Drosophila melanogaster as a model host for studying Pseudomonas

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Aug 13, 2009 - Conservation of host signaling pathways and tissue physiology between Drosophila melanogaster and mammals allows for the modeling of ...
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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. of Microbiology and Molecular Genetics, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, USA. Correspondence should be addressed to L.G.R. ([email protected]).

3Department

© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

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 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 determined 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 extraordinarily broad host range, infecting vertebrates, insects, nematodes, 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 respiratory, 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 examining 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 systematic 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 extensively 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 site Injury site (local)

Bacterial presence/ infection Wound site and blood stream infection

Injector pumping

Systemic

Blood stream infection Excellent

Controlled inoculum load; less amenable to large-scale screen

Feeding

Intestinal (local)

Intestinal Fair colonization/infection

Feeding solution manipulation, Constant load of 104–105 amenable to large-scale screen; CFUs per fly constant feeding on concentrated bacteria

© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

Method Needle pricking

Reproducibility Pros and cons Bacterial kinetics Good Facile, permits assessment of both Exponential local and systemic effects, and bacterial dissemination potential; amenable to large-scale screen

pathogens capable of causing fly infections assayed to date, Pseudomonas species are some of the most virulent, with a lethal dose being less than five bacteria per fly when introduced in the hemolymph13,24. 1. Thoracic or abdominal needle pricking involves the use of a tungsten needle, dipped in a bacterial suspension, to inflict a wound in the fly thoracic cuticular epithelium and underlying muscle (Fig. 1a) or in the abdominal epithelium (arrow in Fig. 1b). We and others have shown that this method introduces bacteria locally at the wound site; there the bacteria proliferate and subsequently disseminate throughout the fly body over a period of time12,14,15,19. This method creates a wound infection and allows for the assessment of both local and systemic effects. It has been used extensively to inoculate a variety of microbes into the fly thorax (Table 2), including the non-pathogenic bacteria Escherichia coli and Micrococcus luteus21, a whole array of bacterial human pathogens25, as well as fungal human pathogens, such as Cryptococcus neoformans, Candida albicans and Aspergilus fumigatus26–28. To study the wound infections that result from trauma, the needle pricking assay with a low dose of B100 bacteria per fly would be most appropriate. This assay offers several advantages in the assessment of highly virulent strains, as low numbers of bacterial cells, introduced primarily locally, subsequently spread systemically. Thus, the ability of a bacterial strain to colonize, invade and evade host immune responses at the site of injury, and eventually systemically, can be assessed. For example, thoracic needle pricking inoculation enabled us to study host responses at the site of injury and infection, and permitted the assessment of early local host responses during the interaction of P. aeruginosa with the fly musculature12. However, if abdominal pricking is used, the thoracic local muscle homeostatic response to injury and infection12 (see below) can not be assessed. 2. Injector pumping produces primarily systemic inoculation, as the bacteria are injected directly in the Drosophila hemolymph through injector-mediated pumping of bacterial inoculum (Fig. 1d). This method allows for the assessment of systemic effects, as it promptly distributes bacteria throughout the fly body12, whereas the small diameter pulled-glass capillary tip inflicts only a minimal wound (Fig. 1d). In addition, the injector pumping method enables inoculation using precise doses of bacteria12,22 and viruses29,30. Injector-mediated pumping and a low dose of B100 bacteria per fly could be used for bacteremia studies. High doses (up to 107 1286 | VOL.4 NO.9 | 2009 | NATURE PROTOCOLS

Exponential

TABLE 2 | Partial list of microbes studied in Drosophila. Needle Injector pricking pumping Feeding Gram negative Agrobacterium tumefaciens56 Erwinia carotovora56,57 Enterobacter cloacae56 Escherichia coli21,22,56 Burkholderia cepacia22 Salmonella typhimurium22 Mycobacterium marinum22 Mycobacterium smegmatis58 Pseudomonas aeruginosa3,8,12,15–17,19,20 Pseudomonas entomophila24,59 Serratia marcescens11,55 Vibrio cholerae31,60 Neisseria sp.23 Gram positive Enterococcus faecalis22,32,61 Staphylococcus aureus22,61,62 Micrococcus luteus21,56 Listeria monocytogenes22 Streptococcus pneumoniae22 Bacillus subtilis63 Bacillus megaterium63 Streptococcus pyogenes62 Streptococcus sp.23 Propionibacterium acnes23 Actinomyces sp.23 Staphylococcus sp.23 Rothia sp.23 Micrococcus roseus64 Fungi Candida albicans27,61,65 Cryptococcus neoformans26 Aspergillus fumigatus66–68 Mucor circinelloides68 Rhizopus oryzae68 Cunninghamella bertholletiae68 Fusarium moniliforme69 Scedosporium sp.69 Metarhizium anisopliae28 Beauveria bassiana70,71

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Figure 1 | Drosophila infection experimental setup. (a) For the needle pricking assay, flies and the lid of a 1.5-ml tube with the bacterial suspension are placed on a CO2 pad. The flies are pricked (red circle) with a tungsten needle that has been dipped into the bacterial solution. (b,c) The dorsolateral thorax is punctured with a relatively thick (0.2 mm main body) needle (arrows delineate the diameter of the needle). (d) For the injector pumping assay, flies are placed on the CO2 pad and the pulled-glass capillary tip is inserted into the fly thorax (red circle) introducing a precise volume of bacterial solution in the fly’s body. (e,f) The dorsolateral 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).

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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 differentially during infection (see Experimental 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 intestinal 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 disseminates 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 pathogenesis, 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 background 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 susceptibility 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

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CFUs per fly

Survival (%)

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Survival (%)

Figure 2 | Parameters that affect mortality a b c and growth rate in the needle pricking assay. 1-d old 100 100 100 Males 7-d old Typical results of mortality (a–e) and growth Females 80 80 80 15-d old rate (f) assessment in the needle pricking 30-d old 60 60 60 assay. (a) Two wild-type strains of different Oregon-RS 40 40 40 genetic background differ in resistance to Canton-S 20 20 20 infection. (b) Young flies (1 and 7 d old) 0 0 0 are more resistant to infection than older 30 32 34 36 38 40 42 30 35 40 45 50 0 100 200 300 400 500 flies (15 or 30 d old). (c) Male and female flies of the Oregon RS genotype show differences d e f 100 100 8 18°C in resistance to infection with the low 7 21°C 80 80 virulence Pseudomonas aeruginosa isolate CF5. 6 25°C 5 60 (d) Three P. aeruginosa isolates differ markedly in 60 4 CF5 their virulence potential. (e,f) Temperature 40 40 25°C 3 PA14 21°C has a major function in fly survival rate (e), 2 20 20 PA2 1 and growth rate, as shown in flies infected 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. aeruginosa correlates with the ability of the bacteria to proliferate3. Therefore, monitoring percentage survival and bacterial proliferation 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

MATERIALS REAGENTS

. Bacterial strains: Wild-type (PA14, PA2 and CF5) and PA14-isogenic mutant P. aeruginosa strains can be obtained from the Rahme and Ausubel Laboratories48, respectively ! CAUTION P. aeruginosa cultures should be autoclaved before disposal. . LB agar plates (Fisher Scientific, cat. no. BP1425, prepared with antibiotics if used) or Pseudomonas Isolation Agar (Difco, cat. no. BD292710). . Antibiotics (rifampicin 0.1 mg ml1, if used)

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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 (quantitative 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 interactions. 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 quantitatively. The non-statistical tool GuiGraph can be used to provide rapid visualization and to aid the interpretation of large geneexpression data sets13,46,47.

. 10 mM MgSO4 (sterilize before use) . Appropriate adult fly lines, Oregon-RS or Canton-S can be availed from the Bloomington Stock Center.

. LB broth (Fisher Scientific, cat. no. BP1427) ! CAUTION Infected flies should be killed by freezing at 20 1C and disposed of as a biohazard material. . Autoclaved water . 99% Ethanol

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. 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) . Stereoscopic microscope ( Stemi 2000-C, Zeiss) equipped with a controllable CO2 flow pad . Fly incubators with high humidity capacity (60–75%), adjustable temperature, and a 12-h light/12-h dark cycle

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 diameter. Once the tips are pulled, they must be ‘backfilled’ with a noncompressible fluid (e.g., mineral oil or silicone) before attachment to the injector. Backfilling can be achieved easily using a 30-guage, 200 needle 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

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(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 studies. For an abdominal inoculation, insert the needle parallel to the anteroposterior body axis in the dorsolateral abdominal cuticle close to the junction between the thorax and the abdomen (white arrow in Fig. 1b). Such positioning will avoid injury of the fly heart. Place the pricked fly aside using the paintbrush, and dip the needle into the bacterial suspension 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 bacteria 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. ? 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.

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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 ml1 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 ml1 of ampicillin and 0.05 mg ml1 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 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 1292 | VOL.4 NO.9 | 2009 | NATURE PROTOCOLS

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

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