The Legionella pneumophila Effector Protein,LegC7, Alters Yeast Endosomal Trafficking

The intracellular pathogen, Legionella pneumophila, relies on numerous secreted effector proteins to manipulate host endomembrane trafficking events during pathogenesis, thereby preventing fusion of the bacteria-laden phagosome with host endolysosomal compartments, and thus escaping degradation. Upon expression in the surrogate eukaryotic model Saccharomyces cerevisiae, we find that the L. pneumophila LegC7/YlfA effector protein disrupts the delivery of both biosynthetic and endocytic cargo to the yeast vacuole. We demonstrate that the effects of LegC7 are specific to the endosome:vacuole delivery pathways; LegC7 expression does not disrupt other known vacuole-directed pathways. Deletions of the ESCRT-0 complex member, VPS27, provide resistance to the LegC7 toxicity, providing a possible target for LegC7 function in vivo. Furthermore, a single amino acid substitution in LegC7 abrogates both its toxicity and ability to alter endosomal traffic in vivo, thereby identifying a critical functional domain. LegC7 likely inhibits endosomal trafficking during L. pneumophila pathogenesis to prevent entry of the phagosome into the endosomal maturation pathway and eventual fusion with the lysosome.     

Legionella pneumophila Subversion of Host Vesicular Transport by SidC Effector Proteins

Tethering proteins play a key role in vesicular transport, ensuring that cargo arrives at a specific destination. The bacterial effector protein SidC and its paralog SdcA have been described as tethering factors encoded by the intracellular pathogen Legionella pneumophila. Here, we demonstrate that SidC proteins are important for early events unique to maturation of vacuoles containing Legionella and discover monoubiquitination of Rab1 as a new SidC‐dependent activity. The crystal structure of the SidC N‐terminus revealed a novel fold that is important for function and could be involved in Legionella adaptations to evolutionarily divergent host cells it encounters in natural environments.      

Inhibition of Host Vacuolar H+-ATPase Activity by a Legionella pneumophila Effector

Legionella pneumophila is an intracellular pathogen responsible for Legionnaires'' disease. This bacterium uses the Dot/Icm type IV secretion system to inject a large number of bacterial proteins into host cells to facilitate the biogenesis of a phagosome permissive for its intracellular growth. Like many highly adapted intravacuolar pathogens, L. pneumophila is able to maintain a neutral pH in the lumen of its phagosome, particularly in the early phase of infection. However, in all cases, the molecular mechanisms underlying this observation remain unknown. In this report, we describe the identification and characterization of a Legionella protein termed SidK that specifically targets host v-ATPase, the multi-subunit machinery primarily responsible for organelle acidification in eukaryotic cells. Our results indicate that after being injected into infected cells by the Dot/Icm secretion system, SidK interacts with VatA, a key component of the proton pump. Such binding leads to the inhibition of ATP hydrolysis and proton translocation. When delivered into macrophages, SidK inhibits vacuole acidification and impairs the ability of the cells to digest non-pathogenic E. coli. We also show that a domain located in the N-terminal portion of SidK is responsible for its interactions with VatA. Furthermore, expression of sidK is highly induced when bacteria begin to enter new growth cycle, correlating well with the potential temporal requirement of its activity during infection. Our results indicate that direct targeting of v-ATPase by secreted proteins constitutes a virulence strategy for L. pneumophila, a vacuolar pathogen of macrophages and amoebae.     

Lipidation by the Host Prenyltransferase Machinery Facilitates Membrane Localization of Legionella pneumophila Effector Proteins

The intracellular human pathogen Legionella pneumophila translocates multiple proteins in the host cytosol known as effectors, which subvert host cellular processes to create a membrane-bound organelle that supports bacterial replication. It was observed that several Legionella effectors encode a prototypical eukaryotic prenylation CAAX motif (where C represents a cysteine residue and A denotes an aliphatic amino acid). These bacterial motifs mediated posttranslational modification of effector proteins resulting in the addition of either a farnesyl or geranylgeranyl isoprenyl lipid moiety to the cysteine residue of the CAAX tetrapeptide. Lipidation enhanced membrane affinity for most Legionella CAAX motif proteins and facilitated the localization of these effector proteins to host organelles. Host farnesyltransferase and class I geranylgeranyltransferase were both involved in the lipidation of the Legionella CAAX motif proteins. Perturbation of the host prenylation machinery during infection adversely affected the remodeling of the Legionella-containing vacuole. Thus, these data indicate that Legionella utilize the host prenylation machinery to facilitate targeting of effector proteins to membrane-bound organelles during intracellular infection.     

The Actin Nucleator Cobl Is Controlled by Calcium and Calmodulin

Actin nucleation triggers the formation of new actin filaments and has the power to shape cells but requires tight control in order to bring about proper morphologies. The regulation of the members of the novel class of WASP Homology 2 (WH2) domain-based actin nucleators, however, thus far has largely remained elusive. Our study reveals signal cascades and mechanisms regulating Cordon-Bleu (Cobl). Cobl plays some, albeit not fully understood, role in early arborization of neurons and nucleates actin by a mechanism that requires a combination of all three of its actin monomer–binding WH2 domains. Our experiments reveal that Cobl is regulated by Ca²⁺ and multiple, direct associations of the Ca²⁺ sensor Calmodulin (CaM). Overexpression analyses and rescue experiments of Cobl loss-of-function phenotypes with Cobl mutants in primary neurons and in tissue slices demonstrated the importance of CaM binding for Cobl’s functions. Cobl-induced dendritic branch initiation was preceded by Ca²⁺ signals and coincided with local F-actin and CaM accumulations. CaM inhibitor studies showed that Cobl-mediated branching is strictly dependent on CaM activity. Mechanistic studies revealed that Ca²⁺/CaM modulates Cobl’s actin binding properties and furthermore promotes Cobl’s previously identified interactions with the membrane-shaping F-BAR protein syndapin I, which accumulated with Cobl at nascent dendritic protrusion sites. The findings of our study demonstrate a direct regulation of an actin nucleator by Ca²⁺/CaM and reveal that the Ca²⁺/CaM-controlled molecular mechanisms we discovered are crucial for Cobl’s cellular functions. By unveiling the means of Cobl regulation and the mechanisms, by which Ca²⁺/CaM signals directly converge on a cellular effector promoting actin filament formation, our work furthermore sheds light on how local Ca²⁺ signals steer and power branch initiation during early arborization of nerve cells—a key process in neuronal network formation.     

Activation of the Legionella pneumophila LegK7 Effector Kinase by the Host MOB1 Protein

Legionella pneumophila infects alveolar macrophages and can cause life-threatening pneumonia in humans. Upon internalization into the host cell, L. pneumophila injects numerous effector proteins into the host cytoplasm as a part of its pathogenesis. LegK7 is an effector kinase of L. pneumophila that functionally mimics the eukaryotic Mst kinase and phosphorylates the host MOB1 protein to exploit the Hippo pathway. To elucidate the LegK7 activation mechanism, we determined the apo structure of LegK7 in an inactive form and performed a comparative analysis of LegK7 structures. LegK7 is a non-RD kinase that contains an activation segment that is ordered, irrespective of stimulation, through a unique β-hairpin-containing segment, and it does not require phosphorylation of the activation segment for activation. Instead, bacterial LegK7 becomes an active kinase via its heterologous molecular interaction with the host MOB1 protein. MOB1 binding triggers reorientation of the two lobes of the kinase domain, as well as a structural change in the interlobe hinge region in LegK7, consequently reshaping the LegK7 structure into an ATP binding-compatible closed conformation. Furthermore, we reveal that LegK7 is an atypical kinase that contains an N-terminal capping domain and a hydrophilic interlobe linker motif, which play key roles in the MOB1-induced activation of LegK7.     

Legionella pneumophila Effector RomA Uniquely Modifies Host Chromatin to Repress Gene Expression and Promote Intracellular Bacterial Replication

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Highlights? Upon infection, L. pneumophila secretes RomA, a SET domain-containing methyltransferase ? RomA triggers H3K14 trimethylation, causing a switch from gene activation to repression ? ChIP-seq identified 4,870 H3K14 methylated promoter regions, including innate immune genes ? RomA SET domain is required for efficient intracellular replication of L. pneumophila     

The Legionella pneumophila rpoS Gene Is Required for Growth within Acanthamoeba castellanii

To investigate regulatory networks in Legionella pneumophila, the gene encoding the homolog of the Escherichia coli stress and stationary-phase sigma factor RpoS was identified by complementation of an E. coli rpoS mutation. An open reading frame that is approximately 60% identical to the E. coli rpoS gene was identified. Western blot analysis showed that the level of L. pneumophila RpoS increased in stationary phase. An insertion mutation was constructed in the rpoS gene on the chromosome of L. pneumophila, and the ability of this mutant strain to survive various stress conditions was assayed and compared with results for the wild-type strain. Both the mutant and wild-type strains were more resistant to stress when in stationary phase than when in the logarithmic phase of growth. This finding indicates that L. pneumophila RpoS is not required for a stationary-phase-dependent resistance to stress. Although the mutant strain was able to kill HL-60- and THP-1-derived macrophages, it could not replicate within a protozoan host, Acanthamoeba castellanii. These data suggest that L. pneumophila possesses a growth phase-dependent resistance to stress that is independent of RpoS control and that RpoS likely regulates genes that enable it to survive in the environment within protozoa. Our data indicate that the role of rpoS in L. pneumophila is very different from what has previously been reported for E. coli rpoS.     

Swiprosin-1 Is a Novel Actin Bundling Protein That Regulates Cell Spreading and Migration

Protein functions are often revealed by their localization to specialized cellular sites. Recent reports demonstrated that swiprosin-1 is found together with actin and actin-binding proteins in the cytoskeleton fraction of human mast cells and NK-like cells. However, direct evidence of whether swiprosin-1 regulates actin dynamics is currently lacking. We found that swiprosin-1 localizes to microvilli-like membrane protrusions and lamellipodia and exhibits actin-binding activity. Overexpression of swiprosin-1 enhanced lamellipodia formation and cell spreading. In contrast, swiprosin-1 knockdown showed reduced cell spreading and migration. Swiprosin-1 induced actin bundling in the presence of Ca²⁺, and deletion of the EF-hand motifs partially reduced bundling activity. Swiprosin-1 dimerized in the presence of Ca²⁺ via its coiled-coil domain, and a lysine (Lys)-rich region in the coiled-coil domain was essential for regulation of actin bundling. Consistent with these observations, mutations of the EF-hand motifs and coiled-coil region significantly reduced cell spreading and lamellipodia formation. We provide new evidence of how swiprosin-1 influences cytoskeleton reorganization and cell spreading.     

Molecular Mimicry by an F-Box Effector of Legionella pneumophila Hijacks a Conserved Polyubiquitination Machinery within Macrophages and Protozoa

The ability of Legionella pneumophila to proliferate within various protozoa in the aquatic environment and in macrophages indicates a remarkable evolution and microbial exploitation of evolutionarily conserved eukaryotic processes. Ankyrin B (AnkB) of L. pneumophila is a non-canonical F-box-containing protein, and is the only known Dot/Icm-translocated effector of L. pneumophila essential for intra-vacuolar proliferation within both macrophages and protozoan hosts. We show that the F-box domain of AnkB and the ⁹L¹⁰P conserved residues are essential for intracellular bacterial proliferation and for rapid acquisition of polyubiquitinated proteins by the Legionella-containing vacuole (LCV) within macrophages, Dictyostelium discoideum, and Acanthamoeba. Interestingly, translocation of AnkB and recruitment of polyubiquitinated proteins in macrophages and Acanthamoeba is rapidly triggered by extracellular bacteria within 5 min of bacterial attachment. Ectopically expressed AnkB within mammalian cells is localized to the periphery of the cell where it co-localizes with host SKP1 and recruits polyubiquitinated proteins, which results in restoration of intracellular growth to the ankB mutant similar to the parental strain. While an ectopically expressed AnkB-⁹L¹⁰P/AA variant is localized to the cell periphery, it does not recruit polyubiquitinated proteins and fails to trans-rescue the ankB mutant intracellular growth defect. Direct in vivo interaction of AnkB but not the AnkB-⁹L¹⁰P/AA variant with the host SKP1 is demonstrated. Importantly, RNAi-mediated silencing of expression of SKP1 renders the cells non-permissive for intracellular proliferation of L. pneumophila. The role of AnkB in exploitation of the polyubiquitination machinery is essential for intrapulmonary bacterial proliferation in the mouse model of Legionnaires'' disease. Therefore, AnkB exhibits a novel molecular and functional mimicry of eukaryotic F-box proteins that exploits conserved polyubiquitination machinery for intracellular proliferation within evolutionarily distant hosts.     

Chemical Genetics Reveals Bacterial and Host Cell Functions Critical for Type IV Effector Translocation by Legionella pneumophila

Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host–pathogen interactions.     

Balamuthia mandrillaris, Free-Living Ameba and Opportunistic Agent of Encephalitis, Is a Potential Host for Legionella pneumophila Bacteria

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of granulomatous encephalitis in humans and other mammalian species. Other free-living amebas, such as Acanthamoeba and Hartmannella, can provide a niche for intracellular survival of bacteria, including the causative agent of Legionnaires' disease, Legionella pneumophila. Infection of amebas by L. pneumophila enhances the bacterial infectivity for mammalian cells and lung tissues. Likewise, the pathogenicity of amebas may be enhanced when they host bacteria. So far, the colonization of B. mandrillaris by bacteria has not been convincingly shown. In this study, we investigated whether this ameba could host L. pneumophila bacteria. Our experiments showed that L. pneumophila could initiate uptake by B. mandrillaris and could replicate within the ameba about 4 to 5 log cycles from 24 to 72 h after infection. On the other hand, a dotA mutant, known to be unable to propagate in Acanthamoeba castellanii, also did not replicate within B. mandrillaris. Approaching completion of the intracellular cycle, L. pneumophila wild-type bacteria were able to destroy their ameboid hosts. Observations by light microscopy paralleled our quantitative data and revealed the rounding, collapse, clumping, and complete destruction of the infected amebas. Electron microscopic studies unveiled the replication of the bacteria in a compartment surrounded by a structure resembling rough endoplasmic reticulum. The course of intracellular infection, the degree of bacterial multiplication, and the ultrastructural features of a L. pneumophila-infected B. mandrillaris ameba resembled those described for other amebas hosting Legionella bacteria. We hence speculate that B. mandrillaris might serve as a host for bacteria in its natural environment.     

Surface Translocation by Legionella pneumophila: a Form of Sliding Motility That Is Dependent upon Type II Protein Secretion

Legionella pneumophila exhibits surface translocation when it is grown on a buffered charcoal yeast extract (BCYE) containing 0.5 to 1.0% agar. After 7 to 22 days of incubation, spreading legionellae appear in an amorphous, lobed pattern that is most manifest at 25 to 30°C. All nine L. pneumophila strains examined displayed the phenotype. Surface translocation was also exhibited by some, but not all, other Legionella species examined. L. pneumophila mutants that were lacking flagella and/or type IV pili behaved as the wild type did when plated on low-percentage agar, indicating that the surface translocation is not swarming or twitching motility. A translucent film was visible atop the BCYE agar, advancing ahead of the spreading legionellae. Based on its abilities to disperse water droplets and to promote the spreading of heterologous bacteria, the film appeared to manipulate surface tension and, as such, acted like a surfactant. Indeed, a sample obtained from the film rapidly dispersed when it was spotted onto a plastic surface. L. pneumophila type II secretion (Lsp) mutants, but not their complemented derivatives, were defective for both surface translocation and film production. In contrast, mutants defective for type IV secretion exhibited normal surface translocation. When lsp mutants were spotted onto film produced by the wild type, they were able to spread, suggesting that type II secretion promotes the elaboration of the Legionella surfactant. Together, these data indicate that L. pneumophila exhibits a form of surface translocation that is most akin to “sliding motility” and uniquely dependent upon type II secretion.The genus Legionella was established in 1977, following the isolation of Legionella pneumophila from patients with a form of pneumonia now known as Legionnaires'' disease (33). Today, L. pneumophila is recognized as a common cause of both community-acquired and nosocomial pneumonia (84). Legionellosis occurs sporadically and in large outbreaks, with the largest outbreak occurring as recently as 2003 and encompassing 800 suspected and 449 confirmed cases (43). L. pneumophila is especially pathogenic for the elderly and the immunocompromised, large and growing segments of the population (39, 84), and recent studies have been highlighting the growing significance of travel-associated Legionnaires'' disease (107). L. pneumophila is a gram-negative, gammaproteobacterium that is widespread in natural and manufactured water systems (22, 39, 93). Infection occurs after the inhalation of Legionella-contaminated water droplets originating from a wide variety of aerosol-generating devices (39). Alarmingly, outbreaks can occur following the airborne spread of L. pneumophila over distances of >10 km from cooling towers or scrubbers (86). Within its aquatic habitats, L. pneumophila survives over a wide temperature range and grows on surfaces, in biofilms, and as an intracellular parasite of protozoa (9, 39, 110). Within the mammalian lung, the organism has the ability to attach to and invade macrophages and epithelia (27, 106, 113). Among the processes that promote L. pneumophila growth in both the environment and the mammalian lung are Lsp type II protein secretion, Dot/Icm type IVB protein secretion, and Lvh type IVA protein secretion (5, 25, 31, 106). Other key surface features of L. pneumophila are polar flagella that promote swimming motility and type IV pili that help mediate adherence (53, 103, 113). In addition to exporting proteins onto its surface into the extracellular milieu, and/or into host cells, L. pneumophila also secretes a siderophore and pyomelanin pigment that help mediate iron assimilation (23). We now report that L. pneumophila has the ability to translocate or spread across an agar surface. This new form of Legionella “motility” did not require the action of flagella, pili, or type IV secretion but was associated with the export of a surfactantlike material and an intact type II secretion system.     

The surfactant of Legionella pneumophila Is secreted in a TolC-dependent manner and is antagonistic toward other Legionella species

When Legionella pneumophila grows on agar plates, it secretes a surfactant that promotes flagellum- and pilus-independent "sliding" motility. We isolated three mutants that were defective for surfactant. The first two had mutations in genes predicted to encode cytoplasmic enzymes involved in lipid metabolism. These genes mapped to two adjacent operons that we designated bbcABCDEF and bbcGHIJK. Backcrossing and complementation confirmed the importance of the bbc genes and suggested that the Legionella surfactant is lipid containing. The third mutant had an insertion in tolC. TolC is the outer membrane part of various trimolecular complexes involved in multidrug efflux and type I protein secretion. Complementation of the tolC mutant restored sliding motility. Mutants defective for an inner membrane partner of TolC also lacked a surfactant, confirming that TolC promotes surfactant secretion. L. pneumophila (lspF) mutants lacking type II protein secretion (T2S) are also impaired for a surfactant. When the tolC and lspF mutants were grown next to each other, the lsp mutant secreted surfactant, suggesting that TolC and T2S conjoin to mediate surfactant secretion, with one being the conduit for surfactant export and the other the exporter of a molecule that is required for induction or maturation of surfactant synthesis/secretion. Although the surfactant was not required for the extracellular growth, intracellular infection, and intrapulmonary survival of L. pneumophila, it exhibited antimicrobial activity toward seven other species of Legionella but not toward various non-Legionella species. These data suggest that the surfactant provides L. pneumophila with a selective advantage over other legionellae in the natural environment.     

Rab41 Is a Novel Regulator of Golgi Apparatus Organization That Is Needed for ER-To-Golgi Trafficking and Cell Growth

Background

The 60⁺ members of the mammalian Rab protein family group into subfamilies postulated to share common functionality. The Rab VI subfamily contains 5 Rab proteins, Rab6a/a’, Rab6b, Rab6c and Rab41. High-level knockdown of Rab6a/a’ has little effect on the tightly organized Golgi ribbon in HeLa cells as seen by fluorescence microscopy. In striking contrast, we found Rab41 was strongly required for normal Golgi ribbon organization.

Methods/Results

Treatment of HeLa cells with Rab41 siRNAs scattered the Golgi ribbon into clustered, punctate Golgi elements. Overexpression of GDP-locked Rab41, but not wild type or GTP-locked Rab41, produced a similar Golgi phenotype. By electron microscopy, Rab41 depletion produced short, isolated Golgi stacks. Golgi-associated vesicles accumulated. At low expression levels, wild type and GTP-locked Rab41 showed little concentration in the Golgi region, but puncta were observed and most were in ruffled regions at the cell periphery. There was 25% co-localization of GTP-locked Rab41 with the ER marker, Sec61p. GDP-locked Rab41, as expected, displayed an entirely diffuse cytoplasmic distribution. Depletion of Rab41 or overexpression of GDP-locked Rab41 partially inhibited ER-to-Golgi transport of VSV-G protein. However, Rab41 knockdown had little, if any, effect on endosome-to-Golgi transport of SLTB. Additionally, after a 2-day delay, treatment with Rab41 siRNA inhibited cell growth, while overexpression of GDP-locked Rab41, but not wild type or GTP-locked Rab41, produced a rapid, progressive cell loss. In double knockdown experiments with Rab6, the Golgi ribbon was fragmented, a result consistent with Rab41 and Rab6 acting in parallel.

Conclusion

We provide the first evidence for distinctive Rab41 effects on Golgi organization, ER-to-Golgi trafficking and cell growth. When combined with the evidence that Rab6a/a’ and Rab6b have diverse roles in Golgi function, while Rab6c regulates mitotic function, our data indicate that Rab VI subfamily members, although related by homology and structure, share limited functional conservation.     

Actin Recruitment to the Chlamydia Inclusion Is Spatiotemporally Regulated by a Mechanism That Requires Host and Bacterial Factors

The ability to exit host cells at the end of their developmental growth is a critical step for the intracellular bacterium Chlamydia. One exit strategy, extrusion, is mediated by host signaling pathways involved with actin polymerization. Here, we show that actin is recruited to the chlamydial inclusion as a late event, occurring after 20 hours post-infection (hpi) and only within a subpopulation of cells. This event increases significantly in prevalence and extent from 20 to 68 hpi, and actin coats strongly correlated with extrusions. In contrast to what has been reported for other intracellular pathogens, actin nucleation on Chlamydia inclusions did not ‘flash’, but rather exhibited moderate depolymerization dynamics. By using small molecule agents to selectively disrupt host signaling pathways involved with actin nucleation, modulate actin polymerization dynamics and also to disable the synthesis and secretion of chlamydial proteins, we further show that host and bacterial proteins are required for actin coat formation. Transient disruption of either host or bacterial signaling pathways resulted in rapid loss of coats in all infected cells and a reduction in extrusion formation. Inhibition of Chlamydia type III secretion also resulted in rapid loss of actin association on inclusions, thus implicating chlamydial effector proteins(s) as being central factors for engaging with host actin nucleating factors, such as formins. In conclusion, our data illuminate the host and bacterial driven process by which a dense actin matrix is dynamically nucleated and maintained on the Chlamydia inclusion. This late stage event is not ubiquitous for all infected cells in a population, and escalates in prevalence and extent throughout the developmental cycle of Chlamydia, culminating with their exit from the host cell by extrusion. The initiation of actin recruitment by Chlamydia appears to be novel, and may serve as an upstream determinant of the extrusion mechanism.     

Caenorhabditis elegans as an Alternative Model Host for Legionella pneumophila,and Protective Effects of Bifidobacterium infantis

The survival times of Caenorhabditis elegans worms infected with Legionella pneumophila from day 7.5 or later after hatching were shorter than those of uninfected worms. However, nematodes fed bifidobacteria prior to Legionella infection were resistant to Legionella. These nematodes may act as a unique alternative host for Legionella research.Legionella pneumophila, an environmental bacterium naturally found in fresh water, is the major causative agent of Legionnaires'' disease (7). Fresh water amoebas, a natural host of Legionella, have been used as an infection model to study invasion of Legionella into human macrophages and subsequent intracellular growth (15). However, analyses using these protozoa have inevitably concentrated on the intracellular lifestyle of L. pneumophila. The fate of Legionella organisms in nonmammalian metazoans had not been described (10) until a very recent report by Brassinga et al. (6).Numerous authors have reported Caenorhabditis elegans to be a suitable model to investigate virulence-associated factors of human pathogens (2, 8, 11, 14, 16, 20, 23, 24, 30, 31, 33). In the present study, we examined whether C. elegans can serve as an alternative host for L. pneumophila. Although the nematocidal activity of Legionella has been described recently, the nematodes in the previous study were infected with the pathogen on buffered charcoal yeast extract (BCYE) agar plates, which can support Legionella growth (6). In contrast, our experiments were independently performed on simple agar plates to exclude the possibility that the inoculated pathogen would have proliferated regardless of whether it had successfully infected the nematodes and derived nutrition from the hosts. Garsin et al. showed that nutrition available in agar plates does influence the virulence of pathogens on the medium (9). Furthermore, some pathogens produce toxic metabolites on nutrient medium in situ (3), and thus, we also avoided this possibility. Moreover, we focused on the effects of worm age, since Legionella is prone to infect elderly people.Age at infection is likely one of the most important determinants of disease morbidity and mortality (18). Since Legionella organisms are prone to infect elderly people opportunistically, infections in young and older nematodes were compared. Furthermore, survival curves were compared between worms fed Escherichia coli OP50 (OP), an international standard food for these organisms, and those fed bifidobacteria prior to infection with Legionella organisms, since lactic acid bacteria exert beneficial effects on human and animal health (21).