genome dynamics in legionella: the basis of...

Genome Dynamics in Legionella: The Basisof Versatility and Adaptation to IntracellularReplication

Laura Gomez-Valero and Carmen Buchrieser

Institut Pasteur, Biologie des Bacteries Intracellulaires and CNRS UMR 3525, 75724 Paris, France

Correspondence: [email protected]

Legionella pneumophila is a bacterial pathogen present in aquatic environments that cancause a severe pneumonia called Legionnaires’ disease. Soon after its recognition, it wasshown that Legionella replicates inside amoeba, suggesting that bacteria replicating in en-vironmental protozoa are able to exploit conserved signaling pathways in human phagocyticcells. Comparative, evolutionary, and functional genomics suggests that the Legionella–amoeba interaction has shaped this pathogen more than previously thought. A complexevolutionary scenario involving mobile genetic elements, type IV secretion systems, andhorizontal gene transfer among Legionella, amoeba, and other organisms seems to takeplace. This long-lasting coevolution led to the development of very sophisticated virulencestrategies and a high level of temporal and spatial fine-tuning of bacteria host–cell interac-tions. We will discuss current knowledge of the evolution of virulence of Legionella from agenomics perspective and propose our vision of the emergence of this human pathogen fromthe environment.

Legionellosis or Legionnaires’ disease is a se-vere pneumonia caused by bacteria belong-

ing to the genus Legionella. The first member ofthis genus, Legionella pneumophila, was recog-nized as a human pathogen during an outbreakof severe pneumonia in 1976 in Philadelphia, PA(Fraser et al. 1977; McDade et al. 1977). Sincethen, outbreaks occur every year, in particularin the industrialized world, and legionellosisremains an up-to-date health problem. Thirty-five years after the discovery of Legionella pneu-mophila, the genus Legionella has grown consid-erably as it now contains 59 species (http://old.dsmz.de/microorganisms/bacterial_nomencla-ture_info.php?genus¼Legionella), all of which

are ubiquitous, environmental bacteria. Al-though 24 different species of Legionella havebeen isolated at least once from humans, L. pneu-mophila is the major cause of Legionnaires’ dis-ease worldwide as it causes more than 90% of thediagnosed cases. Legionella longbeachae is thesecond cause of legionellosis, responsible for3.9% of the cases (Yu et al. 2002; Newton et al.2010). Interestingly, the epidemiology is differ-ent in Australia and New Zealand, where L. pneu-mophila accounts for only 45.7% but L. long-beachae for 30.4% of the cases. Thus, researchfocuses mainly on L. pneumophila and L. long-beachae. In contrast, little or nothing is knownabout the other Legionella species with respect to

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theirenvironmental distribution, ecology, or thecapacity to trigger human disease. All Legionellaspecies, except L. longbeachae, which is presentmainly in compost and potting mix, are found infreshwater environments (Whiley and Bentham2011).

Legionella are intracellular bacteria whosenatural hosts are aquatic protozoa, in whichthese bacteria replicate and are protected fromharsh environmental conditions (Rowbotham1980). Legionella are able to parasitize at least20 different species of amoeba, two species ofciliated protozoa, and one slime mold (Lau andAshbolt 2009), but are associated most frequent-ly with amoeba belonging to the genera Acan-thamoeba, Hartmanella, and Naegleria (Fields1996). Interestingly, not all Legionella will growin the same amoebal host (Rowbotham 1986)and certain amoeba seem also to be resistant toLegionella infection (Atlan et al. 2012). Thus,Legionella are broad host-range protozoan par-asites but some species, in particular L. pneumo-phila, are also pathogens of humans. The vehicleof transmission to humans is aerosols loadedwith Legionella. Changes in the human lifestylein the last decade led to an increased number ofartificial water systems such as air conditioningunits, cooling towers, showers, and fountains,where water is given off in a fine spray providingthe bacterium with the possibility to reach hu-mans as an accidental host. However, althoughLegionella is able to infect humans, human in-fection is a dead end for Legionella replication, asperson-to-person transmission has never beenreported. Therefore, the evolution of virulencetraits in L. pneumophila and L. longbeachae hasresulted from the interaction with environmen-tal protozoa.

The capacity of Legionella to infect its natu-ral hosts, amoeba or ciliated protozoa but alsohuman macrophages, indicates that the mecha-nisms developed by this bacterium to infect loweukaryotes can also be used for infection of cellsof high eukaryotes like humans as was proposedthe first time by Rowbotham in 1980 (Rowbo-tham 1980). This also suggests that virulencefactors of Legionella probably target conservedeukaryotic pathways, allowing Legionella to in-fect many phagocytic eukaryotic cells like differ-

ent aquatic protozoa, ciliates, and human cells.Therefore, the identification of what are thesefactors, how they have been acquired, and whatare their functions, is fundamental to decipherhost–pathogen interactions and the virulencestrategies used by intracellular pathogens.

In recent years, it has been shown that severalintracellular bacterial pathogens are able to enterand replicate in environmental amoebae, similarto what is known for Legionella (Molmeret et al.2005). This observation has led to the idea thatfree-living amoebae are the “Trojan horses” ofthe microbial world, as they have a role in sur-vival, replication, and transmission of bacteria(Winiecka-Krusnell and Linder 1999). The in-teraction between both organisms provides bac-teria with the capacity to infect human cells andto become human pathogens. Furthermore, ge-nome analyses and comparative genomics ofamoeba-associated bacteria revealed that com-mon virulence factors are present in bacteria liv-ing with amoebae (Schmitz-Esser et al. 2010).This finding suggested that the genes codingfor these virulence factors may be interchangedand that they may thus be part of mobile geneticelements. The complete set of mobile geneticelements of a genome is commonly called “mo-bilome” (Siefert 2009), a term we will use here-after. Particularly in bacteria living with amoe-bae, this mobilome seems to be highly dynamic.Because L. pneumophila is probably the best-studied amoeba-associated bacterium, it has be-come a model to study the role of amoebal hostsin bacterial pathogenesis and the acquisition ofvirulence factors from this host–pathogen rela-tionship.

In this review, we will present the state-of-the-art of the evolution of virulence in Legionellafrom a genomics perspective and how currentdata increased our knowledge about Legionellavirulence evolution and host–pathogen inter-action and acquisition of virulence factors ingeneral.

THE Legionella GENOMES ARE GENETICALLYDIVERSE

L. pneumophila strains Philadelphia, Paris, andLens were the first Legionella genomes se-

L. Gomez-Valero and C. Buchrieser

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quenced (Cazalet et al. 2004; Chien et al. 2004).Each strain contains one circular chromosomethat is characterized by an average G þ C con-tent of about 38% and by a size between 3.3 and3.5 Mb. In each strain �3000 protein-codinggenes are predicted, representing a coding ca-pacity of 88% (Table 1A). Comparative geno-mics revealed that �300 genes (�10%) are spe-cific for each strain. Taking into account thatthese strains belong to the same species andserogroup, the genomic diversity among themis relatively high. Further analysis of the genecontent of 217 L. pneumophila strains using aLegionella DNA array confirmed that the genecontent of the L. pneumophila genome is highlydiverse (Cazalet et al. 2008). Both studies sug-gested that the main source of diversity amongthe Legionella genomes is mobile genetic ele-ments and horizontal gene transfer among L.

pneumophila strains, but also among strains be-longing to different Legionella species, otherbacterial species, and most surprisingly, proba-bly also between Legionella and their eukaryotichosts (Cazalet et al. 2004, 2008).

Recent years have seen five new L. pneumo-phila genomes sequenced providing the possi-bility to study their diversity and evolution indepth (Table 1A) (Steinert et al. 2007; D’Auriaet al. 2010; Schroeder et al. 2010; Gomez-Valeroet al. 2011b). Comparative analyses of the eightavailable genomes showed that the core genomecontains 2405 orthologous groups of genes, andeach genome when compared with the sevenothers contains between 154 and 271 strain-spe-cific genes (Fig. 1). Furthermore, more than1000 genes are not present in all eight genomesand thus also belong to the flexible gene contentor accessory genome. In contrast, the L. long-

Table 1. General features of the Legionella genome sequences published

(A) L. pneumophila

Paris Lens Philadelphia Corby Alcoy 130b Lorraine HL06041035

Chromosome size (Mb) 3.5 3.3 3.4 3.6 3.5 3.5 3.5 3.5G þ C content (%) 38 38 38 38 38 38 38 38Number of genes 3178 3034 3083 3290 3197 3293 3170 3184Number of protein-coding

genes3079 2921 2999 3193 3191 3288 3080 3079

Pseudogenes 45 59 32 44 ? ? 37 53tRNA 43 43 43 44 ? 42 44 4316S/23S/5S 3/3/3 3/3/3 3/3/3 3/3/3 3/3/3 3/3/3 3/3/3 3/3/3Coding density (%) 87 87 88 87 86 87 87Plasmids 1 1 0 0 0 0 1 0

(B) L. longbeachae

NSW 150# D-4968þ ATCC39462þ 98072þ C-4E7þChromosome size (Kb) 4.0 4.0 4.1 4.0 3.9G þ C content (%) 37.1 37.0 37.0 37.0 37Number of genes 3660 3557 – – –16S/23S/5S 4/4/4 4/4/4 4/4/4 4/4/4 4/4/4No. of contigs . 0.5–300 kb Complete 13 64 65 63N50 contig sizea Complete – 138 kb 129 kb 134 kbPercentage of coverageb 100% 96.3 96.3 93.4 93.1Number of SNP with NSW150 – 1900 1611 16 853 16 820Plasmids 1 1 0 1 1

aN50 contig size, calculated by ordering all contig sizes and adding the lengths (starting from the longest contig) until the

summed length exceeds 50% of the total length of all contigs.bFor SNP detection: #, completed sequence; þ, draft sequence, not completed; SNP, single nucleotide polymorphism.

Legionella Evolution and Virulence

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beachae genomes, five of which have been se-quenced to date (Cazalet et al. 2010; Kozaket al. 2010), have a slightly larger genome sizeof 3.9–4.1 Mb and show much lower diversityamong them (Table 1B). When comparing twostrains of L. longbeachae belonging to differentserogroups (Sg), the overall DNA polymor-phism is less than 0.4%, whereas this polymor-phism is �2% between L. pneumophila strainsof the same Sg (Cazalet et al. 2010). Interesting-ly, despite the fact that L. pneumophila and L.longbeachae cause the same human disease,high divergence of these two species was ob-served. Only 2290 (65.2%) L. longbeachae genesare orthologous to L. pneumophila genes, where-as 1222 (34.8%) are L. longbeachae specific withrespect to L. pneumophila Paris, Lens, Philadel-phia, and Corby (Cazalet et al. 2010; Gomez-Valero et al. 2011b). The different genome anal-yses, when combined, revealed that the accessorygenome of Legionella, containing approximately3500 genes when eight L. pneumophila and fiveL. longbeachae genomes are analyzed, is largeand, most interestingly, the main part seems to

be transferable, thus constituting the mobilomeof the genus.

THE MOBILOME OF Legionella HASA DIVERSE ORIGIN

The Legionella mobilome comprises plasmids,integrative conjugative elements, insertion se-quences, and genomic-island-like regions likemost of the prokaryotic genomes. However, italso has a particular feature, which is a large arrayof so-called eukaryotic-like genes that were iden-tified during the L. pneumophila genome anal-yses of strains Paris and Lens in 2004 (Cazaletet al. 2004). These genes code for proteins thatshow the strongest similarity, not to prokaryoticbut to eukaryotic proteins, or encode motifsknown to be implicated in protein–protein in-teractions, which are present onlyor primarily ineukaryotes. The eukaryotic-like proteins werepredicted to mimic host proteins to allow intra-cellular replication of Legionella and are thusgood candidates for being implicated in host–pathogen interactions (Cazalet et al. 2004). In-terestingly, many of these potential virulencefactors never had been identified in a prokary-otic genome before the discovery in L. pneumo-phila, suggesting that L. pneumophila may havedeveloped specific mechanismsto cross talk withits eukaryotic hosts (Bruggemann et al. 2006).The presence of a high number of eukaryotic-like proteins in the genus Legionella was furtherconfirmed when analyzing different L. pneumo-phila and L. longbeachae genomes (de Felipeet al. 2005; Cazalet et al. 2010; Kozak et al.2010; Schroeder et al. 2010; Gomez-Valeroet al. 2011b). Thus, the Legionella genomes re-flect the interaction of this bacterium withamoeba, which is probably the driving force inthe evolution of Legionella pathogenicity.

Although few eukaryotic-like genes werethen also identified in other bacteria like Ricket-tsia belli or Wolbachia (Ogata et al. 2006; Walk-er et al. 2007), Legionella is the bacterium withthe widest variety of eukaryotic-like proteinsand eukaryotic domain-carrying proteins; afinding that still holds true today, despite thehundreds of different bacterial genome se-quences that have been sequenced and analyzed

Paris Lens

Philadelphia

Alcoy

Lorraine

HL06041035211 220

178

271

207217

Corby

154

Coregenome

2405

Figure 1. Shared and specific gene content of eightL. pneumophila genomes. Each petal represents a ge-nome with an associated color. The number in thecenter of the diagram represents the orthologousgroups of genes shared by all the genomes. The num-ber inside of each individual petal corresponds to thespecific genes of each genome with nonorthologousgenes in any of the other genomes. The orthologousgroups were defined using the program PanOCT(Fouts et al. 2012) (the parameters used were e-value�1 � 1025, percent identity �35, and length of thematch �75).



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since then. In 2004, more than 60 of these pro-teins were described in the genomes of strainsParis and Lens (Cazalet et al. 2004) and in 2005,in strain Philadelphia (de Felipe et al. 2005).Later, the analysis of the distribution of thesegenes among more than 200 L. pneumophilastrains using DNA arrays showed that the con-servation of certain eukaryotic-like genes with-in the species L. pneumophila was high, indicat-ing strong environmental selection pressures fortheir preservation (Cazalet et al. 2008).

Recently, the sequencing and analyses of fiveadditional L. pneumophila genomes revealedthat over half of the eukaryotic-like proteinsare present in all eight L. pneumophila strains(Gomez-Valero et al. 2011a,b). We thus proposethat these conserved eukaryotic-like proteinshave probably allowed the ancestor of L. pneu-mophila to better adapt to the intracellular en-vironment, but now most of them are evolvingas part of the core genome. Interestingly, ap-proximately 30% of the proteins containing eu-karyotic motifs are also present in the speciesL. longbeachae (Cazalet et al. 2010; Gomez-Va-lero et al. 2011a). These proteins might be partof the core of eukaryotic-like proteins essen-tial for the genus Legionella for its intracellularreplication. However, simultaneously, a flexiblepool of eukaryotic-like proteins is present in theLegionella genomes. It represents more than halfof the eukaryotic-like proteins identified to datein different L. pneumophila strains. The fact thateven phylogenetically very closely related strainslike the L. pneumophila strains HL06041035 andParis present some differences in their reper-toire of eukaryotic-like proteins suggests thatthe acquisition and loss of these genes is anongoing process that has taken place multipletimes and further underlines the high dynamicsand plasticity of the Legionella genomes. In ad-dition to horizontal gene transfer a process ofconvergent evolution might be important in theevolution of Legionella as, although many of theproteins containing eukaryotic motifs are notconserved between the species L. pneumophilaand L. longbeachae, the same eukaryotic motifslike F-box, U-box, ankyrin, or serine thereonineprotein kinase (STPK) motifs are found in bothspecies (Cazalet et al. 2010). A similar observa-

tion has been made when the genome sequenceof the amoeba symbiont “Candidatus Amoebo-philus asiaticus” has been analyzed. Interesting-ly, many of the eukaryotic motifs identified inLegionella are also present in this bacterium(Schmitz-Esser et al. 2010). Most interestingly,an enrichment analysis comparing the fractionof all functional protein domains among 514bacterial proteomes revealed that all genomesof bacteria replicating in amoeba were enrichedin protein domains that are predominantlyfound in eukaryotic proteins (Schmitz-Esseret al. 2010). The most highly enriched domainswere ANK repeats, LRR, SEL1 repeats, and F-and U-box domains, all of which are also pre-sent in L. pneumophila and L. longbeachae. Tak-en together, genome analyses of different bac-teria associated with amoeba undertaken sincethe L. pneumophila genome sequence had beenpublished in 2004, showed that bacteria thatcan exploit amoebae as hosts share a set of eu-karyotic domain proteins that are most proba-bly important for bacteria–host–cell interac-tions, despite their different lifestyles and theirlarge phylogenetic diversity. Thus, a global, con-vergent adaptation mechanism to intracellularparasitism seems to exist in amoeba-associatedbacteria.

EUKARYOTIC-LIKE PROTEINS OF LegionellaARE VIRULENCE FACTORS THAT MIMICHOST PROTEINS

One important prediction from the genomeanalysis of strains Paris and Lens was that theeukaryotic-like proteins of Legionella are secret-ed virulence factors able to interfere with manydifferent host-signaling pathways by mimick-ing host proteins (Cazalet et al. 2004; Brug-gemann et al. 2006). Therefore, many groupsstarted functional studies focusing on theseproteins and within a short period of time theimportance of the eukaryotic-like proteins forthe virulence of Legionella was shown (de Felipeet al. 2008; Nora et al. 2009; Hubber and Roy2010). The main secretion system of Legionella,essential for intracellular growth, is the type IVBDot/Icm secretion system (Marra and Shuman1992; Berger and Isberg 1993). Using many dif-



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ferent methods it has been shown that at least275 L. pneumophila proteins are secreted by thissystem (Campodonico et al. 2005; de Felipeet al. 2005; Shohdy et al. 2005; de Felipe et al.2008; Burstein et al. 2009; Heidtman et al. 2009;Zhu et al. 2011), which represents nearly 10%of the protein-coding genes predicted in theL. pneumophila genomes. At least 75 of theseare eukaryotic-like proteins or carry eukaryoticdomains and, for certain of these, it has alsobeen shown experimentally that they interferewith host–cell signaling pathways (Table 2).

The diverse motifs identified, like ankyrinrepeats, F-box and U-box domains, STPK do-mains, coil-coiled domains, SET, and Sel1 orSec-7 domains indicate also very diverse func-tional roles of these proteins. This fact is, forexample, reflected in the very high number ofdifferent posttranslational modifications Le-gionella is able to induce in its hosts. To date, ithas been shown that L. pneumophila proteins areable to ubiquitinate, phosphorylate, lipidate,glycosylate, AMPylate, DeAMPylate, phospho-cholinate, and dephosphoryl-cholinate specifichost proteins to modulate multiple host path-ways to the pathogen’s advantage (Rolando andBuchrieser 2012). Recently, a new posttransla-tional modification induced by L. pneumophilawas reported. The Legionella-secreted effector,RomA, was shown to be a SET domain-contain-ing methyltransferase that uniquely modifieshost chromatin to repress gene expression andpromote efficient intracellular bacterial replica-tion (Rolando et al. 2013). Interestingly, a com-bination of eukaryotic domains was predicted inseveral of the eukaryotic-like proteins, leading toa modular protein structure. One example isAnkB/LegAU13/Ceg27/Lpg2144/Lpp2082 thatcontains three different eukaryotic protein do-mains: an F-box domain, an ankyrin domain,and a CaaX motif (Table 2). The F-box domaininteracts with the SKP1 component of the SCF1ubiquitin ligase complex and allows Legionellato exploit the host ubiquitination machinery,whereas the ankyrin motif, a widespread pro-tein–protein interaction motif, probably in-teracts with the cellular target like Parvin B de-scribed for strain Paris (Price et al. 2009; Lommaet al. 2010). The F-box domain, as well as the two

ankyrin protein–protein interaction domains,is essential for the biological function of AnkB.The CaaX motif exploits the host prenyltrans-ferase machinery to facilitate membrane lo-calization of L. pneumophila effector proteins(Ivanov et al. 2010; Price et al. 2010). Anothermodular protein is SdhA/Lpg0376, a Dot/Icm-secreted virulence factor that contains a GRIPand a coiled-coil domain (Table 2). SdhA is re-quired for growth within macrophages and pro-tection from host–cell death (Laguna et al.2006). Furthermore, the Legionella-containingvacuole is actively stabilized by the SdhA proteinduring intracellular replication, which affordsthe bacterium protection from cytosolic hostfactors that degrade bacteria and initiate im-mune responses (Creasey and Isberg 2012).AnkY/LegA9/Lpg0402 is also a modular pro-tein as it contains an STPK domain and an an-kyrin motif. However, to date, its functional roleand putative implication in virulence has notbeen shown.

A specific example for the amazing tem-poral and spatial fine-tuning Legionella hasevolved in its interactions with the host cell isthe Dot/Icm-secreted effector LubX/Lpg2830.LubX (for Legionella U-box) is a protein con-taining two U-box domains (U-box1 and U-box2) similar to eukaryotic E3 ubiquitin ligasesthat function as a ubiquitin ligase in conjunc-tion with host UbcH5a or UbcH5c E2 enzymesand mediates polyubiquitination of cellularClk1 (Cdc2-like kinase) (Kubori et al. 2008).U-box1 seems to be critical for ubiquitin liga-tion, whereas the U-box2 domain interacts withthe substrate. Recently, it was shown that thesecreted L. pneumophila effector protein SidHis a second target of LubX within host cells. LubXdirectly binds and polyubiquitinates SidH invitro and mediates its proteasomal degradationin infected cells. LubX is the first example of abacterial metaeffector that regulates spatiallyand temporally the expression level of anothereffector (Kubori et al. 2010). The identificationof this mechanism points to the fact that theremight be many different levels of regulationamong the large number of Legionella effectorsas well as between the effectors and their hosttargets.



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Table 2. List of selected substrates of the Dot/Icm secretion system in L. pneumophila and L. longbeachae strains

L. pneumophila strains L. longbeachae strains

Phila 130b

Pari

s

Lens

Cor

by

Alc

oy

Lorr

aine

HL0

6041

035

NSW

150

D-4

968

ATC

C39

462

9807

2

C-4

E7

Name Product or associated motif Function

Legi

onel

la c

ore

gen

ome

lpg0103 llo3312 vipF Amino-terminal acetyltransferase, GNAT Putative modulation of host protein trafficking

lpg0257 llo2362 sdeA Multidrug resistance protein Adhesion, invasion

lpg0376 llo0548 sdhA GRIP, coiled-coil Putative antiapoptotic

lpg0405 llo2845 - Spectrin Unknown

lpg0422 llo2801 legY Putative glucan 1,4-α-glucosidase Unknown

lpg0483 llo2705 ankC/legA12 Ankyrin Unknown

lpg0515 llo3224 legD2 Phytanoyl-CoA dioxygenase Unknown

lpg1426 llo1791 vpdC Patatin Unknown

lpg1483 llo1682 legK1 STPK Modulation of host immune signaling

lpg1661 llo1691 - Putative N-acetyl transferase Unknown

lpg1950 llo1397 ralF Sec7 domain Protein recruitment to the LCV

lpg2222 llo1443 lpnE Putative β-lactamase (sel1 and TTR domains) Host cell entry

lpg2298 llo1707 ylfA/legC7 Coiled-coil Putative modulation of host protein trafficking

lpg2300 llo0584 ankH/legA3/ankW Ankyrin, NF-κB inhibitor Intracelullar proliferation

lpg2322 llo0570 ankK/legA5 Ankyrin Unknown

lpg2456 llo0365 ankD/legA15 Ankyrin Unknown

lpg2556 llo2218 legK3 STPK Unknown

lpg2832 llo0214 - Hypothetical protein Unknown

lpg2936 llo0081 - rRNA methyltransferase E Unknown

L. p

neum

ophi

la c

ore

gen

ome

lpg0038 ankQ/legA10 Ankyrin Unknown

lpg0403 ankG/ankZ/ legA7 Ankyrin Unknown

lpg0436 ankJ/legA11 Ankyrin Intracellular proliferation

lpg0695 ankN/ankX/legA8 Ankyrin Modulation of vesicular transport

lpg1488 lgt3/legc5 Coiled-coil Cytotoxicity

lpg1701 ppeA/legC3 Coiled-coil Putative modulation of host protein trafficking

lpg1718 ankI/legAS4 Ankyrin methyltransferase Unknown

lpg1884 ylfB/legC2 Coiled-coil Putative modulation of host protein trafficking

lpg1962 lirB/ppiB/rotA Rotamase Unknown

lpg1976 pieG/legG1 Regulator of chromosome condensation Unknown

lpg1978 setA Putative glucosyltransferase Putative modulation of host protein trafficking

lpg2137 legK2 Ca-depPK ER recruitment to the LCV

lpg2144 ankB/legAU13/ ceg27 Ankyrin, F-box, CaaX Recruitment of polyubiquitinated proteins to the LCV

lpg2176 legS2 Sphingosine-1-phosphate lyase Autophagy

lpg2215 legA2 Ankyrin Unknown

lpg2410 vpdA Patatin domain Unknown

lpg2452 ankF/legA14/ceg31 Ankyrin Unknown

lpg2793 lepA Effector protein A Release of bacteria

lpg2999 legP Astacin protease Unknown

Continued

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Table 2. Continued

L. pneumophila strains L. longbeachae strains

Phila 130b

Pari

s

Lens

Cor

by

Alc

oy

Lorr

aine

HL0

6041

035

NSW

150

D-4

968

ATC

C39

462

9807

2

C-4

E7

Name Product or associated motif Function

L. lo

ngbe

acha

e co

re g

enom

e

llo0037 - Ankyrin Unknownllo1168 - Ankyrin Unknownllo1371 - Ankyrin, coiled-coil Unknownllo2668 - Ankyrin Unknownllo3081 - Ankyrin, patatin-like phospholipase Unknownllo3093 - Ankyrin, STPK Unknownllo0114 - LRR Unknownllo1681 - STPK Unknownllo1427 - F-Box Unknownllo0448 - U-Box Unknownllo2643 - PPR, Coiled-coil Unknownllo2200 - TTL Unknownllo2327 - SH2 Unknownllo2352 - PAM2 Unknownllo1196 - Snare Unknownllo0793 - Phosphatidylinositol-4-phosphate 5-kinase Unknownllo2329 - Ras-related small GTPase, Miro-like domain Unknownllo2249 - Miro-like domains Unknownllo1892 - Putative immunoglobulin I-set domain Unknown

Legi

onel

la a

cces

sory

gen

ome

llo0990 - Ankyrin Unknownllo3116 - LRR Unknownllo3118 - LRR Unknown

lpg0041 - Putative metalloprotease Unknownlpg0171 legU1 F-box motif Interaction with host ubiquitination machinerylpg0402 ankY/legA9 Ankyrin, STPK Unknownlpg1328 legT Thaumatin domain Unknownlpg1355 sidG Coiled-coil Interaction with dot/icms system—translocationlpg1684 - Unknown Unknownlpg1890 legLC8 LRR, coiled-coil Unknownlpg1947 lem16 Spectrin Unknownlpg1948 legLC4 LRR, coiled-coil Unknownlpg2224 ppgA Regulator of chromosome condensation Unknownlpg2400 legL7 LRR Unknownlpg2830 lubX/legU2 U box motif Polyubiquitination of host proteinslpg2862 Lgt2/legC8 Glucosyltransferase Cytotoxicity

Filled squares represent presence, white squares absence of a gene. ER, endoplasmatic reticulum; LCV, Legionella-containing vacuole; STPK, seronine threonine protein kinase; LLR, leucine rich repeat; TTL, tubulin-tyrosine ligase; SH2, Src homology 2; PAM, PCI/PINT-associated module; Rab1 GAP, GTPase-activating pro-tein; TTR, tetratricopeptide repeats; Ca-depPK, calmodulin-dependent protein kinase.

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EUKARYOTIC-LIKE PROTEINS WITNESSTHE LONG-LASTING COEVOLUTIONOF Legionella WITH EUKARYOTIC CELLS

The presence of these many eukaryotic-like pro-teins in the genome of amoeba-associated bac-teria raises the question of the origin of thispart of the genome. Are these eukaryotic-likegenes laterally acquired or are they the result ofadaptive changes generating motifs similar tothe eukaryotic ones? The latter is known as “con-vergent evolution” in contrast to horizontal genetransfer (HGT). Although both can lead to sim-ilar proteins, similarity resulting from HGT isdetectable at the primary sequence level, where-as convergent evolution more frequently leads tosimilar tridimensional structures but not neces-sarily to similar amino acid sequences. Legion-ella possesses all necessary factors for acquiringgenes laterally as these bacteria are naturallycompetent and a complete recombination ma-chinery is present, thus it is conceivable thateukaryotic-like proteins have been acquired byHGT. To analyze this question, different phylo-genetic analyses of some of these eukaryotic-likeproteins have been performed revealing a clus-tering of the Legionella eukaryotic-like proteinswith eukaryotic sequences, further supportingthe hypothesis that these were acquired by HGTfrom eukaryotic organisms like amoeba (de Fe-lipe et al. 2005; Nora et al. 2009; Lurie-Wein-berger et al. 2010; Gomez-Valero et al. 2011a).An example is the enzyme arylamine N-acetyl-transferase (NAT) encoded by lpp2516 in strainParis. The L. pneumophila NAT is an atypicalmember of the arylamine N-acetyltransferasefamily that allows L. pneumophila to detoxifyaromatic amine chemicals and to grow in theirpresence (Kubiak et al. 2012). This enzymeprobably allows L. pneumophila that is exposedto many chemicals in its natural and man-madeenvironments, to better adapt and survive inthese environments (Kubiak et al. 2012). Phylo-genetic reconstruction shown in Figure 2A indi-cates that horizontal acquisition of this genefrom ciliates is the most probable origin of thisenzyme in Legionella (Kubiak et al. 2012).

Another convincing example is the eukary-ote-like sphingosine-1-phosphate lyase of L.

pneumophila that is encoded by lpp2128 instrain Paris. It has been shown that the L. pneu-mophila Spl is a Dot/Icm-secreted effector thatis able to complement the sphingosine-sensitivephenotype of Saccharomyces cervisiae and thatcolocalizes with host–cell mitochondria (Deg-tyar et al. 2009). Figure 2B shows a phylogeneticreconstruction of Spl sequences that we haveundertaken. The L. pneumophila Spl proteinsequence clearly falls into the eukaryotic cladeof SPL sequences and those of aquatic protozoaare the closest phylogenetic relatives, suggestingacquisition of this gene by HGT from an amoe-ba host. Most interestingly, Legionella also en-codes proteins not present in any prokaryotesequenced to date, as amoebae are the only or-ganisms in which we identified sequences withsignificant similarity. An example is Lpg1684that codes a protein of unknown function andbelongs to the eukaryotic D123 protein family.Eukaryotic-like proteins may have been ac-quired also from othereukaryotic organisms likeviruses. For example, the eukaryotic-like pro-tein Lpg2416 is an ankyrin-containing proteinwhose best homolog is present in the Acanth-amoeba polyphaga mimivirus, a giant virus in-fecting Acanthamoeba that are also the hosts ofLegionella (Lurie-Weinberger et al. 2010). Fur-thermore, recent studies have shown that theseeukaryotic-like proteins can also be exchangedamong different amoeba-related bacteria, add-ing complexity to the possible evolutionary sce-narios. For example, the sequencing and analy-sis of the genome of L. drancourtii have iden-tified numerous cases of exchange between theintracellular bacteria of the order Legionellalesand Chlamydiales (Gimenez et al. 2011). How-ever, for several eukaryotic-like proteins, a cleardescription of their phylogenetic history re-mains difficult, as the acquisition events mighthave been too ancient for the phylogenetic sig-nal in the sequences to be preserved. Moreover,in some of these proteins, the eukaryotic se-quence is restricted to a motif and therefore toa very short region, for which phylogenetic anal-yses are less evident. Furthermore, only few ge-nome sequences of aquatic amoeba and otherprotozoa that are most probably the main do-nors are currently present in the databases. Only



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100A Gallus gallus

Meleagris gallopavo

Taeniopygia guttata

Monodelphis domestica

Ciona intestinalis

Naegleria gruberi

Dictyostelium fasciculatum

Dictyostelium purpureum

Paramecium tetraurelia

L. pneumophila strains (Lpp2516)

Nectria haematococca

Aspergillus niger

Magnaporthe oryzae

Talaromyces stipitatus

Penicillium chrysogenum

Neosartorya fischeri

Ajellomyces capsulatus

Arthroderma otae

Trichophyton rubrum

Pelodictyon phaeoclathratiforme

Chlorobphaeobacteroides

Prosthecochloris aestuarii

Chlorobium ferrooxidans

Chlorolimicola

Candidatus Nitrospira defluvii

Cellvibrio japonicus

Geobacter bemidjiensis

Kribbella flavida

Gordonia neofelifaecis

Pseudonocardia sp.

Ralstonia eutropha

Burkholderia xenovorans

Enterobacter cloacae

Pseudomonas fluorescens

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Figure 2. Phylogenetic tree of selected eukaryotic-like proteins of Legionella spp. (A) Phylogeny of the enzymearylamine N-acetyltransferase (NAT) (Lpp2516) of L. pneumophila. (B) Phylogeny of the sphingosine-1 phos-phate lyse Lpp2128 of L. pneumophila. Homolog sequences were recruited from a database containing onlycompleted genome sequences. Selected representatives of all eukaryotic groups and one representative of eachbacterial species are included in the analyses. After blastp only significant hits were recruited (e-value ,10 �1024), and only one hit for each species was retained. The alignments were performed with T-coffee (expresso)for Lpp2616 and Muscle for Lpp2128, and followed by manual curation. Phylogenies were reconstructed using adistance method (NJ) with 1000 bootstrap replicates. The corresponding support values are shown in each node(values lower than 50 are not represented). Bars represent 20% and 10% of estimated phylogenetic divergence,respectively. (Legend continues on following page.)



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once the gap of genome sequences of aquaticprotozoa in the databases is filled will we beable to fully understand the origin and historyof these eukaryotic-like proteins.

Independently of the origin of these eukary-otic-like genes, their mechanism of integrationremains unknown. How are these eukaryoticgenes without any similarity to prokaryotic se-quences integrated in the Legionella genome?

Many eukaryotic genes have introns, but theirprokaryotic counterparts do not. How can theseintrons be processed or how did they “disap-pear?” It is tempting to propose that this hap-pened not via HGT but via “horizontal RNAtransfer.” Indeed, the L. pneumophila genomesencode a reverse transcriptase that could beimplicated in this process. However, the inte-gration mechanism remains to be discovered.

Euglenozoa (5)100

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Choanozoa (2)

Metazoa (5)

Fung1 (16)

Roseiflexus castenholzii

Haliangium ochraceum

Myxococcus spp. (2)

Burkholderia (2)

Tetrahymena thermophila

Paramecium tetraurelia

Legionella spp. (Lpp2128)

Entamoeba spp. (3)

Mycetozoa (4)

Phaeodactylum tricornutum

Oomycetes (4)

Plants (6)

Cyanidioschyzon merolae

Symbiobacterium thermophilum

Stigmatella aurantiaca

Prokaryotes (82)

Dictyostelium fasciculatum

Haliscomenobacter hydrossis

Naegleria gruberi

Thermomonospora curvata

B

Figure 2. (Continued). The clustering of Legionella and eukaryotes was confirmed in both cases by in-parallellikelihood reconstructions.



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Moreover, there is evidence for the presence of asecretion signal in the carboxy terminal of theDot/Icm substrates (Nagai et al. 2005; Kuboriet al. 2008; Huang et al. 2010); therefore, acqui-sition of this signal is also a challenge for theseproteins to be secreted. Thus, although we startto discern how these eukaryotic proteins haveappeared in the Legionella genomes, manyquestions with respect to the detailed mecha-nism of acquisition and integration remain un-answered.

THE Legionella ACCESSORY GENES AREPART OF THE MOBILOME

The most evident component of the Legionellaaccessory genome is the plasmids. The strainsParis, Lens, and Lorraine each contain a plasmidof 132, 60, and 150 kb, respectively (Cazaletet al. 2004; Chien et al. 2004). Likewise, L. long-beachae strain NSW150 possesses a plasmid of72 kb, and also strain D-4968 seems to containa plasmid (Kozak et al. 2010). These repliconsshow a heterogeneous distribution among thedifferent genomes, as the same plasmids or par-tially similar plasmids are present in differentstrains (Cazalet et al. 2008). Likewise, a compar-ative analysis of these plasmids revealed that the30.4 kb Tra region present in the plasmid ofL. pneumophila strain Paris shows much higheridentity with the Tra region located on theL. longbeachae plasmid, than with those of theother L. pneumophila strains sequenced (Go-mez-Valero et al. 2011b). Interestingly, regionscoding proteins homologous to Tra proteinsprobably coding for a conjugative machineryare present in all Legionella plasmids. They aresimilar to F-type T4SS that allow the synthesisof a long and flexible pilus for conjugation inliquid and solid media (Lawley et al. 2003).However, such a system is also found in a chro-mosomal localization in the L. pneumophilastrain Philadelphia and in L. longbeachae strainNSW150. In both strains, this region is insertedin a tRNA gene next to an integrase and it isbordered by flanking repeats. The presence ofthese elements suggests that these T4SSs aremobile and that their heterogeneous distribu-tion is the result of the lateral movement of these

plasmids. Furthermore, these conjugative ele-ments can be found in different plasmids or canbe completely or partially present in the chro-mosome, indicating that they might have thecapacity to integrate and excise from the Legion-ella genomes. Indeed, this has been shown forthe region carrying the Lvh genes. Lvh is a ge-nomic-island-like region that encodes a T4ASSimplicated in conjugation and in virulence-re-lated phenotypes under laboratory conditionsmimicking the spread of Legionnaires’ diseasefrom environmental niches (Ridenour et al.2003; Bandyopadhyay et al. 2007). This regioncan be integrated in the chromosome but canalso excise in a site-specific manner to exist as alow copy number plasmid that is undoubtedlytransferable among strains (Doleans-Jordheimet al. 2006). Indeed, it is present in five of theeight sequenced L. pneumophila strains, in L.longbeachae strain D-4968 (Kozak et al. 2010),and according to hybridization results also part-ly in Legionella rubrilucens (Cazalet et al. 2008).Interestingly, in the Legionella strains where theLvh T4SS is not present, another Tra system, a P-type T4SS that codes for short and rigid pili thatallow surface mating for conjugation, is presentin the same chromosomal position (Gomez-Va-lero et al. 2011b). This remarkable diversity andmobility of conjugative elements probably cod-ing T4SSs in Legionella and their presence in allstrains, suggests a role in the bacterial adapta-tion to the different environments and hosts.

An intriguing feature of these probably mo-bile regions is their association with genes cod-ing for homologs of a putative phage repressorprotein (PrpA) and for homologs of LvrA, LvrB,and LvrC, first described for the Lvh region ofL. pneumophila. LvrC is a homolog of CsrA, anRNA-binding protein crucial for the regulationof the switch between the replicative and trans-missive phases of L. pneumophila (Molofsky andSwanson 2004). It is tempting to assume that theCsrA homolog encoded on each of these regionsis implicated in the regulation of the mobilityand/or the integration or excision of these is-lands. Perhaps under certain conditions it isadvantageous for Legionella to have multiplecopies of these plasmids to achieve a higher ex-pression of these genes or for promoting high



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frequencies of DNA interchange to rapidlyadapt to new conditions. This idea is furtherunderlined by the observation that the presenceof homologous Lvr loci is not only limited to theLvh or Tra regions, but they are also present inother genomic locations that seem to be mobile.Thus, large parts of the accessory genome ofLegionella are mobile, and it seems that theirmobility is regulated by a particular mechanismthat still remains elusive.

CONCLUSIONS: THE Legionella ACCESSORYGENOME IS PART OF A GLOBAL MOBILOMECONFERRING VERSATILITY

One of the best-known characteristics of obli-gate, intracellular bacteria is a reduction of thegenome size as many genes become dispensablegiven the stability of the intracellular environ-ment they inhabit. In addition, there is a minorprobability of DNA exchange as they live in a

closed niche (Moya et al. 2008). Extreme exam-ples are the symbiontic bacteria Buchnera aphi-dicola, an insect endosymbiont found in theaphid Cinaria cedri with a genome size of 416 Kbcoding solely for 357 protein-coding genes (Pe-rez-Brocal et al. 2006) or the obligate intracellu-lar parasite Rickettsia prowazekii, the causativeagent of epidemic typhus with a 1.1 Mb genome(Andersson et al. 1998). However, this does nothold true for amoeba-associated bacteria likeLegionella, as no signs that they follow a reduc-tive evolutionary path are present in their ge-nomes, although Legionella are probably repli-cating exclusively intracellularly. Furthermore,Legionella have highly dynamic genomes and alarge flexible gene pool is present in the genuscomprising plasmids, mobile genetic elements,and genes probably transferred from their hosts.This large mobilome confers to these bacteriatheir high versatility and capacity to adapt tomany different environmental conditions and

Legionella

Genomic islandsPlasmids

Eukaryotic- likeproteins

Transposons

Globalmobilome

Mimivirus

Amoeba

NucleusOther bacteria

Coregenome

Mobilome

Figure 3. Model of the genetic flow inside amoeba. The accessory genome of Legionella, other intracellularbacteria, mimivirus and amoeba are contributing to a “global mobilome” that is accessible to the differentorganisms living inside amoeba to adapt to different conditions and allows versatility in the host–pathogeninteractions.



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to infect diverse hosts like amoeba, protists,probably algae, and humans. Thus, the intracel-lular environment provided by amoeba is differ-ent from the environment in which symbiontslike (e.g., Buchneria or Rickettsia live), probablybecause amoeba may host different bacteria andviruses at the same time, allowing exchange ofgenetic material among a variety of organisms.We thus propose that amoeba and aquatic pro-tozoa form communities with a large pool ofexchangeable genetic material, and have a largecommon accessory genome, derived from thehost and from the diverse intracellular organ-isms that inhabit them as parasites or symbionts(Fig. 3). This big “global mobilome” allows theconstant acquisition of DNA and provides alarge repertoire of diverse functions for adapt-ing to changing environmental conditions. Thislarge mobile gene pool combined with the highgenome dynamics provide the basis of versatili-ty and adaptability in Legionella and allow suc-cessful intracellular parasitism in many differenthosts.

ACKNOWLEDGMENTS

This work received financial support from theInstitut Pasteur, the Centre National de la Re-cherche (CNRS), the Institut Carnot-PasteurMI, the Fondation pour la Recherche Medicale(FRM), grant No. DEQ20120323697, ANR-10-LABX-62-IBEID, and the ANR-10-PATH-004project “MobilGenomics,” in the frame ofERA-NET PathoGenoMics. We are most grate-ful to Philippe Glaser for critical reading of themanuscript and for fruitful discussions.

REFERENCES

Andersson SG, Zomorodipour A, Andersson JO, Sicheritz-Ponten T, Alsmark UC, Podowski RM, Naslund AK, Er-iksson AS, Winkler HH, Kurland CG. 1998. The genomesequence of Rickettsia prowazekii and the origin of mito-chondria. Nature 396: 133–140.

Atlan D, Coupat-Goutaland B, Risler A, Reyrolle M, Sou-chon M, Briolay J, Jarraud S, Doublet P, Pelandakis M.2012. Micriamoeba tesseris nov. gen. nov. sp: A new taxonof free-living small-sized Amoebae non-permissive to vir-ulent Legionellae. Protist 163: 888–902.

Bandyopadhyay P, Liu S, Gabbai CB, Venitelli Z, SteinmanHM. 2007. Environmental mimics and the Lvh type IVA

secretion system contribute to virulence-related pheno-types of Legionella pneumophila. Infect Immun 75: 723–735.

Berger KH, Isberg RR. 1993. Two distinct defects in intra-cellular growth complemented by a single genetic locus inLegionella pneumophila. Mol Microbiol 7: 7–19.

Bruggemann H, Cazalet C, Buchrieser C. 2006. Adaptationof Legionella pneumophila to the host environment: Roleof protein secretion, effectors and eukaryotic-like pro-teins. Curr Opin Microbiol 9: 86–94.

Burstein D, Zusman T, Degtyar E, Viner R, Segal G, Pupko T.2009. Genome-scale identification of Legionella pneumo-phila effectors using a machine learning approach. PLoSPathog 5: e1000508.

Campodonico EM, Chesnel L, Roy CR. 2005. A yeast geneticsystem for the identification and characterization of sub-strate proteins transferred into host cells by the Legionellapneumophila Dot/Icm system. Mol Microbiol 56: 918–933.

Cazalet C, Rusniok C, Bruggemann H, Zidane N, MagnierA, Ma L, Tichit M, Jarraud S, Bouchier C, Vandenesch F,et al. 2004. Evidence in the Legionella pneumophila ge-nome for exploitation of host cell functions and highgenome plasticity. Nat Genet 36: 1165–1173.

Cazalet C, Jarraud S, Ghavi-Helm Y, Kunst F, Glaser P, Eti-enne J, Buchrieser C. 2008. Multigenome analysis iden-tifies a worldwide distributed epidemic Legionella pneu-mophila clone that emerged within a highly diversespecies. Genome Res 18: 431–441.

Cazalet C, Gomez-Valero L, Rusniok C, Lomma M, Der-vins-Ravault D, Newton HJ, Sansom FM, Jarraud S, Zi-dane N, Ma L, et al. 2010. Analysis of the Legionella long-beachae genome and transcriptome uncovers uniquestrategies to cause Legionnaires’ disease. PLoS Genet 6:e1000851.

Chien M, Morozova I, Shi S, Sheng H, Chen J, Gomez SM,Asamani G, Hill K, Nuara J, Feder M, et al. 2004. Thegenomic sequence of the accidental pathogen Legionellapneumophila. Science 305: 1966–1968.

Creasey EA, Isberg RR. 2012. The protein SdhA maintainsthe integrity of the Legionella-containing vacuole. ProcNatl Acad Sci 109: 3481–3486.

D’Auria G, Jimenez-Hernandez N, Peris-Bondia F, Moya A,Latorre A. 2010. Legionella pneumophila pangenome re-veals strain-specific virulence factors. BMC Genomics 11:181.

de Felipe KS, Pampou S, Jovanovic OS, Pericone CD, Ye SF,Kalachikov S, Shuman HA. 2005. Evidence for acquisi-tion of Legionella type IV secretion substrates via inter-domain horizontal gene transfer. J Bacteriol 187: 7716–7726.

de Felipe KS, Glover RT, Charpentier X, Anderson OR, ReyesM, Pericone CD, Shuman HA. 2008. Legionella eukary-otic-like type IV substrates interfere with organelle traf-ficking. PLoS Pathog 4: e1000117.

Degtyar E, Zusman T, Ehrlich M, Segal G. 2009. A Legionellaeffector acquired from protozoa is involved in sphingo-lipids metabolism and is targeted to the host cell mito-chondria. Cell Microbiol 11: 1219–1235.

Doleans-Jordheim A, Akermi M, Ginevra C, Cazalet C, KayE, Schneider D, Buchrieser C, Atlan D, Vandenesch F,Etienne J, et al. 2006. Growth-phase-dependent mobility



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



of the lvh-encoding region in Legionella pneumophilastrain Paris. Microbiology 152: 3561–3568.

Fields BS. 1996. The molecular ecology of Legionellae. TrendsMicrobiol 4: 286–290.

Fouts DE, Brinkac L, Beck E, Inman J, Sutton G. 2012.PanOCT: Automated clustering of orthologs using con-served gene neighborhood for pan-genomic analysis ofbacterial strains and closely related species. Nucleic AcidsRes 40: e172.

Fraser DW, Tsai TR, Orenstein W, Parkin WE, Beecham HJ,Sharrar RG, Harris J, Mallison GF, Martin SM, McDadeJE, et al. 1977. Legionnaires’ disease: Description ofan epidemic of pneumonia. N Engl J Med 297: 1189–1197.

Gimenez G, Bertelli C, Moliner C, Robert C, Raoult D, Four-nier PE, Greub G. 2011. Insight into cross-talk betweenintra-amoebal pathogens. BMC Genomics 12: 542.

Gomez-Valero L, Rusniok C, Cazalet C, Buchrieser C.2011a. Comparative and functional genomics of Legion-ella identified eukaryotic like proteins as key players inhost–pathogen interactions. Front Microbiol 2: 208.

Gomez-Valero L, Rusniok C, Jarraud S, Vacherie B, Rouy Z,Barbe V, Medigue C, Etienne J, Buchrieser C. 2011b. Ex-tensive recombination events and horizontal gene trans-fer shaped the Legionella pneumophila genomes. BMCGenomics 12: 536.

Heidtman M, Chen EJ, Moy MY, Isberg RR. 2009. Large-scale identification of Legionella pneumophila Dot/Icmsubstrates that modulate host cell vesicle trafficking path-ways. Cell Microbiol 11: 230–248.

Huang L, Boyd D, Amyot WM, Hempstead AD, Luo ZQ,O’Connor TJ, Chen C, Machner M, Montminy T, IsbergRR. 2010. The E Block motif is associated with Legionellapneumophila translocated substrates. Cell Microbiol 13:227–245.

Hubber A, Roy CR. 2010. Modulation of host cell functionby Legionella pneumophila type IV effectors. Annu RevCell Dev Biol 26: 261–283.

Ivanov SS, Charron G, Hang HC, Roy CR. 2010. Lipidationby the host prenyltransferase machinery facilitates mem-brane localization of Legionella pneumophila effectorproteins. J Biol Chem 285: 34686–34698.

Kozak NA, Buss M, Lucas CE, Frace M, Govil D, Travis T,Olsen-Rasmussen M, Benson RF, Fields BS. 2010. Viru-lence factors encoded by Legionella longbeachae identifiedon the basis of the genome sequence analysis of clinicalisolate D-4968. J Bacteriol 192: 1030–1044.

Kubiak X, Dervins-Ravault D, Pluvinage B, Chaffotte AF,Gomez-Valero L, Dairou J, Busi F, Dupret JM, BuchrieserC, Rodrigues-Lima F. 2012. Characterization of an ace-tyltransferase that detoxifies aromatic chemicals in Le-gionella pneumophila. Biochem J 445: 219–229.

Kubori T, Hyakutake A, Nagai H. 2008. Legionella trans-locates an E3 ubiquitin ligase that has multiple U-boxes with distinct functions. Mol Microbiol 67: 1307–1319.

Kubori T, Shinzawa N, Kanuka H, Nagai H. 2010. Legionellametaeffector exploits host proteasome to temporally reg-ulate cognate effector. PLoS Pathog 6: e1001216.

Laguna RK, Creasey EA, Li Z, Valtz N, Isberg RR. 2006. ALegionella pneumophila-translocated substrate that is re-

quired for growth within macrophages and protectionfrom host cell death. Proc Natl Acad Sci 103: 18745–18750.

Lau HY, Ashbolt NJ. 2009. The role of biofilms and protozoain Legionella pathogenesis: Implications for drinking wa-ter. J Appl Microbiol 107: 368–378.

Lawley TD, Klimke WA, Gubbins MJ, Frost LS. 2003. F factorconjugation is a true type IV secretion system. FEMSMicrobiol Lett 224: 1–15.

Lomma M, Dervins-Ravault D, Rolando M, Nora T, NewtonHJ, Sansom FM, Sahr T, Gomez-Valero L, Jules M, Hart-land EL, et al. 2010. The Legionella pneumophila F-boxprotein Lpp2082 (AnkB) modulates ubiquitination ofthe host protein parvin B and promotes intracellular rep-lication. Cell Microbiol 12: 1272–1291.

Lurie-Weinberger MN, Gomez-Valero L, Merault N, Glock-ner G, Buchrieser C, Gophna U. 2010. The origins ofeukaryotic-like proteins in Legionella pneumophila. Int JMed Microbiol 300: 470–481.

Marra A, Shuman HA. 1992. Genetics of Legionella pneumo-phila virulence. Annu Rev Genet 26: 51–69.

McDade JE, Shepard CC, Fraser DW, Tsai TR, Redus MA,Dowdle WR. 1977. Legionnaires’ disease: Isolation of abacterium and demonstration of its role in other respi-ratory disease. N Engl J Med 297: 1197–1203.

Molmeret M, Horn M, Wagner M, Santic M, Abu Kwaik Y.2005. Amoebae as training grounds for intracellular bac-terial pathogens. Appl Environ Microbiol 71: 20–28.

Molofsky AB, Swanson MS. 2004. Differentiate to thrive:Lessons from the Legionella pneumophila life cycle. MolMicrobiol 53: 29–40.

Moya A, Pereto J, Gil R, Latorre A. 2008. Learning how tolive together: Genomic insights into prokaryote-animalsymbioses. Nat Rev Genet 9: 218–229.

Nagai H, Cambronne ED, Kagan JC, Amor JC, Kahn RA,Roy CR. 2005. AC-terminal translocation signal requiredfor Dot/Icm-dependent delivery of the Legionella RalFprotein to host cells. Proc Natl Acad Sci 102: 826–831.

Newton HJ, Ang DK, van Driel IR, Hartland EL. 2010. Mo-lecular pathogenesis of infections caused by Legionellapneumophila. Clin Microbiol Rev 23: 274–298.

Nora T, Lomma M, Gomez-Valero L, Buchrieser C. 2009.Molecular mimicry: An important virulence strategy em-ployed by Legionella pneumophila to subvert host func-tions. Future Microbiol 4: 691–701.

Ogata H, La Scola B, Audic S, Renesto P, Blanc G, Robert C,Fournier PE, Claverie JM, Raoult D. 2006. Genome se-quence of Rickettsia bellii illuminates the role of amoebaein gene exchanges between intracellular pathogens. PLoSGenet 2: e:76.

Perez-Brocal V, Gil R, Ramos S, Lamelas A, Postigo M, Mi-chelena JM, Silva FJ, Moya A, Latorre A. 2006. A smallmicrobial genome: The end of a long symbiotic relation-ship? Science 314: 312–313.

Price CT, Al-Khodor S, Al-Quadan T, Santic M, Habyari-mana F, Kalia A, Kwaik YA. 2009. Molecular mimicry byan F-box effector of Legionella pneumophila hijacks aconserved polyubiquitination machinery within macro-phages and protozoa. PLoS Pathog 5: e1000704.

Price CT, Al-Quadan T, Santic M, Jones SC, Abu Kwaik Y.2010. Exploitation of conserved eukaryotic host cell far-



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



nesylation machinery by an F-box effector of Legionellapneumophila. J Exp Med 207: 1713–1726.

Ridenour DA, Cirillo SL, Feng S, Samrakandi MM, CirilloJD. 2003. Identification of a gene that affects the efficien-cy of host cell infection by Legionella pneumophila in atemperature-dependent fashion. Infect Immun 71: 6256–6263.

Rolando M, Buchrieser C. 2012. Post-translational modifi-cations of host proteins by Legionella pneumophila: Asophisticated survival strategy. Future Microbiol 7: 369–381.

Rolando M, Sanulli S, Rusniok C, Gomez-Valero L, Bertho-let C, Sahr T, Margueron R, Buchrieser C. 2013. Legion-ella pneumophila effector RomA uniquely modifies hostchromatin to repress gene expression and promote intra-cellular bacterial replication. Cell Host Microbe 13: 395–405.

Rowbotham TJ. 1980. Preliminary report on the pathoge-nicity of Legionella pneumophila for freshwater and soilamoebae. J Clin Pathol 33: 1179–1183.

Rowbotham TJ. 1986. Current views on the relationshipsbetween amoebae, legionellae and man. Isr J Med Sci 22:678–689.

Schmitz-Esser S, Tischler P, Arnold R, Montanaro J, WagnerM, Rattei T, Horn M. 2010. The genome of the amoebasymbiont “Candidatus Amoebophilus asiaticus” revealscommon mechanisms for host cell interaction amongamoeba-associated bacteria. J Bacteriol 192: 1045–1057.

Schroeder GN, Petty NK, Mousnier A, Harding CR, VogrinAJ, Wee B, Fry NK, Harrison TG, Newton HJ, ThomsonNR, et al. 2010. Legionella pneumophila strain 130b pos-sesses a unique combination of type IV secretion systems

and novel Dot/Icm secretion system effector proteins.J Bacteriol 192: 6001–6016.

Shohdy N, Efe JA, Emr SD, Shuman HA. 2005. Pathogeneffector protein screening in yeast identifies Legionellafactors that interfere with membrane trafficking. ProcNatl Acad Sci 102: 4866–4871.

Siefert JL. 2009. Defining the mobilome. Methods Mol Biol532: 13–27.

Steinert M, Heuner K, Buchrieser C, Albert-WeissenbergerC, Glockner G. 2007. Legionella pathogenicity: Genomestructure, regulatory networks and the host cell response.Int J Med Microbiol 297: 577–587.

Walker T, Klasson L, Sebaihia M, Sanders MJ, Thomson NR,Parkhill J, Sinkins SP. 2007. Ankyrin repeat domain-en-coding genes in the wPip strain of Wolbachia from theCulex pipiens group. BMC Biol 5: 39.

Whiley H, Bentham R. 2011. Legionella longbeachae andlegionellosis. Emerg Infect Dis 17: 579–583.

Winiecka-Krusnell J, Linder E. 1999. Free-living amoebaeprotecting Legionella in water: The tip of an iceberg?Scand J Infect Dis 31: 383–385.

Yu VL, Plouffe JF, Pastoris MC, Stout JE, Schousboe M,Widmer A, Summersgill J, File T, Heath CM, PatersonDL, et al. 2002. Distribution of Legionella species andserogroups isolated by culture in patients with sporadiccommunity-acquired legionellosis: An international col-laborative survey. J Infect Dis 186: 127–128.

Zhu W, Banga S, Tan Y, Zheng C, Stephenson R, Gately J,Luo ZQ. 2011. Comprehensive identification of proteinsubstrates of the Dot/Icm type IV transporter of Legion-ella pneumophila. PLoS ONE 6: e17638.



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2013; doi: 10.1101/cshperspect.a009993Cold Spring Harb Perspect Med Laura Gomez-Valero and Carmen Buchrieser Adaptation to Intracellular Replication

: The Basis of Versatility andLegionellaGenome Dynamics in

Subject Collection Bacterial Pathogenesis

Bacteriophage Components in Modern MedicineTherapeutic and Prophylactic Applications of

Sankar Adhya, Carl R. Merril and Biswajit BiswasContact-Dependent Growth Inhibition SystemsMechanisms and Biological Roles of

C. Ruhe, et al.Christopher S. Hayes, Sanna Koskiniemi, Zachary

PathogenesisVaccines, Reverse Vaccinology, and Bacterial

Isabel Delany, Rino Rappuoli and Kate L. Seiband Its Implications in Infectious DiseaseA Genome-Wide Perspective of Human Diversity

Jérémy Manry and Lluis Quintana-Murci

Strategies Persistent InfectionSalmonella and Helicobacter

Denise M. Monack

Host Specificity of Bacterial PathogensAndreas Bäumler and Ferric C. Fang

Helicobacter pyloriIsland of PathogenicitycagEchoes of a Distant Past: The

al.Nicola Pacchiani, Stefano Censini, Ludovico Buti, et

Epithelial Cells Invasion of IntestinalShigellaThe Inside Story of

Nathalie Carayol and Guy Tran Van Nhieu

RNA-Mediated Regulation in Pathogenic Bacteria

al.Isabelle Caldelari, Yanjie Chao, Pascale Romby, et for Stealth Attack

Weapons and Strategies−−Brucella and Bartonella

Christoph DehioHouchaima Ben-Tekaya, Jean-Pierre Gorvel and

and PathogenesisThe Pneumococcus: Epidemiology, Microbiology,

TuomanenBirgitta Henriques-Normark and Elaine I.

BarriersConcepts and Mechanisms: Crossing Host

al.Kelly S. Doran, Anirban Banerjee, Olivier Disson, et

Pathogenesis of Meningococcemia

Lécuyer, et al.Mathieu Coureuil, Olivier Join-Lambert, Hervé

ReplicationVersatility and Adaptation to Intracellular

: The Basis ofLegionellaGenome Dynamics in

Laura Gomez-Valero and Carmen BuchrieserChlamydial Intracellular Survival Strategies

Engel, et al.Robert J. Bastidas, Cherilyn A. Elwell, Joanne N. Pathogenesis

IntracellularFrancisella tularensisMechanisms of

Jean Celli and Thomas C. Zahrt

http://perspectivesinmedicine.cshlp.org/cgi/collection/ For additional articles in this collection, see

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