various time points
post-bacterial inoculation. Uninfected, injured flies should be used as controls.
(ii) To store samples for RNA extraction, grind 5–20 flies in 0.5 ml of Trizol reagent and store extracts at
80 1C until
proceeding with qRT-PCR or microarray protocols12,51. To prepare samples for protein analysis, grind 5–20 flies in
0.2 ml of PBS or lacZ buffer for western blot or b-galactosidase assays, respectively52.

 TIMING
Step 1, Fly preparation: fly cultures: 14 d; newly eclosed fly collection: 2–5 d; and fly aging at 25 1C: 5 d; total preparation
21–24 d
Steps 2–8, Bacterial preparation: 1 d
Step 9, Fly inoculation assay: 1 h
Step 10, Pathogenicity assessment: fly survival and bacterial growth follow up: 1–4 d

? TROUBLESHOOTING
Steps 1 and 10B(ii): Fly-food contamination during fly preparation
Fly food can become contaminated with bacteria and/or fungi. The propensity for food contamination seems to depend mostly
on fly age and general fitness. The conventional food used to rear and maintain flies contains 0.11% (wt/vol) Tegosept and
0.36% (vol/vol) propionic acid to prevent fly-food contamination. Nevertheless, food or fly colonies may become contaminated,
as evidenced from visible growth of microbes on fly food or by plating flies on LB agar. If contamination is detected,
investigators should carry out the procedures described in Box 1.
Contamination with bacterial endosymbiots, such as Wolbachia and Spiroplasma, is not detectable by the methods outlined in
Box 1. However, skewed wild-type male mortality may signal endosymbiont contamination. The presence of a male/female ratio
o1:1 in a wild-type fly population should be considered as an indicator of contamination. Verification of Wolbachia presence
requires PCR amplification of Wolbachia sequences53. The addition of 0.2 mg ml
1 tetracycline to the food for one or more
generations can help control Wolbachia contamination. However, to avoid the introduction of more biological variables due to
the use of tetracycline, the treated fly population should be expanded in the absence of antibiotics before any are used in
experiments.

BOX 1 | DEALING WITH CONTAMINATED FLIES OR FLY FOOD

(i) Transfer sets of 20–30 young flies into fresh food vials and incubate at 25 1C.
(ii) Remove flies after 4 d to eliminate the source of contamination.
(iii) If significant contamination persists in the progeny of the originally transferred flies (F1), transfer the next generation of flies (F2) once
they have reached adulthood to food containing one or more antibiotics (e.g., 0.1 mg ml
1 of ampicillin and 0.05 mg ml
1 of kanamycin).
(iv) After 4 d, transfer the F2 flies to food without antibiotics, allowing them to potentially reestablish their normal microbial flora; collect the
descendents of the F2 flies to be used for infection.
(v) Test for food contamination by inspecting and plating food on LB agar plates as described above in Step 10B.
(vi) Test for persistent fly contamination by grinding untreated flies, as described in Step 10B of the main Procedure, and plating the extract on
LB plates.

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Step 10: Reproducibility of survival and bacterial growth assays

The most reproducible mode of infection is the injector pumping assay, as it allows the investigator to control the injection
volume. Injection of 9.2 nl of P. aeruginosa (5  107 cells/ml) delivers 120–140 bacteria per fly. In contrast, the needle
pricking assay does not permit a precise injection dose, and thus bacterial numbers vary from 25 to 120 cells per fly using a
5  107 cells per ml of P. aeruginosa inoculum. To reduce cell number variability, the tungsten needle should be coated with fly
hemolymph just before infecting each fly group. This can be accomplished by first pricking ten healthy flies, discarding these
flies, and then proceeding with the immersion of the needle in the bacterial inoculum to achieve fly infection as described
above. In addition, use multiple groups of ten flies per experiment to increase the statistical power of the differences in fly
survival kinetics and bacterial proliferation among experimental conditions.

ANTICIPATED RESULTS
The use of fly mutants with compromised immune response or bacterial mutants that show attenuated virulence will shorten or
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

prolong average fly survival, respectively, usually by 2–10 h when incubated at 21 1C (ref 12). P. aeruginosa-infected flies die at
a temperature-controlled rate (Fig. 2e). Using either the needle pricking or the injector pumping method, flies will die
approximately 18 h post-inoculation if flies are inoculated with B100 cells and the assay is carried out at 29 1C; 50% mortality
will be observed by 25, 40 and 80 h if flies are incubated at 25, 21 or 18 1C, respectively (Fig. 2e). Setting the temperature to
21 1C, instead of 25 1C, may enable the identification of moderately attenuated virulent bacterial strains that would otherwise
be missed (Fig. 2e).
It is expected that virulence-attenuated bacterial mutants will produce decreased host mortality, and that such mutants will
not reach the same titers or their growth kinetics may be slower compared with the parental strain in vivo3. However, depending
on which virulence functions have been mutated, mutants may cause varying degrees of lethality in Drosophila. Similarly,
different clinical or environmental P. aeruginosa isolates would be expected to cause varying degrees of lethality3 and to show
differential proliferation rates in Drosophila. Using either the needle pricking or the injector pumping assay to inject B100
bacteria per wild-type fly, bacterial CFUs will start increasing by B6 h post-inoculation. Although bacterial proliferation is
exponential and its rate may be temperature-dependent, changes in host sensitivity across different temperatures should not be
ignored49,50. A titer of 107 bacteria per fly can be attained within B24 h when infected flies are incubated at 25 1C, or within
48 h when flies are incubated at 21 1C (Fig. 2f). Different P. aeruginosa strains can show significant differences in growth rate
within flies and can have differing titer levels that produce 100% fly lethality18. For example, although innate immunity mutant
flies are expected to enable greater bacterial growth over time, attenuated virulence bacterial mutants are expected to show
decreased growth in wild-type flies.
P. aeruginosa infection through needle pricking or injector pumping elicits a cascade of host responses. Needle pricking
infection elicits both local and systemic responses12,13,15. When flies are injected in the thorax, prominent rapid responses at
the injury site include induction of muscle and cytoskeleton genes, peaking 3–6 h post inoculation, followed by a systemic
response, including activation of the Imd pathway, which regulates antimicrobial peptides (e.g., diptericin which is highly
induced at B6 h post-injury and infection). The expression of Toll pathway-regulated genes is observed at B18 h
post-infection, whereas other defense genes controlled by the JAK/STAT pathway are highly expressed at B6 h
post-infection13,21,44. Of course, the infection method and the microbe inoculated can affect the type and timing of host
responses. In this regard, virulent and avirulent strains or P. aeruginosa mutants differ in the extent to which they induce
skeletal muscle (SM) and antimicrobial peptide (AMP) genes12,13. Indeed, virulence-attenuated P. aeruginosa isolates and
PA14-isogenic mutant strains show an increase in SM and AMP gene expression following infection12,13, compared with the
levels elicited by the virulent PA14 parental strain. However, this effect cannot be generalized to include all strains attenuated
in virulence. A comparative quantitative assessment of host responses can reveal large or small effects on gene expression.
Differences in bacterial virulence potential could be related to small, yet biologically significant differences in host expression
levels12,13,51. Thus, host responses could be used as biomarkers of a susceptible or non-susceptible host–pathogen interaction.
For example, both AMP and SM gene downregulation typifies a susceptible interaction caused by a virulent P. aeruginosa
strain12,13. Although additional signatures and assays may be used, including various cellular immunity assays52,54,55
to assess pathogenicity, AMP and SM gene function are the only host functions thus far shown to be relevant for
P. aeruginosa infection12,13.
The innate immunity defenses and tissue physiology in flies and mammals, and the virulence mechanisms used by
P. aeruginosa to infect these phylogenetically diverse hosts, are remarkably conserved5,7. These features, combined with the
genetic tractability of Drosophila, permit large-scale screening for both host3 and bacterial19,20 genes that promote or limit the
initiation and progression of infection. The needle-pricking assay allows for the identification of P. aeruginosa virulence factors
needed to establish a progressive and systemic lethal infection in Drosophila. Meanwhile, the injector pumping assay is
appropriate for identifying virulence factors necessary for systemic lethal infection. Regardless, both needle pricking and
injector pumping assays elicit a cascade of host responses involving known signaling pathways that mediate the immune
potentiation response12,13 as well as many pathways not previously implicated in response to bacterial infection that may

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 PROTOCOL

potentially favor or limit the initiation and progression of pathogenesis12,13. It is worth noting that even low-magnitude
changes in gene expression can be biologically significant if they cluster together with genes of similar function, if they are
functionally validated, and if the data are reproducible12,13,51.
As noted earlier, assessing lethality and bacterial proliferation allows investigators to gain insights into the progression of
the host–pathogen interaction between D. melanogaster and the human opportunistic pathogen P. aeruginosa, and to identity
both pathogen and host genetic factors that enhance or restrict pathogenesis. Such insights have the potential to show
mechanisms of P. aeruginosa pathogenesis and host resistance. Moreover, profiling and comparing host responses mediated by
virulent or avirulent P. aeruginosa strains should allow investigators to gain insights into the host responses that mediate
susceptible versus non-susceptible host–pathogen interactions and to identify interaction-specific signatures. The
P. aeruginosa–Drosophila system permits the genetic manipulation of both the pathogen and host partner and allows
the probing of mechanistic interactions between P. aeruginosa virulence factors and components of critical signaling pathways,
thus providing insights into how these components function to restrict or promote infection. Such insights may advance our
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

understanding of human infections and lead to new approaches for the control of P. aeruginosa infections in human patients.

ACKNOWLEDGMENTS This work was partially supported by the research grants,
Shriners #8892 and R01AI063433. Y.A. was supported by Shriners research
fellowship #8508.
AUTHOR CONTRIBUTIONS Y.A. and L.G.R. wrote the paper. Y.A. performed the
experiments.
Published online at http://www.natureprotocols.com/
Reprints and permissions information is available online at http://npg.nature.com/
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