25 Alternative models in microbial pathogens

25 Alternative Models in Microbial Pathogens Man-Wah ‘Department of Medicine, Department

Tan’

and

Frederick

M Ausube12

ofGenetics, and Department ofMicrobiology and Immunology, Stanford University School Stanford, CA 94305, USA; 2Department of Genetics, Harvard Medical School and of Molecular Biology, Massachusetts General Hospital, Boston, MA 02 I 14, USA

CONTENTS

Introduction P. aeruginosa-multi-host system Other models of host-pathogen interactions Conclusion Appendix I

++++++

INTRODUCTION The struggle for survival is one of the most potent forces that drives evolution. To survive, an organism needs to overcome a variety of biotic and abiotic insults. The most common biotic insult comes in the form of antagonistic interactions between organisms, such as interactions between bacterial pathogens and their hosts. Bacterial pathogens have developed a variety of offensive and defensive weapons to defeat their hosts. To defend themselves against pathogens, plant and animal hosts have evolved the ability to recognize the aggressors and have developed a relay system to communicate this information within the cell so that appropriate defenses can be mounted. Ideally, to make full use of powerful genomic methods to uncover the pathogen-derived virulence determinants and the host-derived defense factors, both the pathogen and its host should be amenable to genetic analysis and their genomes completely sequenced. This would permit both the host and pathogen to be genetically altered and the effects of these alterations on pathogenesis to be readily tested. The availability of complete genome sequences of both the host and pathogen also provides a unique opportunity to utilize DNA chip technology to perform coordinate genome-wide gene expression analyses on the host and pathogen as they interact.

METHODS IN MICROBIOLOGY, ISBN O-12-521531-2

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31 All rights

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Recent work has shown that there are common mechanisms underlying host-pathogen interactions: pathogens use similar virulence factors to infect divergent hosts (reviewed in Finlay, 1999; Finlay and Falkow, 1997). Similarly, the innate immune response to pathogen appears to be ancient and conserved in plants, vertebrates, and invertebrates (reviewed in Tan and Ausubel, 2000; Wilson et al., 1997). Our laboratories took advantage of the conservation of these mechanisms in host-pathogen interactions by developing a non-vertebrate multi-host pathogenesis system. This has enabled us to use genome-wide strategies to dissect the molecular basis of host-pathogen interactions. The first multi-host pathogenesis system developed was based on the ability of Pseudomonas aeruginosa to infect and/or kill divergent hosts. P. aeruginosa is a ubiquitous Gram-negative bacterium and an important pathogen in cystic fibrosis patients, or in patients whose immune system is compromised by medical intervention, infection or burn. The strain used in our studies is PA14, which was originally isolated from the blood of a burn wound patient. PA14 was subsequently shown to infect a variety of plant species, including Arubidopsis thalianu (Rahme et al., 1995, 1997, 2000), and to kill invertebrate hosts such as the soil nematode Caenorhabditis eleguns (Mahajan-Miklos et al., 1999; Tan et al., 1999a,b; Tan and Ausubel, 2000) and insects, such as Gulleriu mellonella (Jander et al., 2000) and Drosophila melanoguster (G. Lau, S. Mahajan-Miklos, E. Perkins and L. Rahme, personal communication). Because pathogenesis in these hosts involves a shared set of P. aeruginosu virulence determinants (see below and reviewed in Finlay, 1999; Mahajan-Miklos et al., 2000; Rahme et al., 2000; Tan and Ausubel, 2000), this multi-host system allows the entire P. aeruginosa genome to be scanned systematically, efficiently and economically for any gene that affects pathogenesis in vivo. This is accomplished by screening a mutagenized library of P. aeruginosu for less pathogenic mutants following infection of individual mutant clones on individual plants or insects or feeding mutant clones of bacteria growing on separate petri plates to C. elegans. In addition, in the case of A. thuliana, C. elegans and D. melunogaster, host immunity mutants can be isolated by screening for host mutants that are either more resistant or more susceptible to pathogen attack. The concept of using non-vertebrate hosts to dissect pathogen- and host-derived factors has recently been extended to include other pathogens and hosts. For example, the gram-negative pathogens Salmonella entericu, Serratiu murscesens, Burkholderiu cepucia and B. pseudomallei and the gram-positive pathogens Enterococcus faecalis and E. faecium have also been shown to kill C. elegans (Aballay et al., 2000; Garsin et al., 2000; Tan and Ausubel, 2000; J. Jedelloh, personal communication). The cellular slime mold Dictyostelium discoideum has also been shown to be an effective host for P. ueruginosu 6. Putatzki, H. Ennis and R. Kessin, personal communication) and Legionella pneumophila (Solomon et al., 2000) and certain strains of the plant pathogens Erwiniu caratovora and E. chrysunthemi have been shown to infect D. melanogaster (Basset et al., 2000). In this chapter, we describe protocols for assessing the virulence of various pathogens using simple non-vertebrate hosts. 462

++++++

f’. AERUGINOSA-MULTI-HOST

SYSTEM

A. thaliana There are many species of plants that are susceptible to P. aeruginosa (Table 25.1, Cho et al., 1975; Schroth et al., 1977). In A. thulianu, P. aeruginosu PA14 causes severe soft-rot symptoms that correspond to bacterial proliferation in the leaves (Plotkinova et al., 2000; Rahme et al., 2000). P. uertcginosa PA14 can invade A. thulium leaves directly through the stomata without the requirement for wounding or mechanical infiltration. PA14 primarily colonizes the intercellular spaces, causing disruption of plant cell walls and membrane structures. Ultimately, PA14 causes a systemic infection that is characterized by basipetal movement along the vascular parenchyma of the leaf, resulting in rotting of the petiole and central bud and death of the plant. Distinctive features of P. ueruginosa pathogenesis are that the surface of mesophyll cell walls adopt an unusual convoluted or undulated appearance, that PA14 cells orient themselves perpendicularly to the outer surface of mesophyll cell walls, and that PA14 cells make circular perforations, approximately equal to the diameter of P. aeruginosu, in mesophyll cell walls. Like other plant pathogens, the ability of P. ueruginosu PA14 to cause disease symptoms and proliferate in A. thuliunu leaves is ecotype (wild-type variety) specific. For example, 5 days after inoculation into ecotype Llagostera (Ll) with lo3 cfu cm-2 leaf surface area, 3 x lo7 cfu cmP2 leaf surface area could be recovered from these leaves. However, similar inoculation in ecotype Argentat (Ag) leaves did not produce any disease symptom, and the bacteria were only able to grow to a density of 3 x lo5 cfucmP2 leaf surface area, 5 days post-inoculation (Rahme et al., 1995). Moreover, virulence is also determined by the genotype of the pathogen. Proliferation in Arubidopsis Ll leaves of isogenic strains of PA14 that have lesions in the gucA, plcS, or toxA genes was significantly less than their parental wild-type, attaining a density of 6 x 103, 1 x lo5 and 2 x lo6 cfu cme2 leaf surface area, respectively (Rahme et al., 1995). Similarly, P. ueruginosu strains PAK and PA01 were also less virulent compared to PA14, with the maximal levels of growth reached in Ll Arubidopsis at 6 x lo4 and 8 x 105, respectively (Rahme et al., 2000). P. ueruginosu PA14 also causes soft-rot symptoms to develop when inoculated into the midrib of lettuce leaf stems, and unlike the infection of A. thuliunu, both PAK and PA01 strains were also infectious in lettuce (Rahme et al., 1997). The severity of symptom development in lettuce directly correlated with the extent of growth in A. thuliunu. Because of the ease of testing several strains on a single lettuce leaf stem, a lettuce screen (described in Rahme et al., 1997) was used to identify P. uerlnginosu PA14 transposon insertion mutants that failed to elicit disease symptoms. In principle, one can use any of the plant species listed in Table 25.1 for the above screen. The lettuce screen led to the identification of nine mutants out of 2500 prototrophic mutants tested. Importantly, all nine mutants identified from the plant screen also exhibited reduced pathogenicity when tested in a burned mouse pathogenicity model at a dose of 5 x lo5 cfu (Rahme et al., 1997).

463

Table

25. I Plant species that can be infected

by

P. oeruginosa Cruciferae Arabidopsis thaliana Lettuce (Lactucu sativa ‘Great Lakes’ and ‘Romaine’) Rutabaga (Brassicu campestris) Brussels sprouts (Brassica olerucea gemmifera Umbelliferae Celery (Apium graveolens var. Dulce) Carrot (Daucus carrota var. sativa) Solanaceae Tomato (Lycopersicon eculentum) Potato tuber (Solarium ttlberosum ‘Whiterose’) Cucurbitaceae Cucumber (Cucumis sativus)

The protocol described below (Protocol 1) to quantify P. aemginosa PA14 pathogenicity in A. thaliana is based on the method described by Rahme et al. (1997). It is a variant of a leaf-infiltration assay developed for P. syringae (Davis et al., 1991; Dong et al., 1991). Conditions used for growing A. thulium are as follows: Germinate A. thulium ecotype Llagostera (LB seeds (available from the Arubidopsis Biological Resource Center, Columbus, OH) and grow in Metromix 200 (W.R. Grace, Inc.) in a climate-controlled greenhouse (20 f 2”C, relative humidity 70 f 5%) with supplemental fluorescent lighting (16 h photo period, 150300 PErn-’ s-r). After 10 days to 2 weeks, transplant seedlings into fresh soil and incubate in a growth chamber at 22°C with a 12 h photo period and a light intensity of loo-150 ~EmP2s-‘, but at the same temperature and relative humidity as the greenhouse. Protocol

I

1. Grow individual bacterial strains or mutants aerobically overnight at 37°C to saturation in 5ml King’s B (Appendix 1, King et al., 1954) or Luria broth (LB) (Miller, 1972). 2. Pellet 1 ml of each overnight culture in a microcentrifuge for 1 minute. Remove the supernatant and resuspend the cells in 1 ml of 10m~ MgSO*. Pellet again and resuspend cells in 1 ml of 10 mM MgS04. Dilute the cells 1: 10 into 10 mM MgS04 and measure absorbance at 600 nm (OD6a0). Dilute this suspension to OD6a0 = 0.002 with 10 mM MgS04 (corresponding to a bacterial density of approximately 4-5 x lo3 per cm2 of leaf area). 3. To inoculated leaves with the bacterial suspension, force the bacterial suspension through the stomata1 openings on the abaxial surface 464

(underside) of mature leaves of 6-week-old A. thaliuna ecotype Llagostera (LB using a 1 ml syringe without a needle. Infiltrate control plants with sterile 10 mM MgSO*. 4. Label the inoculated leaves by attaching tape to toothpicks, writing the inoculum used on the tape, and inserting the toothpicks into the soil on the clockwise side of the inoculated leaves. 5. Incubate inoculated plants in a growth chamber at 28-30°C and 90 to 100% relative humidity. 6. Bacterial growth at each time point is measured by determining the average of the logarithm of the number of colony forming units in five leaf discs. Each leaf disc is obtained by punching with a 0.28 cm2 cork borer outside the initial infiltration site. Grind the leaf discs in 10 mM MgS04 in Eppendorf tubes using a plastic pestle. The number of viable bacteria is determined by plating appropriate dilutions on King’s B medium supplemented with appropriate antibiotics.

Insect P. aeruginosa has also been reported to be a pathogen of insects (Bulla et al., 1975). For example, the 50% lethal dose (LDs& of P. aeruginosa when injected into the hemolymph of the greater wax moth, Galleriu melloneIlu larvae is fewer than 10 bacteria (Jander et al., 2000; Jarosz, 1995; Lysenko, 1963). P. ueruginosu PA14 is also highly virulent when fed to the diamondback moth, Plutella xylostella (Jander et al., 2000). Additionally, P. ueruginosu strains PA14 and PA01 kill adult D. melunoguster following an abdominal prick using a pin dipped in a suspension of P. aeruginosu (G. Lau, S. Mahajan-Miklos, E. Perkins and L. Rahme, personal communication). Recently, our laboratory showed that there is a positive correlation between virulence of P. ueruginosu PA14 mutants in the wax moth caterpillars and mice, suggesting that these insects would serve as a good model system to identify and characterize bacterial genes involved in mammalian pathogenesis Uander et al., 2000). This result further suggests that the use of insects as hosts can be extended to identify virulence determinants of other human pathogens that infect insects, such as the bacterial pathogens Proteus vulgaris, P. mirubilis, Serrutia marcescens, and the fungal pathogens, Fusarium oxysporum and Aspergillus fumigutus (Chadwick et al., 1990; Jander et al., 2000; Tanada and Kaya, 1993). The following is a protocol (Protocol 2) for infecting G. mellonella larvae with P. ueruginosa and for determining the 50% lethal dose (LD,,). Protocols for infecting Drosophila are given later.

Protocol

2

1. Grow from a single colony P. ueruginosu PA14 wild-type or mutant in LB containing 100 pg ml-i rifampicin at 37°C overnight. Dilute this overnight culture 1: 100 in LB and grow to an optical density at 600 nm (OD6& of 0.3 to 0.4. 465

2. Pellet the culture and resuspend in 10 mM M&SO,. After dilution to an OD600 of 0.1 in 10m~ MgSO.+, make IO-fold serial dilutions of the bacterial suspension in 10 mM MgS04, 2 mg ml-i rifampicin. 3. Use a 10 nl Hamilton syrin e to inject 5 ~1 aliquots of the serial ! dilutions (containing from 10 to 0 bacteria) into separate 5th ins&r G. mellonella larvae via the hindmost left proleg (Figure 25.1). Inject 10 larvae at each dilution, three replicates per dilution. As a negative control, inject in triplicate, 10 larvae per replicate, 5 ~1 each of 10 mM MgSO*, 2 mg ml-’ rifampicin. G. melloneZla larvae can be obtained from Van der Horst Wholesale, St. Marys, OH. The addition of rifampicin prevents infection by bacteria naturally present on the surface of the larvae. 4. Score the larvae as live or dead after 60 h at 25°C. The normally white larvae became mellanotic (black) about 5 h before they die and are readily distinguished from live ones. 5. To determine the LDsa, use a computer program such as Systat to fit a curve to the infection data of the following form: Y = A + (1 - A)/(1 + exp[B - G. In(X)]>, where X is the number of bacteria injected, Y is the fraction of larvae killed by infections, and B and G are parameters which are varied for optimal fit of the curve to the data points.

C. elegans P. aeruginosa kills C. elegans by at least three largely distinct mechanisms, which are dependent on growth conditions and the genotype of the bacteria. Strain PA14, when grown in low salt medium (NG), kills worms over a period of 2-3 days (‘slow killing’) by an infection-like process that

Figure 25.1. Injection of a C. mellonella 5th instart larva with a Hamilton the hindmost left proleg (arrow).

466

syringe via

correlates with the accumulation of bacteria in the worm gut (Tan et al., 1999a). When PA14 is grown in a high salt medium (PGS), it kills worms within 4-24 h (‘fast-killing’) by the production of low molecular weight toxin(s) (Mahajan-Miklos et al., 1999). Another strain of P. aevuginosa, PAOl, kills rapidly by yet another mechanism. When PA01 is grown on brain-heart infusion agar, worms become paralyzed within 4 h upon contact with the bacterial lawn (Darby et al., 1999). There are several host-associated factors that affect the susceptibility of nematodes to bacteria-mediated killing. Gravid adult hermaphrodites are more susceptible than adult males due to embryos hatching from within the gravid adults (Tan et al., 1999a). This non-specific effect can be eliminated by using either C. elegans males or temperature sensitive sterile hermaphrodite mutants, such as fer-2 or glp-4 (available from Caenorhabditis Genetics Center at the University of Minnesota). C. elegans mutants with defects in feeding and defecation are also more susceptible to P. ueruginosu slow killing. C. elegans is a filter-feeder; taking in liquid with bacteria and then spitting out the liquid while retaining and grinding up the bacteria when they reach the terminal bulb (Avery, 1993). C. elegans mutants defective in grinding, such as eat-23 and phm-2, receive a higher bacteria inoculum than wild-type because they allow the entry of more intact pathogens into the intestines. Consequently, these mutants are more sensitive to pathogen-mediated killing (M.-W. Tan, G. Alloing and F.M. Ausubel, unpublished data). C. elegans defecates at regular intervals by a series of sequential muscle contractions. A defect in any of these steps leads to failure to remove intestinal contents at regular intervals and causes a ‘constipated’ phenotype. Constipated mutants, such as aex-2 and uric-25, which retain pathogens in the intestines longer are also more sensitive than wild-type worms to pathogen-mediated killing (M.-W. Tan and F.M. Ausubel, unpublished data). In addition to P. ueruginosu, several other bacterial species have been reported to be pathogens of C. eleguns (Aballay et al., 2000; Garsin et al., 2000; Tan and Ausubel, 2000). These include several human pathogens such as B. cepaciu, S. rnurcescens, S. typhimuriurn, E. fuecalis and E. faecium. Protocol 3 describes conditions used to assay C. eleguns slow killing by P. ueruginosu strain PA14 (Tan et al., 1999a). This protocol is also suitable for testing B. cepacia and S. murcescens. Other variations or pathogenspecific conditions are summarized in Table 25.2. Protocols for general growth and maintenance of C. eleguns are described in Sulston and Hodgkin (1987). Protocol

3

1. Seed 3.5 mm diameter NG agar (a modification of the NGM agar described in Sulston and Hodgkin, 1987, Appendix 1) plates with 10 ~1 of an overnight bacterial culture. 2. Incubate the plates at 37°C for 24 h. Equilibrate plates to ambient temperature (23-25°C) for several hours prior to adding 30-40 worms at a specific developmental stage. Usually l-day old hermaphrodites or the final stage larvae (L4) are used. For statistical analysis, have 467

three-four replicates per trial. Use E. coli OP50 as a negative control. 3. Incubate plates at 25°C and score for the number of dead worms every 4-6 h (after the initial 24 h) until all the worms exposed to the wild-type pathogen are dead. A worm is considered dead if it does not respond to touch. Exclude from analysis worms that die as a result of getting stuck to the walls of petri plates. This can be minimized by ensuring that there is no condensate on the walls prior to the addition of worms. 4. The LT50 (time required to kill 50% of the nematodes) can be determined as follows: For each replicate, a curve can be fitted to the data with the aid of a computer program such as SYSTAT, using the equation: proportion of worms killed at time Ti = A + (1 -A)/ (1 + exp[B - G x In (hours after exposure, Ti)]), where A is the fraction of worms that died in an OP50 control experiment, and B and G are parameters which are varied for optimal fit of the curve to the data points. Once B and G have been determined, LTso can be calculated by the formula, I&,

= exp(B/G)

x (1 - 2 x A)(“G)

Table 25.2 C. elegons killing assays for various pathogens P. aeruginosa PA14 fast killing assay (Mahajan-Miklos et al., 1999) 1. Inoculate 5 ml of LB with single colony of P. aerugitzosa PA14 and grow at 37°C overnight. 2. Spread the center of a 3.5 cm diameter plate containing 5 ml PGS agar (see Appendix 1) with 5 ~1 of the overnight culture. Incubate plates for 24 h at 37°C. 3. After equilibrating the plates to room temperature, add 30-40 L4-stage hermaphrodite C. elegans to each plate and incubate at 25°C. 4. Assay for worm mortality at 4-h intervals. Note: For this assay, it is important to use fresh PGS plates (l-7 days after pouring). Worms used for this assay must be cultivated on OP50 that has not turned ‘slimy’ or mucoidy. Slimy plates give inconsistent results. P. aeruginosa PA01 paralysis assay (Darby et al., 1999) 1. Grow P. aeruginosa PA01 overnight at 37 “C in Brain Heart Infusion (BHI) broth. 2. Dilute the overnight culture loo-fold in fresh BHI broth, then spread 400 ~1 of the dilution over the entire surface of BHI agar plates (10 cm diameter). Incubate the plates for 24 h at 37°C. 3. Wash nematodes off culture plates with M9 buffer (pH 6.5). Spin down the nematodes and resuspend in a minimal volume of M9 buffer. Seed the P. aeruginos a lawn with nematodes by placing droplets of the suspension onto the bacterial lawn and incubate at 21-23°C. The droplets will dry within 30 minutes. 468

4. Examine the worms after 4 h; worms that do not move spontaneously or do not respond to touch are scored as paralyzed. E. fuecaIis OGlRF and V583 and E. faecium E007 (Garsin et al., 2000) 1. Inoculate 2 ml of BHI medium with a single colony of the test strain and grow at 37 “C for 4 h or overnight. 2. Spread 10 1.11 of this culture on BHI agar (with appropriate antibiotics that permit the growth of test Enterococcus strains but prevent the growth of E. coli OP50) in 3.5 cm tissue culture plate. 3. After incubating the plates overnight at 37”C, bring to room temperature for 2 to 5 h before the addition of C. elegans. Pick 20-30 C. elegans L4 hermaphrodites from E. coli OP50 lawns to the Enterococcus lawn and incubate at 25°C. 4. Score worm mortality over time by examining the plates at approximately 24 h intervals for 150-200 h. Both E. faecalis and E. fuecium either prevent the hatching of eggs or kill the hatchlings. Therefore, the production of progeny does not obscure the killing assay. S. typhimurium (Aballay et al., 2000) 1. Inoculate 5 ml of LB with a single colony of test strain and grow at 37°C overnight. 2. Spread 10 1.11of the overnight culture on NG agar media (Appendix 1) in 3.5 cm diameter plates. Incubate the seeded plates at 37°C for 2-12 h. Longer incubation times can negatively affect the killing assay. 3. After equilibrating the plates to room temperature, add lo-20 L4-stage hermaphrodite C. eleguns to each plate and incubate at 20-25°C. 4. Transfer the originally seeded worms daily to fresh plates, which are prepared in the same manner as described above, until the end of their reproductive stage. (This step is necessary because the killing is relatively slow, thus permitting the seeded worms (PO) to produce Fl generation that would develop into adults making it impossible to distinguish them from the PO parents. The transferring step can be eliminated when C. eleguns males or temperature sensitive sterile mutants, such as fey-1 or glp-4, are used.) 5. Examine the plates daily for 8-10 days for dead worms.

+a++++

OTHER MODELS INTERACTIONS

OF HOST-PATHOGEN

Recently, the concept of using genetically amenable hosts to study human pathogen has been extended to a system that uses the free-living unicellular organism, Dictyostelium discoideum, and its interaction with an intracellular pathogen, Legionella pneumophila (Solomon and Isberg,

469

2000; Solomon ef al., 2000). Similarly, a system to study the interactions between plant pathogens from the genus Erwinia and their host/vector, D. melanogasfer have been developed (Basset et al., 2000).

Legionella

pneumophila

and Dictyostelium

discoideum

L. pneumophila, the causative agent of Legionaires disease, is found naturally as parasites of freshwater amoebae of the genera Acanfhamoeba and Nuegleria (Rowbotham, 1980). L. pneumophila enjoys a wide host range: more than 13 species of amoebae and two species of ciliated protozoa can support its growth (reviewed in Fields, 1996). In humans, it replicates in macrophages. L. pneumophilu is able to grow intracellularly in D. discoideum, requiring the same genes that it requires for growth in macrophages and amoeba, such as the dot genes (Gao et al., 1997; Segal and Shuman, 1999; Solomon et al., 2000). The following protocol (Protocol 4) for quantifying growth of L. pneumophila in D. discoideum in liquid culture is based on Solomon et al. (2000). Techniques for maintaining D. discoideum axenically in liquid medium or as plaques on a lawn of Klebsiella aerogenes are detailed by Sussman (1987). Protocol

4

1. Culture D. discoideum axenically in a shaken flask in HL-5 liquid medium (Appendix 1). When cells are at the exponential growth phase, pellet cells by spinning at 6009 for 5 minutes. Resuspend in an equal volume of PBS buffer. Pellet cells again by a 5 min spin at 6OOg, then remove PBS by aspiration. Resuspend cells in MB medium (Appendix 1) and plate D. discoideum as an adherent monolayer in 24-well tissue culture dishes at a density of lo6 cellsml-‘. Incubate cells at 25.5”C. Allow plates to equilibrate for 4 h before the addition of bacteria. 2. Grow L. pneumophila at 37°C for 48h on CYE plates (Appendix 1). Alternatively, L. pneumophila can also be grown at 37°C in AYE liquid medium (same as CYE without agar and charcoal) to an OD600 of 3 to 3.5, to allow maximal infectivity. Pellet bacterial cells by spinning for 5 min at 16000g. Resuspend bacterial cells in MB medium; the concentration of bacteria is determined by assuming that OD6a0 = 1.0 is equivalent to lo9 bacteria ml-‘. 3. Infect D. discodeum at a multiplicity of infection (MOB of approximately 1 : 1. 4. Quantify the number of viable L. pneumophila and D. discoideum each day by measuring colony-forming units (CFU) or plaque-forming units (PFU), respectively. a. Quanfifafion on L. pneumophila. Add 0.02% saponin (Sigma S-4521) to infected wells to lyse D. discoideum and to release the intracellular bacteria. Prepare a dilution series of the lysed D. discoideum and plate on CYE plates. Incubate CYE plates at 37°C for 3 to 4 days before counting the colonies (CFU). b. Quanfifafion of D. discoideum. Makeadilutionseriesof harvested D. discoideum in PBS and plate on lawns of K. aerogenes. K. aerogenes can 470

be cultured in LB and plated on SM /5 agar medium (Appendix 1). Incubate plates at 21°C and count the number of plaques (PFU) 3 to 4 days after plating.

Drosophila

melanogaster

and Erwinia

Studies on the Drosophila immune responses have yielded great insights into how a host responds to microbial infections. Recently, it was shown that the signaling pathways in Drosophila defense response are strikingly similar to innate immune responses in mammals (reviewed in Hoffmann et al., 1999). Studies on bacterial infection of Drosophila have relied on direct introduction of bacteria into the body cavity by injection or pricking, or by ingestion of bacteria by the fly larvae. Interestingly, it appears that different pathways may be involved in activating antibacterial responses, depending on the mode of infection. For example, in the absence of physical injury, hemocytes play a significant role in activating a systemic antibacterial response, whereas introduction of bacteria by pricking triggers other pathways that bypass the requirement of hemocytes (Basset et al., 2000). Here we describe both the pricking and feeding protocols for infecting Drosophila. Grow from a single colony the bacterial strains in LB overnight at 37°C. Pellet the overnight bacterial culture. Anesthetize 2-4-damld adult flies with C02. Prick the thorax of the flies with a thin needle that has been dipped into a concentrated bacterial pellet (approximately 4 x 10” cellsmll’) from an overnight culture in LB (Basset et al., 2000). Alternatively, inject 0.15 ul stationary phase culture of bacteria, diluted 1: 10 in Drosophila Ringer’s solution, into the thorax using a thin pulled glass pipette (Hedengren et al., 1999). Transfer injected fly to fresh vials and assess for survivals at regular intervals. Transfer surviving flies to fresh vials every third day. Do not include flies that die within 3 h of injection in the analysis (Lemaitre et al., 1996). A similar procedure can be used for third instar larvae as well. The following feeding protocol was first used to demonstrate the infection of Drosophila larvae by two phytopathogens, Evwinia ca~otovo~u cavotovora and E. chrysanthemi paradisiaca (Basset et al., 2000). Mix the following in a 2 ml microfuge tube: (1) 200 ~1 of a concentrated bacterial pellet from overnight culture in LB; (2) 400 ~1 of crushed banana; and (3) approximately 200 third instar larvae. Cover the tube with a foam plug and incubate at room temperature. After 30 min, transfer the mixture to a standard corn-meal fly medium and incubate at 29°C. Larvae infected by the two species of Erwinia do not die, however, 90% of the treated larvae show induction of genes encoding antimicrobial peptides (Basset et al., 2000).

++++++

CONCLUSION The underlying mechanisms of interactions between bacterial pathogens and their animal and plant hosts are complex. The recent development of 471

alternate non-vertebrate hosts that are amenable to genetic analysis are providing important tools to dissect complex host-pathogen interactions. An important consideration when interpreting results obtained from all the systems described in this chapter is that the temperature used to assess pathogenesis in all the non-vertebrate hosts ranges from 25”C-3O”C, whereas many mammalian pathogens appear to express virulence determinants only at relatively high temperatures such as 37°C. None the less, the common offensive strategies employed by a wide range of prokaryotic pathogens and the conservation of multiple aspects of the innate immune response of their eukaryotic hosts suggest that the analysis of non-vertebrate/bacterial-pathogen interactions will have global relevance.

References Aballay, A., Yorgey, I? and Ausubel, F. M. (2000). Salmonella typhimurium proferates and establishes a persistent infection in the intestine of Caenorhabditis elegans. CUYY.Biol. 10, 1539-1542. Avery, L. (1993). The genetics of feeding in Caenorhabditis elegans. Genetics 133, 897-917. Basset, A., Khush, R. S., Braun, A., Gardan, L., Boccard, F., Hoffmann, J. A. and Lemaitre, B. (2000). The phytopathogenic bacteria Erwinia carotovora infects Drosophila and activates an immune response. Proc. Nat1 Acad. Sci. USA 97, 3376-3381. Bulla, L. A., Rhodes, R. A. and St. Julian, G. (1975). Bacteria as insect pathogens. Ann. Rev. Microbial. 29, 163-190. Chadwick, J. S., Caldwell, S. S. and Chadwick, I? (1990). Adherence patterns and virulence for Galleria mellonella larvae of isolates of Serratia marcescens. J. Invertebr. Pathol. 55, 133-134. Cho, J. J., Schroth, M. N., Kominos, S. D. and Green, S. K. (1975). Ornamental plants as carriers of Pseudomonas aeruginosa. Phytopathology 65, 425-431. Darby, C., Cosma, C. L., Thomas, J. H. and Manoil, C. (1999). Lethal paralysis of Caenorhabditis elegans by Pseudomonas aeruginosa. PYOC.Nat1 Acad. Sci. USA 96, 15202-15207. Davis, K. R., Schott, E. and Ausubel, F. M. (1991). Virulence of selected phytopathogenic pseudomonads in Arabidopsis thaliana. Mol. Plant-Microbe. Interact.

4,477-488. Dong, X., Mindrinos, M., Davis, K. R. and Ausubel, F. M. (1991). Induction of Arabidopsis defense genes by virulent and avirulent Pseudomonas syringae strains and by a cloned avirulence gene. Plant Cell 3, 61-72. Feeley, J. C., Gibson, R. J., Gorman, G. W., Langford, N. C., Rasheed, J. K., Mackel, D. C. and Baine, W. B. (1979). Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J. Clin. Microbial. 10, 437-441. Fields, B. S. (1996). The molecular ecology of legionellae. Trends Microbial. 4,

286-290. Finlay, B. B. (1999). Bacterial disease in diverse hosts. Cell 96, 315-318. Finlay, B. B. and Falkow, S. (1997). Common themes in microbial pathogenicity revisited. Microbial. Mol. Biol. Rev. 61, 136-169. Gao, L. Y., Harb, 0. S. and Abu Kwaik, Y. (1997). Utilization of similar mechanisms by Legionella pneumophila to parasitize two evolutionarily distant host cells, mammalian macrophages and protozoa. Inject. Immun. 65,4738-4746.

472

Garsin, D. A., Sifri, C. D., Mylonakis, E., Qin, X., Singh, K. V., Murray, B. E., Calderwood, S. B. and Ausubel, F. M. (2001). A simple model host for identifying Gram-positive virulence factor. Pm. Nat1 Acad. Sci. USA in press. Hedengren, M., Asling, B., Dushay, M. S., Ando, I., Ekengren, S., Wihlborg, M. and Hultmark, D. (1999). Relish, a central factor in the control of humoral but not cellular immunity in Drosophila. Mol. Cell. 4, 827-837. Hoffmann, J. A., Kafatos, F. C., Janeway, C. A. and Ezekowitz, R. A. (1999). Phylogenetic perspectives in innate immunity. Science 284,1313-1318. Jander, G., Rahme, L. G. and Ausubel, F. M. (2000). Positive correlation between virulence of Pseudomonas aeruginosa mutants in mice and insects. 1. Bacferiol. 182, 3843-3845. Jarosz, J. (1995). Interaction of P. aeruginosa proteinase with the non-self response of insects. Cyfobios. 83, 71-84. King, E. O., Ward, M. K. and Raney, D. E. (1954). Two simple media for the demonstration of phycocyanin and fluorescin. J. Lab. Clin. Med. 44, 301-307. Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J. M. and Hoffmann, J. A. (1996). The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86, 973-983. Lysenko, 0. (1963). The mechanisms of pathogenicity of Pseudomonas aeruginosa (Schroeter) Migula 1. The pathogenicity of strain N-06 for the larvae of the Greater Wax Moth, Galleria mellonella (Linnaeus). 1. lnsecf Pafkol. 5, 78-82. Mahajan-Miklos, S., Rahme, L. G. and Ausubel, F. M. (2000). MicroReview: elucidating the molecular mechanisms of bacterial virulence using non-mammalian hosts. Mol. Microbial. 37, 981-988. Mahajan-Miklos, S., Tan, M.-W., Rahme, L. G. and Ausubel, F. M. (1999). Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosaCaenorkabdifis elagans pathogenesis model. Cell 96, 47-56. Miller, J. H. (1972). Experiments in Molecular Genetics. 2nd edition., Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press. Plotkinova, J. M., Rahme, L. G. and Ausubel, F. M. (2000). l’athogenesis of the human opportunistic pathogen Pseudomonas aeruginosa PA14 in Arabidopsis. Plant Pkysiol. 124, 1766-1744. Rahme, L. G., Ausubel, F. M., Cao, H., Drenkard, E., Goumnerov, B. C., Lau, G. W., Mahajan-Miklos, S., Plotnikova, J., Tan, M. W., Tsongalis, J., Walendziewicz, C. L. and Tompkins, R. G. (2000). Plants and animals share functionally common bacterial virulence factors. Proc. Nat1 Acad. Sci. USA 97, 8815-8821. Rahme, L. G., Stevens, E. J., Wolfort, S. F., Shao, J., Tompkins, R. G. and Ausubel, F. M. (1995). Common virulence factors for bacterial pathogenicity in plants and animals. Science 268, 1899-1902. Rahme, L. G., Tan, M. W., Le, L., Wong, S. M., Tompkins, R. G., Calderwood, S. B., and Ausubel, F. M. (1997). Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors. Proc. Nat1 Acad. Sci. USA 94, 13245-13250. Rowbotham, T. J. (1980). Preliminary report on the pathogenicity of Legionella pneumopkila for freshwater and soil amoebae. 1. Clin. Pafkol. 33, 1179-1183. Schroth, M. N., Cho, J. J., Green, S. K. and Kominos, S. D. (1977). Epidemiology of Pseudomonas aeruginosa in agricultural areas. In: Pseudomonas aeruginosa: Ecological Aspects and Patient Colonization, (ed. V. M. Young), Raven Press, pp.l-29. New York. Segal, G. and Shuman, H. A. (1999). Legionella pneumopkila utilizes the same genes to multiply within Acanfkamoeba casfellanii and human macrophages. Infect. lmmun. 67,2117-2124.

473

Solomon, J. M. and Isberg, R. R. (2000). Growth of Legionella pneomophila in Dicfyosfelium discoideum: a novel system for genetic analysis of host-pathogen interactions. Trends Microbial. 8, 478-480. Solomon, J. M., Rupper, A., Cardelli, J. A. and Isberg, R. R. (2000). Intracellular growth of Legionella pneumophila in Dicfyosfelium discoideum, a system for genetic analysis of host-pathogen interactions. Infect. Immun. 68, 2939-2947. Sulston, J. and Hodgkin, J. (1987). Methods. In The Nematode Caenorhabdifis elegans. (W. B. Wood, ed.), pp. 587-606. Cold Spring Harbor, New York. Sussman, M. (1987). Cultivation and synchronous morphogenesis of Dicfyosfelium under controlled experimental conditions. Methods Cell Biol. 28, 9-29. Tan, M.-W. and Ausubel, F. M. (2000). Caenorhabdifis elegans: a model genetic host to study Pseudomonas aeruginosa pathogenesis. Cum Opin. Microbial. 3, 29-34. Tan, M.-W., Mahajan-Miklos, S. and Ausubel, F. M. (1999a). Killing of Caenorhabdifis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis. Proc. Nat1 Acad. Sci. USA 96, 715-720. Tan, M.-W., Rahme, L. G., Sternberg, J. A., Tompkins, R. G. and Ausubel, F. M. (1999b). Pseudomonas aeruginosa killing of Caenorhabdifis elegans used to identify l? aeruginosa virulence factors. Proc. Nat1 Acad. Sci. USA 96, 2408-2499. Tanada, Y. and Kaya, H. (1993). Insect Pathology. Academic Press,San Diego, Calif. Wilson, I., Vogel, J. and Somerville, S. (1997). Signalling pathways: A common theme in plant and animals? Curr. Biol. 7, R175-R178.

King’s B medium (King et al., 1954) 1.0% Difco Proteose Peptone #3 0.15% K,HPO,, anhydrous 1.5% glycerol adjust pH to 7.0 with HCl. After autoclaving, add sterile MgS04 to 5 mM

NG agar 0.3% NaCl 0.35% Bacto Peptone (Difcol 1 ml I-’ cholesterol (5 mg ml-’ in 95% ethanol) 2% Bacto Agar (Difco) After autoclaving, add the following sterile solutions: 1 ml 1-l CaC& 1 M 1 ml 1-l MgS04 1 M 25 ml 1-l KH2P04 1 M, adjusted to pH 6.0 with solid KOH. PGS agar 1.0% Bacto Peptone (Difco) 1 .O% glucose 1.0% NaCl 2.74% sorbitol 1.7% Bacto Agar (Difco)

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Dispense 5 ml agar per 3.5 cm diameter plate. Use after 1 day. Store the remainder at 4°C and use within 1 week. CYE agar (Feeley et al., 1979) 0.2% activated charcoal (Norit A or Norit SG) 1.0% yeast extract 1.7% agar (Difco) After autoclaving, add filter-sterilized 0.04% L-cysteine HCl . HZ0 and 0.025% ferric pyrophosphate. Adjust the medium to pH 6.9 by adding 4 to 4.5 ml of 1 N KOH. SM/5 agar 0.2% glucose 0.2% Bacto Peptone (Difco) 0.2% yeast extract 0.02% MgS04 . 7H20 0.19% KH2P04 0.1% K2HP04 2% agar pH = 6.4 HL5 Medium 1.4% glucose 0.7% yeast extract 1.4% peptone* 0.095% Na2HP04. 7H20 0.05% KH2P04 pH = 6.5 Autoclave (a) glucose, (b) yeast extract and peptone and (c) Na and K phosphates in three separate solutions. * Thiotone (BBL) and Bacteriological peptone (Oxoid) are preferred. Difco proteose peptone tend to give variable results that is batch dependent (Sussman, 1987). MB medium 20 mM ME [2(Wmorpholinoethanesulfonic 0.7% yeast extract 1.4% Thiotone E peptone

475

acid)], pH 6.9