Surrogate hosts: protozoa and invertebrates as models for studying pathogen-host interactions

Int. J. Med. Microbiol. 293, 321 ± 332 (2003) ¹ Urban & Fischer Verlag http: // www.urbanfischer.de/journals/ijmm

Mini-Review Surrogate hosts: protozoa and invertebrates as models for studying pathogen-host interactions Michael Steinerta, Matthias Leippe b, Thomas Roederb a b

Institut f¸r Molekulare Infektionsbiologie, Universit‰t W¸rzburg, W¸rzburg, Germany Zentrum f¸r Infektionsforschung, Universit‰t W¸rzburg, W¸rzburg, Germany

Received July 10, 2003 ¥ Revision received September 9, 2003 ¥ Accepted September 18, 2003

Abstract Animal models, primary cell culture systems and permanent cell lines have provided important information on virulence properties of pathogenic microorganisms. Recently, it has been shown that some inherent limitations of such models can be circumvented by using non-vertebrate hosts such as Caenorhabditis elegans, Drosophila melanogaster and Dictyostelium discoideum. These new models are helpful to follow infection processes at the molecular level. Persuasive support comes from the fact that processes such as phagocytosis, cell signaling or innate immunity can be studied in these surrogate hosts. This review describes the establishment and application of each of the three aforementioned and genetically tractable hosts. In addition, we will report on a number of approaches that led to the identification of host cell factors which influence the susceptibility of the hosts to infection. Key words: Drosophila ± Caenorhabditis ± Dictyostelium ± infection ± Pseudomonas ± Legionella

Introduction The analysis of factors influencing the virulence of pathogens is one of the cornerstones of experimental infection biology. The approach relies on infection models in which properties such as colonization, host cell invasion, toxin production etc. can be monitored. With the completion of genome projects host models capable of testing thousands of different mutants became mandatory. Mammalian model

organisms such as rat, mouse or pig cannot serve this purpose due to the complexity of experimental approaches, exceptional housing requirements, exorbitant costs, and obvious ethical reasons. Nonvertebrate models have the potential to fill this gap because their culture is simple, inexpensive, they require only minimal laboratory space, and ethical restrictions are not evident. The idea of using genetically tractable hosts to study virulence mechanisms arose in the late 70s of the last century

Corresponding author: Prof. Dr. Matthias Leippe, Zoologisches Institut der Universit‰t Kiel, Olshausenstr. 40, D24098 Kiel, Germany. E-mail: [email protected]

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(Depraitere and Darmon, 1978). The unicellular organism Dictyostelium discoideum was proposed as a model host, e.g. for the study of infection by intracellular pathogens such as Legionella pneumophila. In the meantime, also the potential of more complex, multicellular organisms has been recognized as host models. Among these invertebrates, the two model organisms, the fruit fly Drosophila melanogaster and the soil nematode Caenorhabditis elegans are the most important ones. Like Dictyostelium, they are amenable to genetic manipulation which allows studying complex genetic aspects of pathogen-host interactions. Both organisms are easily culturable in the laboratory and hundreds or even thousands of bacterial strains can be tested simultaneously. This holds true in particular for C. elegans which feeds on bacteria and can easily be adapted to large scale cultures in multiple-well formats. Conclusively, all three organisms have the potential to reveal basic aspects of host-pathogen interactions using saturating genetic screens from both sides, the host and the pathogen. In the following, we will focus on the peculiarities and advantages of each of the three systems.

Caenorhabditis elegans, a surrogate host with less than thousand cells Since C. elegans was chosen by the Nobel laureate Sidney Brenner as a model organism for the study of ontogeny and behavior (1974), it is one of the few workhorses in genetics and developmental biology. C. elegans combines several features that are responsible for its versatility. It is a small translucent soil nematode with a length of about 1 mm (Fig. 1). In the laboratory, it is fed on a lawn of the E. coli strain OP50. These simple growth conditions and the rapid generation time are main advantages of this animal model. Adult hermaphrodites can produce between 250 and 300 eggs that develop within 2 ± 3 days. Worms can be stored frozen in liquid nitrogen and they can be handled in different ways allowing on the one hand mass-production in liquid culture and on the other hand growth on multi-well plates for industrial screening purposes. Like all nematodes, C. elegans shows a stereotype cell lineage leading to exactly 959 somatic cells in adults. Beside its role as a model organism for developmental biologists and geneticists, C. elegans obtained additional interest because the class Nematoda contains parasites of man, house stock and economically important plants.

Fig. 1. Structure and major anatomical organization of the nematode Caenorhabditis elegans. The intestinal and the genital tract are susceptible to bacterial infections. Usually, bacteria are ingested through the muscular pharynx, where most of them are killed by mechanical disruption. In the intestine, the food is further processed and resorbed. This part of the intestinal tract is the main entrance for pathogenic bacteria.

C. elegans was the first metazoan organism the entire genome of which has been sequenced (The C. elegans sequencing consortium, 1998) revealing a total of 19,000 genes, more than half as much as found in the human genome (35,000). A huge number of different mutant strains is available through the Caenorhabditis Genetics Centre (CGC, University of Minnesota, http://biosci.umn.edu/CGC/CGChome page.htm) and genetic mutants can be produced with relative ease. Most compelling is that almost every gene is susceptible to RNAi-based gene silencing, an approach that can be performed with hundreds of genes in parallel (Ashrafi et al., 2003). Bacteria are ingested through a complex pharynx and killed and lysed in the intestine (Fig. 1). For infection studies, the E. coli strain OP50 may be replaced by the bacterial strain of interest and the survival rate as well as the production of offspring can be quantified. Notably, under natural conditions, only very few microorganisms colonize C. elegans. Among them are the endoparasitic fungus Drechmeria coniospora (cited in (Ewbank, 2002))

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and the coryneform bacterium Microbacterium nematophilum (Hodgkin et al., 2000). C. elegans was established as a model host for pathogenic bacteria by research from the Ausubel laboratory focusing on Pseudomonas aeruginosa as a pathogen (Mahajan-Miklos et al., 1999; Garsin et al., 2001). This choice was not surprising because P. aeruginosa is a bacterium that displays a very broad host specificity in that it infects plants, insects and mammals. More recently, a number of grampositive (Bacillus megaterium, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pneumoniae) and gram-negative bacteria (Burkholderia pseudomallei, Salmonella typhimurium, Serratia marcescens) as well as the yeast Cryptococcus neoformans were introduced in the system (Alegado et al., 2003). Using biofilm-producing Yersinia pestis strains that physically blocked bacterial intake Darby et al. (2002) showed a different mechanism of pathogenesis also leading to small numbers of progeny. Although its versatility to act as a host for various human pathogens has been demonstrated, not every bacterium can be studied here because the successful infection of the worm is required to monitor the virulence of the putative pathogen. Some models are discussed in more detail below.

Pseudomonas aeruginosa infection of C. elegans P. aeruginosa was the first bacterial pathogen that was demonstrated to infect C. elegans. The Ausubel group pioneered this field by using the clinical isolate PA14. It became evident that P. aeruginosa kills the worms using two different mechanisms. Whereas Pseudomonas grown under conditions of high osmolarity are able to kill C. elegans already after hours, bacteria grown under conditions of low osmolarity colonize the intestine and kill the worm after several days. In a large screen of Pseudomonas mutant strains impaired in killing C. elegans, Ausubel and coworkers (Mahajan-Miklos et al., 1999) found the production and secretion of phenazines as activities required for quick killing. Mutant strains impaired in the production of these phenazines show a reduced virulence not only in C. elegans but also in the murine model. It is believed that higher phenazine concentrations give rise to the production of reactive oxygen species (ROS). Worms with an increased ability to detoxify ROS show a reduced susceptibility to the quick killing by P. aeruginosa, strengthening the notion that indeed phenazines act by producing ROS. In a second screen, the same group (Tan et al., 1999) identified mutant strains impaired in the ™slow killing∫.

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Among them are those that have a defect in one of the quorum-sensing systems, the LasR system. Other bacterial ™virulence factors∫ such as the protease/ chaperone MucD are also required for killing the nematode (Yorgey et al., 2001). Another Pseudomonas strain, the PAO1 strain shows an alternative strategy to kill C. elegans (Darby et al., 1999). Under conditions of high cell density, the bacteria secrete a toxin into their environment that paralyzes the worms within few hours. Although the nature of the toxin is still enigmatic, it was shown that the quorum-sensing systems LasR and RhlR are necessary for this mode of killing. However, a direct contact between pathogen and host is apparently not required. A large C. elegans screen of worms resistant to this paralysis revealed that egl-9 mutant strains are resistant to PAO1-mediated paralysis. It is known that egl-9 mutants are defective in egglaying. However, the molecular mechanism that confers resistance on these mutants remains to be elucidated. In general, a transcriptomal response of C. elegans to P. aeruginosa infections requires activation of the p38 MAPK signaling pathway (Kim et al., 2002).

Salmonella typhimurium infection of C. elegans S. typhimurium, a pathogen that has a narrow host specificity in that it is apparently well adapted to its intracellular life in mammals, has also the potential to infect and kill C. elegans. But unlike as in its murine host, the bacteria remain extracellular during infection of the worm. Following infection, the median life span of the worms was reduced to one quarter of that observed with unchallenged individuals. The bacteria were able to colonize the intestine and hence to survive and replicate within this hostile environment. Interestingly, mutants that exhibit a reduced virulence in mice, e.g. those that are dysregulated with respect to iron, osmotic stress and stationary growth (mutations in fur-1, ompR and dS/rpoS, respectively), were found to be less virulent also in C. elegans (Labrousse et al., 2000). It has been shown that S. typhimurium infections of the intestine triggers gonadal programmed cell death (Aballay and Ausubel, 2001). As impairment of the programmed cell death machinery of C. elegans leads to hypersensitivity towards Salmonella infections, this gonadal cell death might be recognized as one of the mechanisms to combat infections. The response of the nematode requires Salmonella LPS and is mediated by a MAPK signaling pathway (Aballay et al., 2003).

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Other bacterial infections of C. elegans In addition to the bacteria mentioned above, a number of other interesting host-pathogen systems were studied in detail. Like P. aeruginosa, Serratia marcescens is an opportunistic human pathogen with a broad host specificity. S. marcescens is able to kill C. elegans, although not as quick as P. aeruginosa. Comparable to the situation with Pseudomonas, two types of killing, a quick, toxin-based and a slow mechanism have been observed. Again, the slow mechanism results from an infection of the intestine leading to death after a few days. First screens are in progress and revealed already a number of mutant Serratia strains impaired in bacterial virulence (Kurz and Ewbank, 2000). The potential agent of biological warfare Burkholderia pseudomallei kills C. elegans by a UV-labile toxin. Mutations in the worm's L-type Ca2‡-channel provoke an increased susceptibility towards this infection indicating that this toxin acts via disruption of Ca2‡-signaling (O'Quinn et al., 2001). Bacterial toxins that are produced by Bacillus thuringiensis strains are widely used as specific insecticides. Interestingly, a number of B. thuringiensis strains kill C. elegans. As in insects, the effect of the toxin is limited to the intestine, where it results in regression of the microvilli and shrinkage of intestinal cells, leading eventually to cell death (Borgonie et al., 1995). Enterococcus faecalis is also pathogenic to C. elegans and the killing activity depends on the quorum-sensing system as already known from Pseudomonas (Sifri et al., 2002). C. elegans might also be a model to study the effects of biofilmgenerating bacteria as shown for Yersinia pestis (Darby et al., 2002). Yeast infections of C. elegans In contrast to the huge amount of information available regarding the host-pathogen interactions of C. elegans with various bacteria, almost nothing is known about the ability of fungal pathogens of man to infect C. elegans. A fungus-C. elegans model would open a novel avenue to study the factors enabling fungal pathogens to infect the human host, a problem with increasing importance due to the constantly rising number of immuno-compromised persons. Ausubel and coworkers (Mylonakis et al., 2002) developed a model for fungal pathogenesis using the yeast Cryptococcus neoformans. C. elegans can use yeasts such as Cryptococcus laurentii or C. kuetzinglii as a sole food source. Interestingly, a diet on C. kuetzinglii leads to an increased life span of C. elegans when compared with worms fed on

their normal food source, E. coli OP50. In contrast, the human pathogen, C. neoformans, is able to kill C. elegans. Remarkably, C. neoformans strains impaired in virulence towards humans were also impaired in their ability to kill C. elegans. Genes previously shown to be involved in virulence towards mammals were also involved in killing C. elegans. Among them are genes associated with signal transduction pathways such as PKA1, PKR1, RAS1 and GPA1, or the a-mating type. These results support the notion that this simple model host is well-suited to study fungal virulence factors (Mylonakis et al., 2002).

Drosophila melanogaster, common concepts in flies and vertebrates Drosophila melanogaster, another and even more important workhorse for developmental and genetic studies during the last century is amenable to all kinds of genetic manipulations. Drosophila was the second metazoan organism the entire genome of which has been sequenced (Adams et al., 2000). About 14,000 genes have been identified, most of them with homologues in man. A large number of mutant strains is available for research (i.e. The Bloomington Stock Center at the University of Indiana, http://flystocks.bio.indiana.edu/). Drosophila functions as a model for insect vectors that are responsible for the spread of major human diseases such as malaria. In the last years it became apparent that the outstanding features of this model system predestinate Drosophila to serve as a surrogate host in infection biology. Moreover, the fly has become one of the most important models for the study of the innate immune system (Hoffmann and Reichart, 2002). Drosophila is able to react to different kinds of infections caused by gram-positive or gramnegative bacteria, fungi or parasitic protozoa and metazoa. The most prominent reaction is the production and release of a large armory of antimicrobial peptides with different and complementary target cell spectra. In addition to the release of antimicrobial peptides, a melanization response can combat microbial intruders. Beside this humoral response, blood cells, more accurately the hemocytes, can phagocytose or encapsulate invaders (Hoffmann et al., 1999). Microbes and their cell wall constituents are recognized by so-called pattern recognition molecules (Hultmark, 2003). This event activates one out of two major signal transduction pathways, the Toll pathway and the imd pathway.

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This started almost 30 years ago in the laboratory of Hans Boman (Boman et al., 1972; Flyg et al., 1980). Currently, a Drosophila-Pseudomonas model and models for infections caused by the protozoa Plasmodium and Crithidia are available. The establishment of other Drosophila models for important human pathogens is currently in work and their detailed analysis should yield important information during the next years.

Pseudomonas infections of Drosophila

Fig. 2. Major immune-responsive organs of the fruit fly larva. The fruit fly can activate two different systems in response to an infection. Whereas the fat body and the hemocytes are responsible for the systemic response, the trachea and the intestinal organs (salivary glands, intestinal tract, malpighian tubules) can launch a local response restricted to the site of infection

Both signal transduction pathways are well-studied and have close homologues in mammals (Imler and Hoffmann, 2000; Tzou et al., 2002). A major advantage is the huge number of mutants available, which are defective in different aspects of the immune response. In addition new and sophisticated, RNAi-based technologies enable a quick and local silencing of any gene of interest, an invaluable tool not available in any other model organism (Kalidas and Smith, 2002). A very promising approach for the characterization and study of infections has recently been introduced into the field. It is based on visualization of the expression of antimicrobial peptides following infection. Visualization is possible using flies carrying a construct consisting of the promotor of one of these antimicrobial peptide genes and the green fluorescent protein (GFP). For all major antimicrobial peptides, these reporter lines are available. These allow visualization of the response of the flies to infections with different types of bacteria or fungi (Tzou et al., 2000). These Drosophila models might be of special interest for the study of bacterial infections at barrier epithelia and may substantially contribute to our understanding of basic aspects of the immune system (Fig. 2). In addition to its role as a model for the study of infections in general, several more specific Drosophila models were introduced in the last few years.

Recently, Drosophila was established as a model host for P. aeruginosa infections. As mentioned before, this bacterium shows a very broad hostspecificity covering organisms as diverse as plants, insects and mammals. Infection of Drosophila with P. aeruginosa kills the flies approximately 24 h post infection. In an elegant study, D'Argentio and colleagues (2001) used a number of different Pseudomonas strains to test their ability to infect and kill flies. They found an interesting correlation between twitching motility of bacteria and virulence. In particular, infection with those strains that have mutations in the pil and chp genes, potentially encoding proteins involved in signal transduction pathways, were found to result in delayed or abolished killing. Fauvarque and colleagues (2002) performed similar screens for Pseudomonas strains impaired in their ability to kill Drosophila. They found that the type III secretion system is of great importance for this feature of pathogenic Pseudomonas strains. Mutants derived from the clinical isolate P. aeruginosa CHA that are defective in parts of the type III secretion machinery show a reduced killing potential compared with the parental strain. Notwithstanding, Chugani and coworkers (2001) identified the quorum-sensing signal pathway of Pseudomonas as being important for the virulence towards Drosophila. Repression of the quorumsensing pathway observed in the qscR mutant leads to an augmented killing efficacy. Concordantly, quorum-sensing pathways are required for proper biofilm formation of the bacteria and impairments in this pathway resulted in an uncontrolled proliferation. Parasite infections of Drosophila Protozoan parasites are responsible for a variety of infectious diseases in man. Among them, several are of great medical importance. Drosophila has been shown to cope with bacterial and fungal infections, but only little is known regarding its ability to combat invaders of protozoan origin. Boulanger and

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colleagues (2001) have shown that Drosophila generates an immune response following infection with the flagellated parasite Crithidia. Crithidia species are known to be specific parasites of invertebrates, in which they live and replicate in the gut. Oral infection can provoke the systemic immune response carried out by the fat body but does not impair the survival of the flies. In contrast to this type of infection, injection into the body cavity can kill the flies rapidly depending on the isolate injected. Interestingly, a yet unidentified peptide with a molecular mass of 3 kDa is specifically synthesized in response to flagellate infections. Other flagellates (kinetoplastids) such as Trypanosoma and Leishmania were also found to induce an immune response opening the possibility to study host-pathogen interactions of these medically important parasites as well. Malaria, one of the most threatening infectious disease of man, kills over two million people a year and about half a billion people are infected. It represents a devastating public health menace. The causative agent of malaria tropica, Plasmodium falciparum, is transmitted from one human host to the other by a mosquito vector, Anopheles. To study the parasite-host interactions within the vector, the mosquitoes are not well-suited as their genetics and as their handling in the laboratory is far from being optimal. Recently, Schneider and Shahabuddin (2000) reported that a close relative of P. falciparum, P. gallinaceum, which causes malaria in birds, can develop in Drosophila. Infection via the normal route, i.e. by a blood meal from infected hosts, was not successful, presumably because the gametocytes are unable to cross midgut-associated barriers. A similar result was observed when ookinetes were fed to the flies. However, injection of ookinetes into the hemolymph triggered normal development in the fly up to the early sporozoites. Appearance of the different stages in the fly shows a similar time course as in mosquitoes. Notably, the sporozoites were not able to enter the salivary glands of the fly. Importantly, sporozoites have to mature in these organs to become highly infective. Accordingly, infection of chicken with Drosophila-derived sporozoites was possible but with a much lower efficacy when compared with sporozoites that had developed in mosquitoes. It should be mentioned that the cellular immune system of Drosophila is able to attack the plasmodia and to clear most of them from the hemolymph. Although only a small portion of the plasmodia life cycle can be recapitulated in the fly, the availability of numerous mutant Drosophila strains, defective in the expression of genes involved in the immune response of the fly, enable researchers

to study in detail at least the concomitant hostparasite interactions within a small window of development of this most important human parasite.

Dictyostelium discoideum, a unicellular surrogate host Amoeboid organisms represent a major part of the protozoan world and are phylogenetically diverse. They are primitive, actively phagocytosing eukaryotic cells, many of which use bacteria as a major nutrient source. Amoebae exert mechanisms which may reflect recognition, internalization and destruction of foreign material at a very archaic level and may be comparable to those found in phagocytic cells of higher organisms (Leippe, 1999). Moreover, amoeboid cells are involved in the uptake and intracellular digestion of foreign material in many invertebrates and, primitively, the mechanism of phagocytosis represents the engulfment of food particles by such cells. The idea that amoeboid cells and their inherent phagocytic activity has been retained during evolution of animals as an internal defensive systems was already proposed by Metchnikoff. One may suppose that amoebae possess an array of potent antimicrobial molecules acting in synergy to combat bacterial growth inside their phagosomes (Bruhn and Leippe, 2001). Bacteria that are pathogenic to amoebae and man, e.g. Legionella pneumophila, an important bacterial pathogen that resides in fresh water amoebae and human macrophages, must circumvent the molecular antimicrobial arsenal of both types of phagocytic cells. The lower eukaryote Dictyostelium discoideum provides a unique experimental system for studying cell-type determination, signal transduction, spatial patterning, cytoskeleton dynamics and cellular infection processes. The so-called social amoeba lives on deciduous forest soil and feeds on bacteria and yeast. In the laboratory, Dictyostelium can be grown in axenic liquid medium. The cells which have a doubling time of eight hours in liquid culture divide by binary fission. Upon starvation, Dictyostelium cells aggregate upon chemotaxis in response to relayed cAMP signals. Approximately 105 cells form a multicellular organism that in its slug form can migrate towards light and orient in a thermotactic gradient. The differentiation process culminates in the production of a fruiting body consisting of a rigid stalk and spore cells. The spores enable Dictyostelium to survive unfavourable conditions. The developmental transition allows the analysis of signal

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transduction pathways, several of which are highly conserved in metazoa (Zada-Hames and Ashworth, 1978; Loomis, 1996; Noegel and Schleicher, 2000). The phylogenetic position of Dictyostelium is still a matter of debate. It has been suggested that its evolutionary path separated before the branching of metazoa and fungi but after that of the plant kingdom (Baldauf et al., 2000). The 34-Mb genome of Dictyostelium shares greater similarity with metazoan than with fungal genomes. The six chromosomes are expected to carry 11,000 genes (Glˆckner et al., 2002). It is remarkable that the Dictyostelium genome is approximately seven times larger than the genome of E. coli (4.6 Mb) and more than 90 times smaller than the one of man (3.15 Gb). To provide the basis for genome-wide investigations an international Dictoystelium genome project was initiated (http://www.uni-koeln.de/dictyostelium/) and is expected to be completed by the year 2003. The experimental versatility of Dictyostelium allows to analyze the molecular basis of many fundamental cellular processes. The isolation of mutants is facilitated by the fact that Dictyostelium is haploid. Moreover, there is a growing number of experimental tools that can be applied to this organism. Among them are gene inactivation and gene replacement by homologous recombination, inhibition of synthesis of gene products by antisense RNA, restriction-enzyme-mediated integration (REMI), transposon-tagging-like mutagenesis, and expression of GFP-fusion proteins (Noegel and Schleicher, 2000). Dictyostelium can internalize a wide range of particles including latex beads, yeast, food bacteria, and different pathogenic bacteria. However, one disadvantage is that Dictyostelium does not survive temperatures above 27 8C. For some pathogens which express their virulence factors at higher temperature this limitation may be critical (Solomon and Isberg, 2000). The uptake mechanisms of particles in Dictyostelium are very similar to phagocytosis in macrophages or neutrophils. In both cases, particle adhesion to the cell surface induces local polymerization of the actin cytoskeleton, pseudopodial extension, formation of a phagocytic cup and internalization of the particle into the phagosome. As it has been observed that certain bacterial pathogens such as Salmonella invade epithelial cells via macropinocytosis and that dendritic cells macropinocytose constitutively, research in this area has recently been intensified. Due to the ease of genetic manipulation Dictyostelium already contributed significantly to our current understanding regarding the regulation of macropinocytosis. The similarities between Dictyostelium and mammalian cells are also apparent in membrane traffick-

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ing, endocytic transit and sorting events (Maniak, 1999; Solomon and Isberg, 2000; Cardelli, 2001). Collectively, the availability of cellular markers, the knowledge of cell signaling pathways, the progress of the genome sequencing project and the potential for genetic studies suggests D. discoideum as a valuable model system for studying the intracellular pathogenesis of infectious agents.

Legionella infection of Dictyostelium Legionella pneumophila is the etiological agent of a severe life-threatening pneumonia in humans named Legionnaires' disease. In their aquatic environment, the gram-negative bacteria replicate intracellularly within free-living protozoa. During human infection Legionella invades into and grows within human alveolar macrophages (Abu Kwaik, 1998; Steinert et al., 2002b). Interestingly, Legionella utilizes similar mechanisms to infect protozoa and human macrophages. Multiplication of Legionella results in a host cell that is filled with bacteria. Nutrient depletion in the host cell results in a phenotypic

Fig. 3. Transmission electron micrograph of Legionella pneumophila within a single vacuole of Dictyostelium discoideum after 48 h of co-incubation. Bar ˆ 1 mm.

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change of Legionella from non-motile, dividing bacteria to highly motile and infectious bacteria. In order to establish Dictyostelium as an infection model for Legionella, we and others analyzed intracellular growth and subcellular localization of different Legionella strains and mutants (H‰gele et al., 2000; Solomon et al., 2000). The infection studies revealed that virulent Legionella species grow intracellularly in axenically grown D. discoideum cells (Fig. 3). Whereas amoebae in suspension were relatively resistant, amoebae grown as adherent monolayers were susceptible to Legionella infection. Infection of amoebae results in their lysis. Dictyostelium and infected macrophages both harbour replicating legionellae within organelle-studded vesicles that are associated with rough endoplasmic reticulum as demonstrated by transmission electron microscopy. Co-localization studies with GFP-tagged bacteria and antibodies directed against specific lysosomal markers (DdLIMP) indicated that the bacteria inhibit the phagosome-lysosome fusion (H‰gele et al., 2000). These data are in good agreement with observations made with human macrophages and natural amoebic hosts. Further support for Dictyostelium as a representative host model system for Legionella infections comes from the testing of various well-established Legionella mutants. Experiments with the FlaA-, the Mip-, the ligA-, FliA-negative mutants as well as many mutants of the dot/icm genes gave similar results in D. discoideum, other amoebae and macrophages (Solomon et al., 2000; Heuner et al., 2002; Steinert et al., 2002a). Infection of Dictyostelium by other pathogens To evaluate whether D. discoideum can be employed as a general infection model representative bacterial species which employ different pathogenic strategies were tested. Among these is the facultative intracellular pathogen of professional phagocytes Mycobacterium avium which is one of the primary health threats to patients with AIDS. Additionally, S. typhimurium which is an intracellular pathogen of non-phagocytic intestinal epithelial cells has been tested. Moreover, the extracellular pathogen P. aeruginosa which is known to infect many evolutionary diverse hosts including plants, insects, acanthamoebae and the infection model C. elegans (see above) has been examined. Invasion assays performed with these bacterial species demonstrated that M. avium is able to multiply within Dictyostelium. S. typhimurium also invaded Dictyostelium but was degraded shortly after uptake. Accompanying electron and fluo-

rescence microscopy of infected Dictyostelium cells revealed that M. avium replicates within vacuoles. Light microscopy during the course of infection showed that multiplication of M. avium within Dictyostelium results in host cell lysis (Skriwan et al., 2002). Co-culture assays on agar plates demonstrated inhibition of Dictyostelium growth by P. aeruginosa. Isogenic mutants of Pseudomonas deficient in the las quorum-sensing system were almost as inhibitory as the wild type, while the rhl quorum-sensing mutants permitted growth of Dictyostelium cells. In particular, rhamnolipids were shown to induce lysis of the amoebae. Moreover, the reduced virulence of antibiotic-resistant mutants of P. aeruginosa revealed corresponding results in the rat model of acute pneumonia and the Dictyostelium model (Cosson et al., 2002). Infections of Dictyostelium with obligate intracellular endocytobionts Pathogenic and symbiotic microorganisms share many common strategies and, in some cases even a common evolutionary history (Hentschel et al., 2000; Steinert et al., 2000). In order to analyze pathogenic and symbiotic interactions in the same host organism, Dictyostelium was co-cultured with obligate intracellular endosymbionts of Acanthamoeba spp. as well. The application of fluorescent in situ hybridization (FISH) revealed that the nonculturable strains Neochlamydia sp. TUME1, Parachlamydia sp. UWE25 and the b-proteobacterium UWC6 grow intracellularly within Dictyostelium (Skriwan et al., 2002). In accordance with previous observations in Acanthamoeba, the endosymbionts TUME1 and UWE25 were tightly enclosed by membraneous structures. For the b-proteobacterium UWC6, a transient residence within vacuoles and a persistent residence within the cytoplasm were detected. Interestingly, the strains TUME1 and UWC6 developed a stable intracellular stage after two days and the infection did not result in host cell lysis. Instead the host cells harbouring these endocytobionts were able to continue growth. Co-infection studies with these endocytobionts and L. pneumophila suggest that the endocytobionts may protect the host amoeba from the attack by pathogenic Legionella. Host cell factors of Dictyostelium relevant for infection An important reason for the development of the Dictyostelium infection model was the possibility to

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systematically analyze infection-relevant host cell factors. Especially cellular factors involved in entry, replication and release of the bacteria can be examined using this model. For this purpose, specific cellular inhibitors and Dictyostelium mutants were applied in phagocytosis and infection assays. These studies demonstrated that tyrosine kinase activity, Factin and the membrane-associated protein villidin, and the action of calreticulin, a protein from the lumen of the endoplasmic reticulum, appear to be involved in Legionella uptake (Fajardo et al., unpublished data). Moreover, a Dictyostelium strain carrying an insertion in Gb, a subunit of trimeric G proteins, was shown to be slightly reduced in growth compared to the parental strain. In contrast, the myoA/B double myosin I mutant, the coronin mutant and the profilin mutant were more permissive for intracellular growth of Legionella (Solomon et al., 2000). The null-mutants of the actin-binding protein comitin, which is located on vesicle and Golgi membranes in Dictyostelium and mammalian cells, support the intracellular growth of bacteria suggesting that comitin-dependent processes may be involved in the host cell defense system (Schreiner et al., 2002). Additional support for this hypothesis comes from the observation that S. typhimurium transiently survives within comitin-minus cells but not within the wild-type strain of Dictyostelium (Skriwan et al., 2002). The inability of S. typhimurium to infect wild-type Dictyostelium cells corresponds with the inability of comparable strains to survive within human macrophages (Vladoianu et al., 1990). The natural resistance-associated macrophage protein 1 (Nramp1) is a major determinant of natural resistance to intracellular infections in mice and man (Bellamy, 1999). The Dictyostelium Nramp1, which was cloned recently encodes a highly homologous protein of 53 kDa with 11 putative transmembrane domains. Cells transfected with an Nramp1 antisense gene construct displayed a reduced phagocytosis rate for E. coli, L. pneumophila and M. avium. Even more interesting is that the inhibition of Nramp1 synthesis results in a host cell phenotype that is more permissive for intracellular growth of L. pneumophila and M. avium (Perachino et al., unpublished data). These results implicate that Dictyostelium may be used complementary to macrophages to elucidate the function of Nramp1.

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Conclusions Nowadays it is generally accepted that the ability of an organism to protect itself against potential pathogens and to combat infection is not a privilege of vertebrates. On the contrary, highly effective defense mechanisms and many potent effector molecules that kill prokaryotic and/or eukaryotic invaders have been found in invertebrates. The evaluation of pathogenicity and virulence factors of a broad variety of microorganisms using either surrogate or vertebrate hosts revealed astonishingly similar results. None of the surrogate hosts described here possesses an adaptive immune system, but basic aspects of innate immunity including recognition, adherence and signal transduction are remarkably conserved. The largest body of evidence concerning the immune system of non-vertebrate hosts has certainly been obtained for Drosophila: A plethora of genes were detected that code for antimicrobial peptides, many of which have been discovered originally in other insect species. The signalling pathways leading to the expression of the effector molecules that eventually kill invading microorganisms were discovered in Drosophila prior to the identification of homologous systems in mammals. For C. elegans, the knowledge about the constituents of an immune system is scarce. Although some genes putatively coding for antimicrobial peptides and for lysozyme-like proteins have been identified, one has to admit that it has not been proven unequivocally that the soil nematode possesses an internal defensive system at all. Nevertheless, the enormous number of mutants available compared to mice predestinates C. elegans as a surrogate host. Traditionally, defensive systems are restricted to metazoa. However, already the classical observations of Metchnikoff suggested that amoeboid cells are main players in cellular immunity of animals and that free-living amoebae may be the progenitors of professional phagocytes. As a consequence, the ancient principle of killing microbes by more or less specific proteins most likely has evolved before the advent of metazoa, and amoebae may be a rich source of such antimicrobial molecules. Much can be learned from the Dictyostelium system as far as the fight between phagocytic cells and intracellular pathogens is concerned, and particular mutants may unravel steps that are critical for the outcome of the encounter, i.e. survival of host cell or pathogen. All three surrogate hosts allow high-throughput studies either for the identification of bacterial virulence factors or host factors involved in its response to the infection. Although each of the

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systems has its specific limitations, a large number of different pathogenic microorganisms can be studied in any one of those systems. The cellular analysis of how bacteria colonize their hosts, facilitate the breakdown of dietary macromolecules, stimulate the expression of host genes and shape the development of the host presumably will have important implications for medical treatments. Conclusively, surrogate hosts such as C. elegans, Drosophila and Dictyostelium and the molecular technology adapted to them may considerably facilitate research in the growing field of infection biology. Acknowledgements. The work was supported by the Deutsche Forschungsgemeinschaft (LE 1075/2-3, RO 1241/2-3, STE 838/3-2).

References Aballay, A., Ausubel, F. M.: Programmed cell death mediated by ced-3 and ced-4 protects Caenorhabditis elegans from Salmonella typhimurium-mediated killing. Proc. Natl. Acad. Sci. USA 98, 2735 ± 2739 (2001). Aballay, A., Drenkard, E., Hilbun, L. R., Ausubel, F. M.: Caenorhabditis elegans innate immune response triggered by Salmonella enterica requires intact LPS and is mediated by a MAPK signaling pathway. Curr. Biol. 13, 47 ± 52 (2003). Abu Kwaik, Y.: Fatal attraction of mammalian cells to Legionella pneumophila. Mol. Microbiol. 30, 689 ± 695 (1998). Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A., Gocayne, J. D., Amanatides, P. G., Scherer, S. E., Li, P. W., Hoskins, R. A., Galle, R. F., George, R. A., Lewis, S. E., Richards, S., Ashburner, M., Henderson, S. N., Sutton, G. G., Wortman, J. R., Yandell, M. D., Zhang, Q., Chen, L. X., Brandon, R. C., Rogers, Y. H., Blazej, R. G., Champe, M., Pfeiffer, B. D., Wan, K. H., Doyle, C., Baxter, E. G., Helt, G., Nelson, C. R., Gabor, G. L., Abril, J. F., Agbayani, A, An, H. J., Andrews-Pfannkoch, C., Baldwin, D., Ballew, R. M., Basu, A., Baxendale, J., Bayraktaroglu, L., Beasley, E. M., Beeson, K. Y., Benos, P. V., Berman, B. P., Bhandari, D., Bolshakov, S., Borkova, D., Botchan, M. R., Bouck, J., Brokstein, P., Brottier, P., Burtis, K. C., Busam, D. A., Butler, H., Cadieu, E., Center, A., Chandra, I., Cherry, J. M., Cawley, S., Dahlke, C., Davenport, L. B., Davies, P., de Pablos, B., Delcher, A., Deng, Z., Mays, A. D., Dew, I., Dietz, S. M., Dodson, K., Doup, L. E., Downes, M., DuganRocha, S., Dunkov, B. C., Dunn, P., Durbin, K. J., Evangelista, C. C., Ferraz, C., Ferriera, S., Fleischmann, W., Fosler, C., Gabrielian, A. E., Garg, N. S., Gelbart, W. M., Glasser, K., Glodek, A., Gong, F., Gorrell, J. H., Gu, Z., Guan, P., Harris, M., Harris, N. L., Harvey, D., Heiman, T. J., Hernandez, J. R., Houck, J., Hostin, D., Houston, K. A., Howland,

T. J., Wei, M. H., Ibegwam, C., Jalali, M., Kalush, F., Karpen, G. H., Ke, Z., Kennison, J. A., Ketchum, K. A., Kimmel, B. E., Kodira, C. D., Kraft, C., Kravitz, S., Kulp, D., Lai, Z., Lasko, P., Lei, Y., Levitsky, A. A., Li, J., Li, Z., Liang, Y., Lin, X., Liu, X., Mattei, B., McIntosh, T. C., McLeod, M. P., McPherson, D., Merkulov, G., Milshina, N. V., Mobarry, C., Morris, J., Moshrefi, A., Mount, S. M., Moy, M., Murphy, B., Murphy, L., Muzny, D. M., Nelson, D. L., Nelson, D. R., Nelson, K. A., Nixon, K., Nusskern, D. R., Pacleb, J. M., Palazzolo, M., Pittman, G. S., Pan, S., Pollard, J., Puri, V., Reese, M. G., Reinert, K., Remington, K., Saunders, R. D., Scheeler, F., Shen, H., Shue, B. C., Siden-Kiamos, I., Simpson, M., Skupski, M. P., Smith, T., Spier, E., Spradling, A. C., Stapleton, M,. Strong, R., Sun, E., Svirskas, R., Tector, C., Turner, R., Venter, E., Wang, A. H., Wang, X., Wang, Z. Y., Wassarman, D. A., Weinstock, G. M., Weissenbach, J., Williams, S. M., Woodage,T,. Worley, K. C., Wu, D., Yang, S., Yao, Q. A., Ye, J., Yeh, R. F., Zaveri, J. S., Zhan, M., Zhang, G., Zhao, Q., Zheng, L., Zheng, X. H., Zhong, F. N., Zhong, W., Zhou, X., Zhu, S., Zhu, X., Smith, H. O., Gibbs, R. A., Myers, E. W., Rubin, G. M., Venter, J. C.: The genome sequence of Drosophila melanogaster. Science 287, 2185 ± 2195 (2000). Alegado, R. A., Campbell, M. C., Chen, W. C., Slutz, S. S., Tan, M. W.: Characterization of mediators of microbial virulence and innate immunity using the Caenorhabditis elegans host-pathogen model. Cell. Microbiol. 5, 435 ± 444 (2003). Ashrafi, K., Chang, F. Y., Watts, J. L., Fraser, A. G., Kamath, R. S., Ahringer, J., Ruvkun, G.: Genomewide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421, 268 ± 272 (2003) Baldauf, S. L., Roger, A. J., Wenk-Siefert, I., Doolittle, W. F. A.: A kingdom-level phylogeny of eukaryotes based on combined protein data. Science 290, 972 ± 977 (2000). Bellamy, R.: The natural resistance-associated macrophage protein and susceptibility to intracellular pathogens. Microbes Infect. 1, 23 ± 27 (1999). Boman, H. G., Nilsson, I., Rasmuson, B.: Inducible antibacterial defence system in Drosophila. Nature 237, 232 ± 235 (1972). Borgonie, G., Van Driessche, R., Leyns, F., Arnaut, G., De Waele, D., Coomans, A.: Germination of Bacillus thuringiensis spores in bacteriophagous nematodes (Nematoda: Rhabditida). J. Invertebr. Pathol. 65, 61 ± 67 (1995). Boulanger, N., Ehret-Sabatier, L., Brun, R., Zachary, D., Bulet, P., Imler, J. L.: Immune response of Drosophila melanogaster to infection with the flagellate parasite Crithidia spp. Insect Biochem. Mol. Biol. 31, 129 ± 137 (2001). Brenner, S.: The genetics of Caenorhabditis elegans. Genetics 77, 71 ± 94 (1974). Bruhn, H., Leippe, M.: Membrane-permeabilizing polypeptides of amoebae ± constituents of an archaic animicrobial system. Zoology 104, 3 ± 11 (2001).

Surrogate hosts Cardelli, J.: Phagocytosis and macropinocytosis in Dictyostelium: phosphoinositide-based processes, biochemically distinct. Traffic 2, 311 ± 320 (2001). Chugani, S. A., Whiteley, M., Lee, K. M., D'Argenio, D., Manoil, C., Greenberg, E. P.: QscR, a modulator of quorum-sensing signal synthesis and virulence in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 98, 2752 ± 2757 (2001) Cosson, P., Zulianello, L., Join-Lambert, O., Faurisson, F., Gebbie, L., Benghezal, M., van Delden, C., Curty, L. K., Kˆhler, T.: Pseudomonas aeruginosa virulence analyzed in a Dictyostelium discoideum host system. J. Bacteriol. 184, 3027 ± 3033 (2002). Darby, C., Cosma, C. L., Thomas, J. H., Manoil, C.: Lethal paralysis of Caenorhabditis elegans by Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 96, 15202 ± 15207 (1999) Darby, C., Hsu, J. W., Ghori, N., Falkow, S.: Caenorhabditis elegans: plague bacteria biofilm blocks food intake. Nature 417, 243 ± 244 (2002). D'Argenio, D. A., Gallagher, L. A., Berg, C. A., Manoil, C.: Drosophila as a model host for Pseudomonas aeruginosa infection. J. Bacteriol. 183, 1466 ± 1471 (2001) Depraitere, C., Darmon, M.: Growth of ™Dictyostelium discoideum∫ on different species of bacteria. Ann. Microbiol. B 129, 451 ± 461 (1978) Ewbank J. J.: Tackling both sides of the host-pathogen equation with Caenorhabditis elegans. Microbes Infect. 4, 247 ± 256 (2002) Fauvarque, M. O., Bergeret, E., Chabert, J., Dacheux, D., Satre, M., Attree, I.: Role and activation of type III secretion system genes in Pseudomonas aeruginosainduced Drosophila killing. Microb. Pathog. 32, 287 ± 295 (2002) Flyg, C., Kenne, K., Boman, H. G.: Insect pathogenic properties of Serratia marcescens: phage-resistant mutants with a decreased resistance to Cecropia immunity and a decreased virulence to Drosophila. J. Gen. Microbiol. 120, 173 ± 181 (1980) Garsin, D. A., Sifri, C. D., Mylonakis, E., Qin, X., Singh, K. V., Murray, B. E., Calderwood, S. B., Ausubel, F. M.: A simple model host for identifying Grampositive virulence factors. Proc. Natl. Acad. Sci. USA 98, 10892 ± 10897 (2001) Glˆckner, G., Eichinger, L. Szafranski, K., Pachebat, J. A., Bankier, A. T., Dear, P. H., Lehmann, R., Baumgart, C., Parra, G., Abril, J. F., Guigo, R., Kumpf, K., Tunggal, B., Cox, E., Quail, M. A., Platzer, M., Rosenthal, A., Noegel, A. A.; Dictyostelium Genome Sequencing Consortium: Sequence and analysis of chromosome 2 of Dictyostelium discoideum. Nature 418, 79 ± 85 (2002) H‰gele, S., Kˆhler, R., Merkert, H., Schleicher, M., Hacker, J., Steinert, M.: Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella. Cell. Microbiol. 2, 165 ± 171 (2000).

331

Hentschel, U., Steinert, M., Hacker, J.: Common molecular mechanisms of symbiosis and pathogenesis. Trends Microbiol. 8, 226 ± 231 (2000). Heuner, K., Dietrich, C., Skriwan, C., Steinert, M., Hacker, J.: Influence of the alternative sigma(28) factor on virulence and flagellum expression of Legionella pneumophila. Infect. Immun. 70, 1604 ± 1608 (2002) Hodgkin, J., Kuwabara, P. E., Corneliussen, B.: A novel bacterial pathogen, Microbacterium nematophilum, induces morphological change in the nematode C. elegans. Curr. Biol. 10, 1615 ± 1618 (2000) Hoffmann, J. A., Kafatos, F. C., Janeway, C. A., Ezekowitz, R. A.: Phylogenetic perspectives in innate immunity. Science 284, 1313 ± 1318 (1999). Hoffmann, J. A., Reichart, J.-M.: Drosophila innate immunity: an evolutionary perspective. Nature Immunol. 3, 121 ± 126 (2002) Hultmark, D.: Drosophila immunity: paths and patterns. Curr. Opin. Immunol. 15, 12 ± 19 (2003) Imler, J.-L., Hoffmann, J. A.: Signaling mechanisms in the antimicrobial host defense of Drosophila. Curr. Opin. Microbiol. 3, 16 ± 22 (2000) Kalidas, S., Smith, D. P.: Novel genomic cDNA hybrids produce effective RNA interference in adult Drosophila. Neuron 33, 177 ± 184 (2002) Kim, D. H., Feinbaum, R., Alloing, G., Emerson, F. E., Garsin, D. A., Inoue, H., Tanaka-Hino, M., Hisamoto, N., Matsumoto, K., Tan, M. W., Ausubel, F. M.: A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity. Science 297, 623 ± 626 (2002) Kurz, C. L., Ewbank, J. J.: Caenorhabditis elegans for the study of host-pathogen interactions. Trends Microbiol. 8, 142 ± 144 (2000) Labrousse, A., Chauvet, S., Couillault C., Kurz, C. L., Ewbank, J. L.: Caenorhabditis elegans is a model host for Salmonella typhimurium. Curr. Biol. 10, 1543 ± 1545 (2000) Leippe, M.: Antimicrobial and cytolytic polypeptides of amoeboid protozoa ± effector molecules of primitive phagocytes. Dev. Comp. Immunol. 23, 267 ± 279 (1999) Loomis, W. F.: Genetic networks that regulate development in Dictyostelium cells. Microbiol. Rev. 60, 135 ± 150 (1996). Mahajan-Miklos, S., Tan, M. W., Rahme, L. G., Ausubel, F. M.: Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosaCaenorhabditis elegans pathogenesis model. Cell 96, 47 ± 56 (1999) Maniak, M.: Endocytic transit in Dictyostelium discoideum. Protoplasma 210, 25 ± 30 (1999). Mylonakis, E., Ausubel, F. M., Perfect, J. R., Heitman, J., Calderwood, S. B.: Inducible antibacterial defense system in C. elegans. Proc. Natl. Acad. Sci. USA 99, 15675 ± 15680 (2002) Noegel, A. A., Schleicher, M.: The actin cytoskeleton of Dictyostelium: a story told by mutants. J. Cell Sci. 113, 759 ± 766 (2000).

332

M. Steinert et al.

O'Quinn, A. L., Wiegand, E. M., Jeddeloh, J.: Burkholderia pseudomallei kills the nematode Caenorhabditis elegans using an endotoxin-mediated paralysis. Cell. Microbiol. 3, 381 ± 394 (2001) Schneider, D., Shahabuddin, M.: Malaria parasite development in a Drosophila model. Science 288, 2376 ± 2379 (2000). Schreiner, T., Mohrs, M. R., Blau-Wasser, R., von Krempelhuber, A., Steinert, M., Schleicher, M., Noegel, A. A.: Loss of the F-actin binding and vesicleassociated protein comitin leads to a phagocytosis defect. Euk. Cell 1, 906 ± 914 (2002) Sifri, C. D., Mylonakis, E., Singh, K. V., Qin, X., Garsin, D. A., Murray, B. E., Ausubel, F. M., Calderwood, S. B.: Virulence effect of Enterococcus faecalis protease genes and the quorum-sensing locus fsr in Caenorhabditis elegans and mice. Infect Immun. 70, 5647 ± 5650 (2002) Skriwan, C., Fajardo, M., H‰gele, S., Horn, M., Wagener, M., Michel, R., Krohne, G., Schleicher, M., Hacker, J., Steinert, M.: Various bacterial pathogens and symbionts infect the amoeba Dictyostelium discoideum. Int. J. Med. Micobiol. 291, 615 ± 624 (2002) Solomon, J. M., Isberg, R. R.: Growth of Legionella pneumophila in Dictyostelium discoideum: a novel system for genetic analysis of host-pathogen interactions. Trends Microbiol. 10, 478 ± 480 (2000) Solomon, J. M., Ruper, A., Cardelli, J. A., Isberg, R. R.: Intracellular growth of Legionella pneumophila in Dictyostelium discoideum, a system for genetic analysis of host-pathogen interactions. Infect. Immun. 68, 2939 ± 2947 (2000) Steinert, M., Hentschel, U., Hacker, J.: Symbiosis and pathogenesis: evolution of the microbe-host interaction. Naturwissenschaften 87, 1 ± 11 (2000) Steinert, M., H‰gele, S., Skriwan, C., Grimm, D., Fajardo, M., Heuner, K., Schleicher, M., Hentschel, U., Ludwig, W., Marre, R., Hacker, J.: Interaction of Legionella pneumophila with Dictyostelium discoideum. In: Legionella ± 5th International Conference on Legionella (R. Marre, Y. Abu Kwaik, C. Barlett,

N. P. Cianciotto, B. S. Fields, M. Frosch, J. Hacker, P. C. L¸ck, eds.), pp. 161 ± 164. ASM-Press, Washington 2002a Steinert, M., Hentschel, U., Hacker, J.: Legionella pneumophila: an aquatic microbe goes astray. FEMS Microbiol. Rev. 743, 149 ± 162 (2002b) Tan, M. W., Mahajan-Miklos, S., Ausubel, F. M.: Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis. Proc. Natl. Acad. Sci. USA 96 , 715 ± 720 (1999) Tan, M. W., Rahme, L. G., Sternberg, J. A., Tompkins, R. G., Ausubel, F. M.: Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors. Proc. Natl. Acad. Sci. USA 96, 2408 ± 2413 (1999) The Caenorhabditis elegans sequencing consortium: Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282, 2012 ± 2018 (1998) Tzou, P., De Gregorio, E., Lemaitre, B.: How Drosophila combats microbial infection: a model to study innate immunity and host-pathogen interactions. Curr. Opin. Microbiol. 5, 102 ± 110 (2002) Tzou, P., Ohresser, S., Ferrandon, D., Capovilla, M., Reichhart, J. M., Lemaitre, B., Hoffmann, J. A., Imler J. L.: Tissue-specific inducible expression of antimicrobial peptide genes in Drosophila surface epithelia. Immunity 13, 737 ± 748 (2000) Vladoianu, I.-R., Chang, H. R., Pechere, J. C.: Expression of host resistance to Salmonella typhi and Salmonella typhimurium: bacterial survival within macrophages of murine and human origin. Microb. Pathog. 8, 83 ± 90 (1990). Yorgey, P., Rahme, L. G., Tan, M. W., Ausubel, F. M.: The roles of mucD and alginate in the virulence of Pseudomonas aeruginosa in plants, nematodes and mice. Mol. Microbiol. 41, 1063 ± 1076 (2001) Zada-Hames, I. M., Ashworth, J. M.: The cell cycle during the vegetative stage of Dictyostelium discoideum and its response to temperature change. J. Cell Sci. 32, 1 ± 20 (1978).