Update on Legionnaires’ disease: pathogenesis, epidemiology, detection and control

doi:10.1111/j.1365-2958.2010.07086.x

Mol Microbiol. Author manuscript; available in PMC 2011 April 1.

Published in final edited form as:

Mol Microbiol. 2010 April; 76(1): 1–11.

Published online 2010 February 10. doi: 10.1111/j.1365-2958.2010.07086.x

PMCID: PMC2914503

NIHMSID: NIHMS219258

Update on Legionnaires’ disease: pathogenesis, epidemiology, detection and control

Hubert Hilbi,¹ Sophie Jarraud,² Elizabeth Hartland,³ and Carmen Buchrieser^4,⁵^*

¹Institute of Molecular Life Sciences, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland

²Centre National de Référence des Legionella, Université de Lyon, INSERM U851, Faculté de Médecine, IFR 128, Lyon, France

³Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia

⁴Institut Pasteur, Biologie des Bactéries Intracellulaires, Paris, France

⁵CNRS URA 2171, Paris, France

^* For correspondance: Institut Pasteur Biologie des Bactéries Intracellulaires 28, rue du Dr. Roux, 75724 Paris Cedex 15, France Tel: (33-1)-45-68-83-72 Fax: (33-1)-45-68-87-86 ; Email: carmen.buchrieser/at/pasteur.fr

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Summary

Legionellosis or Legionnaires’ disease is an emerging and often-fatal form of pneumonia that is most severe in elderly and immunocompromised people, an ever-increasing risk group for infection. In recent years, the genomics of Legionella spp. has significantly increased our knowledge of the pathogenesis of this disease by providing new insights into the evolution and genetic and physiological basis of Legionella–host interactions. The 7^th international conference on Legionella, Legionella 2009, illustrated many recent conceptual advances in epidemiology, pathogenesis and ecology. Experts in different fields presented new findings on basic mechanisms of pathogen–host interactions and bacterial evolution, as well as the clinical management and environmental prevalence and persistence of Legionella. The presentations revealed remarkable facts about the genetic and metabolic basis of the intracellular lifestyle of Legionella and reported on its striking ability to manipulate host cell processes by molecular mimicry. Together, these investigations will lead to new approaches for the treatment and prevention of Legionnaires’ disease.

Keywords: Legionella, pathogenesis, epidemiology, detection, control

Introduction

It is 32 years since Legionella pneumophila was recognized as a human pathogen and the cause of a severe pneumonia known as Legionnaires’ disease (McDade et al., 1977). The discovery that L. pneumophila is ubiquitous in aquatic environments and exists as an intracellular parasite of protozoa has provided a link between bacterial ecology and human disease (Rowbotham, 1980) (Figure 1). As Legionella infection is not spread between humans (Fields et al., 2002), environmental monitoring of potable water, cooling towers and related sources is crucial to control the incidence of disease. Today, more than 50 different Legionella species have been described, and the number is steadily growing, as illustrated by the recent description of a new genus, Legionella dresdenensis sp. nov., which was isolated from the river Elbe near Dresden in Germany (Luck et al., 2009).

International meetings on Legionella are organized every four years to communicate new insights into its biology and the management and prevention of human infection. The 7th International Conference in this series, Legionella 2009, was held in Paris, at the Institut Pasteur from October 13-17, 2009. The aim was to discuss the latest research findings on Legionella spp. and on improved methods for the control of Legionella infections in humans. A wide range of current research fields was covered, such as epidemiology and clinical aspects, pathogenesis and immunology, genetics and genomics, ecology and evolution, physiology, regulation, and biochemistry. The attendance of 575 participants from over 50 countries underscored the high interest and relevance of the topic. Excellent scientific presentations and many informal discussions during the four-day meeting contributed to a broader understanding of Legionella biology and virulence. In the following sections, we will highlight many of the new and interesting results presented at the meeting.

Epidemiology and clinical aspects of Legionnaires disease

Legionnaires’ disease (LD) is a serious and potentially life-threatening illness. Elderly adults, smokers and people with weakened immune systems are particularly susceptible. The first part of the conference covered clinical aspects of LD with a specific focus on immunocompromised patients. A prospective study undertaken by Pedro-Botet (Hospital Germans trias I Pujol, Badalona, Spain) that included 157 immunocompromised (IC) and 302 control patients showed that hospital-acquired LD was significantly more frequent in IC patients. In addition, clinical complications and respiratory failure were more frequent and mortality was higher in IC and severely ill patients (unpublished results). Similar results were reported in several oral and poster presentations showing that the predisposing factors for LD have not changed over the years. Mixed infections with different strains of the same species have been studied in chronic infections, such as those caused by Mycobacterium tuberculosis or Helicobacter pylori. Mireia Coscolla (University of Valencia, Paterna, Spain) presented evidence that different L. pneumophila variants can be present simultaneously during acute LD (unpublished results). This might be due to co-infection with different strains from the same environmental source or, alternatively, to two independent infections in a very short period.

In order to identify and trace the strains causing LD, it is important to identify and type Legionella strains in the patient and the environment rapidly and accurately. Real-time PCR assays allow rapid diagnosis of LD and are of great importance in elucidating the role of pathogenic non-pneumophila Legionella species. Massimo Mentasti (Health Protection Agency, London, UK) showed that the sensitivity of newly developed real-time PCR assays for detection and typing of Legionella in respiratory samples was superior to classical culture methods. An exception is the urinary antigen detection test, but this detects only L. pneumophila serogroup 1. In conclusion, real-time PCR coupled with Sequence-Based Typing applied directly to respiratory samples is a valuable tool to diagnose the disease rapidly, allowing timely and effective treatment (Coscolla and Gonzalez-Candelas, 2009; Ginevra et al., 2009).

One major point discussed was the efficacy of current surveillance schemes used for detection of travel-associated LD that has been developed by the European Working Group for Legionella Infections (EWGLINET) since 1987 and by the CDC in the USA since 2005. These surveillance methods resulted in a substantial increase in the detection of travel-associated LD in Europe and USA (85% from 2005 to 2008). Frances Graham (University of Canterbury, Wellington, New Zealand) reported the distinctive legionellosis epidemiology in New Zealand and Australia, where L. longbeachae and L. pneumophila are equally prevalent in LD, in contrast to the rest of the world, where L. pneumophila serogroup 1 predominates. .

Typing and detection for epidemiological surveillance and rapid diagnosis

Comparative genomics recently showed that L. pneumophila strain Paris is a worldwide epidemic and endemic clone (Cazalet et al., 2008). Timothy Harrison (Health Protection Agency, London, UK) reported and EWGLI global picture of strain distribution in human disease and the environment (unpublished results). Data for 3200 isolates from 36 countries distinguished around 700 distinct sequence types (ST) within the species L. pneumophila. Strains belonging to ST1 were most frequent in human disease (12.7%) and present in 18 different countries. Other common STs included ST47, ST23, ST42, ST62 and ST37. While ST1 accounted for 21.4% of environmental isolates and only 7.4% of clinical isolates, ST47 and ST23 accounted for 13.6% and 10.7% of clinical isolates but only 0.4% and 1.5% of environmental isolates. Interestingly, more than 50% of the isolates belonged to just 14 STs. These studies confirm that certain environmental isolates of L. pneumophila are more likely to cause disease than others (Borchardt et al., 2008; Cazalet et al., 2008; Ginevra et al., 2008; Harrison et al., 2009; Kozak et al., 2009). Understanding the molecular basis for these differences represents an important challenge and could lead to more precise definitions of and discrimination between environmental and highly pathogenic strains.

The different prevalence of STs in clinical and environmental isolates was also examined by Jerome Etienne (National Centre for Legionella, Lyon, France), who reported on the possible impact of host-related risk factors on strain distribution. Elderly, immunocompromised women (under immunosuppressive therapies or with a history of cancer) were found to be more frequently infected with the L. pneumophila Paris clone (ST1 or ST1-related isolates), compared to sporadic isolates. The particular tropism of the Paris clone for immunocompromised patients might explain why more than 30% of hospital-acquired LD in France is caused by this strain. In contrast, the L. pneumophila Lorraine clone (ST47, MAb France/Allentown) is mainly responsible for sporadic, community-acquired cases. This study clearly shows that both host-related risk factors and strain specific features contribute to LD.

Valeria Gaïa (Instituto Cantonale di Microbiologia, Bellinzona, Switzerland) presented the use of MALDI-TOF MS (matrix assisted laser desorption ionization - time of flight mass spectrometry) to identify Legionella species, which is a potentially interesting approach for diagnosis in clinical samples. However, quantitative PCR (qPCR) appears to be the method of choice to monitor Legionella in water systems. The interpretation of PCR results from environmental samples remains a challenge, since PCR methods do not differentiate DNA from live and dead bacteria, leading to false positive results. An international multi-centre study presented by John V. Lee (Health Protection Agency, London, UK) used qPCR to monitor Legionella in artificial water systems and reported the first qPCR thresholds (unpublished results). Patrick Yang (CDC, Atlanta, USA) described the development of a Real Time Transcription-Mediated Amplification (TMA) assay that preferentially amplifies RNA (present only in viable cells) and showed that compared to culture, the sensitivity and specificity of the TMA assays were 100% (29/29) and 82.9% (68/82), respectively (unpublished results). Detection and quantification of viable Legionella cells though combined use of ethidium monoazide (EMA) or propydium monoazide (PMA) staining and real-time PCR was proposed by several other teams in the poster session. These new results give us hope that new, rapid and accurate techniques will soon be developed for better surveillance of Legionella in water.

Host response and susceptibility to Legionella infection

The host response and susceptibility to L. pneumophila was examined at the meeting in the context of both human disease and mouse models of infection. Tom Hawn (University of Washington, Seattle, USA) presented data obtained in collaboration with Annelies Verbon on the genetic susceptibility of humans to LD using patient data from the large flower show outbreak in the Netherlands in 1999. Besides the common polymorphism in TLR5 that introduces a premature stop codon leading to a 2-fold increased risk of LD (Hawn et al., 2003), polymorphism (T359C) in TLR6, which recognises bacterial lipoproteins, was also strongly associated with LD, even though there did not seem to be a functional consequence of this polymorphism in terms of cytokine induction in vitro (unpublished data). Further investigation of SNPs that affect innate immune function, including the identification of further TLR and Nod variants, might shed more light on the genetic susceptibility of humans to LD.

In contrast to macrophages, dendritic cells undergo rapid apoptosis upon infection with L. pneumophila. Catarina Noguiera and Craig Roy (Yale University, New Haven, USA) showed how the cell death pathways are activated in dendritic cells. Bax/Bak are pro-apoptotic mediators that induce cytochrome C release, caspase activation and cell death, while Bcl-2 is an anti-apoptotic/pro-survival protein. Dendritic cells prepared from Bax/Bak-deficient mice or Bcl-2 overproducing mice support L. pneumophila replication by inhibiting apoptosis, but do not impact on bacterial replication in macrophages (Nogueira et al., 2009). A possible role for apoptosis in vivo was supported by data obtained using Bcl-2 overproducing mice infected with L. pneumophila. These mice exhibited increased bacterial loads in the lungs, spleen and lymph nodes compared to wild-type mice (unpublished data). In addition, L. pneumophila producing the anti-apoptotic effector AnkG from Coxiella burnetii replicated robustly and inhibited apoptosis in dendritic cells (unpublished data). Together, these results suggest that apoptosis might be an additional innate immune response that contributes to the restriction of L. pneumophila replication in the host.

A longstanding theory on the mechanism of contact-dependent cytotoxicity mediated by L. pneumophila at high multiplicities of infection was challenged by work from Tatiana Silveira and Dario Zamboni (University of São Paulo, São Paulo, Brazil). Pore formation was originally thought to result from the insertion of multiple Dot/Icm channels in the host membrane upon bacterial contact (Kirby et al., 1998). Tatiana Silveira presented evidence suggesting that, in mammalian cells at least, the pores result from activation of the host cell inflammasome in response to bacterial flagellin (Silveira and Zamboni, 2010). This conclusion was based on the fact that L. pneumophila mutants and other Legionella species lacking flagellin do not induce pore formation and that caspase-1 deficient murine macrophages are resistant to pore formation regardless of Dot/Icm activity (Silveira and Zamboni, 2010). Thus, activation of the inflammasome appears largely responsible for a key virulence trait of L. pneumophila.

Further work on mouse susceptibility to L. pneumophila infection by Juliane Lippmann and Bastian Opitz (Charité University Medicine, Berlin, Germany) examined the molecular basis of type I interferon restriction of L. pneumophila replication. Endogenous type I interferon suppresses L. pneumophila replication in epithelial cells and macrophages, and macrophages derived from type I interferon receptor-deficient mice are permissive for L. pneumophila replication. Type I IFN production in Legionella-infected macrophages was dependent on IRF3, but independent of MyD88 (Lippmann et al., 2008; Monroe et al., 2009). IRF3 deficiency enhances L. pneumophila replication (unpublished data). The type I interferon dependent immunity regulated GTPases (IRGs) are also induced by L. pneumophila and type I IFN-dependent bacterial growth restriction is less efficient in cells lacking IRGM1 (unpublished data). Despite this interesting host-pathogen interaction, type I interferon signalling does not seem to play a role in suppressing L. pneumophila replication in mice (Monroe et al., 2009).

Pathogen-host interactions

The biogenesis of the unusual Legionella-containing vacuole (LCV) and the consequences of L. pneumophila infection for the host cell were examined in several presentations. After uptake by macrophages, L. pneumophila avoids endosome fusion and actively intercepts vesicle trafficking in the secretory pathway of the infected cell. The critical role of the Dot/Icm type IV secretion system (T4SS) in LCV biogenesis and intracellular replication of L. pneumophila continues to stimulate immense research interest in mechanisms of substrate secretion and translocation and in the cell biology of Dot/Icm effectors. Joseph Vogel (Washington University, St Louis, USA) presented evidence that some Dot/Icm effectors contain two C-terminal secretion signals and proposed that these two distinct sequences provide temporal regulation of effector translocation (unpublished data). Both Youssef Abu Kwaik (University of Louisville, Buffalo, USA) and Mariella Lomma and Carmen Buchrieser (Pasteur Institute, Paris, France) presented new work on the importance of the host cell ubiquitination pathway for LCV biogenesis. Several Dot/Icm effectors translocated into infected cells carry F-box domains, which in eukaryotic cells mediate the interaction of a protein substrate with the host cell ubiquitination machinery, the SCF (Skp1/Cullin1/F-box) ubiquitin ligase complex. Both groups found that the SCF machinery is needed for LCV biogenesis and L. pneumophila replication, and that the Dot/Icm F-box protein AnkB/Lpp2082 mediates an interaction with Skp1 and induces the ubiquitination of proteins around the LCV (Price et al., 2009; unpublished results). The Buchrieser lab further found that Lpp2082 is needed for virulence in mice and that Lpp2082 induces the de-ubiquitination of a focal adhesion protein ParvB, which was important for L. pneumophila replication (unpublished results).

Further developments in LCV biogenesis were presented by Zhao Qing Luo (Purdue University, Indiana, USA). Despite the fact that the LCV largely avoids interaction with endosomes and maintains a neutral pH, several components of the vacuolar ATPase, usually a late endosomal marker, are known to be associated with the LCV (see below). To examine the recruitment and function of the v-ATPase on the LCV, the Luo lab screened for Dot/Icm proteins that are toxic to yeast at neutral pH. They identified SidK, which interacts directly with the v-ATPase and inhibits ATP hydrolysis, although the vacuolar pH does not change in cells infected with a ΔsidK, mutant suggesting that other effectors are involved in pH maintenance (unpublished results).

Hubert Hilbi (University of Zürich, Zürich, Switzerland) provided an update on L. pneumophila effector proteins that bind to phosphoinositide (PI) lipids, which are pivotal regulators of eukaryotic signal transduction and membrane dynamics. The PI-binding effectors anchor via mono-phosphorylated PIs to the LCV membrane, where they recruit ER vesicles (SidC), function as guanine nucleotide exchange factor (GEF) for the Rab1 GTPase (SidM), or interact with the PI phosphatase OCRL1 (LpnE) (Weber et al., 2009).

New Dot/Icm effectors were introduced by Elizabeth Hartland (University of Melbourne, Melbourne, Australia). In collaboration with Trevor Lithgow (Monash University, Melbourne, Australia), the Hartland lab showed that L. pneumophila produces a protein with all the hallmarks of a mitochondrial carrier protein (unpublished data). Since mitochondrial carrier proteins are believed to be exclusive to eukaryotes and critical to the evolution of mitochondria from prokaryotes, the function of this Dot/Icm effector in L. pneumophila-host interactions might turn out to be very interesting. E. Hartland also presented an update on the CD39 family of ecto-nucleoside triphosphate diphosphohydrolases (NTPDase) and showed that Lpg1905 from L. pneumophila is a structural and functional mimic of mammalian NTPDases (Vivian et al., 2009). This member of the L. pneumophila eukaryotic proteins hydrolyses both ATP and GTP, and this activity is important for L. pneumophila replication within the LCV and for mouse lung infection (Sansom et al., 2008).

The issue of effector redundancy was tackled by Tamara O’Connor and Ralph Isberg (Tufts University, Boston, USA). Their previous work showed that an L. pneumophila mutant lacking 46 Dot/Icm substrates did not exhibit any replication defect and that LCV biogenesis depends on intercepting vesicles trafficking in at least two pathways. L. pneumophila replication is severely affected in cells with reduced levels of both Sec22 and Bet5, (Dorer et al., 2006). The Isberg lab used RNAi to silence one trafficking pathway while screening for mutants defective for replication. The identification of one essential L. pneumophila gene in each pathway led to the construction of double mutants that showed a replication defect in wild type amoebae, though not in macrophages (unpublished data). Nevertheless, this approach lends substantial weight to the argument that the LCV interacts with vesicles from different sources and that subsets of effector proteins act on specific trafficking pathways.

Craig Roy (Yale University, New Haven, USA) presented investigations into the spatial and temporal regulation of Dot/Icm effector function. The first Dot/Icm effector, RalF, which was identified by the Roy lab, has a eukaryotic Sec7 domain that functions as a guanine exchange factor (GEF) for the host GTPase, Arf1 (Nagai et al., 2002). The C-terminus of RalF contains a 20 amino acid translocation signal that is connected to a novel structural region that appears to cap the active site of the GEF and mediate subcellular localization of the effector (Amor et al., 2005). Other effectors, DrrA/SidM, Lpg2603 and Lpg1101, were shown to have a conserved C-terminus that mediates localization of GFP fusion proteins to the host plasma membrane when the gene fusions are expressed ectopically in mammalian cells. Conserved amino acid substitutions at invariant residues in this region abolished plasma membrane localization of these effectors, suggesting that this region is involved in localizing these effectors to the plasma membrane-derived vacuole in which L. pneumophila initially resides (unpublished data). Thus, in addition to contributing to Dot/Icm-mediated translocation into host cells, the C-terminus of these proteins might determine subcellular localization and spatial regulation of effector protein function.

Dot/Icm dependent MAPK signalling is induced upon infection of macrophages (Shin et al., 2008), although the consequences of MAPK activation for L. pneumophila replication are unknown. Ralf Isberg (Tufts University, Boston, USA) reported that L. pneumophila inhibits the equivalent of the MAPK pathway in amoebae by inducing expression of DupA, a tyrosine kinase/dual specificity phosphatase that is likely a negative regulator of MAPK signalling (Li et al., 2009). (Li et al., 2009). Ralf Isberg also reported that the Sel1-repeat protein EnhC is required to maintain cell wall integrity and that ΔenhC mutants leak peptidoglycan by-products during macrophage infection, thereby stimulating Nod1 (unpublished data). This phenomenon explains why ΔenhC mutants exhibit retarded growth 24 h after infection in macrophages but not amoebae, which lack Nod1 (Liu et al., 2008). EnhC interacts with Lpg0663, a soluble lytic transglycosylase (Slt) involved in peptidoglycan synthesis and inhibits its activity (unpublished data). In other ongoing work, the Isberg lab is using serial passage over multiple generations to test the idea that L. pneumophila growth in amoebae has simultaneously selected for growth in macrophages. Interestingly, the results so far suggest that optimal growth in amoebae does not confer optimal growth in macrophages (unpublished data).

Metabolism and virulence

In the first talk in the session on secretion systems, Nicholas Cianciotto (Northwestern University, Chicago, USA) provided an update on the L. pneumophila type II secretion system (T2SS) Lsp that is implicated in virulence and in nutrient acquisition. The L. pneumophila type II “secretome” consists of more than 25 proteins, including various degradative enzymes such as phospholipases, proteases, a ribonuclease and a chitinase. N. Cianciotto presented recent findings on the role of the Lsp T2SS in virulence, survival at low temperatures and surface movement of L. pneumophila. Surface translocation of the bacteria coincides with the production of a translucent film and is dependent on the Lsp system, but not on the twin arginine transporter (Tat) or Dot/Icm system (Stewart et al., 2009). Furthermore, this type of movement does not require flagella or type IV pili that are needed for “swarming” or “twitching motility”. Uncovering how surface translocation works will be an interesting and important challenge.

Secreted L. pneumophila phospholipases were reviewed by Antje Flieger (Robert Koch Institute, Wernigerode, Germany). L. pneumophila produces at least 15 enzymes with phospholipase and lysophospholipase activity. The enzymes are grouped into the Lsp-secreted GDSL hydrolases (e.g. PlaA, PlaC), the Dot/Icm-translocated patatin-like hydrolases (e.g. VipD) and the PlaB family, a novel class of bacterial lyso/phospholipases. Recent work from the Flieger group characterized PlaB as a cell-associated haemolytic phospholipase A with a catalytic serine-aspartate-histidine triad (Bender et al., 2009). PlaB hydrolyzes the eukaryotic membrane constituent phosphatidylcholine, suggesting a role for the enzyme in bacterial virulence.

L. pneumophila exhibits a biphasic life cycle characterized by physiologically and morphologically distinct states; i.e., the bacteria alternate between a replicative, non-motile form and an infectious, flagellated form. Insight into physiological aspects of the life cycle was summarized by Michele Swanson (University of Michigan Medical School, Ann Arbor, USA, whose group demonstrated that the transition from the replicative to the transmissive state responds to metabolic cues, including shortage of amino acids and inhibition of fatty acid biosynthesis (Edwards et al., 2009). The transition is regulated by the “stringent response” pathway through guanosine 3′,5′ bipyrophosphate (ppGpp). Formation of this second messenger requires the two ppGpp synthases RelA and SpoT, which respond to amino acid starvation or inhibition of fatty acid biosynthesis, respectively (Dalebroux et al., 2009).

Morphological aspects of the L. pneumophila developmental cycle were covered by Paul Hoffman (University of Virginia Health Systems, Charlottesville, USA). At late stages during infection of host cells, L. pneumophila differentiates into a resilient, cyst-like form termed “mature intracellular form” (MIF), which contains poly-β-hydroxybutyrate (PHB) storage granules and is highly infectious. Recently, the small DNA-binding regulatory protein integration host factor (IHF) was found to be required for MIF formation in a HeLa cell model and to promote replication in the fresh water amoeba Acanthamoeba castellanii (Morash et al., 2009).

L. pneumophila not only responds to metabolic cues to regulate virulence and transmission, but also targets host cell basic metabolism and protein biosynthesis. In his talk, Yury Belyi (Gamaleya Research Institute, Moscow, Russian Federation) reported that L. pneumophila produces three glucosyltransferases (Lgt1-3) that are differentially regulated and, thus, might play specific roles in L. pneumophila virulence. Lgt-1 efficiently O-glucosylates the eukaryotic elongation factor 1A (eEF1A) using UDP-glucose as a co-substrate. More recently, Lgt-1 was also found to glucosylate the translation factor Hsp70 subfamily B suppressor (Hbs1) (Belyi et al., 2009). These modifications of components of the translation machinery inhibit protein biosynthesis in the target cell and cause its subsequent death.

Pathogen-environment interactions

L. pneumophila evolved to persist and replicate in different niches, including protozoa, nematodes and biofilms. The social soil amoeba Dictyostelium discoideum has emerged as a powerful, genetically tractable model to study interactions with L. pneumophila. Olga Shevchuk from the group of Michael Steinert (Technical University Braunschweig, Braunschweig, Germany) presented results from a proteomic analysis of Legionella-containing vacuoles (LCVs) in D. discoideum amoebae (Shevchuk et al., 2009). 2D gel electrophoresis and MALDI-TOF MS identified 137 LCV host proteins, including protein kinase C inhibitor (PkiA), cysteine protease inhibitor (CpiA) and subunits of the vacuolar proton ATPase, the latter of which is targeted by the L. pneumophila effector protein SidK (see above). Furthermore, comparative proteomics of LCVs harbouring either amoebae-resistant L. pneumophila Corby or amoebae-sensitive L. hackeliae revealed distinct protein patterns, such as a preferential association of Rho GDP dissociation inhibitor (RdiA) or the 14-3-3 protein (FttB) on L. hackeliae LCVs. A proteome analysis of isolated LCVs was also reported by Hubert Hilbi (University of Zürich, Zürich, Switzerland). Here, LCVs in D. discoideum were purified by immuno-magnetic separation using a primary antibody against an Icm/Dot substrate exclusively localizing to LCVs and a secondary antibody coupled to magnetic beads (Urwyler et al., 2009). The proteome was determined by liquid chromatography/ tandem ESI MS and revealed more than 560 host proteins, including a number of small GTPases implicated in endosomal or secretory vesicle trafficking. Thus, LCVs communicate not only with the early secretory pathway, but also with the late secretory pathway and with early and late endosomal trafficking pathways.

Many different species of amoebae and ciliated protozoa are natural hosts of L. pneumophila. Raphael Garduño (Dalhousie University, Halifax, Canada) introduced the fresh water ciliate Tetrahymena tropicalis as a model to study differentiation and dissemination of L. pneumophila. L. pneumophila exhibits Dot/Icm T4SS-dependent survival but does not replicate in T. tropicalis food vacuoles, and the bacteria are expelled in resilient, infectious pellets (Berk et al., 2008). Furthermore, stationary phase bacteria exhibit Dot/Icm-independent differentiation into MIFs during transit through T. tropicalis (Faulkner et al., 2008).

A search for predominant and novel protozoan hosts for L. pneumophila was reported by Rinske Valster from the laboratory of Dick van der Kooij (KWR Watercycle Research Institute, Nieuwegein, The Netherlands). Growth of L. pneumophila was used to amplify and identify putative host protozoa from different water types all related to engineered water systems in temperate regions (unpublished results). Hartmannella vermiformis was confirmed as a predominant host for L. pneumophila in the tested water types. In addition, 18S rRNA gene sequence analysis revealed predominance of Echinamoeba thermarum and Diphylleia rotans in several tests with growth of L. pneumophila, while Sphaeroeca volvox was identified as a protozoan that does not sustain L. pneumophila growth. An extensive screen for L. pneumophila-resistant protozoa in environmental samples, presented by Howard Shuman (Columbia University, New York, USA), revealed that a wide variety of morphologically and phylogenetically-distinct protozoa graze on L. pneumophila (unpublished results).

A first analysis of the interaction of L. pneumophila with nematodes was presented by Ann Karen Brassinga (University of Manitoba, Winnipeg, Canada) working in the laboratory of Costi Sifri (University of Virginia Health Systems, Charlottesville, USA). Interestingly, L. pneumophila non-invasively colonizes and replicates in the intestinal tract of Caenorhabditis elegans, leading to the death of the worm (Brassinga et al., 2009). The susceptibility to L. pneumophila is regulated by innate immune signalling pathways involving p38 mitogen-activated protein kinase and insulin/insulin growth factor-1 receptor. Survival of C. elegans is not affected by the bacterial Dot/Icm system, but L. pneumophila lacking a functional T4SS promotes germ line apoptosis. L. pneumophila is excreted in a cyst-like form that is morphologically similar to PHB-containing MIFs. Thus, nematodes might serve a natural host for L. pneumophila and as a reservoir for dissemination of a resilient, highly virulent form of the pathogen.

L. pneumophila colonizes and persists in biofilms, which are complex communities of different bacterial species embedded in an extracellular matrix. Shin-ichi Yoshida (Kyushu University, Fukuoka, Japan) reported that L. pneumophila forms biofilms more efficiently than any of the other 38 Legionella spp. tested, (Piao et al., 2006). Moreover, the morphology of L. pneumophila within biofilms varies according to the temperature: at 37 °C or 42 °C filamentous bacteria formed a thick and dense mycelial biofilm mat, while at 25 °C rod shaped bacteria produced a biofilm with a loose, pillar-like architecture. Research on biofilm colonization and formation by L. pneumophila has been hampered in the past either because ill-defined bacterial communities were used or because biofilm formation was analyzed in a complex medium, which does not recapitulate the oligotrophic state of a natural biofilm. Sophie Pécastaings and Christine Roques (Laboratory of Industrial Microbiology, Toulouse, France) introduced a new method to produce mono-species L. pneumophila biofilms in a minimal medium containing a mixture of mineral salts and a low concentration carbon source, which sustains growth of sessile but not planktonic bacteria (unpublished results). The biofilms reached a thickness of approximately 300 μm and remained stable for at least three weeks. Analysis by laser scanning confocal microscopy showed typical characteristics of 3D biofilms containing a carbohydrate matrix.

While some cell-cell interactions in the environment are beneficial, bacteria also compete and might kill each other. An interesting aspect of this topic was presented on several posters by Yann Héchard and his group (University of Poitiers, Poitiers, France). These studies showed that a peptide from Staphylococcus warneri strain RK, termed warnericin RK, displays a very narrow range of antimicrobial activity and almost exclusively kills Legionella spp. Recent studies revealed that warnericin RK is an amphiphilic, α-helical peptide that has hemolytic activity (Verdon et al., 2009). Warnericin RK has detergent-like activity and, thus, the high sensitivity of Legionella spp. towards detergents might provide a rational for the high selectivity of the toxin.

Gene regulation

The study of the regulatory networks governing the life cycle and virulence of L. pneumophila is a very active research field. Some aspects of this regulatory network were summarized by Klaus Heuner (Robert Koch Institut, Berlin, Germany), who focused on the regulation of flagella gene expression in L. pneumophila. Production of a long, monopolar flagellum is a prominent feature of virulent, transmissive Legionella. Phenotypic and transciptome analysis of regulatory mutants in the genes encoding three sigma factors (RpoN, FleQ, FliA) and a two-component system (FleR/FleS), revealed that FleQ (sigma 54) is the master regulator of flagella gene expression and that it regulates gene expression RpoN-dependent and RpoN-independent. FliA induces expression of late flagellar genes leading to the complete synthesis of the flagellum (Albert-Weissenberger et al., 2007; Sahr et al., 2009).

With respect to the L. pneumophila life cycle, flagella gene expression is one of the last steps. Important regulatory elements upstream are a two-component system, LetA/LetS (Hammer et al., 2002) and the RNA-binding protein CsrA (Fettes et al., 2001; Molofsky and Swanson, 2003). Tobias Sahr and Carmen Buchrieser (Institut Pasteur, Paris, France) showed that these two important regulators are linked through two small non-coding RNAs (ncRNA), RsmY and RsmZ (Sahr et al., 2009). Expression of RsmY and RsmZ is induced by LetA and then these small ncRNAs sequester CsrA to release it from their targets. These are the first ncRNAs described in L. pneumophila and together with the results obtained by Rasis and Segal (Rasis and Segal, 2009), who identified in addition targets of CsrA, this establishes their crucial importance for L. pneumophila virulence.

Another element of the L. pneumophila stationary phase gene regulatory network is the lqs (Legionella quorum sensing) gene cluster. This locus encodes the autoinducer synthase LqsA, the putative cognate sensor kinase LqsS and the response regulator LqsR. LqsA is a pyridoxal-5′-phosphate-dependent enzyme that produces a diffusible α-hydroxyketone signalling molecule (3-hydroxy-pentadecan-4-one) termed LAI-1 (Legionella autoinducer-1). André Tiaden (University of Zürich, Switzerland) presented a poster demonstrating that lqsA, lqsS and lqsR regulate phagocyte interactions, extracellular filaments and a genomic “fitness” island (Tiaden et al., 2010).

Xavier Charpentier and Howard Shuman (Columbia University, USA) presented new results regarding the response of L. pneumophila to genotoxic stress. The SOS response is used by most Gram-positive and Gram-negative bacteria to overcome DNA damage by inducing DNA repair and mutagenesis. However, genetic and phenotypic evidence suggest that L. pneumophila lacks a prototypic SOS response. First, the genomes of the four sequenced L. pneumophila strains lack lexA, the gene coding for the master regulator of the SOS response. Second, when exposed to UV radiations L. pneumophila fails to induce strong general mutagenesis, a hallmark of the SOS response. Yet, L. pneumophila does respond to UV radiation by inducing competence, a genetically programmed physiological state that confers the ability to take up DNA from the environment. Competence in L. pneumophila is also strongly stimulated by other genotoxic stresses including antibiotics of the fluoroquinolone family, used for the treatment of L. pneumophila infections. By responding to genotoxic stress with limited mutagenesis and the induction of competence, L. pneumophila favours genetic diversity over genetic variability. This parasexual strategy may have enabled L. pneumophila to acquire foreign genes and contributed to its virulence (unpublished results).

Comparative genomics and functional genomics

Genomics has the potential to provide a complete understanding of the genetics, biochemistry, physiology and pathogenesis of a microorganism. Major advances in Legionella genomics and comparative genomics have been achieved following the publication of three L. pneumophila genomes in 2004 (Cazalet et al., 2004; Chien et al., 2004), culminating in the genome comparison of five different L. longbeachae isolates. Carmen Buchrieser (Institut Pasteur, Paris, France) reported on four L. longbeachae genomes belonging to the two different serogroups. Comparison with the genomes of L. pneumophila suggested distinct interactions with host cells, as L. longbeachae possesses a unique repertoire of putative Dot/Icm type IV secretion system effectors that are eukaryotic-like proteins or contain eukaryotic domains and encodes additional secretion systems. However, the Dot/Icm type IV secretion system is also essential for the virulence of L. longbeachae, as a dotA mutant of L. longbeachae is defective in intracellular replication in A. castellanii and in a mouse lung infection model. In contrast to L. pneumophila, L. longbeachae does not encode flagella, thereby providing a possible explanation for differences in mouse susceptibility to infection between the two pathogens, and genome analysis and electron microscopy suggested that L. longbeachae is encapsulated. These species-specific differences might account for the different environmental niches and disease epidemiology for these two Legionella species (Cazalet et al., 2010). These sequences together with the sequence and analysis of a fifth L. longbeachae strain isolated in the USA, reported in a poster presented by Nataia Kozak (CDC, Atlanta, USA) revealed an intriguing feature: L. longbeachae encodes a chemotaxis system but no flagella while L. pneumophila encodes flagella but no chemotaxis system (Cazalet et al., 2010; Kozak et al., 2010). It will be an interesting aspect of future research to understand these particular features of the two main Legionella pathogens. Further genome sequences and analyses will bring important new insight into the genus Legionella, its virulence strategies, biodiversity and niche adaptation. Related to this aspect, Ivan Mozer presented the new web-resources for Legionella sequences available at the Institut Pasteur. This multi-genome browser (http://genolist.pasteur.fr/GenoList/Legionella) together with the Legionella database (http://genolist.pasteur.fr/LegioList) will be an essential and important tool for research for the entire community.

Conclusions and perspectives

Research activity in the area of genomics will continue to advance our knowledge of Legionella evolution and pathogenesis, and will provide a rich source of data for improved detection and diagnostics. Further comparative genomics and experimental work might also help to explain the prevalence of serogroup 1 L. pneumophila as a human pathogen. The new technologies presented in the area of detection and environmental control of Legionella could change the epidemiological landscape of Legionnaire’s disease. The ongoing monitoring of outbreaks, patient characteristics and patient genotypes will continue to provide insight into the circumstances, background and major risk factors for severe Legionella infection. The rapid and exciting developments in diverse aspects of the research field presented at Legionella 2009 has prompted planning for the next Legionella meeting in 2013. This meeting will be held in Melbourne, Australia and hosted by Elizabeth Hartland, University of Melbourne, together with a national organising committee.

Acknowledgements

The authors would like to thank all participants who contributed to this a wonderful meeting. We regret that space limitations do not allow us to report on every topic covered, in particular on the many excellent posters. This meeting was organized by the Institut Pasteur and was supported by EMBO, FEMS, the Institut de Veille Sanitaire, the Dim Maladie Infectieuse and the Centre National de Référence des Legionella, France. We are grateful for the generous support of many sponsors. We would like to thank our colleagues for allowing us to cite unpublished. The funding for research in the authors’ laboratories was provided by the Swiss National Science Foundation (31003A_125369) and the University of Zürich (to HH), the Institut Pasteur, the Centre National de la Recherche (CNRS), NIH Grant 2 R01 AI44212 and the Network of Excellence “Europathogenomics” LSHB-CT-2005-512061, Institut National de la Santé et de la Recherche Médicale (INSERM), University of Lyon, as well as grants to ELH from the Australian National Health and Medical Research Council (NHMRC) and Australian Research Council (ARC).

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