Environmental predators as models for bacterial pathogenesis

Environmental Microbiology (2007) 9(3), 563–575

doi:10.1111/j.1462-2920.2007.01238.x

Minireview Environmental predators as models for bacterial pathogenesis Hubert Hilbi,* Stefan S. Weber, Curdin Ragaz, Yves Nyfeler and Simon Urwyler Institute of Microbiology, ETH Zürich, Wolfgang-Pauli Strasse 10, 8093 Zürich, Switzerland. Summary Environmental bacteria are constantly threatened by bacterivorous predators such as free-living protozoa and nematodes. In the course of their coevolution with environmental predators, some bacteria developed sophisticated defence mechanisms, including the secretion of toxins, or the capacity to avoid lysosomal killing and to replicate intracellularly within protozoa. To analyse the interactions with bacterial pathogens on a molecular, cellular or organismic level, protozoa and other non-mammalian hosts are increasingly used. These include amoebae, as well as genetically tractable hosts, such as the social amoeba Dictyostelium discoideum, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Using these hosts, the virulence mechanisms of opportunistic pathogenic bacteria such as Legionella, Mycobacterium, Pseudomonas or Vibrio were found to be not only relevant for the interactions of the bacteria with protozoa, nematodes and insect phagocytes, but also with mammalian hosts including humans. Thus, non-mammalian model hosts provide valuable insight into the pathogenesis of environmental bacteria. Introduction Bacteria live in harsh environments, characterized by a constant competition for nutrients and the menace of protozoan and metazoan predators. These evolutionary pressures shaped complex bacterial defence strategies and the necessity to establish new replicative niches. To protect themselves from predators, some bacteria form inedible filaments or produce biofilms thus preventing Received 24 October, 2006; accepted 13 December, 2006. *For correspondence. E-mail [email protected]; Tel. (+41) 44 632 4782; Fax (+41) 44 632 1137.

engulfment and phagocytosis, develop mechanisms to survive microbicidal activities, or replicate within and kill protozoa (Matz and Kjelleberg, 2005). Protozoa are primordial phagocytes, which share many features with mammalian phagocytes, particularly macrophages. By fine-tuning their interactions with protozoa, bacteria might become also resistant to bactericidal mammalian macrophages and thereby cause disease in humans (Fig. 1). Environmental protozoa not only select for virulence traits that allow intracellular growth within macrophages, but also serve as protective reservoir and, in the form of intact amoebae or expelled vesicles, facilitate the transmission of infectious agents to humans (Rowbotham, 1980; Berk et al., 1998; reviewed by Brown and Barker, 1999; Greub and Raoult, 2004; Molmeret et al., 2005). Over the last few years, non-mammalian hosts have gained increasing attention as models that harness bacterial adaptation to protozoa and nematodes for molecular pathogenesis studies. Advantages of non-mammalian hosts include their ease of cultivation and short generation times, as well as the availability of elaborate genetic, biochemical and cell biological tools. The hosts most frequently used are fresh water amoebae such as Acanthamoeba castellanii or Hartmannella vermiformis (Brown and Barker, 1999; Greub and Raoult, 2004; Molmeret et al., 2005), the social soil amoeba Dictyostelium discoideum (Solomon and Isberg, 2000; Steinert and Heuner, 2005), the nematode Caenorhabditis elegans (Sifri et al., 2005), and the fruit fly Drosophila melanogaster (Vodovar et al., 2004). Conveniently, amoebae and nematodes are genuine bacterivores, and therefore, naturally phagocytose and feed on pathogenic bacteria. In this review we focus on the use of these hosts to analyse the virulence of well-studied environmental opportunistic pathogens of the genera Legionella, Mycobacterium, Pseudomonas and Vibrio. In many cases, the bacterial factors required for virulence in non-mammalian hosts also play a role in mammalian systems, thus validating the use of these models to study pathogenesis in humans. Bacterial key virulence determinants can be grouped into gene regulation systems, such as alternative sigma factors,

© 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd

564 H. Hilbi et al.

Fig. 1. Environmental niches of pathogenic bacteria and infection of macrophages. Pathogenic bacteria 1 infect and replicate within amoebae and other protozoa, 2 colonize surfaces and grow in biofilms, 3 infect and kill nematodes, and 4 are released from their replicative niches. 5 After transmission, the pathogens infect macrophages of the innate immune system of metazoan organisms. Growth within amoebae affects the physiology and the virulence of pathogenic bacteria and may be a prerequisite to infect macrophages. A given pathogenic bacterium uses specific, conserved strategies to infect and kill various evolutionary distant eukaryotic hosts, including protozoa, nematodes, insects and mammals.

two-component systems or quorum-sensing systems, and ‘effectors’, which determine the interactions with nonmammalian as well as mammalian cells (Table 1). Crucial virulence determinants of Gram-negative pathogens are the type IV secretion systems (T4SS, Legionella), type III secretion systems (T3SS, Pseudomonas), or the recently discovered type VI secretion systems (T6SS, Vibrio). An overview on bacterial and host cell determinants relevant for pathogen–host interactions is given in Table 2. Interactions of opportunistic bacterial pathogens with environmental protozoa Many Gram-negative or Gram-positive bacteria, including the genera Legionella, Mycobacterium, Pseudomonas and Vibrio survive their encounter with free-living protozoa and establish an endosymbiontic or a parasitic relationship with these hosts (Brown and Barker, 1999; Greub Table 1. Functional categories of Legionella and Pseudomonas virulence factors relevant for interactions with non-mammalian hosts.

Gene regulation Adhesion Uptake Replication Toxicity

Legionella

Pseudomonas

RpoS, FliA, LetAS Type IV pili Icm/Dot T4SS, Mip Icm/Dot T4SS Icm/Dot T4SS Lipid A (LpxB)

RpoN, GacAS, Las, Rhl Type IV pili

T3SS, ExoU, Exotoxin A Phenazine pigments, cyanide

and Raoult, 2004; Molmeret et al., 2005). The facultative intracellular bacterium Legionella pneumophila is the causative agent of a severe pneumonia termed Legionnaires’ disease. Approximately one-half of the 50 species comprising the genus Legionella have been associated with human disease, yet in most countries L. pneumophila causes about 90% of all reported cases (Fields et al., 2002). Mycobacterium marinum causes tuberculosis-like infections in fish and amphibians, as well as skin infections such as swimming pool and fish tank granuloma in humans (Primm et al., 2004). Mycobacterium fortuitum is a natural pathogen of insects related to the tuberculosis agent Mycobacterium tuberculosis. Another mycobacterial species, Mycobacterium avium, can cause systemic infections in immunocompromised patients. Legionella spp. and environmental Mycobacterium spp. are ubiquitously present in natural and municipal water sources. However, while an infection with L. pneumophila occurs exclusively by inhalation of contaminated aerosols (Fields et al., 2002), M. marinum, M. fortuitum and M. avium infect humans also through skin abrasions or via the gastrointestinal tract respectively (Primm et al., 2004). Legionella pneumophila replicates within a number of amoeba and ciliated protozoa, as well as within phagocytic and non-phagocytic mammalian cells (Rowbotham, 1980; reviewed by Fields, 1996). Similarly, M. avium, M. marinum and M. fortuitum have been shown to replicate within A. castellanii, while the non-pathogenic species Mycobacterium smegmatis is killed (Cirillo et al.,

© 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 563–575

Predators as pathogenesis models 565 Table 2. Molecular determinants of environmental bacteria and eukaryotic cells relevant for pathogen–host interactions.

Legionella

Amoebaa

Dictyostelium

Icm/Dot T4SS LepA, LepB Mip, PmiA FliA (s28), RpoS (s38) LetA/GacA Type IV pili Tat (TatBC) Lsp T2SS Lipid A (LpxB) Lectin (Gal/GalNac)

Icm/Dot T4SS LepA, LepB, SidC Mip FliA (s28)

Mammalian cellsc

Icm/Dot T4SS

Nramp1 PI(3)K, PI(4)P G protein (b subunit) Coronin Myosin I, actin, other cytoskeletal proteins

Arf1, Sar1, Rab5c Sec22 + TRAPP Cdc48/p97 (ERAD) Proteasome PE-PGRS protein (Mag24) Scavenger receptor (CD36, Peste)

PE-PGRS protein (Mag24) Scavenger receptor (SR-BI, SR-BII)

Las, Rhl GacAS T3SS (regulator) Exotoxin A Phenazine pigments Type IV pili

Las, Rhl RpoN (s54), GacAS T3SS Exotoxin A Phenazine pigments MexEF-OprN (efflux pump)

n.d.d (replication)

PE-PGRS protein (Mag24) Nramp1 Coronin

Pseudomonas

Las, Rhl n.d.d (toxicity)

Las, Rhl T3SS (PscJ) Lipase cytotoxin (ExoU) Rhamnolipids MexEF-OprN (efflux pump)

n.d.d (replication)

Drosophilab

Icm/Dot T4SS SidC Mip, PmiA FliA (s28) LetA/GacA Type IV pili Tat (TatBC) Nramp1 PI(4)P Arf1, Sar1, Rab1 Sec22 + TRAPP Cdc48/p97 (ERAD) Proteasome

Mycobacterium

Vibrio

Caenorhabditis

Vas T6SS Haemolysin-coregulated protein (Hcp-1, Hcp-2)

Las RpoN (s54), GacAS Cyanide Exotoxin A Phenazine pigments MexAB-OprM (efflux pump) Egl-9 Oxidative stress (Age-1, Mev-1, Rad-8) P-glycoprotein

Vas T6SS

a. Acanthamoeba castellanii, Acanthamoeba polyphaga, Hartmanella vermiformis or Tetrahymena pyriformis. b. Drosophila melanogaster Kc167 or S2 cells (macrophage-like phagocytes), or adult flies. c. Primary macrophages (murine bone marrow-derived, human monocyte-derived), macrophage-like cell lines (murine RAW264.7, human HL-60, human U937, human MonoMac6), epithelial cells (HeLa), fibroblasts (human embryonic kidney), or mouse strains. d. n.d., not determined on a molecular level. Bacterial or host cell factors are indicated by regular or bold font style respectively. For details and references see text.

1997). Moreover, M. marinum survives within cysts of Acanthamoeba polyphaga (Steinert et al., 1998). The survival of pathogenic bacteria within protozoa is of particular importance in biofilms, which are complex communities of prokaryotic and eukaryotic organisms linked by manifold metabolic and physical interactions. Legionella pneumophila grows in presence but not in absence of H. vermiformis in defined and undefined bacterial biofilms established under poor nutrient conditions (Murga et al., 2001; Kuiper et al., 2004). Contrarily, in a complex medium supporting axenic replication, L. pneumophila alone adheres to surfaces and forms biofilms. This process coincides with planktonic growth of the bacteria and is partially controlled by the alternative sigma factor FliA (s28) (Mampel et al., 2006). Biofilm formation is favoured at elevated temperatures (37–42°C), where the bacteria appear filamentous and form mycelial mat-like structures (Piao et al., 2006). These studies suggest that in aquatic environments, L. pneumophila replicates preferably if not

exclusively within protozoa. However, L. pneumophila was recently found to grow also by necrotrophy on heatkilled Gram-negative bacteria, biofilm, or A. castellanii, supporting the view that extracellular growth might also be important for persistence of the bacteria in the environment (Temmerman et al., 2006). Amoebae profoundly affect the physiology and antimicrobial susceptibility of L. pneumophila and M. avium. Compared with bacteria grown in broth, L. pneumophila (Cirillo et al., 1994; 1999), as well as M. avium (Cirillo et al., 1997) grown in A. castellanii invade more effectively epithelial cells and macrophages, replicate more efficiently in monocytes or macrophages and are more virulent in mice. Similarly, co-inoculation of L. pneumophila with H. vermiformis or infection with amoebae-grown L. pneumophila also enhances bacterial replication in mouse lungs and mortality in mice (Brieland et al., 1996; 1997). Furthermore, L. pneumophila (Barker et al., 1992; 1995) or M. avium (Miltner and Bermudez,

© 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 563–575

566 H. Hilbi et al. 2000) grown in Acanthamoeba spp. are more resistant to antibiotics or biocides, and the presence of H. vermiformis in a defined biofilm protects L. pneumophila from the biocides chlorine and monochloramine (Donlan et al., 2005). Effects of A. castellanii on the physiology of L. pneumophila are also evident from the observation that the amoebae resuscitate the bacteria from a ‘viable but non-culturable’ state after 125 days’ exposure to water (Steinert et al., 1997). More recently it has become clear that the growth phase of L. pneumophila determines profound physiological changes. Within amoebae, the bacteria adopt a biphasic life style and undergo a reversible transition from a replicative to a transmissive (postexponential growth) state, which is characterized by the expression of genes facilitating transmission to new hosts (reviewed by Molofsky and Swanson, 2004). The opportunistic broad host range pathogen P. aeruginosa is feared as an antibiotic-resistant infectious agent in nosocomial settings, which commonly causes infections in burn victims, cystic fibrosis patients and immunocompromised individuals (Goodman and Lory, 2004). Pseudomonas aeruginosa was found to suppress the growth of A. castellanii at high bacteria to amoeba ratios (100:1) by an unknown mechanism (Wang and Ahearn, 1997). Recently, biofilm formation of P. aeruginosa was analysed with a focus on grazing resistance against protozoa (Matz et al., 2004). Dependent on functional Rhl and Las quorum-sensing systems, type IV pili, flagella, and the polysaccharide alginate, P. aeruginosa formed inedible microcolonies in the presence but not in the absence of grazing protozoan flagellates. Mature biofilms of wild-type but not rhlR/lasR mutant P. aeruginosa exhibited acute cytotoxicity against the protozoa, indicating that the quorum-sensing systems regulate the synthesis of toxins. However, the formation of microcolonies protected only against early protozoan biofilm colonizers but not against late colonizers, such as A. polyphaga and the planktonic ciliate Tetrahymena sp. (Weitere et al., 2005). Moreover, mature biofilms inhibited only the growth of flagellates and A. polyphaga, while Tetrahymena sp. remained unaffected. Vibrio cholerae is the causative agent of cholera, a waterborne devastating diarrhoeal disease, and generally believed to be a strictly extracellular bacterium (Greub and Raoult, 2004). Unexpectedly, V. cholera O139 was recently found to survive and replicate within A. castellanii trophozoites without killing the amoebae (Abd et al., 2005). The bacteria initially localized intracellularly in vacuoles, later on in the cytoplasm and also survived within amoeba cysts. Thus, in relation to A. castellanii, V. cholerae behaved as a facultative intracellular bacterium and, under the experimental conditions used, apparently established a symbiotic relationship with the amoebae.

Replication of Legionella and Mycobacterium within amoebae Intracellular growth of L. pneumophila (Swanson and Hammer, 2000) or M. avium (Cirillo et al., 1997) within amoebae shares many similarities on a cellular and molecular level with growth of the bacteria within macrophages. These findings support the view that adaptation of the bacteria to protozoa is a precondition for their adaptation to macrophages. Legionella pneumophila is taken up by A. castellanii by ‘pathogen-triggered’ phagocytosis (Hilbi et al., 2001). Furthermore, both A. castellanii (Bozue and Johnson, 1996) and H. vermiformis (Venkataraman et al., 1998) take up at least a portion of the bacteria by coiling phagocytosis; however, the relevance of this morphologically distinct phagocytosis for later steps of the infection remains unclear. A 170 kDa galactose/N-acetyl-D-galactosamine (Gal/GalNac) lectin cross-reacting with antiserum against a lectin from Entamoeba histolytica was identified in H. vermiformis as a receptor used by L. pneumophila for invasion (Venkataraman et al., 1997). Blocking the lectin by Gal, GalNac or antibodies against the 170 kDa lectin prevented invasion and intracellular replication of L. pneumophila. Moreover, attachment of the bacteria to the amoebae was found to trigger tyrosine dephosphorylation of the 170 kDa lectin and the cytoskeletal proteins paxilin, vinculin and pp125FAK (Venkataraman et al., 1998). After uptake, L. pneumophila (Bozue and Johnson (1996) or M. avium (Cirillo et al., 1997) inhibit fusion of the phagosomes with lysosomes and form specific ‘Legionella-containing vacuoles’ (LCV) or ‘Mycobacteriumcontaining vacuoles’ respectively. Legionella-containing vacuoles acquire endoplasmic reticulum (ER) membranes in A. castellanii (Bozue and Johnson (1996), as well as in H. vermiformis (Abu Kwaik, 1996). During the last stages of infection in A. polyphaga, the membrane of LCV is disrupted independently of the hydrolytic enzymes secreted by the type II secretion system (T2SS), and the final rounds of bacterial replication occur in the cytoplasm (Molmeret et al., 2004a). Eventually, L. pneumophila kills and lyses A. castellanii (Hägele et al., 1998) and A. polyphaga (Gao and Kwaik, 2000). The mechanism of cell death resembles necrosis rather than apoptosis, because in the dying amoebae neither the exposure of phosphatidylserine on the cell surface nor the fragmentation of nuclear DNA was observed. A key virulence factor of L. pneumophila involved in the interactions with protozoa and mammalian cells is the Icm/Dot (intracellular multiplication/defective organelle transport) T4SS (reviewed by Segal et al., 2005). The Icm/Dot T4SS is a conjugation apparatus that translocates more than 30 ‘effector’ proteins into the host cell, thus preventing fusion of LCV with lysosomes and pro-

© 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 563–575

Predators as pathogenesis models 567

Fig. 2. Legionella pneumophila replicates within a specific vacuole in amoebae. A. The Icm/Dot T4SS of L. pneumophila governs 1 phagocytosis, 2 inhibition of endocytosis, 3 interaction with the early secretory pathway and 5 release from amoebae. Legionella-containing vacuoles accumulate PI(4)P, which is bound by the Icm/Dot-secreted effector protein SidC, and acquire ER markers such as calnexin. Formation of the LCV depends on the activity of the small GTPases Sar1, Arf1 and Rab1 as well as on Icm/Dot-secreted guanine nucleotide exchange factors. 4 Replication of L. pneumophila in LCV does not seem to require the Icm/Dot T4SS. B. Isolated LCV from calnexin-GFP expressing D. discoideum were infected with DsRed-expressing L. pneumophila and labelled for PI(4)P with an anti-PI(4)P antibody (left panels) or with purified GST-SidC fusion protein and an anti-GST antibody (right panels). Scale bar, 2 mm. Modified after Weber and colleagues (2006).

moting interactions with the secretory pathway (Fig. 2A). Formation of the LCV requires the activity of the small GTPases Sar1, Arf1 and Rab1 as well as Icm/Dotsecreted guanine nucleotide exchange factors (reviewed by Nagai and Roy, 2003; Bruggemann et al., 2006a). The Icm/Dot T4SS is required for pathogen-triggered phagocytosis (Hilbi et al., 2001) and intracellular replication in A. castellanii (Segal and Shuman, 1999), as well as for pore formation (Kirby et al., 1998), which was also shown to mediate T2SS-independent cytolysis and egress from A. polyphaga (Molmeret et al. 2002a,b). The function of most Icm/Dot-secreted effector proteins is presently unknown, and the corresponding deletion mutants do not show strong phenotypes in pathogen–host interactions. Contrarily, a L. pneumophila lepA/lepB double mutant is severely defective for the release of intracellular bacteria from A. castellanii without being impaired for intracellular replication (Chen et al., 2004). The Icm/Dot-secreted effectors LepA and LepB share weak similarities with SNARE proteins involved in vesicle trafficking, suggesting

that these proteins likely interfere with this host cell function. Other L. pneumophila secretion systems involved in pathogen–amoeba interactions are the Lsp T2SS (Hales and Shuman, 1999a) and a Tat (twin-arginine translocation) secretion system (De Buck et al., 2005). Furthermore, the peptidyl-prolyl cis/trans isomerase Mip (macrophage infectivity potentiator) is required to infect A. castellanii, H. vermiformis and the protozoan ciliate Tetrahymena pyriformis (Cianciotto and Fields, 1992; Wintermeyer et al., 1995). The alternative sigma factors RpoS (s38) (Hales and Shuman, 1999b) and FliA (s28) (Heuner et al., 2002), and the two-component response regulator LetA (GacA) (GalMor and Segal, 2003; Lynch et al., 2003) regulate the expression of transmissive traits within A. castellanii and macrophages (reviewed by Molofsky and Swanson, 2004). Finally, type IV pili, which are partially required for adherence but not for intracellular replication (Stone and Abu Kwaik, 1998), as well as PmiA, a putative transmembrane protein implicated in pore-forming activity and the forma-

© 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 563–575

568 H. Hilbi et al. tion of LCV (Miyake et al., 2005) govern interactions of L. pneumophila with A. polyphaga. Recently, A. castellanii was used as a selective host to screen a L. pneumophila chromosomal library for icm/dot mutant suppressors (Albers et al., 2005). Several cytotoxic genes were identified, among which a lipid A disaccharide synthase gene (lpxB) was found to be present in two paralogues in the L. pneumophila genome. The lpxB paralogues complemented a conditional E. coli lpxB mutant, indicating that the genes are involved in lipid A biosynthesis, and they are differentially regulated under various environmental conditions (U. Albers, A. Tiaden, T. Spirig and H. Hilbi, unpubl. data). These findings are in agreement with a previous report documenting that L. pneumophila alters the LPS structure upon growth within A. polyphaga (Barker et al., 1993). Dictyostelium as a cellular pathogenesis model While many insights have been gained through the analysis of amoebae infected with environmental bacteria, a major drawback of these models is that most of these protozoa are genetically not tractable. Contrarily, elaborate genetic tools are available for D. discoideum, a haploid social soil amoeba. Dictyostelium discoideum phagocytoses and feeds on bacteria and grows as individual amoebae under rich nutrient conditions. Yet, upon starvation approximately 105 cells aggregate by cAMPcontrolled chemotaxis and, in a complex developmental process, form a fruiting body consisting of spore cells and dead stalk cells (Kessin, 2001). Genetic tools available for D. discoideum include the genomic sequence (Eichinger et al., 2005), plasmids allowing the constitutive or inducible expression of genes, GFP (green fluorescent protein) fusion constructs, targeted deletions of multiple genes, and restriction enzymemediated integration mutagenesis. These features render D. discoideum a powerful protozoan model to study host factors involved in cellular aspects of pathogen–host interactions (Solomon and Isberg, 2000; Steinert and Heuner, 2005). A possible disadvantage of D. discoideum is that the amoebae do not survive temperatures of more than 27°C, which is not compatible with an induction temperature of 37°C required for virulence gene expression of many pathogens. Furthermore, D. discoideum does neither harbour caspases (Golstein et al., 2003) nor the transcription factor NF-kB, and only one paracaspase (showing weak homology to caspases), which is dispensable for developmental cell death (Roisin-Bouffay et al., 2004). Consequently, any interaction with pathogenic bacteria requiring caspases or NF-kB can obviously not be analysed in D. discoideum. Legionella spp. were the first environmental pathogens to be shown to replicate in D. discoideum (Hägele et al.,

2000; Solomon et al., 2000). Legionella pneumophila infects adherent D. discoideum, but not amoebae grown in suspension and also prevents differentiation of D. discoideum into fruiting bodies. However, it is currently not known, whether L. pneumophila specifically interferes with D. discoideum morphogenesis or whether bacterial growth per se is cytotoxic and thus prevents differentiation. Other environmental bacteria that infect and kill D. discoideum include M. marinum (Solomon et al., 2003), M. avium (Skriwan et al., 2002), P. aeruginosa (Cosson et al., 2002; Pukatzki et al., 2002; Skriwan et al., 2002) and V. cholerae (Pukatzki et al. 2006). Replication of Legionella and Mycobacterium within Dictyostelium The mechanism of intracellular replication of L. pneumophila within D. discoideum was found by several criteria to be the same as in A. castellanii, H. vermiformis or macrophages (Solomon and Isberg, 2000). The L. pneumophila Icm/Dot T4SS governs pathogen-triggered phagocytosis by D. discoideum, A. castellanii and macrophages (Hilbi et al., 2001; Weber et al., 2006). Phagocytosis of L. pneumophila does not require phosphatidylinositol-3 kinases (PI(3)K) (Weber et al., 2006) and does not occur through direct fusion with the ER (Lu and Clarke, 2005). Dependent on the Icm/Dot T4SS, L. pneumophila also shows contact-dependent cytotoxicity, forms LCV and replicates within D. discoideum (Solomon et al., 2000; Otto et al., 2004). Furthermore, L. pneumophila growing on agar plates kills D. discoideum, while the amoebae are able to form plaques on lawns of icm/dot mutant L. pneumophila. The LCV prevent fusion with lysosomes and promote association with rough ER, as determined by the acquisition of the ER markers GFP-HDEL and calnexin-GFP (Li et al., 2005; Lu and Clarke, 2005). In D. discoideum an accumulation of the lumenal ER marker calreticulin-GFP on LCV was not observed by fluorescence microscopy, but in macrophages the ER was demonstrated by electron microscopy to fuse with LCV and to exchange lumenal contents (Robinson and Roy, 2006). However, these results rather suggest that EM is a more sensitive method to detect fusion events than identifying differences between amoebae and mammalian cells. In D. discoideum calnexin-positive LCV were found to move along microtubules and undergo a transition from ‘tight’ to ‘spacious’ compartments, while excluding the vacuolar H+-ATPase subunit VatM (Chen et al., 2004; Lu and Clarke, 2005), as well as lysosomal proteins such as DdLIMP (Hägele et al., 2000). Defined D. discoideum mutant strains were analysed for a role in phagocytosis and intracellular growth of

© 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 563–575

Predators as pathogenesis models 569 L. pneumophila or M. marinum. Intracellular growth of L. pneumophila was impaired in D. discoideum lacking the heterotrimeric G-protein b subunit (Solomon et al., 2000) or, more severely, in absence of RtoA, a protein involved in vesicle trafficking (Li et al., 2005). Macroautophagy was found to be dispensable for intracellular replication of L. pneumophila, as D. discoideum mutants lacking components of the macroautophagy machinery (Apg1, -5, -6, -7 or -8) support bacterial growth, and GFP-Apg8 does not colocalize with LCV (Otto et al., 2004). Contrarily, D. discoideum mutants lacking the actin-binding protein coronin enhanced intracellular replication of L. pneumophila (Solomon et al., 2000) or M. marinum (Solomon et al., 2003). Mutants lacking other cytoskeleton-associated proteins such as myosin I (myoA/B) (Solomon et al., 2000), profilin (Hägele et al., 2000) or comitin (Skriwan et al., 2002) are also somewhat more permissive for L. pneumophila, while the absence of a-actinin, LimC/D, or villidin (Fajardo et al., 2004) slightly reduces intracellular growth. Furthermore, deletion of the genes encoding the calcium-binding ER proteins calnexin and calreticulin was reported to reduce intracellular replication of L. pneumophila (Fajardo et al., 2004), which, however, was not observed in another study (Weber et al., 2006). Finally, intracellular replication of L. pneumophila or M. avium was found to be enhanced in mutants lacking the metal cation transporter Nramp1 (natural resistanceassociated membrane protein-1) and, in the case of L. pneumophila, blocked by overexpression of the corresponding gene (Peracino et al., 2006). An analysis of the role of phosphoinositide metabolism in L. pneumophila-infected D. discoideum revealed that deletion or pharmacological inhibition of PI(3)K promotes intracellular replication of L. pneumophila and impairs the transition from tight to spacious LCV (Weber et al., 2006; reviewed by Hilbi, 2006). While PI(3)K inhibitors apparently have no effect on intracellular replication of L. pneumophila within macrophages (Molmeret et al., 2004b), phosphatidylinositol-4 phosphate (PI(4)P) was found to accumulate on LCV in D. discoideum in an Icm/ Dot-dependent manner (Fig. 2B) and was also present on LCV in macrophages (Weber et al., 2006). Moreover, the Icm/Dot-secreted effector protein SidC and its paralogue SdcA specifically bind to PI(4)P in vitro, and SidC accumulates on the LCV membrane in infected D. discoideum. Binding of SidC to LCV was enhanced in absence of PI(3)K, in agreement with the notion that under these conditions the amount of cellular PI(4)P is increased. The alternative sigma factor FliA (s28) was found to be required for intracellular replication of L. pneumophila in D. discoideum (Heuner et al., 2002). Furthermore, while the Icm/Dot-secreted effector proteins LepA and LepB are dispensable for intracellular replication or avoidance of phagosome–lysosome fusion, the effectors promote

a non-lytic exit pathway from D. discoideum (and A. castellanii) but not from macrophages (Chen et al., 2004). These findings indicate that differences exist between the interactions of L. pneumophila with amoebae and macrophages. On the other hand, intracellular replication of M. marinum in D. discoideum as well as in macrophages depends on a functional mag24 gene, encoding a glycine-rich PE-PGRS (polymorphic GC-repetitive sequence) family protein (Solomon et al., 2003). Based on the genomic sequences of the L. pneumophila strains Philadelphia-1 (Chien et al., 2004), Paris and Lens (Cazalet et al., 2004), DNA microarrays were developed representing every gene predicted in the genomes of the different strains. The microarrays were used to analyse gene expression of L. pneumophila during growth in broth and upon infection of A. castellanii (Bruggemann et al., 2006b). These experiments revealed a shift in gene expression upon transmission from the replicative to the transmissive growth phase, reflecting the biphasic life cycle of L. pneumophila. In the replicative phase components of aerobic metabolism, amino acid catabolism and, unexpectedly, the Entner–Doudoroff pathway, as well as a putative glucokinase and a sugar transporter were expressed. The latter findings suggest that L. pneumophila not only utilizes proteins as nutrients but also carbohydrates. Contrarily, in transmissive (postexponential) phase L. pneumophila expresses genes facilitating spread and transmission to a new host, including genes encoding the sigma factor FliA (s28), GGDEF/ EAL regulatory proteins, flagellar apparatus, type IV pilus machinery, as well as many substrates of the Icm/Dot T4SS and Icm/Dot-independent virulence factors (Bruggemann et al., 2006b). Using a fliA deletion mutant, the alternative sigma factor was found to control the flagellar regulon and factors that promote host cell entry and intracellular survival. In a complimentary approach, DNA microarrays covering approximately half of the D. discoideum genome were used to analyse the transcriptional response of the amoebae upon infection with L. pneumophila (wild-type or dotA mutant) or with L. hackeliae, a less virulent Legionella species (Farbrother et al., 2006). Most of the transcriptional changes occurred 24 h post infection, at which time point a set of 131 genes was regulated in a Legionella-specific manner. Functional annotation of the differentially regulated genes revealed that during an infection the Legionella spp. trigger a stress response, interfere with intracellular vesicle trafficking, and exploit host cell metabolism. Interestingly, expression of the rtoA gene, which promotes intracellular replication of L. pneumophila in D. discoideum (Li et al., 2005), was upregulated twofold 24 h post infection (Farbrother et al., 2006).

© 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 563–575

570 H. Hilbi et al. Identification of Pseudomonas and Vibrio virulence factors using Dictyostelium Pseudomonas aeruginosa strain PA14 grown on agar plates kills D. discoideum. Using a plate assay, a number of P. aeruginosa mutants were identified, which no longer kill D. discoideum, but rather allow the amoebae to form plaques on bacterial lawns (Pukatzki et al., 2002). Thus, the quorum-sensing transcription factor LasR, the T3SS component PscJ and the type III-secreted cytolytic lipase ExoU, but not rhamnolipids were found to be required for P. aeruginosa to kill D. discoideum. In a similar plate test P. aeruginosa strain PAO1 inhibited growth of D. discoideum on a lawn of Klebsiella pneumoniae (Cosson et al., 2002). Using this assay, rhl or las quorumsensing mutants were severely or slightly attenuated respectively, and rhamnolipids were found to be required for lysis of the amoebae. The reason for the conflicting results regarding the relevance of rhamnolipids and Las quorum sensing for the virulence of P. aeruginosa is unclear at present. An analysis of different efflux systems identified the MexEF-OprN transporter as another P. aeruginosa virulence factor. Correspondingly, a strain overexpressing MexEF-OprN, which shows a reduced production of secreted virulence factors, including elastase, the phenazine pigment pyocyanin and rhamnolipids, is more permissive for D. discoideum growth and less virulent in a rat pneumonia model (Cosson et al., 2002). A similar plate assay-based approach was recently used to characterize the virulence mechanisms of non-01, non-0139 serogroup V. cholerae V52, a strain that is cytotoxic for D. discoideum (Pukatzki et al., 2006). Several bacterial mutants were obtained by transposon mutagenesis, which no longer kill D. discoideum and thus allow plaque formation on agar plates. Some of the genes identified were found to be required for protein secretion and form a cluster previously designated by bioinformatic analysis as IAHP (IcmF-associated homologous protein) gene cluster. V. cholerae V52 does not harbour a recognizable T4SS or T3SS. Therefore, the IAHP gene cluster constitutes the prototype of a novel secretion system, which was termed ‘Vas’ (virulence-associated secretion) and classified as T6SS. Several Vas-secreted putative effector proteins lacking a hydrophobic N-terminal signal were identified, and a double mutant lacking the Vassecreted identical proteins Hcp (haemolysin-coregulated protein)-1 and -2 was found to be avirulent towards D. discoideum (Pukatzki et al., 2006).

Virulence of Pseudomonas modelled in C. elegans While only a few natural pathogens of nematodes are known, many human pathogens have been shown to kill

C. elegans (Sifri et al., 2005). Among these, P. aeruginosa infects C. elegans, colonizes the intestine and kills the worms. Pseudomonas aeruginosa has a wide host range including plants, protozoa, nematodes, insects and mammals, and therefore, it is of particular interest, whether a common virulence strategy applies to some or all of these hosts. It turns out that the majority of P. aeruginosa virulence factors identified with C. elegans are also required for virulence in amoebae, D. discoideum, D. melanogaster, plants and mice. Thus, P. aeruginosa employs conserved virulence pathways to infect, colonize and kill a variety of hosts. Two distinct killing mechanisms have been described for the interaction of P. aeruginosa with C. elegans. Pseudomonas aeruginosa grown on minimal medium causes ‘slow killing’, which occurs over the course of several days and correlates with the accumulation of the bacteria in the intestine (Tan et al., 1999a). Virulence factors of P. aeruginosa involved in slow killing of C. elegans have been identified by transposon mutagenesis and include quorum-sensing (lasR) and twocomponent (gacAS) regulatory systems, the alternative sigma factor rpoN (s54), as well as exotoxin A, but apparently neither the T3SS nor its secreted effectors (Tan et al., 1999b; Hendrickson et al., 2001). In contrast, P. aeruginosa grown on rich, highosmolarity medium causes ‘fast killing’, which does not require viable bacteria. This type of nematode death proceeds within hours, depends on the presence of RpoN (Hendrickson et al., 2001), and is mediated by the secretion of strain-specific diffusible toxins (Mahajan-Miklos et al., 1999). Transposon mutagenesis revealed that P. aeruginosa PA14 secretes the phenazine pigment pyocyanin, which generates toxic reactive oxygen species (Mahajan-Miklos et al., 1999). A defined P. aeruginosa mutant lacking the phnAB genes encoding anthranilate synthase of the phenazine biosynthesis pathway was completely avirulent. The hypothesis that pyocyanin exerts its toxic effects through a mechanism involving oxidative stress was supported by the findings that the C. elegans mutant age-1, which is resistant to oxidative stress, is also protected from fast killing, while the mev-1 and rad-8 mutants, which are highly sensitive to oxidative stress, are also hypersensitive to fast killing by P. aeruginosa. Another fast killing mechanism was identified in P. aeruginosa PAO1, which under the control of the Las and Rhl quorum-sensing systems produces hydrogen cyanide that paralyses and kills the nematode (Darby et al., 1999; Gallagher and Manoil, 2001). Deletion of the P. aeruginosa hcnC gene, encoding a subunit of hydrogen cyanide synthase, abolished paralytic killing of C. elegans. On the host side, loss-of-function mutations of the C. elegans egl-9 gene (required for normal egg laying) were found to confer resistance to paralytic killing caused

© 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 563–575

Predators as pathogenesis models 571 by P. aeruginosa PAO1 or to exposure to exogenous cyanide. The molecular basis of this resistance mechanism is currently unknown. Drosophila: a model for bacterial virulence and innate immunity Drosophila melanogaster can be used to analyse bacterial virulence and the activation of components of the innate immune system by studying primary macrophage-like phagocytes (haemocytes, differentiated plasmatocytes), phagocytic cell lines, or adult flies pricked with a bacteriaimpregnated needle (reviewed by Vodovar et al., 2004). In adult D. melanogaster, M. marinum initially infects haemocytes, where it blocks vacuolar acidification and replicates intracellularly, before the infection spreads systematically and kills the fly (Dionne et al., 2003). M. marinum lacking the mag24 gene is less virulent for D. melanogaster, similar to what has been observed for infections of D. discoideum (Solomon et al., 2003). Adult D. melanogaster flies are also efficiently killed by P. aeruginosa. In a number of studies, virulence of P. aeruginosa was found to depend on the T3SS, quorum sensing, the GacAS two-component system, phenazine pigments, exotoxin A, as well as type IV pili (reviewed by Vodovar et al., 2004). Furthermore, compared with wildtype flies a D. melanogaster phg1 mutant was equally sensitive to P. aeruginosa, while Phg1 was required to protect the flies from K. pneumoniae (Benghezal et al., 2006). Phg1 is a nine-transmembrane protein superfamily member and was originally identified in D. discoideum due to the specific sensitivity of the corresponding mutant to K. pneumoniae but not to other pathogenic bacteria. These results support the hypothesis that evolutionary distant eukaryotic organisms share conserved factors implicated in the resistance to bacterial infections. Infection of Drosophila phagocytes with Legionella and Mycobacterium In two seminal studies D. melanogaster phagocytic cell lines were used to analyse by RNA interference the requirement of host factors for intracellular replication of L. pneumophila (Dorer et al., 2006) or entry and survival of M. fortuitum (Philips et al., 2005). Intracellular replication of L. pneumophila in D. melanogaster Kc107 cells was found to depend on the bacterial Icm/Dot T4SS and on the expression of the GTPases Arf1, Sar1 or Rab5c, but not Rab7 or Rab1a. Furthermore, the eukaryotic proteasome and cytosolic components of the ER-associated protein degradation (ERAD) pathway, including Cdc48/ p97 or associated cofactors, were also required for intracellular replication. Cdc48/p97 was found to localize to LCV, where the complex participated in the removal of

polyubiquitinated proteins and bacterial substrates. These findings indicate a non-redundant role for several GTPases and for ERAD during intracellular replication of L. pneumophila. In addition, the use of pairs but not single RNAi oligos against many ER-Golgi transport proteins impaired intracellular replication of L. pneumophila. In particular, the simultaneous knockdown of the ER SNARE Sec22 together with Arf1 or with TRAPP (tethering factor transport protein particle) components such as Bet3 or Bet5 abolished intracellular replication. Based on these findings, a model was developed that LCV interfere with and recruit vesicles from different sources of the secretory pathway (Dorer et al., 2006). A genome-wide RNAi screen was performed to identify M. fortuitum factors relevant for entry and survival in D. melanogaster macrophage-like S2 cells (Philips et al., 2005). This screen revealed factors generally required for phagocytosis, including components implicated in vesicle trafficking and actin cytoskeleton organization. Other factors, such as Rab5 or the vacuolar ATPase, were specifically required for an infection with M. fortuitum. Particularly, the CD36 scavenger receptor family member Peste was found to be required for phagocytosis of M. fortuitum, but not other Gram-negative or Gram-positive pathogens. Therefore, Peste seems to function as a pattern recognition receptor during non-opsonic uptake. These findings were extended to mammalian cells by demonstrating that the class B scavenger receptors SC-BI and SC-BII specifically conferred the uptake of M. fortuitum into nonphagocytic cells. Conclusions and perspectives The analysis of bacterial virulence in non-mammalian hosts such as protozoa, D. discoideum, C. elegans or D. melanogaster mirrors many aspects of pathogenesis relevant for mammals, including humans. Genetically tractable non-mammalian hosts are easy to handle experimentally and allow an in-depth analysis of host factors relevant for host–pathogen interactions. Moreover, as most bacterial pathogens can be manipulated genetically, pathogen–host interactions can be studied by reciprocal genetic analysis, allowing the identification of host mutants that suppress the attenuated phenotype of pathogen mutants or vice versa. Possible limitations of nonmammalian models are the absence of relevant pathways or a requirement for experimental conditions, which are incompatible with normal host physiology. In spite of these limitations, environmental predators will continue to provide us with fascinating glimpses into host–pathogen relationships. Finally, a better understanding of these interactions might contribute to the development of novel therapeutic compounds to combat recognized and emerging infectious agents.

© 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 563–575

572 H. Hilbi et al. Acknowledgements The research in our laboratory was made possible by the Swiss National Science Foundation (631-065952), the ETH Zürich (TH 17/02-3), the Commission for Technology and Innovation (CTI/KTI; 6629.2 BTS-LS), the Swiss Federal Office for Energy (SFOE/BFE), the Velux Foundation and the NEMO network with support from the 3R foundation.

References Abd, H., Weintraub, A., and Sandstrom, G. (2005) Intracellular survival and replication of Vibrio cholerae O139 in aquatic free-living amoebae. Environ Microbiol 7: 1003– 1008. Abu Kwaik, Y. (1996) The phagosome containing Legionella pneumophila within the protozoan Hartmannella vermiformis is surrounded by the rough endoplasmic reticulum. Appl Environ Microbiol 62: 2022–2028. Albers, U., Reus, K., Shuman, H.A., and Hilbi, H. (2005) The amoebae plate test implicates a paralogue of lpxB in the interaction of Legionella pneumophila with Acanthamoeba castellanii. Microbiology 151: 167–182. Barker, J., Brown, M.R., Collier, P.J., Farrell, I., and Gilbert, P. (1992) Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation. Appl Environ Microbiol 58: 2420–2425. Barker, J., Lambert, P.A., and Brown, M.R. (1993) Influence of intra-amoebic and other growth conditions on the surface properties of Legionella pneumophila. Infect Immun 61: 3503–3510. Barker, J., Scaife, H., and Brown, M.R. (1995) Intraphagocytic growth induces an antibiotic-resistant phenotype of Legionella pneumophila. Antimicrob Agents Chemother 39: 2684–2688. Benghezal, M., Fauvarque, M.O., Tournebize, R., Froquet, R., Marchetti, A., Bergeret, E., et al. (2006) Specific host genes required for the killing of Klebsiella bacteria by phagocytes. Cell Microbiol 8: 139–148. Berk, S.G., Ting, R.S., Turner, G.W., and Ashburn, R.J. (1998) Production of respirable vesicles containing live Legionella pneumophila cells by two Acanthamoeba spp. Appl Environ Microbiol 64: 279–286. Bozue, J.A., and Johnson, W. (1996) Interaction of Legionella pneumophila with Acanthamoeba castellanii: uptake by coiling phagocytosis and inhibition of phagosomelysosome fusion. Infect Immun 64: 668–673. Brieland, J., McClain, M., Heath, L., Chrisp, C., Huffnagle, G., LeGendre, M., et al. (1996) Coinoculation with Hartmannella vermiformis enhances replicative Legionella pneumophila lung infection in a murine model of Legionnaires’ disease. Infect Immun 64: 2449–2456. Brieland, J.K., Fantone, J.C., Remick, D.G., LeGendre, M., McClain, M., and Engleberg, N.C. (1997) The role of Legionella pneumophila-infected Hartmannella vermiformis as an infectious particle in a murine model of Legionnaires’ disease. Infect Immun 65: 5330–5333. Brown, M.R., and Barker, J. (1999) Unexplored reservoirs of pathogenic bacteria: protozoa and biofilms. Trends Microbiol 7: 46–50.

Bruggemann, H., Cazalet, C., and Buchrieser, C. (2006a) Adaptation of Legionella pneumophila to the host environment: role of protein secretion, effectors and eukaryoticlike proteins. Curr Opin Microbiol 9: 86–94. Bruggemann, H., Hagman, A., Jules, M., Sismeiro, O., Dillies, M.A., Gouyette, C., et al. (2006b) Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila. Cell Microbiol 8: 1228–1240. Cazalet, C., Rusniok, C., Bruggemann, H., Zidane, N., Magnier, A., Ma, L., et al. (2004) Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity. Nat Genet 36: 1165–1173. Chen, J., de Felipe, K.S., Clarke, M., Lu, H., Anderson, O.R., Segal, G., and Shuman, H.A. (2004) Legionella effectors that promote nonlytic release from protozoa. Science 303: 1358–1361. Chien, M., Morozova, I., Shi, S., Sheng, H., Chen, J., Gomez, S.M., et al. (2004) The genomic sequence of the accidental pathogen Legionella pneumophila. Science 305: 1966– 1968. Cianciotto, N.P., and Fields, B.S. (1992) Legionella pneumophila mip gene potentiates intracellular infection of protozoa and human macrophages. Proc Natl Acad Sci USA 89: 5188–5191. Cirillo, J.D., Falkow, S., and Tompkins, L.S. (1994) Growth of Legionella pneumophila in Acanthamoeba castellanii enhances invasion. Infect Immun 62: 3254–3261. Cirillo, J.D., Falkow, S., Tompkins, L.S., and Bermudez, L.E. (1997) Interaction of Mycobacterium avium with environmental amoebae enhances virulence. Infect Immun 65: 3759–3767. Cirillo, J.D., Cirillo, S.L., Yan, L., Bermudez, L.E., Falkow, S., and Tompkins, L.S. (1999) Intracellular growth in Acanthamoeba castellanii affects monocyte entry mechanisms and enhances virulence of Legionella pneumophila. Infect Immun 67: 4427–4434. Cosson, P., Zulianello, L., Join-Lambert, O., Faurisson, F., Gebbie, L., Benghezal, M., et al. (2002) Pseudomonas aeruginosa virulence analyzed in a Dictyostelium discoideum host system. J Bacteriol 184: 3027–3033. Darby, C., Cosma, C.L., Thomas, J.H., and Manoil, C. (1999) Lethal paralysis of Caenorhabditis elegans by Pseudomonas aeruginosa. Proc Natl Acad Sci USA 96: 15202– 15207. De Buck, E., Maes, L., Meyen, E., Van Mellaert, L., Geukens, N., Anne, J., and Lammertyn, E. (2005) Legionella pneumophila Philadelphia-1 tatB and tatC affect intracellular replication and biofilm formation. Biochem Biophys Res Commun 331: 1413–1420. Dionne, M.S., Ghori, N., and Schneider, D.S. (2003) Drosophila melanogaster is a genetically tractable model host for Mycobacterium marinum. Infect Immun 71: 3540– 3550. Donlan, R.M., Forster, T., Murga, R., Brown, E., Lucas, C., Carpenter, J., and Fields, B. (2005) Legionella pneumophila associated with the protozoan Hartmannella vermiformis in a model multi-species biofilm has reduced susceptibility to disinfectants. Biofouling 21: 1–7. Dorer, M.S., Kirton, D., Bader, J.S., and Isberg, R.R. (2006) RNA interference analysis of Legionella in Drosophila cells:

© 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 563–575

Predators as pathogenesis models 573 exploitation of early secretory apparatus dynamics. PLoS Pathog 2: e34. Eichinger, L., Pachebat, J.A., Glockner, G., Rajandream, M.A., Sucgang, R., Berriman, M., et al. (2005) The genome of the social amoeba Dictyostelium discoideum. Nature 435: 43–57. Fajardo, M., Schleicher, M., Noegel, A., Bozzaro, S., Killinger, S., Heuner, K., et al. (2004) Calnexin, calreticulin and cytoskeleton-associated proteins modulate uptake and growth of Legionella pneumophila in Dictyostelium discoideum. Microbiology 150: 2825–2835. Farbrother, P., Wagner, C., Na, J., Tunggal, B., Morio, T., Urushihara, H., et al. (2006) Dictyostelium transcriptional host cell response upon infection with Legionella. Cell Microbiol 8: 438–456. Fields, B.S. (1996) The molecular ecology of Legionellae. Trends Microbiol 4: 286–290. Fields, B.S., Benson, R.F., and Besser, R.E. (2002) Legionella and Legionnaires’ disease: 25 years of investigation. Clin Microbiol Rev 15: 506–526. Gallagher, L.A., and Manoil, C. (2001) Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by cyanide poisoning. J Bacteriol 183: 6207–6214. Gal-Mor, O., and Segal, G. (2003) The Legionella pneumophila GacA homolog (LetA) is involved in the regulation of icm virulence genes and is required for intracellular multiplication in Acanthamoeba castellanii. Microb Pathog 34: 187–194. Gao, L.Y., and Kwaik, Y.A. (2000) The mechanism of killing and exiting the protozoan host Acanthamoeba polyphaga by Legionella pneumophila. Environ Microbiol 2: 79–90. Golstein, P., Aubry, L., and Levraud, J.P. (2003) Cell-death alternative model organisms: why and which? Nat Rev Mol Cell Biol 4: 798–807. Goodman, A.L., and Lory, S. (2004) Analysis of regulatory networks in Pseudomonas aeruginosa by genomewide transcriptional profiling. Curr Opin Microbiol 7: 39–44. Greub, G., and Raoult, D. (2004) Microorganisms resistant to free-living amoebae. Clin Microbiol Rev 17: 413–433. Hägele, S., Hacker, J., and Brand, B.C. (1998) Legionella pneumophila kills human phagocytes but not protozoan host cells by inducing apoptotic cell death. FEMS Microbiol Lett 169: 51–58. Hägele, S., Kohler, R., Merkert, H., Schleicher, M., Hacker, J., and Steinert, M. (2000) Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella. Cell Microbiol 2: 165–171. Hales, L.M., and Shuman, H.A. (1999a) Legionella pneumophila contains a type II general secretion pathway required for growth in amoebae as well as for secretion of the Msp protease. Infect Immun 67: 3662–3666. Hales, L.M., and Shuman, H.A. (1999b) The Legionella pneumophila rpoS gene is required for growth within Acanthamoeba castellanii. J Bacteriol 181: 4879–4889. Hendrickson, E.L., Plotnikova, J., Mahajan-Miklos, S., Rahme, L.G., and Ausubel, F.M. (2001) Differential roles of the Pseudomonas aeruginosa PA14 rpoN gene in pathogenicity in plants, nematodes, insects, and mice. J Bacteriol 183: 7126–7134. Heuner, K., Dietrich, C., Skriwan, C., Steinert, M., and Hacker, J. (2002) Influence of the alternative sigma(28)

factor on virulence and flagellum expression of Legionella pneumophila. Infect Immun 70: 1604–1608. Hilbi, H. (2006) Modulation of phosphoinositide metabolism by pathogenic bacteria. Cell Microbiol 8: 1697–1706. Hilbi, H., Segal, G., and Shuman, H.A. (2001) Icm/Dotdependent upregulation of phagocytosis by Legionella pneumophila. Mol Microbiol 42: 603–617. Kessin, R.H. (2001) Dictyostelium – Evolution, Cell Biology, and the Development of Multicellularity. Cambridge, UK: Cambridge University Press. Kirby, J.E., Vogel, J.P., Andrews, H.L., and Isberg, R.R. (1998) Evidence for pore-forming ability by Legionella pneumophila. Mol Microbiol 27: 323–336. Kuiper, M.W., Wullings, B.A., Akkermans, A.D., Beumer, R.R., and van der Kooij, D. (2004) Intracellular proliferation of Legionella pneumophila in Hartmannella vermiformis in aquatic biofilms grown on plasticized polyvinyl chloride. Appl Environ Microbiol 70: 6826–6833. Li, Z., Solomon, J.M., and Isberg, R.R. (2005) Dictyostelium discoideum strains lacking the RtoA protein are defective for maturation of the Legionella pneumophila replication vacuole. Cell Microbiol 7: 431–442. Lu, H., and Clarke, M. (2005) Dynamic properties of Legionella-containing phagosomes in Dictyostelium amoebae. Cell Microbiol 7: 995–1007. Lynch, D., Fieser, N., Gloggler, K., Forsbach-Birk, V., and Marre, R. (2003) The response regulator LetA regulates the stationary-phase stress response in Legionella pneumophila and is required for efficient infection of Acanthamoeba castellanii. FEMS Microbiol Lett 219: 241– 248. Mahajan-Miklos, S., Tan, M.W., Rahme, L.G., and Ausubel, F.M. (1999) Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosaCaenorhabditis elegans pathogenesis model. Cell 96: 47–56. Mampel, J., Spirig, T., Weber, S.S., Haagensen, J.A., Molin, S., and Hilbi, H. (2006) Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions. Appl Environ Microbiol 72: 2885–2895. Matz, C., and Kjelleberg, S. (2005) Off the hook – how bacteria survive protozoan grazing. Trends Microbiol 13: 302– 307. Matz, C., Bergfeld, T., Rice, S.A., and Kjelleberg, S. (2004) Microcolonies, quorum sensing and cytotoxicity determine the survival of Pseudomonas aeruginosa biofilms exposed to protozoan grazing. Environ Microbiol 6: 218–226. Miltner, E.C., and Bermudez, L.E. (2000) Mycobacterium avium grown in Acanthamoeba castellanii is protected from the effects of antimicrobials. Antimicrob Agents Chemother 44: 1990–1994. Miyake, M., Watanabe, T., Koike, H., Molmeret, M., Imai, Y., and Abu Kwaik, Y. (2005) Characterization of Legionella pneumophila pmiA, a gene essential for infectivity of protozoa and macrophages. Infect Immun 73: 6272–6282. Molmeret, M., Alli, O.A., Radulic, M., Susa, M., Doric, M., and Kwaik, Y.A. (2002a) The C-terminus of IcmT is essential for pore formation and for intracellular trafficking of Legionella pneumophila within Acanthamoeba polyphaga. Mol Microbiol 43: 1139–1150.

© 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 563–575

574 H. Hilbi et al. Molmeret, M., Alli, O.A., Zink, S., Flieger, A., Cianciotto, N.P., and Kwaik, Y.A. (2002b) IcmT is essential for pore formation-mediated egress of Legionella pneumophila from mammalian and protozoan cells. Infect Immun 70: 69–78. Molmeret, M., Bitar, D.M., Han, L., and Kwaik, Y.A. (2004a) Disruption of the phagosomal membrane and egress of Legionella pneumophila into the cytoplasm during the last stages of intracellular infection of macrophages and Acanthamoeba polyphaga. Infect Immun 72: 4040– 4051. Molmeret, M., Zink, S.D., Han, L., Abu-Zant, A., Asari, R., Bitar, D.M., and Abu Kwaik, Y. (2004b) Activation of caspase-3 by the Dot/Icm virulence system is essential for arrested biogenesis of the Legionella-containing phagosome. Cell Microbiol 6: 33–48. Molmeret, M., Horn, M., Wagner, M., Santic, M., and Abu Kwaik, Y. (2005) Amoebae as training grounds for intracellular bacterial pathogens. Appl Environ Microbiol 71: 20–28. Molofsky, A.B., and Swanson, M.S. (2004) Differentiate to thrive: lessons from the Legionella pneumophila life cycle. Mol Microbiol 53: 29–40. Murga, R., Forster, T.S., Brown, E., Pruckler, J.M., Fields, B.S., and Donlan, R.M. (2001) Role of biofilms in the survival of Legionella pneumophila in a model potable-water system. Microbiology 147: 3121–3126. Nagai, H., and Roy, C.R. (2003) Show me the substrates: modulation of host cell function by type IV secretion systems. Cell Microbiol 5: 373–383. Otto, G.P., Wu, M.Y., Clarke, M., Lu, H., Anderson, O.R., Hilbi, H., et al. (2004) Macroautophagy is dispensable for intracellular replication of Legionella pneumophila in Dictyostelium discoideum. Mol Microbiol 51: 63–72. Peracino, B., Wagner, C., Balest, A., Balbo, A., Pergolizzi, B., Noegel, A.A., et al. (2006) Function and mechanism of action of Dictyostelium Nramp1 (Slc11a1) in bacterial infection. Traffic 7: 22–38. Philips, J.A., Rubin, E.J., and Perrimon, N. (2005) Drosophila RNAi screen reveals CD36 family member required for mycobacterial infection. Science 309: 1251–1253. Piao, Z., Sze, C.C., Barysheva, O., Iida, K., and Yoshida, S. (2006) Temperature-regulated formation of mycelial matlike biofilms by Legionella pneumophila. Appl Environ Microbiol 72: 1613–1622. Primm, T.P., Lucero, C.A., and Falkinham, J.O., 3rd (2004) Health impacts of environmental mycobacteria. Clin Microbiol Rev 17: 98–106. Pukatzki, S., Kessin, R.H., and Mekalanos, J.J. (2002) The human pathogen Pseudomonas aeruginosa utilizes conserved virulence pathways to infect the social amoeba Dictyostelium discoideum. Proc Natl Acad Sci USA 99: 3159–3164. Pukatzki, S., Ma, A.T., Sturtevant, D., Krastins, B., Sarracino, D., Nelson, W.C., et al. (2006) Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci USA 103: 1528–1533. Robinson, C.G., and Roy, C.R. (2006) Attachment and fusion of endoplasmic reticulum with vacuoles containing Legionella pneumophila. Cell Microbiol 8: 793–805.

Roisin-Bouffay, C., Luciani, M.F., Klein, G., Levraud, J.P., Adam, M., and Golstein, P. (2004) Developmental cell death in Dictyostelium does not require paracaspase. J Biol Chem 279: 11489–11494. Rowbotham, T.J. (1980) Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. J Clin Pathol 33: 1179–1183. Segal, G., and Shuman, H.A. (1999) Legionella pneumophila utilizes the same genes to multiply within Acanthamoeba castellanii and human macrophages. Infect Immun 67: 2117–2124. Segal, G., Feldman, M., and Zusman, T. (2005) The Icm/Dot type-IV secretion systems of Legionella pneumophila and Coxiella burnetii. FEMS Microbiol Rev 29: 65–81. Sifri, C.D., Begun, J., and Ausubel, F.M. (2005) The worm has turned – microbial virulence modeled in Caenorhabditis elegans. Trends Microbiol 13: 119–127. Skriwan, C., Fajardo, M., Hagele, S., Horn, M., Wagner, M., Michel, R., et al. (2002) Various bacterial pathogens and symbionts infect the amoeba Dictyostelium discoideum. Int J Med Microbiol 291: 615–624. Solomon, J.M., and Isberg, R.R. (2000) Growth of Legionella pneumophila in Dictyostelium discoideum: a novel system for genetic analysis of host–pathogen interactions. Trends Microbiol 8: 478–480. Solomon, J.M., Rupper, A., Cardelli, J.A., and Isberg, R.R. (2000) Intracellular growth of Legionella pneumophila in Dictyostelium discoideum, a system for genetic analysis of host–pathogen interactions. Infect Immun 68: 2939– 2947. Solomon, J.M., Leung, G.S., and Isberg, R.R. (2003) Intracellular replication of Mycobacterium marinum within Dictyostelium discoideum: efficient replication in the absence of host coronin. Infect Immun 71: 3578–3586. Steinert, M., and Heuner, K. (2005) Dictyostelium as host model for pathogenesis. Cell Microbiol 7: 307–314. Steinert, M., Emody, L., Amann, R., and Hacker, J. (1997) Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii. Appl Environ Microbiol 63: 2047–2053. Steinert, M., Birkness, K., White, E., Fields, B., and Quinn, F. (1998) Mycobacterium avium bacilli grow saprozoically in coculture with Acanthamoeba polyphaga and survive within cyst walls. Appl Environ Microbiol 64: 2256– 2261. Stone, B.J., and Abu Kwaik, Y. (1998) Expression of multiple pili by Legionella pneumophila: identification and characterization of a type IV pilin gene and its role in adherence to mammalian and protozoan cells. Infect Immun 66: 1768– 1775. Swanson, M.S., and Hammer, B.K. (2000) Legionella pneumophila pathogenesis: a fateful journey from amoebae to macrophages. Annu Rev Microbiol 54: 567–613. Tan, M.W., Mahajan-Miklos, S., and Ausubel, F.M. (1999a) Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis. Proc Natl 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 Caenorhabditis elegans used to identify P. aeruginosa virulence factors. Proc Natl Acad Sci USA 96: 2408–2413.

© 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 563–575

Predators as pathogenesis models 575 Temmerman, R., Vervaeren, H., Noseda, B., Boon, N., and Verstraete, W. (2006) Necrotrophic growth of Legionella pneumophila. Appl Environ Microbiol 72: 4323–4328. Venkataraman, C., Haack, B.J., Bondada, S., and Abu Kwaik, Y. (1997) Identification of a Gal/GalNAc lectin in the protozoan Hartmannella vermiformis as a potential receptor for attachment and invasion by the Legionnaires’ disease bacterium. J Exp Med 186: 537–547. Venkataraman, C., Gao, L.Y., Bondada, S., and Kwaik, Y.A. (1998) Identification of putative cytoskeletal protein homologues in the protozoan host Hartmannella vermiformis as substrates for induced tyrosine phosphatase activity upon attachment to the Legionnaires’ disease bacterium, Legionella pneumophila. J Exp Med 188: 505–514. Vodovar, N., Acosta, C., Lemaitre, B., and Boccard, F. (2004) Drosophila: a polyvalent model to decipher host–pathogen interactions. Trends Microbiol 12: 235–242.

Wang, X., and Ahearn, D.G. (1997) Effect of bacteria on survival and growth of Acanthamoeba castellanii. Curr Microbiol 34: 212–215. Weber, S.S., Ragaz, C., Reus, K., Nyfeler, Y., and Hilbi, H. (2006) Legionella pneumophila exploits PI(4)P to anchor secreted effector proteins to the replicative vacuole. PLoS Pathog 2: e46. Weitere, M., Bergfeld, T., Rice, S.A., Matz, C., and Kjelleberg, S. (2005) Grazing resistance of Pseudomonas aeruginosa biofilms depends on type of protective mechanism, developmental stage and protozoan feeding mode. Environ Microbiol 7: 1593–1601. Wintermeyer, E., Ludwig, B., Steinert, M., Schmidt, B., Fischer, G., and Hacker, J. (1995) Influence of site specifically altered Mip proteins on intracellular survival of Legionella pneumophila in eukaryotic cells. Infect Immun 63: 4576–4583.

© 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 563–575