Molecular epidemiology, phylogeny and evolution of Legionella

    Molecular epidemiology, phylogeny and evolution of Legionella A. Khodr, E. Kay, L. Gomez-Valero, C. Ginevra, P. Doublet, C. Buchrieser, S. Jarraud PII: DOI: Reference:

S1567-1348(16)30167-8 doi: 10.1016/j.meegid.2016.04.033 MEEGID 2731

To appear in: Received date: Revised date: Accepted date:

14 February 2016 29 April 2016 30 April 2016

Please cite this article as: Khodr, A., Kay, E., Gomez-Valero, L., Ginevra, C., Doublet, P., Buchrieser, C., Jarraud, S., Molecular epidemiology, phylogeny and evolution of Legionella, (2016), doi: 10.1016/j.meegid.2016.04.033

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ACCEPTED MANUSCRIPT Molecular epidemiology, phylogeny and evolution of Legionella

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A. Khodr1,2, E. Kay3, L. Gomez-Valero1,2, C. Ginevra3,4, P. Doublet3, C. Buchrieser1,2, S. Jarraud3,4 1

Institut Pasteur, Unité de Biologie des Bactéries Intracellulaires CNRS UMR 3525, 28, Rue du Dr Roux, 75724, Paris, France 3 CIRI, International Center for Infectiology Research, Inserm, U1111 ; CNRS ; UMR5308, Université Lyon 1 ; École Normale Supérieure de Lyon, Lyon, F-69008, France. 4 French National Reference Center of Legionella, Hospices Civils de Lyon, France.

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Running title: Legionella epidemiology and evolution

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Keywords: Legionella pneumophila; phylogeny; epidemiology; experimental evolution;

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ICEs; HGT; T4ASS.

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ACCEPTED MANUSCRIPT Abstract

Legionella are opportunistic pathogens that develop in aquatic environments where they

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multiply in protozoa. When infected aerosols reach the human respiratory tract they may accidentally infect the alveolar macrophages leading to a severe pneumonia called

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Legionnaires' disease (LD). The ability of Legionella to survive within host-cells is strictly dependent on the Dot/Icm Type 4 Secretion System that translocates a large repertoire of effectors into the host cell cytosol. Although Legionella is a large genus comprising nearly 60 species that are worldwide distributed, only about half of them have been involved in LD cases. Strikingly, the species L. pneumophila alone is responsible for 90% of all LD cases.

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The present review summarizes the molecular approaches that are used for L. pneumophila genotyping with a major focus on the contribution of whole genome

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sequencing (WGS) to the investigation of local L. pneumophila outbreaks and global epidemiology studies. We report the newest knowledge regarding the phylogeny and the evolution of Legionella and then focus on virulence evolution of those Legionella species

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that are known to have the capacity to infect humans. Finally, we discuss the evolutionary forces and adaptation mechanisms acting on the Dot/Icm system itself as well as the role of mobile genetic elements (MGE) encoding T4ASSs and of gene duplications in the evolution

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of Legionella and its adaptation to different hosts and lifestyles.

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ACCEPTED MANUSCRIPT Contents

1. Introduction

3. Genotyping methods and epidemiology

3.2. Subtyping of endemic clones

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3.1. Historical typing methods and today’s standards

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2. Taxonomy and ecology of the genus Legionella

3.3. Whole genome sequencing: a new tool form molecular typing of L. pneumophila 4. Update on the phylogeny of the genus Legionella

5. Evolution of the species Legionella pneumophila and the genus Legionella

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5.1. Impact of recombination on the evolution of Legionella pneumophila 5.2. Experimental evolution of Legionella pneumophila

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5.3. Evolutionary forces acting on the Dot/Icm type IVB secretion systems in the genus Legionella

6. Adaptation of Legionella to the environment and its hosts 6.1. Role of integrative conjugative elements in the evolution of Legionella

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6.1.1. Lvh (Legionella Vir homologue) region 6.1.2. Trb/Tra family of conjugative elements

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6.1.3. Genomic Island associated T4SS family 6.2. Role of gene duplications in the adaptation of Legionella

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6.3. Specific features of Legionella influencing environmental adaptation

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7. Concluding remarks

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ACCEPTED MANUSCRIPT 1. Introduction

Legionella pneumophila is a human pathogen that was recognised only 40 years ago after a large outbreak of pneumonia during a convention of the American Legion, thus

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named Legionnaires’ disease (LD) (Fraser et al., 1977; McDade et al., 1977). Why was a bacterium that causes severe pneumonia only identified in 1977? Firstly, the development

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of man-made water systems producing aerosols (e.g. air conditioning systems, cooling towers, spas etc.) created conditions allowing the direct access of this opportunistic bacterium to human lungs. This fact is thought to have made LD an emerging disease since the 1970’s. Secondly, the fastidious growth of L. pneumophila and the requirement of L-

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cysteine and iron for growth on axenic culture media would have made it difficult to isolate this organism earlier (Feeley et al., 1978). However, the magnitude of the first recognized

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outbreak of LD in Philadelphia in 1977, its mysterious character due to the long lasting search for the causative agent of this disease that was widely reported by the media, combined with the severity of the pneumonia and the group of patients that belonged mostly to the American Legion, made this outbreak an exceptional event. Thus, very

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significant resources were employed for the epidemiological investigation to finally identify the causative agent. Interestingly, only techniques routinely used in the field of Rickettsia,

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also an intracellular bacterium, such as inoculation of both guinea pigs and yolk sacs of egg embryos, allowed Joseph McDade and his collaborators to cultivate and identify

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L. pneumophila (McDade et al., 1977). Once the bacterium was identified and isolated, sero-epidemiological studies were done, which also allowed the recognition of earlier

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outbreaks of LD but also enabled description of Pontiac fever (Blaser, 1977; McDade et al., 1979). Pontiac fever is a milder form of legionellosis, classically described as an influenzalike illness without pneumonia.

Today, L. pneumophila has been identified as one of the three most common causes of severe community-acquired pneumonia (CAP): Legionnaires’ disease accounts for 2%–8% of CAP cases (Bartlett, 2008, 2011; Roig and Rello, 2003). A review of 41 European studies of CAP identified Legionella as the causative agent in 1.9% of outpatients, 4.9% of hospitalised patients and 7.9% of ICU patients (Woodhead, 2002). The exact incidence of LD worldwide is unknown due to difference between countries in surveillance and reporting but also diagnosis of LD. The development of diagnostic tests for the easy detection of L. pneumophila serogroup 1-specific antigens in urine was a significant advance in the diagnosis of legionellosis. In 2014 the European prevalence was 13.5 cases per million inhabitants; most cases (78%) were confirmed by this urinary antigen test (European Centre for Disease Prevention and Control, 2016). The mortality rate remains

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ACCEPTED MANUSCRIPT high (8% to 30% in ICUs and for hospital-acquired LD) despite improved diagnostic and therapeutic management of patients. Known risk factors for LD include increasing age, male gender, smoking, chronic lung disease, diabetes and various conditions associated with immunodeficiency (Phin et al., 2014). LD occurs sporadically and in outbreaks. In

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Europe, most cases (approximately 80%) are community-acquired and sporadic (European Centre for Disease Prevention and Control, 2016). Most of the cases occur during summer

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and early autumn; the yearly incidence of LD seems to be associated with climate changes, such as increased precipitation (Cunha et al., 2015). The sources of infection of sporadic cases are more rarely investigated and identified. However several systems and matrices have been classified as confirmed sources of Legionella (van Heijnsbergen et al., 2015).

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Healthcare and travel-associated outbreaks are mainly related to contaminated water systems (showers and baths, respiratory therapy or air conditioning equipments, spa pools,

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and, less commonly, food display humidifiers). Community outbreaks are predominantly linked to contaminated aerosols from wet cooling systems. As mentioned above, L. pneumophila as several other species of Legionella can cause also Pontiac fever. However the causative bacteria have not been isolated yet from Pontiac fever patients. The

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diagnosis is performed by urinary antigen test or by using serology. While the disease progresses acutely and shows a high attack rate of approximately 95%, the mortality rate is

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zero (Fields et al., 2002).

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Important knowledge about the epidemiology, the clinical presentations and the treatment of LD was readily gained and published soon after its recognition in 1976 (Fraser et al., 1977). However, advances in microbiology have now led to a better understanding of

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the ecological niches, the pathogenesis and the evolution of L. pneumophila. Here, we review the latest knowledge gained on the phylogeny and evolution of L. pneumophila through whole genome sequencing and different molecular approaches employed recently for its study.

2. Taxonomy and ecology of genus Legionella Since Legionnaires’ disease was recognized, isolation and identification of different strains and their characterization led to the establishment of the family Legionellaceae consisting of the single genus Legionella among the subdivision γ2-Proteobacteria (Brenner et al., 1979; Woese, 1987). On the basis of low DNA-DNA hybridization values between some Legionella species, a classification separating the family Legionellaceae in three genera - Legionella, Fluoribacter and Tatlockia was proposed (Garrity et al., 1980).

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ACCEPTED MANUSCRIPT However, additional studies showing that all legionellae studied have 16S ribosomal RNA sequences more than 95% identical did not support this division (Fry et al., 1991). Today, the genus Legionella comprises over 60 species (http://www.bacterio.net/legionella.html); all species have been isolated from environmental samples, and about half of the known

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species were also isolated at least once from patients and have thus been associated with infection. The type species is Legionella pneumophila, corresponding to the first bacterium

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of the genus described that is nowadays also the species responsible for nearly 95% of cases of LD diagnosed worldwide (Fraser et al., 1977; McDade et al., 1977). This species can be subdivided in 16 serogroups but the majority of culture-confirmed LD cases (84% worldwide, 80% in Europe) is caused by L. pneumophila serogroup 1 (Lp1) (Beaute et al.,

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2013; Fields et al., 2002; Yu et al., 2002). In 1988, Brenner et al. subdivided the species Legionella pneumophila in three subspecies (Brenner et al., 1988) - L. pneumophila subsp.

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pneumophila; L. pneumophila subsp. fraseri; L. pneumophila subsp. pascullei. Nevertheless, the tools to distinguish subspecies are not available in routine laboratories and thus these subdivisions are rarely reported. Other species of the genus Legionella also isolated from patients in 2014 in Europe are L. longbeachae (2%), L. micdadei (1%), L. bozemanii (<1%),

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L. macaechernii (<1%), L. sainthelensi (<1%), L. other species (<1%) and L. species not identified (1%) (European Centre for Disease Prevention and Control, 2016). The majority

immune-compromised

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of confirmed infections involving non-pneumophila Legionella species occur in severely patients.

Interestingly,

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et

al.

reported

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distinct

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epidemiological pattern of legionellosis in New Zealand and Australia, where L. longbeachae and L. pneumophila are similarly prevalent in LD (Graham et al., 2011). Infections with L. longbeachae are commonly associated with exposure to contaminated

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composts and potting soils, and have been increasingly reported in Europe in the past ten years (Amodeo et al., 2010; Currie and Beattie, 2015). The comparison of the L. longbeachae genome with that of Lp1 identified many species-specific differences that may account for the different environmental niches and disease epidemiology of these two species. Among these, L. longbeachae encodes several enzymes that might confer the ability to degrade plant material suggesting that L. longbeachae may also be able to interact with plants. Interestingly, L. longbeachae does not encode flagella, a major virulence factor for L. pneumophila, but is encapsulated (Cazalet et al., 2010). Regarding L. pneumophila, the high prevalence of Lp1 strains in human disease as mentioned above, does not seem to be due to its environmental distribution (Doleans et al., 2004). In order to explain this epidemiological predominance, comparative genome analyses of over 200 Legionella strains using macroarrays were performed. One major finding of this study was that the lipopolysaccharide (LPS) biosynthesis gene cluster of sg1 was the only common feature of Lp1 strains suggesting that the specific LPS of sg1 is at least partly responsible for the

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ACCEPTED MANUSCRIPT predominance of sg1 in human disease (Cazalet et al., 2008). These genome analyses further showed that the LPS gene cluster of sg1 strains is present in Lp strains representing different positions in the phylogenetic tree and genetic backgrounds indicating that the LPS cluster is exchanged by HGT (Cazalet et al., 2008; Cazalet et al., 2004; Gomez-Valero et

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al., 2011; Merault et al., 2011).

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A particular form of Legionella bacteria called Legionella-like amoebal pathogens (LLAPs) has been described in 1956 as intracellular parasite of free-living amoebae that cause lysis of the amoeba cells (Drozanski, 1956).Though initially named Sarcobium lyticum (Drozanski, 1991), this species was reclassified within the genus Legionella as L.

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lytica (Hookey et al., 1996). The dichotomy between LLAP and other Legionella spp. is based only on the fact that LLAPs grow poorly or not at all on BCYE agar. Based on 16s

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rRNA sequence, LLAPs clustered within a monophyletic group containing all other species (Birtles et al., 1996; Fry et al., 1991). Today these LLAPs represent five species in the genus Legionella - Legionella lytica (LLAP-3, LLAP-9, LLAP-7), L. drozanskii (LLAP-1), L.

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rowbothamii (LLAP-6), L. fallonii (LLAP-10), and L. drancourtii (LLAP-12) (Adeleke et al., 1996; Adeleke et al., 2001; La Scola et al., 2004). Serological studies suggested that they might be involved in human infections as a co-pathogen (La Scola et al., 2002; Marrie et al.,

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2001 ; McNally et al., 2000).

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Legionella exist as an intracellular parasite of protozoa such as Acanthamoeba, Hartmanella, Tetrahymena and Naegleria (Brieland et al., 1997; Rowbotham, 1980). During

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human infection, Legionella invades and replicates in macrophages, which are considered the primary target of Legionella. Conservation of many signaling pathways of protozoa to human macrophages explains in part the ability of Legionella to infect humans. Through the action of a large number of effector proteins translocated into host cell cytosol via the defect in organelle trafficking/intracellular multiplication (Dot/Icm) type IV secretion system (T4SS), the bacteria modulate host cell vesicular traffic and endosomal maturation pathways. It includes the recruitment of vesicles derived from endoplasmic reticulum (ER) to convert its nascent phagosome into an ER-derived compartment, termed Legionella-containing vacuole (LCV), for survival and replication (Allombert et al., 2013; Isberg et al., 2009). L. pneumophila deliver more than 300 effectors into host cells (Isberg et al., 2009; Newton et al., 2010; Zhu et al., 2011). The exact function of the vast majority of effectors still remains unknown. Many of these effector proteins harbor eukaryotic-like domains or resemble eukaryotic proteins that seem to have been acquired form their eukaryotic hosts via horizontal gene transfer (Gomez-Valero and Buchrieser, 2013).

All Legionella species

analyzed to date harbor a type IVB Icm/Dot secretion system however they have a large

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ACCEPTED MANUSCRIPT non-overlapping effector repertoire (Burstein et al., 2016; Gomez-Valero et al., 2014) (see paragraph 4.3). Legionella uses amoebae as a natural niche for its development, and human cells (such as macrophages, epithelial cells) are thought to be an accidental niche in the normal

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life cycle of the bacteria. Although human-to-human transmission has been thought not to occur, recently the first case of a probable human-to-human transmission of L. pneumophila

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has been reported during a large outbreak in Portugal in 2015 (Correia et al., 2016). However, co-evolution with environmental phagocytic cells is still without doubt the major driving force of the evolution of this bacterium. The replication capacity of different Legionella species in amoeba and human cells correlates with the epidemiological data for

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these species, suggesting that common as well as species-specific mechanisms might be involved in Legionella infection and replication in human cells (Gomez-Valero et al., 2014).

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Although amoeba and human macrophages are conventional cellular models for studying the virulence of L. pneumophila, this bacterium is able to colonize other model organisms such as non-phagocytic cell lines like HeLa cells (Garduno et al., 1998), permissive A/J mice and different cell lines (Brieland et al., 1994), the nematode Caenorhabditis elegans

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(Brassinga and Sifri, 2013), the wax moth wax Galleria melonella (Harding et al., 2012) or the fruit fly Drosophila melanogaster (Sun et al., 2013). In an evolutionary perspective, the

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fact that L. pneumophila is able to multiply in very different phagocytic cells (unicellular protists to the mammalian macrophages) reveals that these bacteria are able to control

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Shuman, 1999).

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cellular pathways which are conserved within its different eukaryotic hosts (Segal and

3. Genotyping methods and epidemiology

3.1. Historical typing methods and today’s standards As mentioned above, L. pneumophila is the most prevalent Legionella species involved in LD. After genus and species identification, the discrimination between Legionella isolates can be performed using several different subtyping methods. These typing methods aim to characterize isolates in order to identify epidemiologically linked LD cases and/or to identify the environmental source of the infection allowing taking corrective actions. Most of the methods are dedicated to L. pneumophila typing, but some of them could also be applied to other Legionella species. Most typing methods used are molecular methods; nevertheless a few phenotyping methods have also been described. For example sets of monoclonal antibodies that allow subtyping L. pneumophila serogroup 1 (Lp1) have

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ACCEPTED MANUSCRIPT been described (Helbig et al., 2002; Joly et al., 1986). First a standard phenotyping scheme was described that allowed dividing Lp1 into 9 subtypes which constitute an interesting first screen during epidemiological investigation and which could also enhance the discriminatory power of an associated genotyping method (Helbig et al., 2002). Later, first

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molecular typing methods have been developed for L. pneumophila that were based on the comparison of DNA band patterns generated by different methods. Finally, methods based

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on sequencing of amplicons have been developed (Aurell et al., 2005; Gaia et al., 2005; Helbig et al., 2003; Pourcel et al., 2007; Ratzow et al., 2007) and more recently, a MALDITOF-MS approach (Fujinami et al., 2010), whole genome mapping (Bosch et al., 2015) and whole genome sequencing-based methods have been described ( see paragraph 3.3). The

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efficiency of a typing method to differentiate bacterial isolates could be evaluated by calculating the discriminatory power (D). The discriminatory power is the average

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probability that the typing system will assign a different type to two unrelated strains randomly sampled in the microbial population of a given taxon and is calculated according to Hunter and Gaston (Hunter and Gaston, 1988). Table 1 sum up the discriminatory power of the typing methods for which it was available.

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Restriction fragment length polymorphisms (RFLP) analysis was one of the first molecular typing methods developed for L. pneumophila. This method has been used as

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the gold standard until recently in some countries such as the UK (Harrison et al., 2007). The method is a restriction endonuclease digest analysis of gDNA in which differences

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between the sequences of the genomic DNAs of strains are detected following electrophoretic separations of the digests using specific probes in a southern blot assay (Saunders et al., 1990). This method has also been used for L. longbeachae typing (Lanser

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et al., 1990). Alternative probes targeting the ribosomal operon have also been used for L. pneumophila typing (Saunders et al., 1991). RFLP typing has a high discriminatory index, but several methods with an equal discriminatory index have been developed later have shown to be more discriminative (e.g. Pulse-field gel electrophoresis (PFGE) (Pruckler et al., 1995)) or are easier to set up (e.g. PCR-based techniques). PFGE was longtime one of the gold standard methods. The method is based on the separation of macro-restriction fragments of the bacterial chromosome generated by digestion with an infrequent cutting site restriction endonuclease by pulse-field gel electrophoresis. PFGE has a high discriminatory index as assessed by several studies that are mainly using the enzyme SfiI as restriction endonuclease (Amemura-Maekawa et al., 2005; Marrie et al., 1999; Riffard et al., 1998). Despite its high discriminatory power, this method suffers some drawbacks: it is time consuming (4 days to obtain results), inter-gel reproducibility is often poor, electrophoresis requires specific equipment and computer-aided imaging analysis is needed, data are difficult to exchange between laboratories making investigations of travel-

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ACCEPTED MANUSCRIPT associated LD cases more difficult. Recently an improved protocol for L. pneumophila typing was described that reduces the total duration of the experiment to 2 days (Chang et al., 2009). Based on the complete genome sequences, new restriction endonucleases for PFGE typing were identified and the electrophoretic parameters were optimized (Zhou et al.,

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2010). PFGE has also been used for L. longbeachae typing (Montanaro-Punzengruber et al., 1999); moreover, an optimized protocol using a double digestion has been described for

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L. anisa typing (Akermi et al., 2006). Despite international efforts for standardization of PFGE typing protocols (restriction endonuclease, plug preparation and electrophoretic parameters) the obtained data remain difficult to exchange.

PCR-based techniques generating band patterns have also been applied for

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L. pneumophila typing. Amplified fragment length polymorphism (AFLP) is one of the methods standardized by the European working group on Legionella infection (EWGLI,

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become in 2012 the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) study group for Legionella infections: ESGLI), as it has a high discriminatory index. Bacterial DNA is digested and specific adapters are ligated to the restriction fragments. These adapters are then used as targets for PCR amplification. The length

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polymorphism of the amplified fragments generated is visualized via an agarose or acrylamide gel electrophoresis (Fry et al., 2005; Fry et al., 1999; Fry et al., 2002). Several

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other PCR-based methods generating DNA-band patterns have been described for L. pneumophila subtyping such as arbitrary primed PCR (AP-PCR) (Gomez-Lus et al.,

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1993; Grattard et al., 1996; Lawrence et al., 1999c), the infrequent-restriction-site PCR (IRS-PCR) (Jakubek et al., 2013; Riffard et al., 1998) or multi-locus Variable-Number Tandem-Repeat (VNTR) analysis (MLVA) which uses the diversity of VNTRs for typing

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(Nederbragt et al., 2008; Pourcel et al., 2003). An MLVA type database has also been created and is available via the website http://bacterial-genotyping.igmors.u-psud.fr/. Conversely to AP-PCR, the amplification step of AFLP, IRS-PCR and MLVA is performed in stringent conditions, which makes inter laboratories reproducibility higher. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOf-MS) for rapid discrimination of Legionella isolates was also evaluated (Fujinami et al., 2010). First tests showed that two sets of L. pneumophila isolates clustered in the same way when typed by mass spectrometry or by PFGE. Nevertheless, only 23 L. pneumophila isolates have been tested and thus this method needs to be evaluated on a larger number of isolates from diverse origins before conclusions on its usefulness can be drawn. However, it seems to be a promising tool as MALDI-TOF-MS data can be generated within few hours after Legionella growth and MALDI-TOF mass spectrometers are increasingly present in microbiology laboratories for bacterial identification purposes.

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ACCEPTED MANUSCRIPT Whole genome mapping have recently been evaluated for L. pneumophila typing (Bosch et al., 2015). The authors have tested this new tool on a panel of 53 related and unrelated isolated including stability and reproducibility testing. They demonstrated the high discriminatory power of the method, which allows a result in less than 24 hours after culture

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growth.

Methods based on the comparison of polymorphisms present in selected DNA

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targets have also been developed. These sequence-based typing (SBT) methods are based on the sequence comparison of several PCR-amplified DNA fragments. For L. pneumophila typing, several targets combinations have been assessed to obtain the most discriminative method (Aurell et al., 2005; Gaia et al., 2005; Gaia et al., 2003; Ratzow

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et al., 2007). Since 2007, a consensus was defined and SBT is the gold standard method recommended by EWGLI for L. pneumophila subtyping. The method is based on the

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sequence comparison of 7 PCR-amplified genes fragments (flaA, pilE, asd, mip, mompS, proA, and neuA). Raw sequence data are submitted to an online database available at the Public

Health

of

England

(PHE)

(http://bioinformatics.phe.org.uk/legionella/legionella_sbt/php/sbt_homepage.php),

website which

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allows the assignment of individual allele numbers to each target. A string of the individual allele numbers separated by commas in a predetermined order defined the allelic profile

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(e.g., 1,4,3,1,1,1,1) (Gaia et al., 2005; Ratzow et al., 2007). A Sequence Type (ST) number is assigned for each allelic profile (e.g. allelic profile 1,4,3,1,1,1,1 = ST1). In March 2016,

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the SBT database included 10792 entries from 60 countries representing 2156 different STs. Isolation of clinical or environmental L. pneumophila strains is not an easy task. This impairs the epidemiological investigations, as both clinical and environmental isolates are

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required for comparisons to find the source of infection. In this respect, SBT as PCR-based methods have an advantage as they can be applied directly to DNA extracted from clinical or environmental samples without the need to isolate the strains. This direct use on DNA samples has been used in few cases, however variable results are reported as in some cases all genes could be amplified and sequenced from the extracted DNA (Fry NK, 2005), but in other cases none or only few of the 7 SBT genes could be amplified and sequenced (Luck et al., 2007). To enhance the sensitivity of SBT directly applied on clinical samples, the addition of a prior amplification step leading to a nested or semi-nested PCR before sequencing of the target genes was reported (Coscolla and Gonzalez-Candelas, 2009; Ginevra et al., 2009). Coscollá and colleagues enhanced the discriminatory index of the method by adding three intergenic regions as new targets to the six target genes of standard SBT, but these targets were not included in the EWGLI definition of sequence types. An optimized protocol of a nested PCR-based SBT is available on the EWGLI site (www.ewgli.org).

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ACCEPTED MANUSCRIPT Sequence-based typing appears to be the method of choice for the exchange of results and as a powerful tool for global epidemiology. Furthermore, the application of the SBT method directly on clinical and environmental samples offers new solutions during epidemiological investigations (Coscolla and Gonzalez-Candelas, 2009; Mentasti et al.,

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2012; Moran-Gilad et al., 2015). The addition of new targets to the standard SBT has shown that the discriminative power of this technique can be enhanced easily (Coscolla and

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Gonzalez-Candelas, 2009, Ratzow et al., 2007). This concept has now been extended to whole genome sequencing data and core genome-MLST approaches that have been

3.2. Subtyping of endemic clones

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described and will be detailed further below (see paragraph 3.3).

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Comprehensive molecular typing of Legionellae around the world allows building up a global picture of strain distributions. Several independent studies have shown that only few genotypes (e.g. ST1, ST23, ST37, ST40, ST47, ST62…) cause most of unrelated culture-proven LD cases (Cazalet et al., 2008; Ginevra et al., 2008; Harrison et al., 2009;

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Lawrence et al., 1999b; Moran-Gilad et al., 2014). Some of these genotypes are distributed

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worldwide (Cazalet et al., 2008).

The collaborative results obtained by members of EWGLI since 2003 using the

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standard SBT scheme showed that a minority of strains causes nearly half of all reported cases of Legionnaires’ disease in Western Europe. However, the high isolation rate of strains with the above mentioned genotypes in clinical samples makes it difficult, and

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frequently even impossible, to demonstrate links between cases and environmental sources of infections during epidemiological investigations. Few studies have described methods allowing the subtyping of some STs (e.g. ST1 or ST62) for a better discrimination of these genotypes. A spoligotyping method was designed to subtype ST1. The CRISPR spacers were amplified using primers located in the direct repeat of the CRISPR array. The presence of these spacers was then revealed by reverse dot blot using 42 probes homologous to CRISPR spacers. A spoligotype was defined by the specific binary code deduced from the presence or absence of each of the 42 spacers. In their study Ginevra et al. were able to discriminate 233 ST1 isolates into 41 different spoligotypes (Ginevra et al., 2012). This method was first developed using a membrane-based hybridization assay and was transferred to use as a microbead-based high-throughput assay (Gomgnimbou et al., 2014)The subtyping of ST62 isolates using sequencing of the CRISPR array was also helpful in differentiating outbreak isolates from

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ACCEPTED MANUSCRIPT unrelated ST62 isolates and to confirm the environmental source of the outbreak (Luck et al., 2015). 3.3 Whole genome sequencing: a new tool for molecular typing of L. pneumophila

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Different studies have been conducted to evaluate the potential of whole genome sequencing (WGS) as a tool for L. pneumophila typing. Data comparisons were based on

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SNP-analyses, core genome comparisons, accessory genome comparisons, mobile genetic element profiling and core genome MLST (cgMLST). The first study describing WGS as a typing tool for L. pneumophila was performed by sequencing 7 isolates (3 clinical and 4 environmental) from an outbreak in the UK in 2003, retrospectively (Reuter et al., 2013).

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The objective of the study was to evaluate the feasibility of using WGS for Legionella outbreak investigations. The results obtained by SNP analyses were concordant with the

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SBT results (which were derived from the WGS data) and with the previous investigation results that linked two clinical isolates with three environmental isolates and excluded one clinical and one environmental isolate from the outbreak. The authors pointed to some limitations of WGS such as the limited number of available L. pneumophila genomes and

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the lack of automated pipelines for genome analysis. The use of WGS for the real-time investigation of a L. pneumophila community

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outbreak has also been used during an outbreak in Québec city in 2012. WGS was performed in comparison with SBT and PFGE (Levesque et al., 2014). All three methods

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gave concordant results and allowed the identification of the environmental source of the outbreak. Recently the use of WGS for the real-time investigation of a nosocomial L. pneumophila outbreak in an Australian hospital was reported (Graham et al., 2014). In

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this study, the genomes of six ST1 isolates were sequenced, four from the hospital (three clinical and one environmental isolate, respectively) and two unrelated isolates. All genomes obtained were compared to the ST1 reference genome (Paris, CR628336). SBT and virulence gene profiling was also performed for the typing of these isolates. WGS was the unique method used in this study that allowed the discrimination of unrelated ST1 isolates from the isolates from the hospital. Unrelated isolates showed more than 1500 different SNPs when compared to the hospital isolates that did not have more than 20 SNPs between two strains. In this study, the authors could link the two clinical isolates of the 2013 outbreak and one clinical isolate from a LD case in 2011 to an environmental isolate from a water sample from the hospital. They have demonstrated the usefulness of WGS for real-time investigation of LD cases and showed the higher discriminatory power of this method compared to the SBT method especially for the typing of endemic clones such as ST1.

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ACCEPTED MANUSCRIPT A large retrospective study by sequencing isolates from the same hospital was conducted recently (Bartley et al., 2016). The clinical isolates as well as 39 environmental ST1 isolates from different parts of the hospital were sequenced. A SNP-based approach as well as mobile genetic element profiling was used to analyze the whole genome

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sequencing data. As in the previous study, few SNP differences were observed between the related clinical and environmental isolates. Despite this small number of differences, the

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authors identified three clonal variants that seem to correspond to different areas of the hospital. The patient isolates were most closely related to isolates from their immediate environments, which might indicate localized geographic microevolution. Nevertheless, the authors also demonstrated a highly stable L. pneumophila population that contaminated the

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hospital with only few differences according to the time of isolation and localization. Thus the authors had doubts about the correlation between the geographic localizations of the

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variants and those of the patients.

Isolates belonging to ST191 that had caused a community LD outbreak in 2012 in Edinburgh, UK have been particularly closely analyzed by performing WGS of 21 isolates from 15 patients (McAdam et al., 2014). First SNP-based comparison of the core genomes

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after removal of three high-density SNP regions supposed to be due to recombination events was performed. This analysis led to the discrimination of four different subtypes

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among the patients. Two subtypes differed by 20 SNPs but had been isolated from the same patient. Secondly an analysis of the accessory genome was performed; in particular

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the gene content of type 4 secretion system (T4SS) related genomic regions was assessed. Variations in the Lvh T4ASS encoding region were identified between isolates. Of note, this analysis allowed the identification of a new L. pneumophila vir homologues (Lvh) encoding

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region (the Lvh region will be detailed further in the article). SNPs were also identified in the T4BSS encoded by the dot/icm genes. The differences between the T4SSs did not enhance the discrimination among the isolates. Finally, two isolates from the same patient, which had an indistinguishable core genome, differed by the presence of a 55Kb element including genes encoding resistance to heavy metals and a 2.7 Kb region encoding 2 hypothetical proteins. This study highlights that using the same dataset different levels of discrimination can be obtained depending on the analyses performed. It also highlights that an outbreak can be due to a polymorphic population of L. pneumophila strains in the environmental water source, leading to a complex distribution of L. pneumophila variants between patients of the outbreak and even within the same patient. Recently a new cgMLST scheme for L. pneumophila typing based on 17 genomes from databases has been proposed where 1521 genes are analyzed. This cgMLST method has been validated on 21 genomes from the databases and evaluated on three epidemiologically unrelated humidifier-associated pediatric LD clusters from Israel. The

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ACCEPTED MANUSCRIPT cgMLST allowed differentiating isolates within the endemic ST1 isolates. The cgMLST seems to provide a high discriminatory power and easily exchangeable results (MoranGilad et al., 2015). Taken together, the different studies discussed show the power of WGS for

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investigations of L. pneumophila outbreaks. Different sequencing and analysis methods were used giving the same or higher discrimination levels as the standard methods used.

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Although, a different discriminatory power was observed when several analysis methods are combined, WGS seems to be the future of molecular typing; from a global epidemiology standpoint, there is a need of standardization of both sequencing methods and sequence data analyses in order to make the data comparable and exchangeable between labs. In

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contrast, for local outbreak investigations, several analysis strategies may be used with the

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same success for the in depth characterization of outbreak related isolates.

4. Update on the phylogeny of the genus Legionella

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Phylogenetic studies of the species Legionella have been scarce and these few studies were based only on one or few genes (Grattard et al., 2006; Ko et al., 2002; Rubin

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et al., 2005). Since these studies were published new Legionella species have been described and the current accessibility to more and more complete genome sequences allows the use of a higher number of genes for phylogenetic reconstruction. The knowledge

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of a whole genome based phylogeny is important as it allows a better understanding of the phylogenetic relationships of the different Legionella species. Recently the whole genome

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sequences of 38 different Legionella species have been published and the genus phylogeny was reconstructed based on a concatenated alignment of 78 quasi-universal bacterial proteins. The results showed that the genus Legionella is divided into 3 clades where L. hackeliae, L. lansingensis and L. micdadei are present in the older one. The major Legionella pathogens (L. pneumophila, L. longbeachae, Legionella bozemanii and L. dumoffi) clustered together and a deep-branching clade consisting of Legionella oakridgensis, Legionella londiniensis and Legionella adelaidensis was observed. Legionella geestiana was considered as a outgroup to the rest of the Legionella species (Burstein et al., 2016). 5. Evolution of the species Legionella pneumophila and the genus Legionella

5.1. Impact of recombination on the evolution of L. pneumophila

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ACCEPTED MANUSCRIPT Early studies applying multi locus enzyme electrophoreses (MEE) supported a clonal population structure of the species L. pneumophila (Selander et al., 1985). Similarly, different sequence-based typing methods showed no evidence of recombination but suggested that the species L. pneumophila is clonal (Edwards et al., 2008). However, large

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population genomics approaches not only identified the occurrence of recombination in the species L. pneumophila but they showed that recombination has a key contribution to the

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genetic diversity of this species. For example recent data demonstrated that 34% to 57% of the genome of L. pneumophila has been involved in recombination events that are an ongoing process (Coscolla et al., 2011). Additionally, two recombination hot spots were identified: the first one is rich in genes related to T4ASS and the second one is rich in

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genes encoding mostly for hypothetical proteins of unknown function (Coscolla et al., 2011). Interestingly, intragenic recombination events were shown to occur also in housekeeping

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genes (pgk, atpD, ffh, metK) (Gomez-Valero et al., 2011), which is indicative of very high recombination rates (Feil et al., 2001).

Recently the index of association that is commonly used to measure the rate of intergenic recombination and the r/m ratio (Recombination /mutation) was calculated. This

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showed that the species L. pneumophila has a recombination value between that of Staphylococcus aureus (clonal) and that of Neisseria menigitidis (highly recombining

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species) (Underwood et al., 2013). Furthermore, to confirm the occurrence of recombination, a reticulate network tree based on the sequences used for the Sequence-based typing

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(SBT) scheme was constructed. Such a network tree allows more explicit representation of the evolutionary history than traditional phylogenetic trees. The tree computed from L. pneumophila data obtained from strains sampled from both environmental sources

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associated with human habitations and from patients with Legionnaires’ disease proofed that many recombination events between lineages occurred. Thus Legionella undergoes significant recombination and both clonal vertical inheritance of genetic material and exchange via recombination play a role in the evolution of L. pneumophila (Underwood et al., 2013). Interestingly, not only intragenic recombination events are occurring but also intergenic recombination events take place. Whole genome comparisons of single nucleotide polymorphisms (SNP) of different strains identified in each genome several regions with very low polymorphism (<0,05%) suggesting the exchange of DNA fragments between these strains. Surprisingly, these regions can be very large. For example between the strains Paris and HL06041035 genomic regions ranging from 10 to 99 kbs or between strains Philadelphia and Paris a region of 213 kb with a SNP rate lower than 0.005% were identified. It was suggested that these exchanges occur by conjugation. Thus a high DNA exchange rate seems to be present, in particular among strains present in the same

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ACCEPTED MANUSCRIPT geographical region (Gomez-Valero et al., 2011). Taken together, the recent results obtained through whole genome sequencing and comparison changed the initial view of a clonal population structure of L. pneumophila and showed that in contrast high

5.2. Experimental evolution of Legionella pneumophila

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recombination rates and DNA exchange via conjugation are key evolutionary mechanism.

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The development of next generation sequencing methods in recent years has allowed to undertake studies that were previously not feasible. Two recent studies in the Legionella field illustrated well these new possibilities of research. Ensminger and colleagues undertook a genome comparison of the classically used L. pneumophila strain

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Philadelphia-1 laboratory strains called JR32 and Lp01 and their derivatives called Lp02 and Lp03 (Rao et al., 2013). This study describes all the mutations associated to these strains and therewith allowed reconstructing their evolutionary history from the ancestral

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Philadelphia-1 strain isolated during the first reported Legionnaires’’ disease outbreak. The study confirms that JR32 and Lp02 derived from genetically identical clones, whereas polymorphisms detected in strains Lp02 and Lp03, both thymidine auxotroph, reveal that

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Lp03 is not a derivate of Lp02 as thought before. By analysing the whole genome sequence of each of these strains, it was shown that the genetic make-up of each laboratory strain

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has indeed changed over time due to numerous lab passages and genetic manipulations. For example, each strain contains large deletions in MGE discussed later in this review, in particular deletions of the Lvh-region and the tra regions (Rao et al., 2013). Furthermore,

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several, different single nucleotide polymorphisms (SNPs) were observed in each strain. The authors also suggest, that the use of strains JR32 and Lp01 for research should

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perhaps be reconsidered. These clones were genetically changed to allow easier strain manipulation and to improve transformation efficiencies with exogenous DNA (Berger and Isberg, 1993; Marra and Shuman, 1989) as compared to the original Philadelphia-1 strain. However, Rao and colleagues showed that the electroporation of plasmids is possible also in the original Philadelphia-1 strains similar to what was shown for the Paris strain isolated from a nosocomial outbreak of legionellosis in a Paris hospital (Lawrence et al., 1999a). This strain has been successfully genetically manipulated several times by using natural competence (Lomma et al., 2010; Rolando et al., 2013; Sahr et al., 2012). These findings led the authors to suggest that the use of the original Philadelphia-1 strain as it is closer to the original clinical background or the Paris strain that has not been engineered, might be a better choice for research on this pathogen (Rao et al., 2013). Finally, re-sequencing of the Philadelphia-1 strain revealed several sequencing errors present in the sequence published in 2004, which should be taken in consideration in the future (Chien et al., 2004; Rao et al., 2013).

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ACCEPTED MANUSCRIPT Ensminger and colleagues conducted another, very interesting study on genome evolution of Legionella using next generation sequencing (Ensminger et al., 2012). To learn what the molecular determinants important for infection of macrophages by L. pneumophila are and therewith to understand how this bacterium adapts to a specific host,

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L. pneumophila was restricted to growth within mouse macrophages for hundreds of generations. The genomic sequence and the infectivity of the resulting strains were

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compared to the original ones. This showed that the evolved strains had accumulated several adaptive mutations as it replicated better in macrophages than the ancestor strain. Indeed several of the mutations correlated with the fitness increase such as those present in the gene coding the flagellar regulator FleN or the lysine biosynthesis pathway encoding

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genes. Both provided an advantage in replication in macrophages. Furthermore, identical mutations were present in different lineages that had undergone the same growth restriction,

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demonstrating parallel evolution due to the selective pressures exerted by the macrophages. These results reveal how the environment may modify a genome and support the hypothesis that cycling through many different eukaryotic hosts allows L. pneumophila to be a broad host-range pathogen. Thus this ecological profile is

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advantageous for infecting many different hosts with the cost to not be highly adapted to any of these hosts. Further similar studies will help us to decipher the virulence factors

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necessary for the infection of human macrophages and the adaptation to different specific hosts, in particular for L. pneumophila as there is high functional redundancy among its

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effectors.

5.3. Evolutionary forces acting on the Dot/Icm type IVB secretion system (T4BSS) in

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the genus Legionella

Legionella are cycling between extra- and intracellular environments, but seem to replicate only within eukaryotic cells like aquatic protozoa. Essential for the adaptation to these different environments is the ability of Legionella to secret proteins via its different secretion systems, in particular via the Dot/ T4BSS. The Dot/Icm T4BSS is similar to the Tra/Trb system of IncI plasmids and is essential for the establishment of a replication permissive vacuole within host cells (Isberg et al., 2009). The Dot/Icm system has the amazing capacity to translocate over 300 effector proteins, but may also transfer DNA (Segal et al., 1999; Vogel et al., 1998). To date several effector proteins have been functionally analysed and their roles in the subversion of many different host-signalling pathways have been elucidated (Hubber and Roy, 2010; Nora et al., 2009; Rolando and Buchrieser, 2012). Secretion of proteins via the Dot/Icm system is essential for the adaptation of Legionella to the intracellular environments it encounters. The description of the different effectors studied to date is not in the scope of this review, thus will discuss

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ACCEPTED MANUSCRIPT here the evolutionary forces and the adaptation mechanisms acting on the Dot/Icm system itself. Already early studies using DNA/DNA hybridization analyses of the Dot/Icm locus, reported that the dot/icm genes are conserved in different L. pneumophila strains but also in

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different Legionella species (Morozova et al., 2004). Whole genome sequencing confirmed this finding and expanded the knowledge of this system as it revealed that the dot/Icm loci

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of strains L. pneumophila Philadelphia, Paris, Lens, Lorraine, Corby and HL 06041035 are not only present but also display a very high nucleotide conservation of 98-100% among orthologs Exceptions are only dotA, icmX that are more divergent (84% nucleotide identity) and icmC of strain Corby as compared to icmC of strain Paris as it is shorter and

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thus shows only 54% nucleotide identity. These results indicate that a strong negative selection acts on the genes constituting the Dot/Icm system (Gomez-Valero et al., 2011). A

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recent study analysing the genome sequences of Legionella hackeliae, Legionella micdadei and Legionella fallonii showed that the Dot/Icm system is also conserved, but that the genes show a lower nucleotide identity than among strains belonging to the same species.

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The degree of conservation of the different Dot/Icm proteins is very variable, ranging from >90% for DotB to proteins without any homology such as IcmR (Gomez-Valero et al., 2014). Surprisingly, the dotA gene is again one of the least conserved genes of the Dot/Icm

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system.

The dotA gene encodes an integral cytoplasmic membrane protein with eight

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hydrophobic transmembrane domains that is essential for L. pneumophila virulence, as bacteria lacking the dotA gene are defective in all virulence activities that require the Dot/Icm secretion system (Roy et al., 1998). Although the DotA protein is essential for

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successful infection of eukaryotic cells, the dotA gene shows higher variation than most of the other dot/icm genes among L. pneumophila strains and also among different Legionella species. The variation found in the dotA gene may be a sign that this gene is a target for host specialization and adaptive evolution, and its sequence variation may reflect the adaptation to different hosts and environments. Analyses of over 300 dotA gene sequences from L. pneumophila strains isolated from natural environments, man-made and clinical environments showed that clinical and man-made environmental strains belong to a sub-set of the genotypes existing in nature. Recombination and strong diversifying selection of the dotA alleles was observed (Costa et al., 2010). Interestingly one gene of the Dot/Icm system, icmR, is not conserved at the sequence level, but is in all different species analysed to date replaced by a nonhomologous gene with functional equivalence. This finding was reported the first time for the species L. micdadei and L. longbeachae (Feldman and Segal, 2004) and later confirmed for 27 additional Legionella species (Feldman et al., 2005). Similarly, a gene

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ACCEPTED MANUSCRIPT encoding a protein with no similarity to any previously described proteins is present in the position of icmR in L. fallonii, suggesting that it is serving as a functional equivalent of icmR of L. pneumophila like it was shown for other species (Gomez-Valero et al., 2014). IcmR is a regulator of IcmQ (another component of the Dot/Icm system) that possesses pore

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forming activity (Dumenil et al., 2004). In contrast, the most highly conserved genes among all different strains investigated are the dotB, icmS, icmW and icmP genes (Gomez-Valero

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et al., 2014). This observation is in line with the finding that, the L. pneumophila dotB, icmS, icmW and icmP genes can functionally replace their homologues in Coxiella burnetii (Zusman et al., 2003).

Taken together, recombination and frequent non-synonymous mutations are the

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evolutionary mechanisms that led to the diversification of the dotA gene. Positive selection seems to act on the icmR gene homologues (Feldman et al., 2005) whereas purifying

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selection is playing a role in the evolution of the highly conserved dotB, icmS, icmW and icmP genes. Furthermore, Burstein and colleagues observed that even though the gene order and orientation of the Dot/Icm systems of different Legionella species is highly conserved, gene insertions, most likely mediated by recombination events may occur

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(Burstein et al., 2016). These mechanisms together may allow the adaptation of the different Legionella strains and species to specific environmental niches and diverse

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cell and the pathogen.

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protozoan hosts and play a role in the evolutionary arms race between the protozoan host

6. Adaptation of Legionella to the environment and its hosts

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6.1. Role of integrative conjugative elements in the evolution of Legionella Bacteriophages, plasmids, and mobile genetic elements (MGE) like integrative conjugative elements (ICE), as well as natural competence are key players in genome plasticity and evolution and may mediate adaption to specific environments and condition (Bellanger et al., 2014; Thomas and Nielsen, 2005). Genome sequencing and comparison revealed that the L. pneumophila genomes show high plasticity and have a very dynamic accessory genome due to horizontal gene transfer and the presence of many MGE (Cazalet et al., 2004; Gomez-Valero and Buchrieser, 2013). Particular interesting is the finding that many of the L. pneumophila MGEs are ICEs that encode different type IVA secretion systems (T4ASS). The first one described was the so-called L. pneumophila vir homologues region (Lvh-region). It contains 11 genes encoding a T4ASS and that was found to be located on a DNA island with a GC content higher than the average of the L. pneumophila chromosome (Segal et al., 1999). Then, the L. pneumophila pathogenicity island 1 (LpPI-1), a 65 kb region encoding a T4ASS homologous to the Tra proteins of the

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ACCEPTED MANUSCRIPT F-plasmid of E. coli was described (Brassinga et al., 2003). It was thought to be specific to strain Philadelphia, but genome comparison by hybridization showed that this LpPI-1 is present in several L. pneumophila strains (Cazalet et al., 2008). Recent whole genome analyses of different L. pneumophila strains revealed that ICE carrying T4ASS are a

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common feature of the Legionella genomes (Gomez-Valero and Buchrieser, 2013; GomezValero et al., 2011). Different but related T4ASS are present on MGE in the genomes and

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three distinct T4SSAs with different prevalence among strains have been described: Lvh, Trb/Tra like T4SS and GI-T4SS (Glöckner et al., 2008; Gomez-Valero et al., 2011; Samrakandi et al., 2002; Segal et al., 1999). The genetic organization and components of these T4ASSs have high similarity to the classical VirB4/D4 conjugative system of

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Agrobacterium tumefaciens (Alvarez-Martinez and Christie, 2009). Here we will focus on the analysis and description of these three different classes of T4ASS as they seem to play

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an important role in the evolution of the Legionella genomes and probably also in the adaptation capacity of Legionella to the different environments this bacterium encounters.

6.1.1. Lvh (Legionella Vir homologue) region

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The Lvh T4ASS is located on a plasmid-like element of 36kb in strain Paris, 45kb in strain Philadelphia and strain Lens. This plasmid-like element can exist integrated in the

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genome or as a multi copy plasmid (Cazalet et al., 2004). Analysis of the mobility of this region showed that it is a phage like element whose excision and integration is growth

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phase dependent (Doleans-Jordheim et al., 2006). In L. pneumophila Lens and Paris the Lvh-region integrates in the tmRNA (Cazalet et al., 2004) and in L. pneumophila strain Philadelphia-1 it is inserted in a tRNA-Arg (Chien et al., 2004). In all but the L. pneumophila

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strain 130b, the Lvh-region exists as a single copy when integrated in the chromosome. In strain L. pneumophila 130b two distinct Lvh-regions are integrated in the genome in close vicinity. Lvh1 and Lvh2 are integrated in the 130b genome on either side of a CRISPR locus and share 92% similarity to each other. Lvh2 is 99% similar to the Lvh of strain Paris suggesting that it was acquired by horizontal gene transfer from this strain (Schroeder et al., 2010). The mobility of the Lvh-region was also deduced from whole genome analyses of the different derivatives of the original Philadephia-1 ancestor strain used as laboratory strains (JR32, Lp01, Lp02, Lp03). Indeed the original Philadelphia-1 strain carried the Lvhregion, but its derivatives Lp01, Lp02 and Lp03 have lost the entire Lvh-region (Rao et al., 2013). In contrast in the JR32 strain, another derivative of Philadelphia-1 strain, the Lvhregion is still present. However, in the J32 linage, a locus belonging to the Trb/Tra type T4ASS is deleted in comparison to the Philadelphia-1 progenitor (Rao et al., 2013). Interestingly, the Lvh-region shows not only intra- but also interspecies mobility since it is

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ACCEPTED MANUSCRIPT present as a chromosomally integrated putative plasmid-like element in L. longbeachae strain D-4968 (Kozak et al., 2010b). The functional role of the Lvh T4ASS in L. pneumophila is not clear. However, it was shown that a mutant in the lvhB2 gene that is part of the T4ASS does not display a

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significant defect in interactions with host cells when the bacteria are grown at 37°C, but it has an approximately 100-fold effect on entry and intracellular replication when the bacteria

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are grown at 30°C, suggesting a role of this element at lower temperatures (Ridenour et al., 2003). Similarly, Bandyopadhyay and colleagues showed that the Lvh-region impacts infection and inclusion in Acanthamoeba castelanii cysts at lower temperatures. Furthermore, they showed that in growth conditions that mimic the aquatic phase of

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L. pneumophila such as water stress (WS) the Lvh T4ASS can functionally complement the defect in entry, delay of phagosome acidification, and intracellular multiplication phenotypes

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of a Dot/Icm mutant (Bandyopadhyay et al., 2007). They thus proposed that the Lvh-region is involved in virulence-related phenotypes under conditions mimicking the spread of Legionnaires' disease from environmental niches. In a recent study Bandyopadhyay and colleagues showed that the VirD4 protein alone encoded in the Lvh-region, is responsible

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for the restoration of the wild-type phenotype of a Dot/Icm mutant (Bandyopadhyay et al., 2013).

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Interestingly, the presence of a small noncoding RNA (sRNA, lpr0035) overlapping the attL site of the Lvh–region of strain Philadelphia (pLp45) was reported. The deletion in

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the 5’ chromosomal junction of pLp45 that is removing the 49-bp direct repeat as well as the sRNA lpr0035, locked this element in the chromosome and abolished its mobility. Amoebae and macrophage infection assays with stationary phase or WS-treated cultures of

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this mutant strain established a role of lpr0035 in the Legionella entry phenotype and in intracellular multiplication in J774 macrophages and A. castellani. However, the authors conclude that the mobility of pLp45 was not required for the entry and intracellular replication phenotype since only the virulence phenotype but not the mobility of pLp45 was complemented when expressing lpr0035 in the deletion mutant (Jayakumar et al., 2012). The different analyses together suggest that the Lvh-region is not important for in vitro growth of L. pneumophila, but it seems to offer adaptive advantages in certain stressful environmental niches like under lower temperatures and water stress. Further analyses will be necessary to fully understand the function of this T4ASS that is circulating among Legionella strains as it is encoded on a MGE.

6.1.2. Trb/Tra family of conjugative elements The Lvh T4ASS is not present in L. pneumophila strain Corby, but a similar T4ASS is integrated in this site (tmRNA) and a second genomic island carrying a T4ASS is

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ACCEPTED MANUSCRIPT integrated in the tRNAPro gene, a site not occupied by a MGE in the strains Paris, Lens or Philadelphia. These two MGEs were named Trb-1 and Trb-2 (Glöckner et al., 2008). Like the Lvh-region, the Trb-1 and Trb-2 regions (42,710 bp and 34,434 bp, respectively) are able to excise from the chromosome forming episomal circles (Glöckner et al., 2008). Trb-1

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encodes for the proteins needed to assemble a functional conjugation/T4ASS and can be transferred horizontally to other L. pneumophila strains by conjugation where it integrates in

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a site-specific manner in the tRNAPro gene. In contrast to the Lvh-region, excision of the Trb-1 island is not growth phase dependent and can take place during intracellular replication in A. castellani. However, Trb-1 is not necessary for intracellular replication of L. pneumophila (Glöckner et al., 2008; Lautner et al., 2013). Recently, two similar genomic

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islands, Trb-3 in L. pneumophila strain Lorraine and Trb-4 in L. longbeachae NSW150 have been identified but it is not known yet whether these elements are also mobile (Gomez-

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Valero et al., 2011). Comparative genomics revealed, that the Lvh-region and all Trb-1 like MGEs of L. pneumophila are associated with a lvrRABC gene cluster (Gomez Valero et al., 2011). The lvrR gene is predicted to code for a transcriptional regulator and LvrC is a homologue of CsrA, an RNA binding protein that is crucial for the regulation of the

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L. pneumophila life cycle (Molofsky and Swanson, 2003). Deletion of the lvrRABC gene cluster from the Trb-1 region of strain Corby showed that these proteins are involved in the

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regulation of the circularisation of the Trb-1 ICE, however the exact mechanism is not known (Lautner et al., 2013).

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The so-called LpPI-1 described in strain Philadelphia-1 belongs also to the Trb/Tra family of conjugative/T4SS (Brassinga et al., 2003). This 65 kb region is inserted in the tRNALys,Arg and is predicted to encode a T4ASS and a set of cargo genes coding for

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transport proteins acetyltransferases, reductases, transposases, integrases, phage related genes and the virulence related magA-msrA locus. MagA is expressed intracellularly during transition from exponential phase to post-stationary phase. MsrA catalyses the reduction of methionine sulfoxide residues to methionine and is an established virulence determinant in E. coli, Neisseria gonorrhoeae and Mycoplasma genitalium (Dhandayuthapani et al., 2001; Moskovitz et al., 1995; Skaar et al., 2002). This ICE, was recently renamed ICE-ßox as studies examining its function and mobility showed that it confers resistance to ß-lactam antibiotics, hydrogen peroxide and bleach in vitro. It also protects L. pneumophila from the immune response of macrophages that produce reactive oxygen species, a protection directly correlated with the NADPH component of macrophages since L. pneumophila carrying the ICE-ßox replicated much better during the first 24 h, than bacteria lacking this element (Flynn and Swanson, 2014). Thus, this ICE may contribute to increased fitness of L. pneumophila in natural environments and confers oxidative stress resistance to this pathogen.

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6.1.3. Genomic Island associated T4SS family An additional class of T4SSs named the genomic island T4SS (GI-like) (Juhas et al., 2007) is present within the L. pneumophila genomes. Two such GI-like islands named

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Legionella genomic island 1 (LGI-1) and LGI-2 were identified in strain 130b (Schroeder et al., 2010). Strain Corby exhibits four such genomic islands associated T4SS named LpcGI-

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1 and 2, LpcGI-Asn and LpcGI-Phe. LpcGI-1 and 2 are also present in strain Paris (GomezValero et al., 2011). Both are mobile and exist in two or three different episomal forms, respectively. As for Trb-1, the excision and transfer of one form named LpcGI-2-A implicates the integrase encoded on this GI. The second form, named LpcGI-2AB seems to

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be transferred by a different conjugation system since its excision is integrase independent. However, for an unknown reason deletion of the two integrases encoded on this island,

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increases the rate of the episomal form. In contrast, LpcGI-Asn and LpcGI-Phe do not encode a T4ASS, little is known about their mobility but they are also integrated in tRNA genes, and nothing is known about their functional role they may play (Lautner et al., 2013). The here analysed, completely sequenced L. pneumophila (Paris, Lens, Corby,

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Alcoy, Lorraine, HL 06041035, 130b), and one L. longbeachae genome (NSW150) encode between one to four GI-T4SS and at least one of these elements is inserted in a tRNAthr site.

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Although, GI-T4SSs are widely conserved, some of those present in L. longbeachae or L. drancourtii miss part of the LvrABC regulatory locus that is in contrast highly conserved in

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the Lvh-region and the Trb/Tra type ICEs. GI-T4SSs are evolutionary distant from T4ASS and T4BSS. They form a separate clade that is a divergent monophyletic group. They lack the essential mobilization genes mob that processes the DNA before and after conjugation

virulence

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and the OriT. In the cargo regions of these LGI-T4SS a number of antibiotic resistance and genes

are

present

additionally

to

variable

amounts

of

RND

(resistance/nodulation/division) efflux systems putatively involved in pathogenicity (Wee et al., 2013). In strain Philadelphia the region carrying the RNDs modules is called pLP100 due to its size (100 kb) and its capacity to excise from the chromosome to form a circular 100kb plasmid-like element (Trigui et al., 2013). It encodes a putative conjugation system and is integrated in a tRNAthr gene. It excises from the chromosome in a growth phase dependent manner, but in contrast to the Lvh-region, the excision is more frequent during exponential than in post-exponential growth phase. Interestingly, excision in postexponential phase is Hfq dependent. pLP100 cargo genes like copA confer higher copper resistance to this strain (Trigui et al., 2013). In contrast, it does not influence Legionella survival during amoebae or macrophages infection (Kim et al., 2009). Taken together, all Legionella strains sequenced to date encode one or several T4ASSs present on MGE that are often ICE. Their implication in Legionella virulence and

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ACCEPTED MANUSCRIPT adaptation seem to be host and strain specific and varies from one type to another. They may confer advantages to Legionella in specific environmental growth conditions, like does the Lvh–region for growth at low temperature and in water stress conditions or the ICE-ßox that confers resistance to ß-lactam antibiotics, hydrogen peroxide and bleach and protects

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L. pneumophila from the immune response of macrophages that produce reactive oxygen species. However, for most of these T4ASSs, no strong virulence phenotype was observed

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and for many the function is not known yet. The presence of these many and various types of MGE encoding T4ASSs in the Legionella genomes is intriguing (table 2 and 3) and thus it is plausible that each plays a different role in the adaption of Legionella to the environment

L. pneumophila fitness need to be found yet.

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and the different hosts, but the exact conditions where each element has an impact on

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6.2 Role of gene duplications in the adaptation of Legionella In addition to horizontal gene transfer (HGT), gene duplication events also shape the bacterial genomes and contribute to evolution (Andersson et al., 2015). Gene duplication

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and subsequent divergence of the extra copy to acquire a new function is one of the main sources of functional diversity and may be associated with adaptation to specific

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environments, development of novel life strategies, or other species-specific adaptations (Andersson et al., 2015; Conant and Wolfe, 2008; Innan and Kondrashov, 2010). Although Legionella has a moderate genome redundancy relative to other bacterial species, genome

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sequencing revealed that certain protein families possess numerous paralogous genes (Cazalet et al., 2004; Chien et al., 2004). For instance, expansion of the Dot/Icm effector

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repertoire, probably through acquisition of foreign genetic material and gene duplications, results in paralogue effector families like in the case of the Sid (substrate of Icm/Dot) effectors (Luo and Isberg, 2004). As an example, the SidE family is comprised of 4 paralogues genes, sdeA, sdeB, sdeC and sidE encoding potentially redundant functions (Bardill et al., 2005; Jeong et al., 2015). The genetic and functional redundancies in the effector arsenal probably promotes intracellular replication of Legionella in a diverse range of protozoan hosts in the environment and it is possible that each paralogue is specific for a particular host cell (Chien et al., 2004; Luo and Isberg, 2004; O'Connor et al., 2011). In addition to duplication of structural genes, a new picture has recently emerged in which Legionella may favor, duplication, divergence, and maintenance of several paralogous global regulatory proteins during evolution. Global regulators allow coordination of cellular functions in response to physiological, metabolic and/or environmental fluctuations and thus are crucial for successful bacterial infection, persistence and

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ACCEPTED MANUSCRIPT dissemination. Thus, duplication of such regulators may lead to more versatile and adapted bacteria than in the case of structural gene duplications (Wang et al., 2011). The nucleoidassociated protein Fis (factor for inversion stimulation) is a central component of such global regulatory networks. Comparative genomic analysis revealed that in all currently

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accessible Legionella genome sequences three Fis paralogs are present (Fis1, Fis2 and Fis3) whereas Gammaproteobacteria usually contain only one single Fis protein (Cazalet et

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al., 2004; Chien et al., 2004; Zusman et al., 2014). Fis is considered as a global regulator with a broad set of regulatory functions such as genome organization, phage transposition, initiation of the replication and gene transcription (Dillon and Dorman, 2010). In particular, Fis regulates the expression of virulence related-genes in many species such as enteric

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bacteria (e.g. Escherichia coli, Salmonella enterica, Shigella, Vibrio cholera and Dikeya dandantii) (Falconi et al., 2001; Goldberg et al., 2001; Kelly et al., 2004; Lautier and Nasser,

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2007; Lenz and Bassler, 2007). It was shown that Fis preferentially binds a 17-bp and ATrich consensus sequence at promoter regions of target genes and can work in concert with other regulators to modulate transcription initiation (Browning et al., 2010). The reconstruction of the evolutionary history of the three Fis proteins of Legionella suggests

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that two duplication events occurred before the divergence of the genus Legionella (Zusman et al., 2014). The three Fis protein sequences share a little less than 50% amino

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acid identity, suggesting a rapid evolution after gene duplication. However, the helix-turnhelix DNA-binding domain is conserved in the three protein sequences as well as the amino

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acids involved in the recognition of the Fis regulatory motif on the promoter region of targets genes (Zusman et al., 2014). Interestingly, Fis1 and Fis3, but not Fis2, are required for optimal intracellular growth in both amoeba and macrophages and Fis1 and Fis3 directly

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repress the level of expression of 18 Dot/Icm effector-encoding genes. Fis1 and Fis3 appear to have common and distinct target genes since some effector genes are mainly repressed by either Fis1 or Fis3 while other sets of genes are repressed by both regulators in a similar manner. In contrast, Fis2 does not seem to modulate the expression of the effector genes tested, at least under the experimental conditions of the study. These results suggest that after gene duplication, the three copies have diverged and have acquired at least in part, distinct regulatory functions. They suggest that maintaining three distinct Fis regulatory proteins may allow fine-tuning gene expression of the large repertoire of Dot/Icm effectors of Legionella. In addition, the three Fis proteins are probably part, with other transcriptional regulators (CpxR, PmrA), of sophisticated regulatory networks that allow proper coordination of virulence-related gene expression and/or to respond to the multiple stimuli that the bacterium encounters inside hosts or in the environment. Fis is not the only regulator present in several copies in the Legionella genomes. In their study, Zusman and collaborators also reported that the two-component system CpxRA, that regulates the

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ACCEPTED MANUSCRIPT expression of several effector-encoding genes as well as dot/icm genes (Altman and Segal, 2008; Gal-Mor and Segal, 2003), has two other paralogs in Legionella longbeachae and L. dumoffii. However, the roles of these multiple CpxRA homologs in some Legionella species remain to be determined.

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Another remarkable feature of the Legionella genomes is the presence of four to seven csrA paralogs depending on strains (Abbott et al., 2015b). For instance, L.

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pneumophila strains Philadelphia and Paris contain 5 and 6 CsrA-like genes, respectively, and up to 7 CsrA copies are identified in the virulent environmental isolate Lp LPE509 (Abbott et al., 2015b) or in L. drancourtii (Zusman et al., 2014). In contrast, L. longbeachae strains NMW150 and D-4968 contain four CsrA copies. CsrA is a pivotal regulator of

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metabolism and virulence that controls the switch from replicative to transmissive phase in L. pneumophila. It was shown that CsrA is absolutely required for efficient bacterial

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replication in broth culture or inside eukaryotic cells and that ir it represses phenotypic traits related to the virulent phase of the L. pneumophila life cycle such as pigmentation, motility, cell shape shortening, stress resistance, sodium sensitivity and cytotoxicity (Fettes et al., 2001; Forsbach-Birk et al., 2004; Molofsky and Swanson, 2003). The CsrA protein acts as a

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global post-transcriptional regulator by directly binding messenger RNA at an ANGGA motif and thus interfering with translation initiation and/or mRNA stability. In L. pneumophila, it

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was shown that CsrA directly repressed the expression of numerous Dot/Icm effector genes (Nevo et al., 2014; Rasis and Segal, 2009).

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Recently, Abbott et al. (2015b) have performed a functional study on one of the csrA paralogues (named CsrR) that, like the canonical CsrA is conserved among L. pneumophila strains. CsrA and CsrR share only 28% amino acid identity but despite significant genetic

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drift, CsrR retained the amino acid residues necessary for its RNA binding activity. However, CsrR appears unlikely to regulate neither transmissive traits commonly studied nor intracellular growth inside eukaryotic cells. In contrast, CsrR seems to be required to enhance survival of L. pneumophila in water since a csrR deletion mutant presents reduced viability after prolonged incubation in hot tap water compared to the wild type strain. In addition, CsrA specifically binds to a ANGGA motif of the csrR mRNA in vitro and represses its translation, creating a global hierarchical regulatory cascade that adds another level of complexity to posttranscriptional regulation in L. pneumophila (Abbott et al., 2015b). The authors speculate that csrA gene duplication and genetic drift result in a new regulatory role for CsrR that retained the mRNA binding function of canonical CsrA but acquired a new cohort of targets that increase the capacity of L. pneumophila to withstand a broad range of environmental conditions. While CsrA promotes intracellular replication inside host cells, CrsR would play a role in the environmental persistence by enhancing its survival under poor-nutrient conditions. Both, the canonical csrA and csrR genes appear to be stably

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ACCEPTED MANUSCRIPT integrated into the chromosome in contrast to the other CsrA paralogs that are associated to T4ASS within integrative and conjugative elements (ICEs). It seems that the number of copies of CsrA depends on the number of genomic island-encoded T4SSs present in the genome of a specific strain (Gomez-Valero et al., 2011). In L. pneumophila Paris strain,

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except the canonical csrA and csrR genes that belong to the core genome, the four other csrA-like gene copies are located on the accessory genome: i) one copy, named LvrC, is

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flanking the Lvh region (Doléans-Jordheim et al. 2006) ; ii) two other CsrA/LvrC-like copies are located at the 5’ end of two distinct genomic island-associated T4ASS (Wee et al. 2013); iii) and the fourth CsrA homolog is carried on the 136 kb-plasmid upstream the Tratype T4ASS (Gomez-Valero et al., 2011) (see table 3). As previously suggested in this

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review, ICE-related LvrC/csrA homologues might be implicated in the regulation of the mobility of these islands (Lautner et al., 2013). Very recently, Swanson and collaborators

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(Abbott et al., 2015a) demonstrated that the CsrA paralog encoded by the ICE-βox element, named CsrT (for CsrA paralog for ICE transfer) decreases conjugative transfer, replication in macrophages and oxidative stress resistance conferred by its cognate mobile element. CsrT seems to play an even broader regulatory role since its ectopic expression also

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reduces motility not only in L. pneumophila but also in Bacillus subtilis probably by binding hag mRNA at an ANGGA as the B. subtillis CsrA does (Abbott et al., 2015a). It is also

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tempting to speculate that these CsrA-like extra-copies might play a role in regulating the expression of their cognate T4ASS-encoding genes that mediate horizontal transfer of

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these ICE elements. To conclude, L. pneumophila ICE CsrA-like regulators seem to play a crucial role in maintenance and spread of these mobile elements however, the specific physiological stimuli, metabolic shift or environmental changing conditions that allow their

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excision and transfer are still unknown. A summary showing the number of duplications of each of the mentioned genes above is shown in table 3. The reason why Legionella duplicated and maintained several copies of such regulatory proteins (transcriptional or post-transcriptional regulators) is not known but it presumably reflects a regulatory plasticity that may be important for the adaptation to different hosts and lifestyles. It would be of interest to determine the role of each paralog, if they have related or redundant functions in L. pneumophila, or if they are differentially expressed in response to signals at specific stages of infection or environmental cues. 6.3. Specific features of Legionella influencing environmental adaptation

One of the major findings in microbiology of the last 10 years was the discovery of the CRISPR-Cas system and the identification of its immune role against invasion by phages or plasmids (Horvath and Barrangou, 2010). It became a breakthrough discovery

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ACCEPTED MANUSCRIPT when it was shown that it could be used as a programmable genome-editing tool (Jinek et al., 2012). Recently, it was shown that this locus might also play a role in infection. In the L. pneumophila strain 130b genome a type II-B CRISPR-Cas locus that contains cas9, cas1, cas2, cas4, and an array with 60 repeats and 58 unique spacers is present. The cas1 and

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cas2 genes of the CRISPR-Cas locus are more expressed during intracellular growth in macrophages as compared to in vitro growth in liquid media. Further analyses showed that

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cas9, cas1, cas4, and CRISPR array mutants grew normally in macrophages and amoeba, in contrast the cas2 gene was required for intracellular growth in amoeba but dispensable for growth in macrophages (Gunderson and Cianciotto, 2013). Cas2 of L. pneumophila is thus one of the rare proteins identified to date that show different functions in human

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macrophages and in protozoan hosts. Further analyses of the function of Cas2 showed that it possesses RNase and DNase activity and that this nuclease activity promotes infection of

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amoeba. Interestingly, it was shown that this activity is also not conserved in all amoeba hosts but differs between amoeba species (Gunderson et al., 2015). Thus the CRISP-Cas locus has an additional role, adaptation of L. pneumophila to specific protozoan hosts to better persist in the environment. As the introduction of cas2 into strain Philadelphia-1, a

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L. pneumophila strain not carrying the CRIISPR-Cas locus caused that strain to be more infectious, there might be a selective advantage to acquire cas2, perhaps via MGEs. Indeed,

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in strain Paris this locus is located on the Lvh-region described earlier, which is a MGE.

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7. Concluding remarks

In the last years, large-scale genome sequencing of many Legionella species and strains

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has greatly improved our knowledge on the evolution, pathogenicity and genetic diversity of the genus Legionella. Furthermore, an increasing number of studies that have been conducted demonstrated that whole genome sequencing allows robust genotyping and without doubt it will become the method of choice for epidemiological investigations in the future. Comparative genomics approaches are equally valuable to shed light on the high plasticity of the Legionella genomes that have lead to the present genetic and pathogenic heterogeneity within this species. Significant differences are observed between strains, showing that numerous genomic rearrangements continue to occur also after speciation. Some sites of syntenic differences are marked by the presence of mobile genetic elements such as ICE or genomic islands that are usually associated with T4ASS. In contrast to the Dot/Icm T4BSS, which is the key for Legionella pathogenesis, the roles of these various T4ASS in virulence or in the adaption to changing environmental conditions remain to be elucidated. However, the data known to date about the known T4ASSs show that they are less decisive for L. pneumophila adaptation and persistence than the T4BSS Dot/Icm

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ACCEPTED MANUSCRIPT system. Another striking feature of the Legionella genomes revealed by comparative genomics is the presence of a large cohort of Dot/Icm effectors, which of many harbour numerous and various protein domains typically present in eukaryotic organisms. A large proportion of these genes is species specific and is correlated with the evolutionary history

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of this bacterium, a pathogen that co-evolved with a variety of protozoan hosts. As the Dot/Icm T4BSS is well conserved among species, even within non-pathogenic strains, it is

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likely that the increased virulence towards humans of some strains is related to this pool of effectors and/or to the sophisticated regulatory circuits that control their expression and not the Dot/Icm secretion system. Thus these many different putative effector proteins represent a promising research topic for future studies on the emergence and evolution of

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pathogenicity.

Acknowledgements

The authors declare that they have no competing interests.

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Work in SJ and PD laboratory is performed within the framework of the LABEX ECOFECT (ANR-11-LABX-0042) of Université de Lyon, within the program “Investissements d’Avenir”

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(ANR-11-IDEX-0007) operated by the French National Research Agency (ANR) and supported by the Institut national de la santé et de la recherche médicale (INSERM), the

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Fondation pour la Recherche Médicale (FRM) grant n° DBI20131228568, the Hospices Civils de Lyon and the General Direction for Health (DGS). Work in CB laboratory is financed by the Institut Pasteur, the Centre National de Recherche

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Scientifique (CNRS), the Institut Carnot-Pasteur MI, the French Region Ile de France (DIM Malinf), the grant n°ANR-10-LABX-62-IBEID, the Infect-ERA project EUGENPATH (ANR13-IFEC-0003-02) and the Fondation pour la Recherche Médicale (FRM) grant N° DEQ20120323697.

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Table legends

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Table 3: Summary table of the distribution of the various ICEs and some of the duplicated genes present in Legionella. To Asses the presence or absence of (Fis, CsrA and SidE) a tblastn was performed using the amino acid sequence of each of these proteins of Legionella pneumophila Paris strain. The “(+)” for Legionella pneumophila Corby and Alcoy corresponds to the presence of a truncated 4th copy of the protein. +: Present, -: absent. The phylogenetic tree is based on the 16S gene after running G-blocks for the alignment

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Reference Fry et al., 1999 Fry et al., 1999 Fry et al., 1999 Fryet al., 1999 Gaia et al., 2005 Gaia et al., 2005 Ginevra et al., 2011 Bosch et al., 2015 NA*

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*NA Not Available

Discriminatory power (D) 0.84 0.896 0.99 0.891 0.94 0.98 0.797 0.87 NA*

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Methods Ribotyping RFLP PFGE AFLP SBT SBT + Mab Spoligotyping Whole genome mapping cgMLST

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Table 1. Discriminatory power of Legionella typing methods

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Insertion site

Size (Kb)

Paris

Nb of copies 1

tmRNA

36

Philadelphia

1

Arg

Lens Wadsworth 130b

1 2

LpVv2 HL06041035

1

tmRNA Inserted upstream and downstream of the CRISPR Locus tmRNA

L. micdadei

ATCC33218

1

tmRNA

L. longbeachae

D-4968 - WGS ACZGv1

1

L. pneumophila

Paris (plasmid) Loraine (plasmid) Lens (plasmid) Philadelphia NSW150 NSW150 (plasmid) D4968-WGS

1 1 1 1 1 1 1

Species L. pneumophila Lvh

L. longbeachae

L. hackeliae L. fallonii

ATCC35250 LHAv2 (Plasmid) ATCC700992 (plasmid) ATCC700992 Chromosome

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tRNA

tRNA

Reference

45 or 50 Lvh1: 25.5 Lvh2: 17.2

(Cazalet et al., 2004) (Chien et al., 2004) This study (Schroeder et al., 2010)

36

This study

32.3

This study

45.2

(Kozak et al., 2010a) (GomezValero et al., 2011)

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Genome Accession Nb and status CR628336 (complete) AE017354 (Complete) CR628337 (complete) FR687201, Draft (145 contigs) EMBL: FQ958211 (complete) EMBL: PRJEB7312 (complete)

tRNA, tRNA Arg tRNA, tRNA (A) ?

28 28.2 32 64 48.7 26.6 ?

1

?

?

This study

ACZG00000000, Draft (13 contigs) CR628338 (complete) FQ958212 (complete) CR628339 (complete) AE017354 (complete) NC_013861 (complete) NC_014544 (complete) ACZG00000000, Draft (13 contigs) EMBL: PRJEB7321

1 1

?

100 78

This study This study

LN614828 (complete) LN614827 (complete)

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Leu

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Gly

tRNA,

Cyst

tRNA

This study

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Alcoy

2

L. longbeachae

NSW150

1

L. pneumophila

Alcoy Corby

1 2

Philadelphia Paris

1 2

Lens Wadsworth 130b

1 2

Lorraine Subspecies HL06041035

1 2

L. longbeachae

D-4968

1

L. drancourti

LLAP12

L. fallonii

ATCC700992

1

L. hackeliae

ATCC35250

L. micdadei

ATCC33218

thr

tRNA thr LpcGI-1: tRNA Met LpcGI-2: tRNA thr tRNA thr lppGI-1: tRNA Met lppGI-2: tRNA thr tRNA thr LpwGI-1: tRNA Arg LpwGI-2: tRNA Pro tRNA Thr LpvGI-1: tRNA Arg LpvGI-2: tRNA Pro tRNA Arg

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pro

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GIT4ASS

L. pneumophila

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Trb (P-type)

39.6 35.3 80

51 45 22 115 45 123 80 59 70.5 98 104 >45

(Lautner et al., 2013) This study (Schroeder et al., 2010) This study

CP000675 (completete)

(GomezValero et al., 2011) (Wee et al., 2013)

NC_013861 (complete)

This study This study

CP001828 complete CP000675 complete AE017354 complete CR628336 complete CR628337 complete CAFM0000000 (Draft, 159 contigs) FQ958210 (complete) FQ958211 (complete)

(Wee et al., 2013) (Wee et al., 2013)

ACZG00000000 (Draft, 13 contigs) ACUL00000000 (Draft, 263 contigs)

This study

EMBL: PRJEB7322 (complete) EMBL: PRJEB7321 (Complete) EMBL: PRJEB7312 (Complete)

LdrGI-1: tRNA pro LdrGI-2: tRNA LdrGI-3:? LdrGI-4: ? pro tRNA

>46 >30 >44 >58 49

1

Arg

89.2

This study

1

Met

37.1

This study

4

tRNA tRNA

FQ958210 (complete) CAFM0000000 (Draft, 159 contigs) CP001828 complete

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Table 3: Summary table of the distribution of the various ICEs and some of the duplicated genes present in Legionella.

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Highlights

- WGS has become the method of choice for L. pneumophila outbreak investigations

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- High recombination rates and DNA exchange via conjugation are key evolutionary mechanism - The Dot/Icm T4BSS well conserved among species is the key for Legionella pathogenesis

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- Legionella species have a large non-overlapping Dot/Icm effector repertoire

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- All Legionella strains sequenced encode one or several T4ASSs present often on ICE

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