Comparative and Functional Genomics of Legionella Identified Eukaryotic Like Proteins as Key Players in Host–Pathogen Interactions

Abstract

Although best known for its ability to cause severe pneumonia in people whose immune defenses are weakened, Legionella pneumophila and Legionella longbeachae are two species of a large genus of bacteria that are ubiquitous in nature, where they parasitize protozoa. Adaptation to the host environment and exploitation of host cell functions are critical for the success of these intracellular pathogens. The establishment and publication of the complete genome sequences of L. pneumophila and L. longbeachae isolates paved the way for major breakthroughs in understanding the biology of these organisms. In this review we present the knowledge gained from the analyses and comparison of the complete genome sequences of different L. pneumophila and L. longbeachae strains. Emphasis is given on putative virulence and Legionella life cycle related functions, such as the identification of an extended array of eukaryotic like proteins, many of which have been shown to modulate host cell functions to the pathogen’s advantage. Surprisingly, many of the eukaryotic domain proteins identified in L. pneumophila as well as many substrates of the Dot/Icm type IV secretion system essential for intracellular replication are different between these two species, although they cause the same disease. Finally, evolutionary aspects regarding the eukaryotic like proteins in Legionella are discussed.

Introduction

Genomics has the potential to provide an in depth understanding of the genetics, biochemistry, physiology, and pathogenesis of a microorganism. Furthermore comparative genomics, functional genomics, and related technologies, are helping to unravel the molecular basis of the pathogenesis, evolution, and phenotypic differences among different species, strains, or clones and to uncover potential virulence genes. Knowledge of the genomes provides the basis for the application of new powerful approaches for the understanding of the biology of the organisms studied.

Although Legionella are mainly environmental bacteria, several species are pathogenic to humans, in particular Legionella pneumophila (Fraser et al., 1977; Mcdade et al., 1977) and Legionella longbeachae (Mckinney et al., 1981). Legionnaires’ disease has emerged in the second half of the twentieth century partly due to human alterations of the environment. The development of artificial water systems in the last decades like air conditioning systems, cooling towers, showers, and other aerosolizing devices has allowed Legionella to gain access to the human respiratory system. When inhaled in contaminated aerosols, pathogenic Legionella can reach the alveoli of the lung where they are subsequently engulfed by macrophages. In contrast to most bacteria, which are destroyed, some Legionella species can multiply within the phagosome and eventually kill the macrophage, resulting in a severe, often fatal pneumonia called legionellosis or Legionnaires’ disease (mortality rate of 5–20%; up to 50% in nosocomial infections; Steinert et al., 2002; Marrie, 2008; Whiley and Bentham, 2011). To replicate intracellularly L. pneumophila manipulates host cellular processes using bacterial proteins that are delivered into the cytosolic compartment of the host cell by a specialized type IV secretion system called Dot/Icm. The proteins delivered by the Dot/Icm system target host factors implicated in controlling membrane transport in eukaryotic cells, which enables L. pneumophila to create an endoplasmic reticulum-like vacuole that supports intracellular replication in both protozoan and mammalian host cells (for a review see Hubber and Roy, 2010).

An interesting epidemiological observation is, that among the over 50 Legionella species described today, strains belonging to the species L. pneumophila are responsible for over 90% of the legionellosis cases worldwide and strains belonging to the species L. longbeachae are responsible for about 5% of human legionellosis cases worldwide (Yu et al., 2002). Surprisingly, this distribution is very different in Australia and New Zealand where L. pneumophila accounts for “only” 45.7% of the cases but L. longbeachae is implicated in 30.4% of the human cases. Furthermore, among the strains causing Legionnaires’ disease, L. pneumophila serogroup 1 (Sg1) alone is responsible for over 85% of cases (Yu et al., 2002; Doleans et al., 2004) despite the description of 15 different Sg within this species. In addition, the characterization of over 400 different L. pneumophila Sg1 strains has shown that only a minority among these is responsible for causing most of the human disease (Edelstein and Metlay, 2009). Some of these clones are distributed worldwide like L. pneumophila strain Paris (Cazalet et al., 2008) others have a more restricted geographical distribution, like the recently described endemic clone, prevalent in Ontario, Canada (Tijet et al., 2010). For the species L. longbeachae two serogroups are described to date (Bibb et al., 1981; Mckinney et al., 1981). L. longbeachae Sg1 is predominant in human disease as it causes up to 95% of the cases of legionellosis worldwide and most outbreaks and sporadic cases in Australia (Anonymous, 1997; Montanaro-Punzengruber et al., 1999). The two main human pathogenic Legionella species, L. pneumophila and L. longbeachae cause the same disease and symptoms in humans (Amodeo et al., 2009), however, there exist major differences between both species in niche adaptation and host susceptibility.

• (i)They are found in different environmental niches, as L. pneumophila is mainly found in natural and artificial water circuits and L. longbeachae is principally found in soil and therefore associated with gardening and use of potting compost (O’Connor et al., 2007). However, although less common, the isolation of L. pneumophila from potting soil in Europe has also been reported (Casati et al., 2009; Velonakis et al., 2009). Human infection due to L. longbeachae is particularly common in Australia but cases have been documented also in other countries like the USA, Japan, Spain, England, or Germany (MMWR, 2000; Garcia et al., 2004; Kubota et al., 2007; Kumpers et al., 2008; Pravinkumar et al., 2010).
• (ii)As described for other Legionella species, person to person transmission of L. longbeachae has not been documented, however, the primary transmission mode seems to be inhalation of dust from contaminated compost or soil that contains the organism (Steele et al., 1990; MMWR, 2000; O’Connor et al., 2007).
• (iii)Furthermore, for L. pneumophila a biphasic life cycle was observed in vitro and in vivo as exponential phase bacteria do not express virulence factors and are unable to replicate intracellularly. The ability of L. pneumophila to replicate intracellularly is triggered at the post-exponential phase by a complex regulatory cascade (Molofsky and Swanson, 2004; Sahr et al., 2009). In contrast, less is known on the L. longbeachae intracellular life cycle and its virulence factors. It was recently shown that unlike L. pneumophila the ability of L. longbeachae to replicate intracellularly is independent of the bacterial growth phase (Asare and Abu Kwaik, 2007) and that phagosome biogenesis is different. Like L. pneumophila, the L. longbeachae phagosome is surrounded by endoplasmic reticulum and does not mature to a phagolysosome; however it acquires early and late endosomal markers (Asare and Abu Kwaik, 2007).
• (iv)Another interesting difference between these two species is their ability to colonize the lungs of mice. While only A/J mice are permissive for replication of L. pneumophila, A/J, C57BL/6, and BALB/c mice are all permissive for replication of L. longbeachae (Asare et al., 2007; Gobin et al., 2009). Resistance of C57BL/6 and BALB/c mice to L. pneumophila has been attributed to polymorphisms in Nod-like receptor apoptosis inhibitory protein 5 (naip5) allele that recognizes the C-terminus of flagellin (Wright et al., 2003; Molofsky et al., 2006; Ren et al., 2006; Lightfield et al., 2008). The current model is that L. pneumophila replication is restricted due to flagellin dependent caspase-1 activation through Naip5-Ipaf and early macrophage cell death by pyroptosis. However, although depletion or inhibition of caspase-1 activity leads to decreased targeting of bacteria to lysosomes, the mechanism of caspase-1-dependent restriction of L. pneumophila replication in macrophages and in vivo is not fully understood (Schuelein et al., 2011).

In the last years, six genomes of different L. pneumophila strains (Paris, Lens, Philadelphia, Corby, Alcoy, and 130b (Cazalet et al., 2004; Chien et al., 2004; Steinert et al., 2007; D’Auria et al., 2010; Schroeder et al., 2010) have been published. The genome sequences of all but strain 130b were completely finished. Furthermore, the sequencing and analysis of four genomes of L. longbeachae have been carried out recently (Cazalet et al., 2010). L. longbeachae strain NSW150 of Sg1 isolated in Australia from a patient was sequenced completely, and for the remaining three strains (ATCC33462, Sg1 isolated from a human lung, C-4E7 and 98072, both of Sg2 isolated from patients) a draft genome sequence was reported. A fifth L. longbeachae strain (D-4968 of Sg1, isolated in the US from a patient) was recently sequenced and the analysis of the genome sequences assembled into 89 contigs was reported (Kozak et al., 2010).

Here we will describe what we learned from the analysis and comparison of the sequenced Legionella strains. We will discuss their general characteristics and then highlight the specific features or common traits with respect to the different ecological niches and the differences in host susceptibility of these two Legionella species. Emphasis will be put on putative virulence and Legionella life cycle related functions. In the last part we will analyze and discuss the possible evolution of the identified virulence factors. Finally, future perspectives in Legionella genomics are presented.

General Features of the L. pneumophila and L. longbeachae Genomes

Legionella pneumophila and L. longbeachae each have a single, circular chromosome with a size of 3.3–3.5 Mega bases (Mb) for L. pneumophila and 3.9–4.1 Mb for L. longbeachae. For both the average G + C content is 38% (Tables 1). The L. pneumophila strains Paris and Lens each contain different plasmids, 131.9 kb and 59.8 kb in size, respectively. In strain Philadelphia-1, 130b, Alcoy, and Corby no plasmid was identified. The L. longbeachae strains NSW10 and D-4986 carry highly similar plasmids of about 70 kb and DNA identity of 99%, strains C-4E7 and 98072 also contain each a highly similar plasmid of 133.8 kb in size. Thus similar plasmids circulate among L. longbeachae strains, but they seem to be different from those found in L. pneumophila.

Table 1.

General features of the sequenced Legionella genomes.

A. Complete and draft genomes of L. pneumophila obtained by classical or new generation sequencing
L. pneumophila
	Paris	Lens	Philadelphia	Corby	Alcoy	130bc
Chromosome size (kb)a	3504 (131.9)b	3345 (59.8)	3397	3576	3516	3490
G + C content (%)	38.3 (37.4)	38.4 (38)	38.3	38	38.4	38.2
No. of genesa	3123 (142)	2980 (60)	3031	3237	3197	3288
No. of protein coding genesa	3078 (140)	2921 (60)	2999	3193	3097	3141
Percentage of CDS (%)	87.9	88.0	90.2	86.8	86.0	87.9
No. of specific genes	225	181	213	144	182	386c
No. of 16S/23S/5S	03/03/03	03/03/03	03/03/03	03/03/03	03/03/03	ND
No. transfer RNA	44	43	43	43	43	42
Plasmids	1	1	0	0	0	0
B. Complete and draft genomes of L. longbeachae obtained by classical or new generation sequencing
L. longbeachae
	NSW 150	D-4968	ATCC33462	98072	C-4E7
Chromosome size (Kb)	4077 (71)	4016 (70)	4096	4018 (133.8)	3979 (133.8)
G + C content (%)	37.1 (38.2)	37.0	37.0	37.0 (37.8)	37 (37.8)
No. of genes	3660 (75)	3557 (61)	–	–	–
No. of 16S/23S/5S	04/04/04	04/04/04	04/04/04	04/04/04	04/04/04
No. of contigs > 0.5–300 kb	Complete	13	64	65	63
N50 contig size*	Complete	–	138 kb	129 kb	134 kb
Percentage of coverage**	100%	96.3	96.3	93.4	93.1
Number of SNP with NSW150	0	1900	1611	16 853	16 820
Plasmids	1	1	0	1	1

^aUpdated annotation; CDS, coding sequence; ^bdata from plasmids in parenthesis; ^cThe 130b sequence is not a manually corrected and finished assembly, thus the high number of specific genes might be due to not corrected sequencing errors; ND, not determined; *N50 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 (half of all bases reside in a contiguous sequence of the given size or more); SNP, single nucleotide polymorphism; **for SNP detection; – not determined.

A total of ∼3000 and 3500 protein-encoding genes are predicted in the L. pneumophila and L. longbeachae genomes, respectively. No function could be predicted for about 40% of these genes and about 20% are unique to the genus Legionella. Comparative analysis of the genome structure of the L. pneumophila genomes showed high colinearity, with only few translocations, duplications, deletions, or inversions (Figures 1A,B) and identified between 6 and 11% of genes as specific to each L. pneumophila strain. Principally, the genomes contain three large plasticity zones, where the synteny is disrupted: a 260-kb inversion in strain Lens with respect to strains Paris and Philadelphia-1, a 130-kb fragment which is inserted in a different genomic location in strains Paris and Philadelphia-1 and the about 50 kb chromosomal region carrying the Lvh type IV secretion system, previously described in strain Philadelphia-1 (Segal et al., 1999). Furthermore, deletions and insertions of several smaller regions were identified in each strain, as well as regions with variable gene content. In contrast, comparison of the completed chromosome sequences of L. pneumophila and L. longbeachae shows that the two Legionella species have a significantly different genome organization (Figure 1C). Moreover only about 65% of the L. longbeachae genes are orthologous to L. pneumophila genes, whereas about 34% of all genes are specific to L. longbeachae with respect to L. pneumophila Paris, Lens, Philadelphia, and Corby (defined by less than 30% amino acid identity over 80% of the length of the smallest protein).

Figure 1.

Synteny plot of the chromosomes of L. pneumophila strains Paris, Lens, Corby, and L. longbeachae NSW150. The plot was created using the mummer software package. (A) Synteny plot of the chromosomes of strains L. pneumophila Paris and Corby (B) and strains L. pneumophila Paris and Lens and (C) strains L. pneumophila Paris and L. longbeachae NSW150. Inversions between the genomic sequences are represented in blue. Genome-wide synteny is disrupted by a 260 kb inversion (blue) and a 130 kb plasticity zone between strain L. pneumophila Paris and Lens. In contrast, synteny between L. pneumophila and L. longbeachae is highly conserved.

Analysis of single nucleotide polymorphisms (SNP) revealed a very low SNP number of less than 0.4% among the four L. longbeachae genomes, which is significantly lower than the polymorphism of about 2% between L. pneumophila Sg1 strains Paris and Philadelphia (Table 1). Comparison of the two L. longbeachae Sg1 genomes (NSW150, ATCC33462) identified 1611 SNPs of which 1426 are located in only seven chromosomal regions mainly encoding putative mobile elements, whereas the remaining 185 SNPs were evenly distributed around the chromosome. A similar number of about 1900 SNPs were identified when comparing strains NSW150 to strain D-4968 (Table 1). In contrast, the SNP number between two strains of different Sg was higher, with about 16000 SNPs present between Sg1 and Sg2 strains (Table 1). This low SNP number and relatively homogeneous distribution of the SNPs around the chromosome suggest recent expansion for the species L. longbeachae (Cazalet et al., 2010). The sequences and their analysis are accessible at http://genolist.pasteur.fr/LegioList/.

To investigate the phylogenetic relationship among the L. pneumophila and L. longbeachae strains we here used the nucleotide sequence of recN (recombination and repair protein-encoding gene) aligned based on the protein alignment. Based on an analysis of 32 protein-encoding genes widely distributed among bacterial genomes, RecN was described as the gene with the greatest potential for predicting genome relatedness at the genus or subgenus level (Zeigler, 2003). As depicted in Figure 2, the phylogenetic relationship among the four L. pneumophila strains is very high, and L. longbeachae is clearly more distant.

Figure 2.

Phylogenetic tree showing the relationship of the sequenced L. pneumophila and L. longbeachae strains based on the recN sequence. The tree was constructed using the recN sequences of each genome and the Neighbor joining method in MEGA. L. longbeachae is indicated without strain designation, as the RecN sequence of all sequenced strains is identical and thus only one representative strain is indicated on the tree. Numbers at branching nodes are percentages of 1000 bootstrap replicates.

Diversity in Secretion Systems and Their Substrates may Contribute to Differences in Intracellular Trafficking and Niche Adaptation

The capacity of pathogens like Legionella to infect eukaryotic cells is intimately linked to the ability to manipulate host cell functions to establish an intracellular niche for their replication. Essential for the ability of Legionella to subvert host functions are its different secretion systems. The two major ones, known to be involved in virulence of L. pneumophila are the Dot/Icm type IV secretion system (T4BSS) and the Lsp type II secretion system (T2SS; Marra et al., 1992; Berger and Isberg, 1993; Rossier and Cianciotto, 2001).

For L. pneumophila type II protein secretion is critical for infection of amebae, macrophages and mice. Analyses of the L. longbeachae genome sequences showed, that it contains all genes to encode a functional Lsp type II secretion machinery (Cazalet et al., 2010; Kozak et al., 2010). Several studies, including the analysis of the L. pneumophila type II secretome indicated that L. pneumophila encodes at least 25 type II secreted substrates (Debroy et al., 2006; Cianciotto, 2009). Although this experimentally defined repertoire of type II secretion-dependent proteins is the largest known in bacteria, it may contain even more than 60 proteins as 35 additional proteins with a signal sequence were identified by in silico analyses (Cianciotto, 2009). A search for homologs of these substrates in the L. longbeachae genome sequences revealed that 9 (36%) of the 25 type II secretion system substrates described for L. pneumophila are absent from L. longbeachae (Table 2). For example the phospholipase C encoded by plcA and the chiA-encoded chitinase, which was shown to promote L. pneumophila persistence in the lungs of A/J mice are not present in L. longbeachae (Debroy et al., 2006). Thus over a third of the T2SS substrates seem to differ between L. pneumophila and L. longbeachae, a feature probably related to the different ecological niches occupied, but also to different virulence properties in the hosts.

Table 2.

Distribution of type II secretion-dependent proteins of L. pneumophila in L. longbeachae.

L. pneumophila						L. longbeachae		Name	Product
Phila	Paris	Lens	Corby	Alcoy	130b*	NSW	D-4968
lpg0467	lpp0532	lpl0508	lpc2877	lpa00713	lpw05741	llo2721	llb2607	proA	Zinc metalloprotease, promotes amebal infection
lpg1119	lpp1120	lpl1124	lpc0577	lpa01742	–	llo1016	llb0700	map	Tartrate-sensitive acid phosphatase
lpg2343	lpp2291	lpl2264	lpc1811	lpa03353	lpw25361	llo2819	llb2504	plaA	Lysophospholipase A
lpg2837	lpp2894	lpl2749	lpc3121	lpa04118	lpw30971	llo0210	llb1661	plaC	Glycerophospholipid:cholestrol transferase
lpg0502	lpp0565	lpl0541	lpc2843	lpa00759	lpw05821	–	–	plcA	Phospholipase C
lpg0745	lpp0810	lpl0781	lpc2548	lpa01148	lpw08251	llo2076	llb3335	lipA	Mono- and triacylglycerol lipase
lpg1157	lpp1159	lpl1164	lpc0620	lpa01801	lpw12111	llo2433	llb2928	lipB	Triacylglycerol lipase
lpg2848	lpp2906	lpl2760	lpc3133	lpa04141	lpw31111	llo0201	llb1671	srnA	Type 2 ribonuclease, promotes amebal infection
lpg1116	lpp1117	lpl1121	lpc0574	lpa01738	lpw11641	–	–	chiA	Chitinase, promotes lung infection
lpg2814	lpp2866	lpl2729	lpc3100	lpa04088	lpw30701	llo0255	llb1611	lapA	Leucine, phenylalanine, and tyrosine aminopeptidase
lpg0032	lpp0031	lpl0032	lpc0032	lpa00041	lpw00321	–	–	lapB	Lysine and arginine aminopeptidase
lpg0264	lpp0335	lpl0316	lpc0340	lpa00461	lpw03521	llo3103	llb2271		Weakly similar to bacterial amidase
lpg2622	lpp2675	lpl2547	lpc0519	lpa03836	lpw28341	–	–		Weakly similar to bacterial cysteine protease
lpg1918	lpp1893	lpl1882	lpc1372	lpa02774	lpw19571	llo3308	llb2032	celA	Endoglucanase
lpg2999	lpp3071	lpl2927	lpc3315	lpa04395	lpw32851	–	–		Predicted astacin-like zink endopeptidase
lpg2644	lpp2697	lpl2569	lpc0495	lpa03870	–	–	–		Some similarity to collagen like protein
lpg1809	lpp1772	lpl1773	lpc1253	lpa02614	lpw18401	llo1104	llb0603		Unknown
lpg1385	lpp1340	lpl1336	lpc0801	lpa02037	lpw13951	llo1474	llb0177		Unknown
lpg0873	lpp0936	lpl0906	lpc2419	lpa01320	lpw09571	llo2475	llb2883		Unknown
lpg0189	lpp0250	lpl0249	lpc0269	lpa00360	lpw02811	–	–		Unknown
lpg0956	lpp1018	lpl0958	lpc2331	lpa01443	lpw10421	llo1935	llb3498		Unknown
lpg2689	lpp2743	lpl2616	lpc0447	lpa03925	lpw29431	llo0361	llb1497	icmX	Linked to Dot/Icm type IV secretion genes
lpg1244	lpp0181	lpl0163	–	–	lpw01541	–	–	lvrE	Linked to Lvh type IV secretion genes
lpg1832	lpp1795	lpl1796	lpc1276	lpa02647	lpw18641	llo1152	llb0546		Weakly similar to VirK
lpg1962	lpp1946	lpl1936	lpc1440	lpa02861	lpw20131	–	–		Putative peptidyl-prolyl cis-trans isomerase
lpg0422	lpp0489	lpl0465	lpc2921	lpa0657	lpw05041	llo2801	llb2523	gamA	Glucoamylase

Substrates in this list are according to Cianciotto (2009); *strain 130b is not a finished sequence and not manually curated. Thus absence of a substrate can also be due to gaps in the sequence; – means not present; NSW means L. longbeachae NSW150.

Indispensible for replication of L. pneumophila in the eukaryotic host cells is the Dot/Icm T4SS (Nagai and Kubori, 2011), which translocate a large repertoire of bacterial effectors into the host cell. These effectors modulate multiple host cell processes and in particular, redirect trafficking of the L. pneumophila phagosome and mediate its conversion into an ER-derived organelle competent for intracellular bacterial replication (Shin and Roy, 2008; Cianciotto, 2009). The Dot/Icm system is conserved in L. longbeachae with a similar gene organization and protein identities of 47–92% with respect to L. pneumophila (Figure 3). This is similar to what has been reported previously for other Legionella species (Morozova et al., 2004). The only major differences identified are that in L. longbeachae the icmR gene is replaced by the ligB gene, however, the encoded proteins have been shown to perform similar functions (Feldman and Segal, 2004; Feldman et al., 2005) and that the DotG/IcmE protein of L. longbeachae (1525 aa) is 477 amino acids larger than that of L. pneumophila (1048 aa; Cazalet et al., 2010). DotG of L. pneumophila is part of the core transmembrane complex of the secretion system and is composed of three domains: a transmembrane N-terminal domain, a central region composed of 42 repeats of 10 amino acid and a C-terminal region homologous to VirB10. In contrast, the central region of L. longbeachae DotG is composed of approximately 90 repeats. Among the many VirB10 homologs present in bacteria, the Coxiella DotG and the Helicobacter pylori Cag7 are the only ones, which also have multiple repeats of 10 aa (Segal et al., 2005). It will be challenging to understand the impact of this modification on the function of the type IV secretion system. A L. longbeachae T4SS mutant obtained by deleting the dotA gene is strongly attenuated for intracellular growth in Acanthamoeba castellanii and human macrophages (Cazalet et al., 2010, and unpublished data), is outcompeted by the wild type strain 24 and 72 h after infection of lungs of A/J mice and is also dramatically attenuated for replication in lungs of A/J mice upon single infections (Cazalet et al., 2010). Thus, similar to what is seen for L. pneumophila, the Dot/Icm T4SS of L. longbeachae is also central for its pathogenesis and the capacity to replicate in eukaryotic host cells.

Figure 3.

Alignment of the chromosomal regions of L. pneumophila and L. longbeachae coding the Dot/Icm type 4 secretion system genes. The comparison shows that all genes are highly conserved (47–92% identity) between L. pneumophila Paris and L. longbeachae. Red arrows, genes conserved between L. pneumophila and L. longbeachae (>47% identity); black arrows, L. pneumophila specific genes compared to L. longbeachae (<35% identity); blue arrows, genes conserved between L. pneumophila and L. longbeachae but located in different places of the genome; green arrows, L. longbeachae specific genes compared to L. pneumophila. Red arrow boxed in green depicts dotG. N-terminal and C-terminal parts of dotG are highly conserved while the central part composed of repeated sequences differs between L. pneumophila and L. longbeachae.

This T4SS is crucial for intracellular replication for Legionella as it secretes an exceptionally large number of proteins into the host cell. Using different methods, 275 substrates have been shown to be translocated in the host cell in a Dot/Icm T4SS dependent manner (Campodonico et al., 2005; De Felipe et al., 2005, 2008; Shohdy et al., 2005; Burstein et al., 2009; Heidtman et al., 2009; Zhu et al., 2011). Table 3 shows the distribution of the 275 Dot/Icm substrates identified in L. pneumophila strain Philadelphia and their distribution in the six L. pneumophila and five L. longbeachae genomes sequenced. Their conservation among different L. pneumophila strains is very high, as over 80% of the substrates are present in all L. pneumophila strains analyzed here. In contrast, the search for homologs of these L. pneumophila Dot/Icm substrates in L. longbeachae showed that even more pronounced differences are present than in the repertoire of type II secreted substrates. Only 98 of these 275 L. pneumophila Dot/Icm substrates have homologs in the L. longbeachae genomes (Table 3). However, the repertoire of L. longbeachae substrates seems also to be quite large, as a search for proteins that encode eukaryotic like domains and contain the secretion signal described by Nagai et al. (2005) and the additional criteria defined by Kubori et al. (2008) predicted 51 putative Dot/Icm substrates specific for L. longbeachae NSW150 (Cazalet et al., 2010) indicating that at least over 140 proteins might be secreted by the Dot/Icm T4SS of L. longbeachae. A similar number of L. longbeachae specific putative eukaryotic like proteins and effectors was predicted for strain D-4968 (Kozak et al., 2010). Examples of effector proteins conserved between the two species are RalF, VipA, VipF, SidC, SidE, SidJ, YlfA LepA, and LepB, which contribute to trafficking or recruitment and retention of vesicles to L. pneumophila (Nagai et al., 2002; Chen et al., 2004; Luo and Isberg, 2004; Campodonico et al., 2005; Shohdy et al., 2005; Liu and Luo, 2007). It is interesting to note that homologs of SidM/DrrA and SidD are absent from L. longbeachae but a homolog of LepB is present. For L. pneumophila it was shown that SidM/DrrA, SidD, and LepB act in cooperation to manipulate Rab1 activity in the host cell. DrrA/SidM possesses three domains, an N-terminal AMP-transfer domain (AT), a nucleotide exchange factor (GEF) domain in the central part and a phosphatidylinositol-4-Phosphate binding domain (P4M) in its C-terminal part. After association of DrrA/SidM with the membrane of the Legionella-containing vacuole (LCV) via P4M (Brombacher et al., 2009), it recruits Rab1 via the GEF domain and catalyzes the GDP–GTP exchange (Ingmundson et al., 2007; Machner and Isberg, 2007). Rab1 is then adenylated by the AT domain leading to inhibition of GAP-catalyzed Rab1-deactivation (Müller et al., 2010). LepB cannot bind AMPylated Rab1 (Ingmundson et al., 2007). Recently it was shown that SidD deAMPylates Rab1 and enables LepB to bind Rab1 to promote its GTP–GDP exchange (Neunuebel et al., 2011; Tan and Luo, 2011). One might assume that other proteins of L. longbeachae not yet identified may perform the functions of DrrA/SidM and SidD. Another interesting observation is, that all except four of the effector proteins of L. pneumophila that are conserved in L. longbeachae are also conserved in all sequenced L. pneumophila genomes (Table 3).

Table 3.

Distribution of 275 Dot/Icm substrates identified in strain L. pneumophila Philadelphia in the 5 sequenced L. pneumophila and 5 sequenced L. longbeachae strains.

L. pneumophila						L. longbeachae					Name	Product
Phila	Paris	Lens	Corby	Alcoy	130b	NSW 150	D-4968	AT	98072	C-4E7
lpg0008	lpp0008	lpl0008	lpc0009	lpa0011	lpw00071	–	–	−	−	−	ravA	Unknown
lpg0012	lpp0012	lpl0012	lpc0013	lpa0016	lpw00111	–	–	−	−	−	cegC1	Ankyrin
lpg0021	lpp0021	lpl0022	lpc0022	lpa0030	lpw00221	llo0047	llb1841	+	+	+	–	Unknown
lpg0030	lpp0030	lpl0031	lpc0031	lpa0040	lpw00311	–	–	−	−	−	ravB	Unknown
lpg0038	lpp0037	lpl0038	lpc0039	lpa0049	lpw00381	–	–	−	−	−	ankQ/legA10	Ankyrin repeat
lpg0041	–	–	lpc0042	lpa0056	–	–	–	−	−	−	–	Putative metalloprotease
lpg0045	lpp0046	lpl0044	lpc0047	lpa0060	lpw00441	–	–	−	−	−	–	Unknown
lpg0046	lpp0047	lpl0045	lpc0048	lpa0062	lpw00451	–	–	−	−	−	–	Unknown
lpg0059	lpp0062	lpl0061	lpc0068	lpa0085	lpw00621	–	–	−	−	−	ceg2	Unknown
lpg0080	lpp0094	–	–	lpa3018	lpw00781	–	–	−	−	−	ceg3	Unknown
lpg0081	lpp0095	–	–	–	lpw00791	–	–	−	−	−	–	Unknown
lpg0090	lpp0104	lpl0089	lpc0109	lpa0132	lpw00881	–	–	−	−	−	lem1	Unknown
lpg0096	lpp0110	lpl0096	lpc0115	lpa0145	lpw00961	llo1322	llb0347	+	+	+	ceg4	Unknown
lpg0103	lpp0117	lpl0103	lpc0122	lpa0152	lpw01031	llo3312	llb2028	+	+	+	vipF	N-terminal acetyl-transferase, GNAT
lpg0126	lpp0140	lpl0125	lpc0146	lpa0185	lpw01261	–	–	−	−	−	cegC2	Ninein
lpg0130	lpp0145	lpl0130	lpc0151	lpa0194	lpw01311	llo3270	llb2073	+	+	+	–	Unknown
lpg0135	lpp0150	lpl0135	lpc0156	lpa0204	lpw01361	llo2439	llb2921	+	+	+	sdhB	Unknown
lpg0160	lpp0224	lpl0224	lpc0242	lpa0322	lpw02541	–	–	−	−	−	ravD	Unknown
lpg0170	lpp0232	lpl0233	lpc0251	lpa0335	lpw02641	llo1378	llb0280	+	+	+	ravC	Unknown
lpg0171	lpp0233	lpl0234	–	–	lpw02651	–	–	−	−	−	legU1	F-box motif
lpg0172	lpp0234	–	lpc0253	lpa0339	lpw02661	–	–	−	−	−	–	Unknown
lpg0181	lpp0245	lpl0244	lpc0265	lpa0388	lpw02761	llo2453	llb2907	+	+	+	–	Unknown
lpg0191	lpp0251	–	–	–	lpw02821	–	–	−	−	−	ceg5	Unknown
lpg0195	lpp0253	lpl0251	lpc0272	lpa0339	lpw02851	–	–	−	−	−	ravE	Unknown
lpg0196	lpp0254	lpl0252	–	–	lpw02861	llo2549	llb2798	+	+	+	ravF	Unknown
lpg0210	lpp0269	lpl0264	lpc0285	lpa0388	lpw02981	–	–	−	−	−	ravG	Unknown
lpg0227	lpp0286	lpl0281	lpc0303	lpa0412	lpw03151	llo2491	llb2864	+	+	+	ceg7	Unknown
lpg0234	lpp0304	lpl0288	lpc0309	lpa0419	lpw03221	llo0425	llb1431	+	+	+	sidE/laiD	Unknown
lpg0240	lpp0310	lpl0294	lpc0316	lpa0428	lpw03291	llo1601	llb0040	+	+	+	ceg8	Unknown
lpg0246	lpp0316	lpl0300	lpc0323	lpa0436	lpw03361	–	–	−	−	−	ceg9	Unknown
lpg0257	lpp0327	lpl0310	lpc0334	lpa0450	lpw03461	llo2362	llb3009	+	+	+	sdeA	Multidrug resistance protein
lpg0260	lpp0332	lpl0313	lpc0337	lpa0456	lpw03491	–	–	−	−	−	–	Unknown
lpg0275	lpp0349	lpl0327	lpc0351/3529	lpa0477	lpw03641	–	–	−	−	−	sdbA	Unknown
lpg0276	lpp0350	lpl0328	lpc0353	lpa0479	lpw03651	llo0327	llb1533	+	+	+	legG2	Ras guanine nucleotide exchange factor
lpg0284	lpp0360	lpl0336	lpc0361	lpa0490	lpw03741	–	–	−	−	−	ceg10	Unknown
lpg0285	lpp0361	lpl0337	lpc0362	lpa0492	lpw03751	–	–	−	−	−	lem2	Unknown
lpg0294	lpp0372	lpl0347	lpc0373	lpa0508	lpw03861	llo0464	llb1386	+	+	+	–	Unknown
lpg0364	lpp0429	lpl0405	lpc2980	lpa0578	lpw04431	–	–	−	−	−	–	Unknown
lpg0365	lpp0430	lpl0406	lpc2979	lpa0580	lpw04441	llo0525	llb1334	+	+	+	–	Unknown
lpg0375	lpp0442	lpl0418	lpc2968	lpa0596	–	–	–	−	−	−	–	Unknown
lpg0376	lpp0443	lpl0419	lpc2967	lpa0597	lpw04591	llo0548	llb1307	+	+	+	sdhA	GRIP, coiled-coil
lpg0390	lpp0457	lpl0433	lpc2954	lpa0613	lpw04721	–	–	−	−	−	vipA	Unknown
lpg0401	lpp0468	lpl0444	lpc2942	lpa0629	lpw04831	llo2582	llb2763	+	+	+	legA7/ceg11	Unknown
lpg0402	–	–	–	–	–	–	–	−	−	−	ankY/legA9	Ankyrin, STPK
lpg0403	lpp0469	lpl0445	lpc2941	lpa0630	lpw04841	–	–	−	−	−	ankG/ankZ/legA7	Ankyrin
lpg0405	lpp0471	lpl0447	lpc2939	lpa0633	lpw04861	llo2845	llb2472	+	+	+	–	Spectrin domain
lpg0422	lpp0489	lpl0465	lpc2921	lpa0657	lpw05041	llo2801	llb2523	+	+	+	legY	Putative Glucan 1,4-alpha-glucosidase
lpg0436	lpp0503	lpl0479	lpc2906	lpa0673	lpw05181	–	–	−	−	−	ankJ/legA11	Ankyrin
lpg0437	lpp0504	lpl0480	lpc2905	lpa0674	lpw05191	–	–	−	−	−	ceg14	Unknown
lpg0439	lpp0505	lpl0481	lpc2904	lpa0678	lpw05201	llo2983	llb2392	+	+	+	ceg15	Unknown
lpg0483	lpp0547	lpl0523	lpc2861	lpa0739	lpw05631	llo2705	llb2623	+	+	+	ankC/legA12	Ankyrin
lpg0515	lpp0578	lpl0554	lpc2829	lpa0776	lpw05951	llo3224	llb2129	+	+	+	legD2	Phytanoyl-CoA dioxygenase domain
lpg0518	lpp0581	lpl0557	lpc2826	lpa0781	lpw05981	–	–	−	−	−	–	Unknown
lpg0519	–	–	–	–	–	–	–	−	−	−	ceg17	Unknown
lpg0621	lpp0675	lpl0658	lpc2673	lpa0975	lpw06951	–	–	−	−	−	sidA	Unknown
lpg0634	lpp0688	lpl0671	lpc2660	lpa0996	lpw07081	llo2574	llb2771	+	+	+	–	Unknown
lpg0642	lpp0696/97	lpl0679	lpc2651	lpa1005	lpw07161	–	–	−	−	−	wipB	Unknown
lpg0695	lpp0750	lpl0732	lpc2599	lpa1082	lpw07721	–	–	−	−	−	ankN/ankX legA8	Ankyrin
lpg0696	lpp0751	lpl0733	lpc2598	lpa1084	lpw07731	–	–	−	−	−	lem3	Unknown
lpg0716	lpp0782	lpl0753	lpc2577	lpa1108	lpw07931	–	–	−	+	+	–	Unknown
lpg0733	lpp0799	lpl0770	lpc2559	lpa1135	lpw08111	llo0831	llb0892	+	+	+	ravH	Unknown
lpg0796	lpp0859	–	–	–	–	–	–	−	−	−	–	Unknown
lpg0898	lpp0959	lpl0929	lpc2395	lpa1360	lpw09801	–	–	−	−	−	ceg18	Unknown
lpg0926	lpp0988	lpl0957	lpc2365	lpa1397	lpw10111	–	–	−	−	−	ravI	Unknown
lpg0940	lpp1002	lpl0971	lpc2349	lpa1415	lpw10251	–	–	−	−	−	lidA	Unknown
lpg0944	lpp1006	–	lpc2345	lpa1421	–	–	–	−	−	−	ravJ	Unknown
lpg0945	lpp1007	lpl1579	lpc2344	lpa1423	lpw10311	–	–	−	−	−	legL1	LLR
lpg0963	lpp1025	lpl0992	lpc2324	lpa1453	lpw10491	llo0934	llb0782	+	+	+	–	Unknown
lpg0967	lpp1029	–	lpc2320	lpa1459	lpw10531	–	–	−	−	−	–	Unknown
lpg0968	lpp1030	lpl0997	lpc2319	lpa1460	lpw10541	–	–	−	−	−	sidK	Unknown
lpg0969	lpp1031	lpl0998	lpc2318	lpa1461	lpw10551	llo3265	llb2078	+	+	+	ravK	Unknown
lpg1083	–	–	–	–	–	–	–	−	−	−	–	Unknown
lpg1101	lpp1101	lpl1100	lpc2154*	lpa1709	lpw11451	–	–	−	−	−	lem4	Unknown
lpg1106	lpp1105	lpl1105	lpc2149	lpa1719	lpw11501	llo1414	llb0239/40	+	+	+	–	Unknown
lpg1108	lpp1108	lpl1108	lpc2146	lpa1724	lpw11531	llo3030	llb2350	+	+	+	ravL	Unknown
lpg1109	lpp1109	–	lpc2145	lpa1725	–	–	–	−	−	−	ravM	Unknown
lpg1110	lpp1111	lpl1114	lpc2142	lpa1728	lpw11571	–	–	−	−	−	lem5	Unknown
lpg1111	lpp1112	lpl1115	lpc2141	lpa1730	lpw11581	llo3126	llb2244	+	+	+	ravN	Unknown
lpg1120	–	–	–	–	lpw11681	–	–	−	−	−	lem6	Unknown
lpg1121	lpp1121	lpl1126	lpc0578	lpa1743	lpw11691	llo1321	llb0348	+	+	+	ceg19	Unknown
lpg1124	lpp1125	lpl1129	lpc0582	lpa1748	lpw11741	llo3206	llb2150	+	+	+	–	Unknown
lpg1129	lpp1130	–	–	–	lpw11801	–	–	−	−	−	ravO	Unknown
lpg1137	lpp1139	lpl1144	lpc0601	lpa1776	lpw11901	llo2404	llb2962	+	+	+	–	Unknown
lpg1144	lpp1146	lpl1150	lpc0607	lpa1785	lpw11971	–	–	−	−	−	cegC3	Unknown
lpg1145	lpp1147	lpl1151	lpc0608	lpa1787	lpw11981	–	–	−	−	−	lem7	Unknown
lpg1147	lpp1149	lpl1153	lpc0610	lpa1789	lpw12001	–	–	−	−	−	–	GCN5-related N-acetyltransferase
lpg1148	lpp1150	lpl1154	lpc0611	lpa1790	lpw12011	–	–	−	−	−	–	Unknown
lpg1152	lpp1154	lpl1159	lpc0615	lpa1795	lpw12061	–	–	−	−	−	ravP	Unknown
lpg1154	lpp1156	lpl1161	lpc0617	lpa1797	lpw12081	llo2487	llb2868	+	+	+	ravQ	Unknown
lpg1158	lpp1160	lpl1165*	lpc0621	lpa1802	lpw12121	–	–	−	−	−	–	Unknown
lpg1166	lpp1168	lpl1174	lpc0631	lpa1819	lpw12211	llo1034	llb0680	+	+	+	ravR	Unknown
lpg1171	lpp1173	lpl1179	lpc0637	lpa1826	–	–	–	−	−	−	–	Spectrin domain
lpg1183	lpp1186	lpl1192	lpc0650	lpa1839	lpw12401	llo2390	llb2978	+	+	+	ravS	Unknown
lpg1227	lpp1235	lpl1235	lpc0696	lpa1899	lpw12861	–	–	−	−	−	vpdB	Unknown
lpg1273	lpp1236	lpl1236	lpc0698	lpa1901	lpw12871	–	–	−	−	−	–	Unknown
lpg1290	lpp1253	–	–	–	–	–	–	−	−	−	lem8	Unknown
lpg1312	–	–	–	–	lpw13261	–	–	−	−	−	legC1	Unknown
lpg1316	–	–	–	–	–	llo1389	llb0269	+	+	+	ravT	Unknown
lpg1317	–	–	–	–	–	–	–	−	−	−	ravW	Unknown
lpg1328	lpp1283	lpl1282	lpc0743	lpa1958	–	–	–	−	−	−	legT	Thaumatin domain
lpg1355	lpp1309	–	–	–	–	–	–	−	−	−	sidG	Coiled-coil
lpg1426	lpp1381	lpl1377	lpc0842	lpa2090	lpw14431	llo1791	llb3606	+	+	+	vpdC	Patatin domain
lpg1449	lpp1404	–	–	–	lpw14671	–	–	−	−	−	–	Unknown
lpg1453	lpp1409	lpl1591	lpc0868	lpa2119	lpw14711	–	–	−	−	−	–	Unknown
lpg1483	lpp1439	lpl1545	lpc0898	lpa2161	lpw15031	llo1682	llb3727	+	+	+	legK1	STPK
lpg1484	lpp1440	lpl1544	lpc0899	lpa2162	lpw15041	–	–	−	−	−	–	Unknown
lpg1488	lpp1444	lpl1540	lpc0903*	lpa2168	lpw15081	–	–	−	−	−	lgt3/legc5	Coiled-coil
lpg1489	lpp1445	lpl1539	lpc0905	lpa2169	lpw15091	–	–	−	−	−	ravX	Unknown
lpg1491	lpp1447	–	–	–	–	–	–	−	−	−	lem9	Unknown
lpg1496	lpp1453	lpl1530	lpc0915	lpa2185	lpw15181	–	–	−	−	−	lem10	Unknown
lpg1551	lpp1508	lpl1475	lpc0972	lpa2253	–	–	–	−	−	−	ravY	Unknown
lpg1578	lpp4178	lpl4143	lpc1002	lpa2292	lpw16011	llo1503	llb0148	+	+	+	–	Unknown
lpg1588	lpp1546	lpl1437	lpc1013	lpa2305	lpw16131	–	–	−	−	−	legC6	Coiled–coil
lpg1598	lpp1556	lpl1427	lpc1025	lpa2317	lpw16231	–	–	−	−	−	lem11	Unknown
lpg1602	lpp1567	lpl1423/26*	lpc1028	lpa2318	lpw16241	–	–	−	−	−	legL2	LRR
lpg1621	lpp1591	lpl1402	lpc1048	lpa2346	lpw16461	llo1014	llb0702	+	+	+	ceg23	Unknown
lpg1625	lpp1595	lpl1398	lpc1052	lpa2350	lpw16511	llo0719	llb1016	+	+	+	lem23	Unknown
lpg1639	lpp1609	lpl1387	lpc1068	lpa2367	lpw16651	–	–	−	−	−	–	Unknown
lpg1642	lpp1612a/b	lpl1384	lpc1071	lpa2371	lpw16681	–	–	−	−	−	sidB	Putative hydrolase
lpg1654	lpp1625	–	lpc1084	lpa2390	–	llo0791	llb0935	+	+	+	–	Unknown
lpg1660	lpp1631	lpl1625	lpc1090	lpa2398	lpw16861	–	–	−	−	−	legL3	LRR
lpg1661	lpp1632	lpl1626	lpc1091	lpa2399	lpw16871	llo1691	llb3715	+	+	+	–	Putative N-acetyl transferase
lpg1666	lpp1637	lpl1631	lpc1096	lpa2408	lpw16921	–	–	−	−	−	–	Unknown
lpg1667	lpp1638	lpl1632	lpc1097	lpa2409	lpw16931	–	–	−	−	−	–	Unknown
lpg1670	lpp1642	lpl1635	lpc1101	lpa2413	lpw16971	–	–	−	−	−	–	Unknown
lpg1683	–	–	lpc1114	lpa2431	–	llo2508	llb2843	+	+	+	ravZ	Unknown
lpg1684	–	–	lpc1115	lpa2432	–	llo2267	llb3113	+	+	+	–	Unknown
lpg1685	–	–	lpc1116	lpa2433	–	llo3208	llb2147	+	+	+	–	Unknown
lpg1687	lpp1656	lpl1650	lpc1118	lpa2437	lpw17121	–	–	−	−	−	mavA	Unknown
lpg1689	lpp1658	lpl1652	lpc1120	lpa2439	lpw17141	llo1697	llb3708	+	+	+	–	Unknown
lpg1692	–	–	lpc1123	lpa2442	–	–	–	−	−	−	–	Unknown
lpg1701	lpp1666	lpl1660	lpc1130	lpa2455	lpw17231	–	–	−	−	−	ppeA/legC3	Coiled-coil
lpg1702	lpp1667	lpl1661	lpc1131	lpa2456	lpw17241	–	–	−	−	−	ppeB	Unknown
lpg1716	lpp1681	lpl1675	lpc1146	lpa2474	lpw17391	–	–	−	−	−	–	Unknown
lpg1717	lpp1682	–	–	–	lpw17401	–	–	−	−	−	–	Unknown
lpg1718	lpp1683	lpl1682	lpc1152	lpa2484	lpw17411	–	–	−	−	−	ankI/legAS4	Ankyrin
lpg1751	lpp1715	lpl1715	lpc1191	lpa2538	lpw17761	llo2314	llb3061	+	+	+	–	Unknown
lpg1752	lpp1716	lpl1716	lpc1192	lpa2539	lpw17771	llo2315	llb3060	+	+	+	–	Unknown
lpg1776	lpp1740	lpl1740	lpc1217	lpa2570	lpw18031	llo1437	llb0214*	+	+	+	–	Unknown
lpg1797	–	–	lpc1239	lpa2599	lpw32931	–	–	−	−	−	rvfA	Unknown
lpg1798	lpp1761	lpl1761	lpc1241	lpa2600	lpw18281	llo0991	llb0731	+	+	+	marB	Unknown
lpg1803	lpp1766	lpl1766	lpc1246	lpa2606	lpw18331	llo2611	llb2729	+	+	+	–	Unknown
lpg1836	lpp1799	lpl1800	lpc1280	lpa2652	lpw18691	–	–	−	−	−	ceg25	Unknown
lpg1851	lpp1818	lpl1817	lpc1296	lpa2675	lpw18871	llo1047	llb0666	+	+	+	lem14	Unknown
lpg1884	lpp1848	lpl1845	lpc1331	lpa2714	lpw19161	–	–	−	−	−	ylfB/legC2	Coiled-coil
lpg1888	lpp1855	lpl1850	lpc1336	lpa2723	lpw19211	–	–	−	−	−	–	Unknown
lpg1890	–	lpl1852	lpc1338	lpa2726	lpw19231	–	–	−	−	−	legLC8	LRR, coiled-coil
lpg1907	lpp1882	lpl1871	lpc1361	lpa2762	lpw19461	llo1240	llb0452	+	+	+	–	Unknown
lpg1924	lpp1899	lpl1888	lpc1378	lpa2783	lpw19631	–	–	−	−	−	–	Unknown
lpg1933	lpp1914	lpl1903	lpc1406	lpa2811	lpw19721	–	–	−	−	−	lem15	Unknown
lpg1947	lpp1930	lpl1917*	–	lpa2835	lpw19951	–	–	−	−	−	lem16	Spectrin domain
lpg1948	–	–	–	–	–	–	–	−	−	−	legLC4	LRR, coiled-coil
lpg1949	lpp1931	lpl1918	lpc1422	lpa2837	lpw19961	–	–	−	−	−	lem17	Unknown
lpg1950	lpp1932	lpl1919	lpc1423	lpa2838	lpw19971	llo1397	llb0259	+	+	+	ralF	Sec7 domain
lpg1953	lpp1935	lpl1922	lpc1426	lpa2842	lpw20041	–	–	−	−	−	legC4	Coiled-coil
lpg1958	lpp1940	–	–	–	–	–	–	−	−	−	legL5	LRR
lpg1959	lpp1941	–	–	lpa2857	lpw20101	–	–	−	−	−	–	Unknown
lpg1960	lpp1942	lpl1934*	lpc1437	lpa2859	lpw20111	llo0565	llb1288	+	+	+	lirA	Unknown
lpg1962	lpp1946	lpl1936	lpc1440	lpa2861	lpw20131	–	–	−	−	−	lirB	Rotamase
lpg1963	–	–	lpc1441/42	lpa2863	–	–	–	−	−	−	pieA/lirC	Unknown
lpg1964	–	–	–	–	–	–	–	−	−	−	pieB/lirD	Unknown
lpg1965	–	–	lpc1443/45	lpa2865	lpw20141	–	–	−	−	−	pieC/lirE	Unknown
lpg1966	lpp1947	–	lpc1446	lpa2867	lpw20151	–	–	−	−	−	pieD/lirF	Unknown
lpg1969	lpp1952	lpl1941	lpc1452	lpa2874	lpw20201	llo3131	llb2239	+	+	+	pieE	Unknown
lpg1972	lpp1955	lpl1950	lpc1459	lpa2884	lpw20291	–	–	−	−	−	pieF	Unknown
lpg1975	lpp1959	lpl1953	lpc1462	lpa2889(1)	lpw20351	–	–	−	−	−	–	Unknown
lpg1976	lpp1959	lpl1953	lpc1462	lpa2889(2)	lpw20351	–	–	−	−	−	pieG/legG1	Regulator of chromosome condensation
lpg1978	lpp1961	lpl1955	lpc1464	lpa2892	lpw20371	–	–	−	−	−	setA	Putative Glyosyltransferase
lpg1986	lpp1967	lpl1961	lpc1469	lpa2898	lpw20431	–	–	−	−	−	–	Unknown
lpg2050	lpp2033	lpl2028	lpc1536	lpa2992	lpw21141	–	–	−	−	−	–	Unknown
lpg2131	–	–	–	–	–	–	–	−	−	−	legA6	Unknown
lpg2137	lpp2076	lpl2066	lpc1586	lpa3060	lpw23101	–	–	−	−	−	legK2	STPK
lpg2144	lpp2082	lpl2072	lpc1593	lpa3071	lpw23181	–	–	−	−	−	ankB/legAU13/ceg27	Ankyrin, F-box
lpg2147	lpp2086	lpl2075	lpc1596	lpa3076	lpw23211	–	–	−	−	−	mavC	Unknown
lpg2148	lpp2087	lpl2076	lpc1597	lpa3077	lpw23221	–	–	−	−	−	–	Unknown
lpg2149	lpp2088	lpl2077	lpc1598	lpa3078	lpw23231	–	–	−	−	−	–	Unknown
lpg2153	lpp2092	lpl2081	lpc1602	lpa3083	lpw23271	–	–	−	−	−	sdeC	Unknown
lpg2154	lpp2093	lpl2082	lpc1603	lpa3086	lpw23281	llo3097	llb2278	+	+	+	sdeC	Unknown
lpg2155	lpp2094	lpl2083	lpc1604	lpa3087	lpw23291	llo3096	llb2279	+	+	+	sidJ	Unknown
lpg2156	lpp2095	lpl2084	lpc1605	lpa3088	lpw23301	llo3095	llb2280	+	+	+?	sdeB	Unknown
lpg2157	lpp2096	lpl2085	lpc1618	lpa3037	lpw23331	–	–	−	−	−	sdeC	Unknown
lpg2166	lpp2104	lpl2093	lpc1626	lpa3107	lpw23451	llo2398	llb2969	+	+	+	lem19	Unknown
lpg2160	lpp2099	lpl2088	lpc1621	lpa3100	lpw23361	llo2645	llb2690	+	+	+	–	Unknown
lpg2176	lpp2128	lpl2102	lpc1635	lpa3118	lpw23561	–	–	–	–	–	legS2	Sphingosine-1-phosphate lyase
lpg2199	lpp2149	lpl2123	lpc1663	lpa3157	lpw23811	–	–	–	–	–	cegC4	Unknown
lpg2200	lpp2150	lpl2124	lpc1664	lpa3158	lpw23821	–	–	–	–	–	cegC4	Unknown
lpg2215	lpp2166	lpl2140	lpc1680	lpa3179	lpw24011	–	–	–	–	–	legA2	Ankyrin
lpg2216	lpp2167	lpl2141	lpc1681	lpa3180	lpw24021	–	–	–	–	–	lem20	Unknown
lpg2222	lpp2174	lpl2147	lpc1689	lpa3191	lpw24081	llo1443	llb0208	+	+	+	lpnE	Putative beta-lactamase (SEL1 domain)
lpg2223	lpp2175	lpl2149*	lpc1691	lpa3196	lpw24091	–	–	–	–	–	–	Unknown
lpg2224	–	–	–	–	–	–	–	–	–	–	ppgA	Regulator of chromosome condensation
lpg2239	lpp2192	–	–	–	lpw24261	–	–	–	–	–	–	Unknown
lpg2248	lpp2202	lpl2174	lpc1717	lpa3237	lpw24371	–	–	–	–	–	lem21	Unknown
lpg2271	lpp2225	lpl2197	lpc1740	lpa3268	lpw24611	llo2530	llb2821	+	+	+	–	Unknown
lpg2298	lpp2246	lpl2217	lpc1763	lpa3296	lpw24841	llo1707	llb3696	+	+	+	ylfA/legC7	Coiled-coil
lpg2300	lpp2248	lpl2219	lpc1765	lpa3298	lpw24871	llo0584	llb1266	+	+	+	ankH/legA3, ankW	Ankyrin, NfkappaB inhibitor
lpg2311	lpp2259	lpl2230	lpc1776	lpa3312	lpw24981	–	–	−	−	−	ceg28	Unknown
lpg2322	lpp2270	lpl2242	lpc1789	lpa3328	lpw25121	llo0570	llb1282	+	+	+	ankK/legA5	Ankyrin
lpg2327	lpp2275	lpl2247	lpc1794	lpa3335	lpw25181	–	–	−	−	−	–	Unknown
lpg2328	lpp2276	lpl2248	lpc1795	lpa3336	lpw25191	–	–	−	−	−	lem22	Unknown
lpg2344	lpp2292	lpl2265	lpc1812	lpa3355	lpw25371	–	–	−	−	−	mavE	Unknown
lpg2351	lpp2300	lpl2273	lpc1820	lpa3367	lpw25461	llo2850	llb2466	+	+	+	mavF	Unknown
lpg2359	lpp2308	lpl2281	lpc1828	lpa3376	lpw25561	llo2856	llb2460	+	+	+	–	Unknown
lpg2370	–	–	–	–	–	–	–	−	−	−	–	HipA fragment
lpg2372	lpp3009	–	lpc3248	lpa4300	–	–	–	−	−	−	–	Unknown
lpg2382	lpp2444	lpl2300	lpc2108	lpa3446	lpw25841	llo1576	llb0071	+	+	+	–	Unknown
lpg2391	lpp2458	lpl2315	lpc2086	lpa3485	lpw26021	–	–	−	−	−	sdbC	Unknown
lpg2392	lpp2459	lpl2316	lpc2085	lpa3486	lpw26041	–	–	−	−	−	legL6	LRR
lpg2400	–	lpl2323	–	–	lpw26121	–	–	−	−	−	legL6	LRR
lpg2406	lpp2472	lpl2329	lpc2070	lpa3506	lpw26191	llo2172	llb3225	+	+	+	lem23	Unknown
lpg2407	lpp2474	–	lpc2069	lpa3507	–	–	–	−	−	−	–	Unknown
lpg2409	lpp2476	lpl2332	lpc2067	lpa3511	lpw26241	–	–	−	−	−	ceg29	Unknown
lpg2410	lpp2479	lpl2334	lpc2065	lpa3513	lpw26261	–	–	−	−	−	vpdA	Patatin domain
lpg2411	lpp2480	lpl2335	lpc2064	lpa3515	lpw26281	llo2227	llb3158	+	+	+	lem24	Unknown
–	lpp2486	–	–	–	–				−	−	–	F-box
lpg2416	–	lpl2339	lpc2057	lpa3527	lpw26351	–	–	−	−	−	legA1	Unknown
lpg2420	–	lpl2343	lpc2056	lpa3529	lpw26391	–	–	−	−	−	–	Unknown
lpg2422	lpp2487	lpl2345	lpc2055	lpa3530	lpw26401	llo1650	llb3763/64	+	+	+	lem25	Unknown
lpg2424	lpp2489	lpl2347	lpc2053	lpa3532	lpw26421	–	–	−	−	−	mavG	Unknown
lpg2425	lpp2491	lpl2348	lpc2051	lpa3537	lpw26431	–	–	−	−	−	mavH	Unknown
lpg2433	lpp2500	lpl2353	lpc2043	lpa3548	lpw26521	–	–	−	−	−	ceg30	Unknown
lpg2434	lpp2501	lpl2355	lpc2042	lpa3550	lpw26531	–	–	−	−	−	–	Unknown
lpg2443	lpp2510	lpl2363	lpc2033	lpa3562	–	–	–	−	−	−	–	Unknown
lpg2444	lpp2511	lpl2364	lpc2032	lpa3563	lpw26641	–	–	−	−	−	mavI	Unknown
lpg2452	lpp2517	lpl2370	lpc2026	lpa3574	lpw26701	–	–	−	−	−	ankF/legA14/ceg31	Ankyrin
lpg2456	lpp2522	lpl2375	lpc2020	lpa3583	lpw26751	llo0365	llb1493	+	+	+	ankD/legA15	Ankyrin
lpg2461	lpp2527	lpl2380	lpc2015	lpa3589	lpw26801	llo1991	llb3433	+	+	+	–	Unknown
lpg2464	–	lpl2384	–	–	lpw26851	–	–	−	−	−	sidM/drrA	Unknown
lpg2465	–	lpl2385	–	–	lpw26861	–	–	−	−	−	sidD	Unknown
lpg2490	lpp2555	lpl2411	lpc1987	lpa3628	lpw27131	–	–	−	−	−	lepB	Coiled-coil, Rab1 GAP
lpg2482	lpp2546	lpl2402	lpc1996	lpa3615	lpw27041	–	–	−	−	−	sdbB	Unknown
lpg2498	lpp2566	lpl2420	lpc1975	lpa3646	lpw27241	–	–	−	−	−	mavJ	Unknown
lpg2504	lpp2572	lpl2426	lpc1967	lpa3658	lpw27301	llo2525	llb2826	+	+	+	sidI/ceg32	Unknown
lpg2505	lpp2573	lpl2427	lpc1966	lpa3659	lpw27311	llo2526	llb2825	+	+	+	–	Unknown
lpg2508	lpp2576	lpl2430	lpc1962/63*	lpa3666	lpw27341	–	–	−	−	−	sdjA	Unknown
lpg2509	lpp2577	lpl2431	lpc1961	lpa3667	lpw27351	llo3097	llb2278	+	+	+	sdeD	Unknown
lpg2510	lpp2578	lpl2432	lpc1960	lpa3668	–	llo3098	llb2276	+	+	+	sdcA	Unknown
lpg2511	lpp2579	lpl2433	lpc1959	lpa3669	lpw27371	–	–	−	−	−	sidC	PI(4)P binding domain
lpg2523	–	–	–	–	lpw27501	–	–	−	−	−	lem26	Unknown
lpg2525	–	–	–	–	–	–	–	−	−	−	mavK	Unknown
lpg2526	lpp2591	lpl2446	lpc1946	lpa3687	lpw27521	–	–	−	−	−	mavL	Unknown
lpg2527	lpp2592	lpl2447	lpc1944	lpa3688	lpw27531	llo3335	llb2002	+	+	+	–	Unknown
lpg2529	lpp2594	lpl2449	lpc1942	lpa3692	lpw27551	llo2238	llb3146	+	+	+	lem27	Unknown
lpg2538	lpp2604	lpl2459	lpc1930	lpa3706	lpw27671	–	–	−	−	−	–	Unknown
lpg2539	lpp2605	lpl2460	lpc1929	lpa3707	lpw27681	llo1348	llb0317	+	+	+	–	Unknown
lpg2541	lpp2607	lpl2462	lpc1927	lpa3710	lpw27701	–	–	−	−	−	–	Unknown
lpg2546	lpp2615	–	lpc1919	lpa3727	lpw27791	–	–	−	−	−	–	Unknown
lpg2552	lpp2622	lpl2473	lpc1911	lpa3738	lpw27871	llo1062	llb0648	+	+	+	–	Unknown
lpg2555	lpp2625	lpl2480	lpc1908	lpa3743	lpw27901	llo2220	llb3170	+	+	+	–	Unknown
lpg2556	lpp2626	lpl2481	lpc1906	lpa3745	lpw27911	llo2218	llb3172	+	+	+	legK3	STPK
lpg2577	lpp2629	lpl2499	lpc0570	lpa3768	lpw28241	–	–	−	−	−	mavM	Unknown
lpg2584	lpp2637	lpl2507	lpc0561	lpa3779	lpw28321	–	–	−	−	−	sidF	Unknown
lpg2588	lpp2641	lpl2511	lpc0557	lpa3784	lpw28361	llo2622	llb2718	+	+	+	legS1	Unknown
lpg2591	lpp2644	lpl2514	lpc0551	lpa3790	lpw28391	llo0626	llb1219	+	+	+	ceg33	Unknown
lpg2603	lpp2656	lpl2526	lpc0539	lpa3807	lpw28521	–	–	−	−	−	lem28	Unknown
lpg2628	lpp2681	lpl2553	lpc0513	lpa3846	lpw28781	–	–	−	−	−	–	Unknown
lpg2637	lpp2690	lpl2562	lpc0503	lpa3859	lpw28871	–	–	−	−	−	–	Unknown
lpg2638	lpp2691	lpl2563	lpc0502	lpa3861	lpw28891	llo2645	llb2690	+	+	+	mavV	Unknown
lpg2692	lpp2746	lpl2619	lpc0444	lpa3929	lpw29461	–	–	−	−	−	–	Unknown
lpg2694	lpp2748	lpl2621	lpc0442	lpa3931	lpw29481	–	–	−	−	−	legD1	Phyhd1 protein
lpg2718	lpp2775	lpl2646	lpc0415	lpa3966	lpw29771	–	–	−	−	−	wipA	Unknown
lpg2720	lpp2777	lpl2648	lpc0413	lpa3968	lpw29791	–	–	−	−	−	legN	cAMP-binding protein
lpg2744	lpp2800	lpl2669	lpc0386	lpa4004	lpw30031	–	–	−	−	−	–	Unknown
lpg2745	lpp2801	lpl2670	lpc0385	lpa4005	lpw30041	llo0308	llb1553	+	+	+	–	Unknown
lpg2793	lpp2839	lpl2708	lpc3079	lpa4063	lpw30471	–	–	−	−	−	lepA	Effector protein A
lpg2804	lpp2850	lpl2719	lpc3090	lpa4076	lpw30591	llo0267	llb1598	+	+	+	lem29	Unknown
lpg2815	lpp2867	lpl2730	lpc3101	lpa4089	lpw30711	llo0254	llb1612	+	+	+	mavN	Unknown
lpg2826	–	lpl2741	lpc3113	lpa4104	lpw30831	–	–	−	−	−	ceg34	Unknown
lpg2828	lpp2882	lpl2743	lpc3115	lpa4109	lpw30851	llo0783	llb0944	+	+	+	–	Unknown
lpg2829	lpp2883/86*	–	–	–	lpw30861	–	–	−	−	−	sidH	Unknown
lpg2830	lpp2887	–	–	–	lpw30881	–	–	−	−	−	lubX/legU2	U-box motif
lpg2831	lpp2888	–	–	–	lpw30891	–	–	−	−	−	VipD	Patatin-like phopholipase
lpg2832	lpp2889	lpl2744	lpc3116	lpa4110	lpw30921	llo0214	llb1656	+	+	+	–	Putative hydrolase
lpg2844	lpp2903	lpl2756	lpc3128	lpa4133	–	–	–	−	−	−	–	Unknown
lpg2862	–	–	–	–	–	–	–	−	−	−	Lgt2/legC8	Coiled-coil
lpg2874	lpp2933	lpl2787	lpc3160	lpa4176	lpw31411	–	–	–	–	–	–	Unknown
lpg2879	lpp2938	lpl2792	lpc3165	lpa4186	lpw31471	llo0192	llb1681	+	+	+	–	Unknown
lpg2884	lpp2943	lpl2797	lpc3170	lpa4193	lpw31531	llo0197	llb1676	+	+	+	–	Unknown
lpg2885	lpp2944	lpl2798	lpc3171	–	lpw31541	–	–	−	−	−	–	Unknown
lpg2888	lpp2947	lpl2801	lpc3174	lpa4199	lpw31571	llo0200	llb1672	+	+	+	–	Unknown
lpg2912	lpp2980	lpl2830	lpc3214	lpa4255	lpw31931	–	–	−	−	−	–	Unknown
lpg2936	lpp3004	lpl2865	lpc3243	lpa4293	lpw32251	llo0081	llb1804	+	+	+	–	rRNA small subunit methyltransferase E
lpg2975	lpp3047	lpl2904	lpc3290	lpa4358	−?	llo3405	llb1930	+	+	+	–	Unknown
lpg2999	lpp3071	lpl2927	lpc3315	lpa4395	lpw32851	–	–	−	−	−	legP	Astacin protease
lpg3000	lpp3072	lpl2928	lpc3316	lpa4397	lpw32861	llo3444	llb1887	+	+	+	–	Unknown

List of substrates is based on Isberg et al. (2009), De Felipe et al. (2008), Ninio et al. (2009), Zhu et al. (2011); AT = ATCC33462; *pseudogene, +? or −? strains 130b, C-4E7 and 98072 are not a finished sequence and not manually curated. Thus absence of a substrate can also be due to gaps in the sequence; shaded in gray, substrates conserved in all L. pneumophila and L. longbeachae genomes.

Taken together the T2SS Lsp and the T4SS Dot/Icm are highly conserved between L. pneumophila and L. longbeachae. However, more than a third of the known L. pneumophila type II- and over 70% of type IV-dependent substrates differ between both species. These species specific, secreted effectors might be implicated in the different niche adaptations and host susceptibilities. Most interestingly, of the 98 L. pneumophila substrates conserved in L. longbeachae 87 are also present in all L. pneumophila strains sequenced to date. Thus, these 87 Dot/Icm substrates might be essential for intracellular replication of Legionella and represent a minimal toolkit for intracellular replication that has been acquired before the divergence of the two species.

Molecular Mimicry is a Major Virulence Strategy of L. pneumophila and L. longbeachae

The L. pneumophila genome sequence analysis has revealed that many of the predicted or experimentally verified Dot/Icm secreted substrates are proteins similar to eukaryotic proteins or contain motifs mainly or only found in eukaryotic proteins (Cazalet et al., 2004; De Felipe et al., 2005). Thus comparative genomics suggested that L. pneumophila encodes specific virulence factors that have evolved during its evolution with eukaryotic host cells such as fresh-water ameba (Cazalet et al., 2004). The protein-motifs predominantly found in eukaryotes, which were identified in the L. pneumophila genomes are ankyrin repeats, SEL1 (TPR), Set domain, Sec7, serine threonine kinase domains (STPK), U-box, and F-box motifs. Examples for eukaryotic like proteins of L. pneumophila are two secreted apyrases, a sphingosine-1-phosphate lyase and sphingosine kinase, eukaryotic like glycoamylase, cytokinin oxidase, zinc metalloprotease, or an RNA binding precursor (Cazalet et al., 2004; De Felipe et al., 2005; Bruggemann et al., 2006). Function prediction based on similarity searches suggested that many of these proteins are implicated in modulating host cell functions to the pathogens advantage (Cazalet et al., 2004). Recent functional studies confirm these predictions.

As a first example, it was shown that L. pneumophila is able to interfere with the host ubiquitination pathway. The L. pneumophila U-box containing protein LubX was shown to be a secreted effector of the Dot/Icm secretion system that mediates polyubiquitination of a host kinase Clk1 (Kubori et al., 2008). Recently, LubX was described as the first example of an effector protein, which targets and regulates another effector within host cells, as it functions as an E3 ubiquitin ligase that hijacks the host proteasome to specifically target the bacterial effector protein SidH for degradation. Delayed delivery of LubX to the host cytoplasm leads to the shutdown of SidH within the host cells at later stages of infection. This demonstrates a sophisticated level of co-evolution between eukaryotic cells and L. pneumophila involving an effector that functions as a key regulator to temporally coordinate the function of a cognate effector protein (Kubori et al., 2010; Luo, 2011). Furthermore, AnkB/Lpp2028, one of the three F-box proteins of L. pneumophila, was shown to be a T4SS effector that is implicated in virulence of L. pneumophila and in recruiting ubiquitinated proteins to the LCV (Al-Khodor et al., 2008; Price et al., 2009; Habyarimana et al., 2010; Lomma et al., 2010).

A second example is the apyrases (Lpg1905 and Lpg0971) encoded in the L. pneumophila genomes. Indeed, both are secreted enzymes important for intracellular replication of L. pneumophila. Lpg1905 is a novel prokaryotic ecto-NTPDase, similar to CD39/NTPDase1, which is characterized by the presence of five apyrase-conserved regions and enhances the replication of L. pneumophila in eukaryotic cells (Sansom et al., 2007). Apart from ATP and ADP, Lpg1905 also cleaves GTP and GDP with similar efficiency to ATP and ADP, respectively (Sansom et al., 2008). A third example is a L. pneumophila homolog of the highly conserved eukaryotic enzyme sphingosine-1-phosphate lyase (Spl). In eukaryotes, SPL is an enzyme that catalyzes the irreversible cleavage of sphingosine-1-phosphate (S1P). S1P is implicated in various physiological processes like cell survival, apoptosis, proliferation, migration, differentiation, platelet aggregation, angiogenesis, lymphocyte trafficking and development. Despite the fact that the function of the L. pneumophila Spl remains actually unknown, the hypothesis is that it plays a role in autophagy and/or apoptosis (Cazalet et al., 2004; Bruggemann et al., 2006). Recently it has been shown that the L. pneumophila Spl is a secreted effector of the Dot/Icm T4SS, that it is able to complement the sphingosine-sensitive phenotype of Saccharomyces cerevisiae. Moreover, L. pneumophila Spl co-localizes to the host cell mitochondria (Degtyar et al., 2009).

Taken together, the many different functional studies undertaken based on the results of the genome sequence analyses deciphering the roles of the eukaryotic like proteins have clearly established that they are secreted virulence factors that are involved in host cell adhesion, formation of the LCV, modulation of host cell functions, induction of apoptosis and egress of Legionella (Nora et al., 2009; Hubber and Roy, 2010). Most of these effector proteins are expressed at different stages of the intracellular life cycle of L. pneumophila (Bruggemann et al., 2006) and are delivered to the host cell by the Dot/Icm T4SS. Thus molecular mimicry of eukaryotic proteins is a major virulence strategy of L. pneumophila.

As expected, eukaryotic like proteins and proteins encoding domains mainly found in eukaryotic proteins are also present in the L. longbeachae genomes. However, between the two species a considerable diversity in the repertoire of these proteins exists. For example Spl, LubX, the three L. pneumophila F-box proteins, and the homolog of one (Lpg1905) of the two apyrases are missing in all sequenced L. longbeachae genomes. In contrast a glycoamylase (Herrmann et al., 2011) and an uridine kinase homolog are present also in L. longbeachae (Cazalet et al., 2010; Kozak et al., 2010; Table 3). However, other proteins encoded by the L. longbeachae genome contain U-box and F-box domains and might therefore fulfill similar functions. Thus, although the specific proteins may not be conserved, the eukaryotic like protein–protein interaction domains found in L. pneumophila are also present in L. longbeachae.

The differences in trafficking between L. longbeachae and L. pneumophila mentioned above might be related to specific effectors encoded by L. longbeachae. A search for such specific putative effectors of L. longbeachae identified several proteins that might contribute to these differences like a family of Ras-related small GTPases (Cazalet et al., 2010; Kozak et al., 2010). These proteins may be involved in vesicular trafficking and thus may account at least partly for the specificities of the L. longbeachae life cycle. L. pneumophila is also known to exploit monophosphorylated host phosphoinositides (PI) to anchor the effector proteins SidC, SidM/DrrA, LpnE, and LidA to the membrane of the replication vacuole (Machner and Isberg, 2006; Murata et al., 2006; Weber et al., 2006, 2009; Newton et al., 2007; Brombacher et al., 2009). L. longbeachae may employ an additional strategy to interfere with the host PI as a homolog of the mammalian PI metabolizing enzyme phosphatidylinositol-4-phosphate 5-kinase was identified in its genome. One could speculate that this protein allows direct modulation of the host cell PI levels.

Interestingly, although 23 of the 29 ankyrin proteins identified in the L. pneumophila strains are absent from the L. longbeachae genome, L. longbeachae encodes a total of 23 specific ankyrin repeat proteins (Table 3). For example, L. pneumophila AnkX/AnkN that was shown to interfere with microtubule-dependent vesicular transport is missing in L. longbeachae (Pan et al., 2008). However, L. longbeachae encodes a putative tubulin–tyrosine ligase (TTL). TTL catalyzes the ATP-dependent post-translational addition of a tyrosine to the carboxy terminal end of detyrosinated alpha-tubulin. Although the exact physiological function of alpha-tubulin has so far not been established, it has been linked to altered microtubule structure and function (Eiserich et al., 1999). Thus this protein might take over this function in L. longbeachae.

Legionella longbeachae is the first bacterial genome encoding a protein containing an Src Homology 2 (SH2) domain. SH2 domains, in eukaryotes, have regulatory functions in various intracellular signaling cascades. Furthermore, L. longbeachae encodes two proteins with pentatricopeptide repeat (PPR) domains. This family seems to be greatly expanded in plants, where they appear to play essential roles in organellar RNA metabolism (Lurin et al., 2004; Nakamura et al., 2004; Schmitz-Linneweber and Small, 2008). Only 12 bacterial PPR domain proteins have been identified to date, all encoded by two species, the plant pathogens Ralstonia solanacearum and the facultative photosynthetic bacterium Rhodobacter sphaeroides. Thus, genome analysis revealed a particular feature of the Legionella genomes, the presence of many eukaryotic like proteins and protein domains, some of which are common to the two Legionella species, others which are specific and may thus account for the species specific features in intracellular trafficking and niche adaptation in the environment.

Surface Structures – A Clue to Mouse Susceptibility to Infection with Legionella

Despite the presence of many different species of Legionella in aquatic reservoirs, the vast majority of human disease is caused by a single serogroup (Sg) of a single species, namely L. pneumophila Sg1, which is responsible for about 84% of all cases worldwide (Yu et al., 2002). Similar results are obtained for L. longbeachae. Two serogroups are described, but L. longbeachae Sg1 is predominant in human disease. Lipopolysaccharide (LPS) is the basis for the classification of serogroups but it is also a major immunodominant antigen of L. pneumophila and L. longbeachae. Interestingly, it has also been shown that membrane vesicles shed by virulent L. pneumophila containing LPS are sufficient to inhibit phagosome–lysosome fusion (Fernandez-Moreira et al., 2006). Results obtained from large-scale genome comparisons of L. pneumophila suggested that LPS of Sg1 itself might be implicated in the predominance of Sg1 strains in human disease compared to other serogroups of L. pneumophila and other Legionella species (Cazalet et al., 2008). A comparative search for LPS coding regions in the genome of L. longbeachae NSW 150 identified two gene clusters encoding proteins that could be involved in production of lipopolysaccharide (LPS) and/or capsule. Neither shared homology with the L. pneumophila LPS biosynthesis gene cluster suggesting considerable differences in this major immunodominant antigen between the two Legionella species. However, homologs of L. pneumophila lipidA biosynthesis genes (LpxA, LpxB, LpxD, and WaaM) are present. Electron microscopy also demonstrated that, in contrast to L. pneumophila, L. longbeachae produces a capsule-like structure, suggesting that one of the aforementioned gene cluster encodes LPS and the other the capsule (Cazalet et al., 2010).

As mentioned in the introduction, only A/J mice are permissive for replication of L. pneumophila, in contrast A/J, C57BL/6, and BALB/c mice are all permissive for replication of L. longbeachae. In C57BL/6 mice cytosolic flagellin of L. pneumophila triggers Naip5-dependent caspase-1 activation and subsequent proinflammatory cell death by pyroptosis rendering them resistant to infection (Diez et al., 2003; Wright et al., 2003; Molofsky et al., 2006; Ren et al., 2006; Zamboni et al., 2006; Lamkanfi et al., 2007; Lightfield et al., 2008). Genome analysis shed light on the reasons for these differences. L. longbeachae does not carry any flagellar biosynthesis genes except the sigma factor FliA, the regulator FleN, the two-component system FleR/FleS and the flagellar basal body rod modification protein FlgD (Cazalet et al., 2010; Kozak et al., 2010). Analysis of the genome sequences of strains L. longbeachae D-4968, ATCC33642, 98072, and C-4E7 as well as a PCR-based screening of 50 L. longbeachae isolates belonging to both serogroups by Kozak et al. (2010) and of 15 additional isolates by Cazalet et al. (2010) did not detect flagellar genes in any isolate confirming that L. longbeachae, in contrast to L. pneumophila does not synthesize flagella. Interestingly, all genes bordering flagellar gene clusters are conserved between L. longbeachae and L. pneumophila, suggesting deletion of these regions from the L. longbeachae genome. This result suggests, that L. longbeachae fails to activate caspase-1 due to the lack of flagellin, which may also partly explain the differences in mouse susceptibility to L. pneumophila and L. longbeachae infection. The putative L. longbeachae capsule may also contribute to this difference.

Quite interestingly, although L. longbeachae does not encode flagella, it encodes a putative chemotaxis system. Chemotaxis enables bacteria to find favorable conditions by migrating toward higher concentrations of attractants. In many bacteria, the chemotactic response is mediated by a two-component signal transduction pathway, comprising a histidine kinase CheA and a response regulator CheY. Homologs of this regulatory system are present in the L. longbeachae genomes sequenced (Cazalet et al., 2010; Kozak et al., 2010). Furthermore, two homologs of the “adaptor” protein CheW that associate with CheA or cytoplasmic chemosensory receptors are present. Ligand-binding to receptors regulates the autophosphorylation activity of CheA in these complexes. The CheA phosphoryl group is subsequently transferred to CheY, which then diffuses away to the flagellum where it modulates motor rotation. Adaptation to continuous stimulation is mediated by a methyltransferase CheR. Together, these proteins represent an evolutionarily conserved core of the chemotaxis pathway, common to many bacteria and archea (Kentner and Sourjik, 2006; Hazelbauer et al., 2008). Homologs of all these proteins are present in the L. longbeachae genomes (Cazalet et al., 2010; Kozak et al., 2010) and a similar chemotaxis system is present in Legionella drancourtii LLAP12 (La Scola et al., 2004) but it is absent from L. pneumophila. The flanking genomic regions are highly conserved among L. longbeachae and all L. pneumophila strains sequenced, suggesting that L. pneumophila, although it encodes flagella has lost the chemotaxis system encoding genes by deletion events.

Thus these two species differ markedly in their surface structures. L. longbeachae encodes a capsule-like structure, synthesizes a very different LPS, does not synthesize flagella but encodes a chemotaxis system. These differences in surface structures seem to be due to deletion events leading to the loss of flagella in L. longbeachae and the loss of chemotaxis in L. pneumophila leading in part to the adaptation to their different main niches, soil, and water.

Evolution of Eukaryotic Effectors – Acquisition by Horizontal Gene Transfer from Eukaryotes?

Human to human transmission of Legionella has never been reported. Thus humans have been inconsequential in the evolution of these bacteria. However, Legionella have co-evolved with fresh-water protozoa allowing the adaptation to eukaryotic cells. The idea that protozoa are training grounds for intracellular pathogens was born with the finding by Rowbotham (1980) that Legionella has the ability to multiply intracellularly. This lead to a new percept in microbiology: bacteria parasitize protozoa and can utilize the same process to infect humans. Indeed, the long co-evolution of Legionella with protozoa is reflected in its genome by the presence of eukaryotic like genes, many of which are clearly virulence factors used by L. pneumophila to subvert host functions. These genes may have been acquired either through horizontal gene transfer (HGT) from the host cells (e.g., aquatic protozoa) or from bacteria or may have evolved by convergent evolution. Recently it has been reported that L. drancourtii a relative of L. pneumophila has acquired a sterol reductase gene from the Acanthamoeba polyphaga Mimivirus genome, a virus that grows in ameba (Moliner et al., 2009). Thus, the acquisition of some of the eukaryotic like genes of L. pneumophila by HGT from protozoa is plausible. ralF was the first gene suggested to have been acquired by L. pneumophila from eukaryotes by HGT, as RalF carries a eukaryotic Sec 7 domain (Nagai et al., 2002). In order to study the evolutionary origin of eukaryotic L. pneumophila genes, we have undertaken a phylogenetic analysis of the eukaryote-like sphingosine-1-phosphate lyase of L. pneumophila that is encoded by lpp2128 described earlier. The phylogenetic analyses shown in Figure 4 revealed that it was most likely acquired from a eukaryotic organism early during Legionella evolution (Degtyar et al., 2009; Nora et al., 2009) as the Lpp2128 protein sequence of L. pneumophila clearly falls into the eukaryotic clade of SPL sequences.

Figure 4.

Phylogenetic tree of a multiple sequence comparison of sphingosine-phosphate lyase proteins present in eukaryotic and prokaryotic genomes. Phylogenetic reconstruction was done with MEGA using the Neighbor-Joining method. Numbers indicate bootstrap values after 1000 bootstrap replicates. The red lines indicate the L. pneumophila sequences that are embedded in the eukaryotic clade. The bar at the bottom represents the estimated evolutionary distance.

We then tested the hypothesis that L. longbeachae might have acquired genes also from plants, which is conceivable as it is found in soil. We thus undertook here a phylogenetic analysis similar to that described above for the L. longbeachae protein Llo2643 that contains PPR repeats, a protein family typically present in plants. A Blast search in the database revealed that homologs of Llo2643 are only found in eukaryotes, in particular in plants and algae. The only prokaryotes encoding this protein are the cyanobacteria Microcoelus vaginatus and Cylindrospermopsis rasiborskii. This rare presence in bacteria is suggestive of a horizontal transfer event from eukaryotes to these bacteria. Figure 5 shows the phylogenetic tree we obtained. The fact that the bacterial proteins group together may also be due to a phenomenon of long branch attraction. Thus, the Llo2643 protein of L. longbeachae appears closer to plant proteins than prokaryotic ones. Once more plant proteins, perhaps from algae, will be in the database, it might become possible to evaluate whether L. longbeachae indeed acquired genes from plants.

Figure 5.

Phylogenetic tree of the protein Llo2643 and their homologs after blastp search. The tree was constructed by the Neighbor-joining method using the program MEGA. The red lines indicate the L. longbeachae sequences that are close to sequences derived from plant genomes. Numbers indicate bootstrap support for nodes from 1000 NJ bootstrap replicates. The bar at the bottom represents the estimated evolutionary distance.

Legionella is not the only prokaryote whose genome shows an enrichment of proteins with eukaryotic domains. Another example is the genome of “Ca. Amoebophilus asiaticus” a Gram-negative, obligate intracellular ameba symbiont belonging to the Bacteroidetes, which has been discovered within an ameba isolated from lake sediment (Schmitz-Esser et al., 2008) has been reported (Schmitz-Esser et al., 2010). In a recent report Schmitz-Esser et al. (2010) show that the genome of this organism also encodes an arsenal of proteins with eukaryotic domains. To further investigate the distribution of these protein domains in other bacteria the authors have undertaken an enrichment analysis comparing the fraction of all functional protein domains among 514 bacterial proteomes (Schmitz-Esser et al., 2010). This showed that the genomes of bacteria for which the replication in ameba has been demonstrated were enriched in protein domains that are predominantly found in eukaryotic proteins. Interestingly, the domains potentially involved in host cell interaction described above, such as ANK repeats, LRR, SEL1 repeats, and F- and U-box domains, are among the most highly enriched domains in proteomes of ameba-associated bacteria. Bacteria that can exploit amebae as hosts thus share a set of eukaryotic domains important for host cell interaction despite their different lifestyles and their large phylogenetic diversity. This suggests that bacteria thriving within ameba use similar mechanisms for host cell interaction to facilitate survival in the host cell. Due to the phylogenetic diversity of these bacteria, it is most likely that these traits were acquired independently during evolutionary early interaction with ancient protozoa.

Conclusion

Legionella pneumophila and L. longbeachae are two human pathogens that are able to modulate, manipulate, and subvert many eukaryotic host cell functions to their advantage, in order to enter, replicate, and evade protozoa or human alveolar macrophages during disease. In the last years genome analyses, as well as comparative and functional genomics have demonstrated that genome plasticity plays a major role in differences in host cell exploitation and niche adaptation of Legionella. The genomes of these environmental pathogens are shaped by HGT between eukaryotes and prokaryotes, allowing them to mimic host cell functions and to exploit host cell pathways. Genome plasticity and HGT lead in each strain and species to a different repertoire of secreted effectors that may allow subtle adaptations to, e.g., different protozoan hosts. Plasmids can be exchanged among strains and phages and deletions of surface structures like flagella or chemotaxis systems has taken place. Thus genome plasticity is major mechanism by which Legionella may adapt to different niches and hosts.

Access to genomic data has revealed many potential virulence factors of L. pneumophila and L. longbeachae as well as metabolic capacities of these bacteria. The increasing information in the genomic database will allow a better identification of the origin and similarity of eukaryotic like proteins or eukaryotic protein domains and other virulence factors. New eukaryotic genomes like that of the natural host of Legionella, A. castellanii are in progress. These additional data will allow studying possible transfer events of genes from the eukaryotic host to Legionella more in depth. Taken together, the progressive increase of information on Legionella as well as on protozoa will allow more complete comparative and phylogenetic studies to shed light on the evolution of virulence in Legionella. However, much work remains to be done to translate the basic findings from genomics research into improved understanding of the biology of this organism. As data are accumulating, new fields of investigation will emerge. Without doubt the investigation and characterization of regulatory ncRNAs will be one such field. Manipulation of host-epigenetic information and investigating host susceptibility to disease will be another. In particular development of high throughput techniques for comparative and functional genomics as well as more and more powerful imaging techniques will accelerate the pace of knowledge acquisition.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.