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J Bacteriol. Nov 2010; 192(22): 6001–6016.
Published online Sep 10, 2010. doi:  10.1128/JB.00778-10
PMCID: PMC2976443

Legionella pneumophila Strain 130b Possesses a Unique Combination of Type IV Secretion Systems and Novel Dot/Icm Secretion System Effector Proteins[down-pointing small open triangle]

Abstract

Legionella pneumophila is a ubiquitous inhabitant of environmental water reservoirs. The bacteria infect a wide variety of protozoa and, after accidental inhalation, human alveolar macrophages, which can lead to severe pneumonia. The capability to thrive in phagocytic hosts is dependent on the Dot/Icm type IV secretion system (T4SS), which translocates multiple effector proteins into the host cell. In this study, we determined the draft genome sequence of L. pneumophila strain 130b (Wadsworth). We found that the 130b genome encodes a unique set of T4SSs, namely, the Dot/Icm T4SS, a Trb-1-like T4SS, and two Lvh T4SS gene clusters. Sequence analysis substantiated that a core set of 107 Dot/Icm T4SS effectors was conserved among the sequenced L. pneumophila strains Philadelphia-1, Lens, Paris, Corby, Alcoy, and 130b. We also identified new effector candidates and validated the translocation of 10 novel Dot/Icm T4SS effectors that are not present in L. pneumophila strain Philadelphia-1. We examined the prevalence of the new effector genes among 87 environmental and clinical L. pneumophila isolates. Five of the new effectors were identified in 34 to 62% of the isolates, while less than 15% of the strains tested positive for the other five genes. Collectively, our data show that the core set of conserved Dot/Icm T4SS effector proteins is supplemented by a variable repertoire of accessory effectors that may partly account for differences in the virulences and prevalences of particular L. pneumophila strains.

Many bacterial pathogens use specialized protein secretion systems to deliver into host cells virulence effector proteins that interfere with the antimicrobial responses of the host and facilitate the survival of the pathogen (5, 10, 22, 76). The components of these secretion systems are highly conserved. Comparative bioinformatic analysis of pathogen genomes revealed an ever-increasing number of proteins that are likely to be translocated virulence effectors. Only a few effectors have been characterized, and their biochemical functions are unknown, yet the identification of translocated effector proteins and their mechanism of action is fundamental to understanding the pathogenesis of many bacterial infections.

Legionella pneumophila is the etiological agent of Legionnaires’ disease, which is an acute form of pneumonia (34, 66). L. pneumophila serogroup 1 accounts for more than 90% of all cases worldwide. Although L. pneumophila is an environmental organism, its ability to survive and replicate in amoebae, such as Acanthamoeba castellanii, has equipped the organism with the capacity to replicate in human cells (45, 58, 68, 80). Following the inhalation of aerosols containing L. pneumophila into the human lung, the bacteria promote their uptake by alveolar macrophages and epithelial cells (21, 44, 71), where they replicate within an intracellular vacuole that avoids fusion with the endocytic pathway (46, 47). L. pneumophila evades endosome fusion by establishing a replicative vacuole that shares many characteristics with the endoplasmic reticulum (ER) (48, 53, 89). The formation of the unique Legionella-containing vacuole (LCV) requires the Dot (defective in organelle trafficking)/Icm (intracellular multiplication) type IV secretion system (T4SS) (85, 91).

Type IV secretion systems are versatile multiprotein complexes that can transport DNA and proteins to recipient bacteria or host cells (19, 36). Based on structural and organizational similarity, three main T4SS classes have been distinguished: T4SSA, T4SSB, and genomic island-associated T4SS (GI-T4SS) (3, 51). The genetic organization and components of T4SSA have high similarity to the classical VirB4/VirD4 transfer DNA (T-DNA) transfer system of Agrobacterium tumefaciens (3). In the sequenced L. pneumophila strains, three distinct T4SSAs with different prevalences among strains have been described: Lvh, Trb-1, and Trb-2 (37, 83, 86). The Lvh (Legionella vir homologues) T4SSA is not required for intracellular bacterial replication in macrophages and amoebae but seems to contribute to infection at lower temperatures and inclusion in Acanthamoeba castellanii cysts (6, 78, 86).

The Dot/Icm T4SSB secretes and translocates multiple bacterial effector proteins into the vacuolar membrane and cytosol of the host cell (31, 70). The functions of the great majority of these proteins are unknown. Several effectors have similarity to eukaryotic proteins or carry eukaryotic motifs (7, 16, 25). They are predicted to allow L. pneumophila to manipulate host cell processes by functional mimicry (31, 70). Many of the effectors have paralogues or belong to related protein families that are likely to have overlapping functions.

Comparative analysis of the recent L. pneumophila genome sequences has revealed their diversity and plasticity (16, 18, 88). This plasticity enables the bacterium to acquire new genetic factors, including new effector proteins that enhance bacterial replication and survival in eukaryotic cells. This has resulted in a diverse species in which 7 to 11% of the genes in each L. pneumophila isolate are strain specific (38). Some of the diversity occurs among genes encoding Dot/Icm effectors, including those within the same family. For example some ankyrin repeat and F-box effector genes are highly conserved, while others vary considerably between L. pneumophila isolates (16, 41, 62, 73, 75). Even though it is not experimentally proven, the subsequent selection of Dot/Icm effectors among different L. pneumophila isolates might reflect their usefulness in host-pathogen interactions, whereby different effector repertoires are maintained during adaptation to different environmental niches or hosts. This may then translate into differences in virulence during opportunistic infection.

In this study, we sequenced the genome of L. pneumophila serogroup 1 strain 130b (ATCC BAA-74, also known as Wadsworth or AA100) (29, 30) and analyzed the sequence for T4SSs and novel Dot/Icm effectors. This analysis established that the strain encodes a unique combination of T4SSs and a set of Dot/Icm effectors that had not been described previously but that are present in a range of clinical and environmental L. pneumophila isolates. The new effectors represent the latest members of an ever-growing list of T4SS substrates and presumably reflect the great capacity of L. pneumophila for adaptation to a variety of hosts.

MATERIALS AND METHODS

Bacterial strains and sequencing.

The sequenced L. pneumophila serogroup 1 strain 130b is a clinical isolate from the Wadsworth Veterans Administration Hospital, Los Angeles, CA (29, 30). The L. pneumophila ΔDotA strain is a dotA insertion mutant (kanamycin resistance) of L. pneumophila strain 130b (84).

L. pneumophila 130b was obtained from Nick Cianciotto, Northwestern University, and was subjected to minimal passages. High-purity chromosomal DNA was prepared for sequencing by proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation (90). The whole genome of 130b was sequenced using paired-end 454 FLX pyrosequencing and assembled using the 454/Roche Newbler assembly program into 274 contigs (N50 contig size, 35,584 bp) from 248,625 sequence reads with an average read length of 157 bp. The contigs were scaffolded using paired reads, with an average pair distance of 3,028 bp, into 11 scaffolds (N50 scaffold size, 2,421,541 bp). The lvh collapsed repeat region was reassembled into two distinct lvh regions by first separating reads that mapped to the previously sequenced 130b lvh region (accession no. AF410854), differentiating lvh reads according to microheterogeneity (single-nucleotide polymorphism [SNP] content), and then using Newbler (with stringent cutoffs) to generate individual lvh assemblies that could be unambiguously positioned within a scaffold in the main assembly. Several contig gaps were closed using PCR and Sanger sequencing in conjunction with manual examination of the individual 454 reads in Consed (39). Unassembled contigs of less than 300 bp were removed from the end of the assembly. This gave a total sequence length of 3,473,547 bp assembled into 145 contigs in 4 scaffolds plus 14 small contigs (114 to 3,270 bp) that could not be scaffolded.

The 130b genome was aligned with the other sequenced L. pneumophila genomes to aid whole-genome comparisons, making the first gene dnaA. An automated annotation was performed on the genome sequence using SUGAR, as previously described (93). Artemis (82) was used to facilitate the manual curation of the sequence and annotation of the effectors and T4SSs.

Bioinformatic analysis.

Pairwise whole-genome comparisons of the 130b genome with the other sequenced L. pneumophila genomes—Lens (accession number CR628337), Philadephila-1 (accession number AE017354), Corby (accession number CP000675), and Paris (accession number CR628336)—and Legionella longbeachae NSW150 (accession number FN650140) were performed using BLASTn and visualized using the Artemis Comparison tool (ACT) (13). Genome comparison figures were made using easyfig (http://easyfig.sourceforge.net/), and the circular diagram was produced with DNAplotter (12).

Bioinformatic analysis of domains and motifs of individual effector protein candidates was performed using the Pfam database (Pfam release 24, HMMER3.0 beta 3 [32]), SMART (version 6 [61]), and the NCBI Conserved Domain Database (version 2.18 [65]).

Phylogenetic analysis.

Thirty genes in the Philadelphia-1 genome were identified from Hidden Markov-Model (HMM) profiles of genes previously identified as conserved across all bacteria (96). Homologues of these genes were then searched for using tBLASTn (2) in the genomes of L. pneumophila strains 130b, Corby, Lens, and Paris and L. longbeachae NSW150. Each gene set was manually checked to ensure all genes were intact, and four genes (dnaG, pyrG, rplF, and rpsJ) that were disrupted by sequencing errors in the unfinished 130b genome were removed from the data set. The remaining 26 single-copy genes (frr, infC, nusA, pgk, rplA, rplB, rplC, rplD, rplE, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC, rpsE, rpsI, rpsK, rpsM, smpB, and tsf) were concatenated for each genome, corresponding to a total of 18.5 kb, and aligned using CLUSTALW2 (59).

A maximum-likelihood (ML) tree was built using PhyML (40). The general time reversible substitution model with gamma-distributed rate variation was used for most of the individual alignments, as suggested by FindModel (74). The transition/transversion ratio, proportion of invariable sites, and gamma distribution parameter were estimated by PhyML.

Strain and plasmid construction.

The plasmids pXDC61, pXDC61-C2 (pXDC61 LegC2), and pXDC61 FabI were a kind gift from Xavier Charpentier and Howard Shuman (Columbia University Medical Center, New York, NY) and were described previously (24). All plasmids, primers, and restriction enzymes used to construct the pXDC61-derived expression vectors for β-lactamase (TEM1) fusions of the putative new L. pneumophila strain 130b effector proteins are listed in Table Table1.1. All genes were PCR amplified from L. pneumophila strain 130b genomic DNA, and the PCR products were digested and ligated into pXDC61. The sequence identities and correct orientation of the inserts were verified by DNA sequencing. The new plasmids were transformed into L. pneumophila strain 130b wild type and ΔDotA by electroporation.

TABLE 1.
Plasmids, cloning primers, and restriction sites used in this study

Translocation assay.

The β-lactamase (TEM1) translocation assay for the identification of Icm/Dot T4SS substrates was adapted from the protocol described by de Felipe et al. (24). Raw264.7 macrophages were cultured in a humidified atmosphere of 5% CO2 at 37°C in RPMI 1640 glutamine medium supplemented with 10% fetal calf serum and Glutamax (Invitrogen). To obtain a confluent cell layer, 7.5 × 104 Raw264.7 macrophages were seeded in 200 μl growth medium per well of a black wall/clear flat-bottom 96-well plate (Becton Dickinson) and cultured overnight, and the medium was replaced with 150 μl fresh growth medium immediately prior to infection. L. pneumophila strain 130b wild type or the ΔDotA mutant harboring the pXDC61-derived TEM1 fusion expression plasmids was inoculated in ACES yeast extract (AYE) broth to an optical density at 600 nm (OD600) of 0.1 from 3-day-old charcoal-yeast extract (CYE) plates and grown in the presence of 6 μg/ml chloramphenicol and 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 37°C for 21 h (post-exponential growth phase) (92). The bacteria were diluted in Raw264.7 macrophage growth medium and added to the macrophages at a multiplicity of infection (MOI) of 40, and the infection was synchronized by centrifugation (900 × g; 10 min). After 1 h of incubation at 37°C, 5% CO2, the supernatant was replaced by 100 μl Hanks’ buffered salt solution (Gibco) supplemented with 20 mM HEPES and 3 mM probenecid, pH 7.4 (HBSS-HP), and 20 μl freshly prepared CFF2-AM β-lactamase substrate (LiveBLAzer FRET-B/G Loading Kit; Invitrogen) was added. After 1 h 45 min of incubation at room temperature in the dark, the cells were washed four times with HBSS-HP. Fluorescence emission at 450 nm and 520 nm was measured from the bottom using a Fluostar Optima plate reader (excitation wavelength, 410 nm; 10-nm band-pass). The translocation rate was calculated as recommended in the LiveBLAzer FRET-B/G Loading Kit manual. Briefly, emission values were first corrected by subtraction of the average background signals recorded for empty wells, and then the 450-nm/520-nm emission ratio for each well was calculated. The translocation rate was expressed as the fold increase of the 450-nm/520-nm emission ratio of each infected cell in correlation with the emission ratio of uninfected cells. Experiments were performed three times.

L. pneumophila pXDC61 ltpG strains were grown for 16 h as described above but without the addition of IPTG. Expression of the TEM1 fusion of LtpG was subsequently induced for 5 h in bacterial liquid culture and, during the first hour of infection, by the addition of 1 mM IPTG.

To verify expression of the TEM1 fusion proteins in Legionella, 0.5 ml of the 21 h-cultures used for infection was harvested, and equal amounts of bacteria, adjusted according to the OD600, were analyzed by Western blotting using a mouse anti-β-lactamase antibody (QED Bioscience Inc.).

Prevalence screen.

Genomic DNA for 54 clinical and environmental L. pneumophila isolates was obtained from the Respiratory and Systemic Infection Laboratory, Health Protection Agency Centre for Infection, London, United Kingdom (see Table S1a in the supplemental material). These isolates were obtained from a range of locations across the United Kingdom and were previously characterized by serogroup, monoclonal antibody (MAb) subgroup, allelic profile, and sequence type (42, 43, 77). Thirty-three isolates were obtained from the Microbiological Diagnostic Unit at the University of Melbourne, as described previously (71), and from the collection of Stacey Yong, Taylor's University College, Malaysia (see Table S1b in the supplemental material). The PCR primers were designed to amplify 400- to 700-bp sequences of the putative effector genes and controls (Table (Table22 ). PCR screening using standard conditions was performed three times, and the results were analyzed by agarose gel electrophoresis. PCR-negative strains from the Australian and Malaysian collections were, in addition, confirmed by Southern hybridization, as described previously (71).

TABLE 2.
Sequences of the primers used for the screen of L. pneumophila isolates

Nucleotide sequence accession number.

The annotated draft genome sequence of L. pneumophila 130b can be obtained from the European Nucleotide Archive under accession number FR687201, and the raw sequence data can be obtained from the Sequence Read Archive under accession number ERA011231.

RESULTS AND DISCUSSION

General features of the L. pneumophila 130b genome.

The genome of L. pneumophila strain 130b was sequenced using 454 technology to approximately 11-fold coverage and assembled into 4 scaffolds (consisting of 145 contigs) and 14 small, unscaffolded contigs (Fig. (Fig.1).1). Analysis of the sequence suggested that the genome consists of a single circular chromosome of approximately 3.5 Mb with an average G+C content of 38%. No plasmids were identified in the genome sequence. The draft sequence was predicted to contain 3,293 coding sequences (CDSs) and 42 tRNAs, which is in good correlation with the other sequenced L. pneumophila strains (Philadelphia, 3.4 Mb, 2,942 CDSs, 43 tRNAs; Lens, 3.3 Mb, 2,947 CDSs, 43 tRNAs; Paris, 3.5 Mb, 3,082 CDSs, 43 tRNAs; Corby: 3.6 Mb, 3,204 CDSs, 44 tRNAs). The genome of 130b is highly syntenic with those of the other sequenced L. pneumophila strains, although, as can be expected from a species showing evidence of high genome plasticity (16), there are a number of insertions and deletions and other regions of difference (RODs) that are evident in whole-genome comparisons (data not shown).

FIG. 1.
Circular map of the L. pneumophila 130b draft genome. From the outside in, the green regions in the first circle show the positions of the T4SSs (detailed in Table Table3)3) and the hypervariable region (shown in detail in Fig. Fig.5). ...

Relationship of 130b to other legionellae.

Phylogenetic analysis was performed using 26 genes from the five sequenced L. pneumophila serogroup 1 genomes (130b, Lens, Philadelphia-1, Corby, and Paris) and L. longbeachae NSW150. These phylogenetic markers—frr, infC, nusA, pgk, rplA, rplB, rplC, rplD, rplE, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC, rpsE, rpsI, rpsK, rpsM, smpB, and tsf—are housekeeping genes that are involved in replication, transcription, translation, or central metabolism processes. These genes were used in a previous study to infer evolutionary relationships between 578 bacterial strains (96). These single-copy housekeeping genes were used to enable a phylogenetic signal to be detected from truly orthologous sequences free from horizontal gene transfer (HGT) or duplication events.

The resulting phylogenetic tree (Fig. (Fig.2)2) showed all the L. pneumophila serogroup 1 strains to be monophyletic, with L. longbeachae as an outgroup. Within the serogroup 1 clade, strains Lens and 130b were grouped together, with a very high bootstrap value (99.8%) in comparison with all the other L. pneumophila strains. This suggests that Lens and 130b have only recently diverged relative to the other L. pneumophila strains. This high-confidence grouping is an interesting result, as Lens was only recently isolated from an epidemic outbreak in France in 2003 and 2004, while 130b is a commonly used laboratory strain that originated from a transtracheal aspirate isolated in the United States in 1978 (16, 29, 30).

FIG. 2.
Phylogeny of Legionella showing the phylogenetic relationship of L. pneumophila 130b to the other sequenced L. pneumophila strains, Corby, Paris, Lens, and Philadelphia-1, with L. longbeachae NSW150 as an outgroup. The tree was built using 26 housekeeping ...

The secretion systems of L. pneumophila 130b.

The main aim of sequencing the 130b genome was to facilitate the analysis of the secretion systems, in particular, the type IV secretion systems, and the Dot/Icm T4SS effector repertoire.

An overview of the protein secretion systems encoded by L. pneumophila 130b is shown in Table Table3.3. The genome encodes the structural components of a putative type 1 secretion system (T1SS), which was first described as the Legionella secretion system (Lss) in L. pneumophila Corby (49), including a putative T1SS-associated regulatory GGDEF family protein, LssE. The functionality of the T1SS in L. pneumophila has yet to be demonstrated.

TABLE 3.
Overview of the L. pneumophila strain 130b secretion systems

Functional analysis of the T2SS of L. pneumophila has been performed mainly in strain 130b; several substrates have been identified, and a contribution to Legionella virulence has been established (20, 79). The genes comprising the T2SS system are not in a single locus but form several clusters scattered around the genome, and this organization is largely conserved in location and sequence among the different L. pneumophila genomes, suggesting strong selection for its retention.

Type IV secretion systems.

L. pneumophila 130b has the complete Dot/Icm T4SSB encoded in five loci in two distinct regions on the chromosome (Fig. (Fig.1).1). Although some degree of sequence variation in the Dot/Icm systems of different strains has been identified, the loci are largely conserved (Fig. (Fig.3A)3A) (69, 86, 91) and are found in syntenic regions in each of the other sequenced L. pneumophila genomes.

FIG. 3.
Organization of the Dot/Icm T4SSB (A), Lvh1 T4SSA (B), and Trb1-like T4SSA (C) loci on the L. pneumophila 130b chromosome. The color coding represents shared gene names.

Lvh T4SSA regions have been identified in the sequenced L. pneumophila strains Philadelphia-1, Paris, and Lens. The genes encoding the Lvh T4SSA are found either on a plasmid-like element or integrated in the chromosome (16, 18, 27). Furthermore, an lvh gene cluster was also found as part of a chromosomally integrated putative plasmid-like element in L. longbeachae strain D-4968, showing interspecies mobility of the lvh region (56). However, in strain 130b, we identified two distinct lvh regions, both of which were integrated into the chromosome (Fig. (Fig.1).1). We found no evidence in the sequence reads that either of these regions was mobilized on plasmid-like elements. Lvh1 (Fig. (Fig.3B)3B) is almost identical (99% DNA identity), except for a few SNPs, to the published lvh region previously sequenced from strain 130b (accession number AF410854 [83]). The SNPs could be due to sequencing errors in either or both sequenced regions or they could be indicative of sequence divergence. The second T4SSA Lvh system in 130b, Lvh2, has 99% sequence identity to the Lvh region in the genome of L. pneumophila Paris. Lvh1 and Lvh2 in 130b have only 92% similarity to each other, and therefore, it seems unlikely that the two Lvh regions arose from a duplication event. Instead, it seems more likely that Lvh2 was acquired via a plasmid-like element highly similar to that found in the Paris strain and was subsequently integrated into the chromosome. The two Lvh loci are located close to each other in the 130b genome (Fig. (Fig.1),1), on either side of the CRISPR locus, and are divergently transcribed. Both are part of a larger ROD that shows variation in each of the sequenced strains.

The Lvh T4SS is not present in L. pneumophila strain Corby. Instead, two similar T4SSAs, Trb-1 and Trb-2, are encoded on separate genomic islands in the strain (37). Both genomic islands can exist integrated into the genome or as episomal plasmids. Glöckner et al. and Samrakandi et al. independently suggested that L. pneumophila 130b contains a Trb-1-like T4SSA (37, 83). The 130b genome sequence confirmed the presence of a T4SSA gene cluster (lpw_22591 to lpw_22831) that shows similarity to the L. pneumophila Corby Trb-1 T4SSA (Fig. (Fig.3C);3C); however, the genetic context in 130b differs (37). Integration of the L. pneumophila Corby Trb-1 genomic island was reported to be specifically into the tRNAPro gene. In contrast, in L. pneumophila 130b, the 22-kb Trb-1-like cluster is found in a nonsynonymous genomic location as part of a larger, approximately 150-kb ROD, and there was no evidence in the sequence reads that it can exist in plasmid-like form in 130b.

In addition to the Dot/Icm T4SSB and the three T4SSA systems, we found two highly similar clusters of 24 genes in the L. pneumophila 130b genome, which resemble clusters of the recently defined GI-T4SS (50). We named these putative new T4SSs Legionella GI-T4SS-1 (LGI-1; lpw_10731 to lpw_10961) and Legionella GI-T4SS-2 (LGI-2; lpw_21631 to lpw_21861) (Fig. (Fig.1).1). Both the LGI-1 and LGI-2 clusters encode homologues of the T4SS components VirD4, VirB4, and TraB and several proteins that have significant similarity to the proteins encoded in the transfer region of several integrative and conjugative elements (ICEs) (Fig. (Fig.4),4), including ICEhin1056 from Haemophilus influenzae (accession number AJ627386 [67]). The first four genes of both LGI clusters encode homologues of the proteins LvrR, LvrA, LvrB, and the CsrA-like global regulator protein LvrC, all of which are found in analogous positions in the Lvh and the Trb-1-like T4SSA systems. This suggests an important role for these proteins and a common mechanism for the regulation of T4SSA and GI-T4SS in L. pneumophila.

FIG. 4.
Organization of the putative genomic-island-associated T4SS gene clusters LGI-1 and LGI-2 on the L. pneumophila 130b chromosome. The color coding shows the sequence and predicted structural homology of the encoded proteins to T4SS-associated proteins, ...

Although it was not previously recognized as a putative T4SS cluster, we found that LGI-1 was conserved in the other sequenced L. pneumophila strains and was in the same genomic location, although there is variation in the gene content immediately flanking LGI-1 in Philadelphia-1, Paris, and Corby. This region was previously found to be heterogeneously distributed among 217 L. pneumophila strains (15, 18), and based on this observation, it was suggested that the region might be mobile.

LGI-2 is part of a 150-kb ROD in 130b, which also encodes the Trb-1-like T4SSA, which is absent from strains Lens and Philadelphia. LGI-2 is highly similar (approximately 87% DNA identity) to regions in the genomes of strains Corby (lpc1857 to lpc1880) and Paris (lpp2375 to lpp2398). However, these LGI-2-like regions are found in a different genomic location, and the flanking regions are highly divergent in all three strains.

Even though the functionality of the two putative new T4SSs has yet to be demonstrated, it is tempting to speculate that they might contribute to the genetic competence and mobilization of genomic islands in L. pneumophila, adding another layer of complexity to the factors that shape the genome plasticity and virulence of L. pneumophila.

The conserved Dot/Icm T4SS effectors.

L. pneumophila has accumulated a large number of T4SS effector proteins. To date, Dot/Icm-dependent translocation has been experimentally proven for 151 effector proteins, and several dozen additional candidate Dot/Icm effectors have been identified by sequence analysis or specialized assays (9, 24, 33, 57, 63, 87, 98). Furthermore, it has been estimated that the total number of effector proteins could be as high as 300 (9). However, only a fraction of the proven effector proteins have been functionally characterized so far, and this analysis is hampered by families of effector protein paralogues that have presumed functional redundancy.

Homologues of 136 of the 151 proven effectors were identified in the L. pneumophila 130b genome. All of the effectors that are not present in strain 130b are also absent from at least one of the other sequenced L. pneumophila strains (Table (Table4).4). It is notable that no effector pseudogenes were identified in the 130b genome, whereas at least one effector pseudogene was identified in each of the other L. pneumophila genomes, which totaled 15 effector pseudogenes across all four strains compared (Table (Table4).4). A core set of 107 intact effector genes is common to all five sequenced L. pneumophila genomes. The conservation of this core set of T4SS effectors could be indicative of their biological importance. Recently, the genome sequences of the L. longbeachae strains D-4968 and NSW150 were reported (14, 56). L. longbeachae encodes a Dot/Icm T4SSB and replicates in a specialized vacuole in macrophages, which shares features with, but is distinct from, the L. pneumophila replicative vacuole (4). L. pneumophila and L. longbeachae encode a common set of effectors, which includes about one-third of the validated L. pneumophila effectors and may constitute the minimum set of effectors required by Legionella species to survive and replicate in macrophages. Once more L. longbeachae strains and other Legionella species, such as Legionella micdadei, are sequenced, the core set of essential effectors will be defined with greater confidence.

TABLE 4.
Presence of known L. pneumophila T4SS effectors in sequenced genomes

The effector-rich hypervariability region of L. pneumophila.

Approximately 30% of the proven effector proteins are absent from at least one of the sequenced L. pneumophila strains (Table (Table4).4). This substantial number of “accessory” effectors is one possible explanation for phenotypic differences in some virulence-associated attributes, such as host cell adherence, intracellular trafficking, and cytotoxicity, that have been reported between L. pneumophila 130b, L. pneumophila Philadelphia JR32, and other L. pneumophila strains (1, 83).

Two regions of high genomic plasticity, rich in effector proteins, were previously described in L. pneumophila (72, 98). These regions partially overlap (Fig. (Fig.5)5) and, in strain 130b, together comprise a 37-kb region located between lpw_20071 and lpw_20361 (Fig. (Fig.1).1). Moreover, whole-genome alignment revealed that this combined region forms part of a larger genomic region that displays considerable divergence among the five sequenced genomes (Fig. (Fig.5)5) and, in 130b, constitutes the 96 kb from lpw_19681 to lpw_20471. In contrast, there are no gene deletions or insertions for more than 30 kb upstream and 48 kb downstream of the region shown in Fig. Fig.55 (data not shown). This suggests that the two regions initially described by Zusman et al. and Ninio et al. (72, 98) constitute the inner core of a much larger 80- to 100-kb region of high genomic plasticity that represents a strain-specific variable effector region (Fig. (Fig.5).5). Adjacent to this large variable region is a conserved tRNAPhe gene, which could represent an insertion site if the region was horizontally acquired. However, no repeat ends or integrase genes were identified in any of the strains, and the presence of a distinct genomic island was not evident from examination of the G+C content of this region in each of the genomes. Notably, many of the insertions and deletions were found to be associated with insertion sequence (IS) elements, as highlighted for 130b in Fig. Fig.5,5, and it is highly likely that these repetitive elements have contributed to the gene flux in this hypervariable region. Many of the putative CDSs in this hypervariable region encode hypothetical proteins, and therefore, it is possible that there are more effectors encoded in the region.

FIG. 5.
A region of high genome variability in L. pneumophila encodes several new and known Dot/Icm T4SS effectors. Shown is a genome comparison of the five sequenced L. pneumophila genomes (130b, Lens, Philadelphia-1, Corby, and Paris). The CDSs for each genome ...

Novel T4SS 130b effector proteins.

Prior to this work, 151 proteins were proven to be substrates of the Dot/Icm T4SS, a number that is unmatched by other bacterial pathogens. Most of the analysis was done using the Philadelphia strain. However, given the high genomic plasticity of the sequenced L. pneumophila strains, this is likely to result in an underestimation of the true number of effectors found in the species. Indeed, strain-specific effector candidates have been predicted in L. pneumophila Lens and Paris, but only three of them have been proven to be translocated (16, 33, 62). Accordingly, we analyzed the genome of L. pneumophila strain 130b for proteins with characteristics of known T4SS effectors, such as eukaryotic-like domains, that are absent from L. pneumophila Philadelphia-1. Our screen identified 16 new putative Dot/Icm T4SS effector proteins (Table (Table55).

TABLE 5.
Putative novel Dot/Icm T4SS effectors and their homologues (>80% identity) in other L. pneumophila strains

Two of these new putative effector proteins, Lpw_20091 and Lpw_20341, are encoded in the inner core of the hypervariable effector-rich region (Fig. (Fig.5).5). The putative effector lpw_20091 encodes a protein containing a predicted filamentation induced by the cyclic AMP (cAMP) (FIC) domain. Homologues of lpw_20091 are also found in the same genomic location in Lens and Corby, but not in Philadelphia-1 or Paris (Fig. (Fig.55 and Table Table5),5), which instead contain the effector gene legL5 (lpg1958 and lpp1940, respectively) at this location. The FIC domain was initially identified in the Escherichia coli cell division protein, Fic-1 (55). Structural similarity to FIC domain proteins was shown for the so-called death on curing (DOC) protein, which is part of a toxin-antidote plasmid addiction system in E. coli (54, 60). FIC domain proteins have recently gained increased attention, as members of this diverse family were found to be translocated virulence factors of several pathogenic bacteria (81, 94, 97). The FIC domain catalyzes the covalent modification of target proteins with AMP, interfering with host cell signaling. The conserved Dot/Icm T4SS effector LegA8/AnkX/AnkN (Lpg0695) belongs to the FIC domain protein family, and its ability to disrupt microtubule-dependent vesicle transport relies on a catalytically active FIC domain (73, 81). The newly identified FIC domain protein Lpw_20091 shows little similarity to LegA8, suggesting that it might have a different host cell target and function.

The second new putative effector, Lpw_20341, is 130b strain specific and encoded next to the LegG1/PieG effector homologue Lpw_20351 (Lpg1975/6 in Philadelphia), which is found in all sequenced L. pneumophila strains (Fig. (Fig.55 and Table Table4).4). Lpw_20341 has some similarity to the ankyrin repeat superfamily of proteins. Ankyrin repeats are predominantly eukaryotic protein-protein interaction motifs but are also found in 29 proteins of different L. pneumophila strains, 14 of which are proven Dot/Icm T4SS effectors (16, 33, 41, 73).

The genes encoding two additional novel ankyrin repeat proteins, Lpw_02301 and Lpw_02381, which have no homologues in the other sequenced L. pneumophila strains, are found in the ROD next to the lvh2 locus in the 130b genome. Lpw_02301 has a predicted amino-terminal peptidase domain. Lpw_02381 is a large, 1,059-amino-acid protein that not only features 3 predicted ankyrin repeats in its N-teminal region, but also contains a predicted human Ras GTPase guanine nucleotide exchange factor (GEF) domain in its carboxyl terminus. The Ras subfamily of Ras GTPases is involved in the regulation of a wide variety of cellular processes, such as cell proliferation, adhesion, movement, division, secretion, and differentiation, which makes the members attractive targets for bacterial effector proteins (28, 64). The only other L. pneumophila protein for which a RasGEF domain has been identified is the effector protein LegG2, which is present in all the sequenced L. pneumophila strains (24).

The 130b genome encodes another ankyrin repeat protein, Lpw_22981, a homologue of which is found in Lens (Lpl2058). Genome comparison indicated that there is also a homologue of this gene in the Philadelphia-1 genome but that it has been fragmented by a deletion and multiple frameshift mutations. The predicted CDSs for the hypothetical proteins encoded by lpg2128, lpg2129, and legA6 (lpg2131) together make up part of the full-length gene homologue. Fragments of an Lpw_22981 gene homologue are also present in Corby (lpc1576) and Paris (lpp2068). The fragmentation is a likely explanation as to why no translocation into host cells was observed for LegA6 (24).

Apart from the family of ankyrin repeat effectors, two other multitudinous effector families in L. pneumophila are formed by leucine-rich-repeat (LRR) and coiled-coil (CC) domain-containing effectors. LRRs are small sequence motifs that mediate protein-protein interactions of various proteins with diverse cellular functions. We identified two putative LRR effector proteins in 130b, Lpw_16311 and Lpw_26201. Lpw_16311 has no homologues in the other sequenced L. pneumophila strains, but the LRR domain has some similarity to other LRR-containing effectors, such as LegL7. Lpw_26201 is encoded next to the Dot/Icm effector Lem23 (Lpw_26191) in another previously identified hypervariable effector-rich genomic region located between lpw_26101 and lpw_26281 (lpg2398 to lpg2411) (98). A homologue of Lpw_26201 is present in L. pneumophila Lens (Lpl2330), and it was shown that Lpl2330 is upregulated during the transmissive phase (52).

A new putative CC effector protein, Lpw_21901, which is found only in L. pneumophila 130b, is encoded in a ROD downstream of LGI2. CC domains induce protein-protein binding by homotypic interaction, and CC effector proteins, including LegC2, LegC7, and VipA, were shown to be involved in the modulation of vesicular trafficking (11, 87).

L. pneumophila 130b encodes several other putative strain-specific effector proteins scattered around the genome, as shown in Fig. Fig.1.1. Lpw_03701 is encoded adjacent to a hypothetical protein, Lpw_03691, and together they represent an insertion in the 130b genome or a deletion in the other L. pneumophila genomes in an otherwise conserved region between the effectors LegG2 (Lpw_03651) and Ceg10 (Lpw_03741). The last 180 amino acids of this unique protein share 67% identity with the carboxyl terminus of an ankyrin repeat protein from L. pneumophila Paris (Lpp0356). However, the ankyrin repeats found in the amino terminus of Lpp0356 are not conserved in Lpw_03701. Instead, the amino terminus has similarity (32% identity) to the uncharacterized protein Lpw_27671, which has homologues in all five L. pneumophila strains. Another insertion in the 130b genome, Lpw_04551, is found in an otherwise conserved region between the proven effectors Lpg0365 (Lpw_04441) and SdhA (Lpw_04591). Lpw_04551 has some similarity to several L. longbeachae proteins, with the putative F-box protein Llb_3234 being the closest homologue (51% identity), and hypothetical proteins encoded in L. pneumophila Paris (Lpp1330; 50% identity) and the obligate intracellular amoebal pathogen Legionella drancourtii LLAP12 (46% identity).

Curiously, L. pneumophila 130b encodes a predicted nucleoside triphosphatase (NTPase) of the NACHT family (Lpw_28221) that has no homologues in other L. pneumophila strains. NACHT domains are found in animal, fungal, and bacterial proteins, and several NACHT domain-containing proteins, for example, Nod1/2 or the NAIPs, are involved in sensing bacterial pathogens and are important effector molecules of the innate immune response (8, 35). The NACHT domain protein NAIP5 is crucial for the activation of immune signaling and restriction of intracellular growth of Legionella in mouse macrophages (26, 95). It will be very interesting to analyze if L. pneumophila 130b might express a NACHT protein, which could enable the manipulation of innate immune recognition.

L. pneumophila 130b possesses several paralogues of genes encoding known effector proteins listed in Table Table4,4, including a paralogue (lpw_00591) of sdbB (lpw_27041). The same set of two sdbB genes is also found in the same genomic locations in Lens (lpl0058 and lpl2402) and Corby (lpc0065 and lpc1996), but Philadelphia-1 and Paris each have only one of the two paralogous genes (lpg2482 and lpp2546, respectively), as the lpw_00591 homologue is absent from these strains.

Adjacent to the effector paralogue sdbB2, the 130b genome encodes another putative effector, Lpw_00581, which has some predicted structural similarity to a protozoan ciliary protein and is also found in L. pneumophila Lens and Corby.

Ten novel 130b effectors are translocated into macrophages.

To investigate if some of the newly identified effector candidates were indeed substrates of the Dot/Icm T4SS, we used the fluorescence-based β-lactamase (TEM1) translocation assay to study effector translocation (17, 24). L. pneumophila 130b expressing N-terminal fusions of TEM1 to several putative effector proteins (see Figure S1 in the supplemental material) was used to infect Raw264.7 macrophages. Translocation of the fusion proteins into the host cells results in the cleavage of the TEM1 substrate CCF2, which is measured as a shift in its fluorescence emission wavelength. Our experiments demonstrated that TEM1 fusions of the effector protein candidates Lpw_00581, Lpw_02301, Lpw_02381, Lpw_03701, Lpw_04551, Lpw_16311, Lpw_20341, Lpw_25791, and Lpw_26201 were translocated into Raw264.7 macrophages in a DotA-dependent manner, while TEM1-Lpw_21901 and TEM1-Lpw_28221 were not translocated into the host cells (Fig. (Fig.6A).6A). Interestingly, the induction of expression of TEM1-Lpw_20091 in Legionella overnight cultures led to a strong growth defect (data not shown). To circumvent this problem, we grew the bacterial cultures without induction for 16 h and then induced expression of TEM1-Lpw_20091 and the positive control TEM1-LegC2 for 5 h and during the first hour of infection (Fig. (Fig.6B)6B) or only parallel to the infection (data not shown). Under these conditions, we observed translocation of TEM1-Lpw_20091 by wild-type bacteria to levels comparable to those of TEM1- effector fusion proteins.

FIG. 6.
L. pneumophila 130b translocates 10 novel Dot/Icm T4SS effector proteins into Raw264.7 macrophages. L. pneumophila 130b wild type (black bars) or the T4S-deficient L. pneumophila 130b ΔDotA mutant (white bars) harboring pXDC61 TEM1-effector gene ...

Altogether, we identified 10 novel T4SS effector proteins, which we called Legionella translocated proteins LtpA to LtpJ (Table (Table5),5), that are not present in L. pneumophila Philadelphia-1. Among them, the FIC domain effector LtpG (Lpw_20091) showed a particular phenotype, as induction of protein expression in liquid cultures resulted in a strong growth defect. No bacterial growth defects upon heterologous expression of the FIC domain effector AnkX were reported. It is still not known if the growth defect depends on the enzymatic activity of the FIC domain or how expression and translocation of endogenous LtpG are regulated during infection.

Prevalence of the novel effector proteins in a collection of Legionella isolates.

The newly identified T4SS effector proteins were selected because they were absent from L. pneumophila Philadelphia. To determine the prevalence of these effectors outside the sequenced prototypic strains, we performed a PCR screen of a large set of environmental and clinical Legionella isolates (summarized in Fig. Fig.7;7; individual results for each strain are shown in Table S2 in the supplemental material). The results for representative PCR-negative strains were confirmed by Southern blotting (data not shown). The set included 87 environmental and clinical L. pneumophila isolates from different serogroups from Malaysia, Australia, and the United Kingdom. Based on this screen, the novel effectors could be divided into two groups (Fig. (Fig.7).7). Group 1 comprised rare effector genes, namely, ltpB, ltpC, ltpE, ltpF, and ltpJ, which were detected in less than 15% of the analyzed strains. In contrast, members of the second group, ltpA, ltpD, ltpG, ltpH, and ltpI, showed higher prevalences of 34% to 62% in the PCR screen. The prevalences of three L. pneumophila Lens homologues of the newly identified effectors ltpA, ltpG, and ltpJ had already been investigated in a seminal multigenome DNA array screen including more than 200 L. pneumophila and non-pneumophila strains (15). In this screen, the gene lpl0057 (an ltpA homologue) was found in 36%, lpl1931 (an ltpG homologue) in 40%, and lpl2330 (an ltpJ homologue) in 8% of all tested strains. These numbers show good correlation with our data (ltpA, 52%; ltpG, 34%; and ltpJ, 7%) and support the reliability of the PCR screen. Our screen revealed no correlation between the presence of the novel effectors and the clinical or environmental origin of the strains. However, there is no scientific evidence that these environmental L. pneumophila strains have less potential to cause Legionnaires’ disease in humans than clinical strains. The results of our screen suggest that there is a prominent group of effectors present in 30 to 60% of the population, which has to be considered when Dot/Icm T4SS-dependent phenotypes are compared between different strains or generalized for the species L. pneumophila.

FIG. 7.
Distribution of the 10 novel Dot/Icm T4SS effectors in 87 L. pneumophila isolates. Genomic DNA from 87 clinical and environmental L. pneumophila isolates was analyzed for the presence of homologues of the novel L. pneumophila 130b effectors ltpA to ltpJ ...

Conclusions.

The genome of L. pneumophila strain Alcoy (accession number CP001828) became available just prior to the submission of the manuscript, and comparative genome analysis confirmed that the Dot/Icm T4SSB is conserved among all the L. pneumophila strains, although this is not the case for the T4SSA systems. Strains Paris, Lens, and Philadelphia-1 do not have the Tra/Trb system, and Corby does not have an Lvh region; however, 130b has both T4SSA systems. Both regions were also recently reported to be present in the genome of Alcoy (23). However, we report here the first identification of two distinct Lvh regions within one strain, and it will be interesting to see if both regions are functional and contribute to virulence. Analysis of the L. pneumophila Alcoy genome revealed that all 107 of the putative core set of L. pneumophila Dot/Icm T4SS effectors identified in this study are present. In addition, the redefined hypervariable region we describe here correlates with a synonymous region in Alcoy (lpa_02788 to lpa_02905). No new Dot/Icm T4SS effectors have been described for strain Alcoy. Of the 10 newly identified and validated T4SS effectors, only LtpA and LtpE possess homologues with moderate similarity in the strain, underlining the high plasticity of the effector pool between L. pneumophila strains. The characterization of the mode of action of the novel effector proteins will reveal if and how these effectors contribute to strain-to-strain variation in virulence.

Supplementary Material

[Supplemental material]

Acknowledgments

We are indebted to Howard Shuman and Xavier Charpentier for the generous gift of pXDC61 and the control plasmids for the TEM1 translocation assay. We thank Anthony Davidson for his assistance with the PCR screen. We are indebted to Stacey Yong for providing Malaysian L. pneumophila isolates for screening.

E.H. is an Australian Research Council (ARC) Future Fellow, and S.A.B. is an ARC Research Fellow. The Pathogen genomics group is funded by The Wellcome Trust. This work was supported by the National Health and Medical Research Council (NHMRC) of Australia, the Wellcome Trust, and the Medical Research Council (MRC).

Footnotes

[down-pointing small open triangle]Published ahead of print on 10 September 2010.

Supplemental material for this article may be found at http://jb.asm.org/.

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