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The Genomic Sequence of the Accidental Pathogen Legionella pneumophila

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Science  24 Sep 2004:
Vol. 305, Issue 5692, pp. 1966-1968
DOI: 10.1126/science.1099776

Abstract

We present the genomic sequence of Legionella pneumophila, the bacterial agent of Legionnaires' disease, a potentially fatal pneumonia acquired from aerosolized contaminated fresh water. The genome includes a 45–kilobase pair element that can exist in chromosomal and episomal forms, selective expansions of important gene families, genes for unexpected metabolic pathways, and previously unknown candidate virulence determinants. We highlight the genes that may account for Legionella's ability to survive in protozoa, mammalian macrophages, and inhospitable environmental niches and that may define new therapeutic targets.

Legionella pneumophila was recognized as a human opportunistic pathogen after its isolation from patients in an outbreak of fatal pneumonia (Legionnaires' disease) at an American Legion convention in Philadelphia (1). L. pneumophila and other members of the genus are found within biofilms and fresh and industrial water systems worldwide, posing a significant public health concern. Inhaled Legionella spp. cause sporadic and epidemic cases of Legionnaires' disease and the flu-like Pontiac fever (2).

L. pneumophila are aerobic Gramnegative, motile, rod-shaped bacteria of the γ-proteobacterial lineage. These intracellular pathogens use the Icm/Dot type IVB secretion system (3, 4) to deliver effector proteins to the host cells (57) that modulate the fate of the phagocytic vacuole by preventing phagosome-lysosome fusion and vacuole acidification, and recruiting vesicles that confer on it properties of the endoplasmic reticulum (8). After proliferating within this compartment, the bacteria destroy the host cell and infect other cells.

The most intriguing aspects of L. pneumophila biology are its extraordinary ability to commandeer the organelle trafficking systems of a wide range of host cells and its ability to survive in harsh environments such as plumbing systems treated with potent biocides. Legionella species are denizens of soil and water with life-styles ranging from highly virulent (L. pneumophila) to non-pathogenic (protozoan symbionts) (2). Genomic comparisons permit the development of testable models for the molecular bases of these properties of Legionella spp. and their ability to adapt to different niches.

The genome of L. pneumophila subsp. pneumophila (serogroup 1), Philadelphia 1 strain, derived from the original 1976 isolate, consists of a single circular chromosome of 3,397,754 base pairs (bp) (GenBank accession code AE017354) with 38% G+C content (Fig. 1 and table S1) (9, 10).

Fig. 1.

Circular map of L. pneumophila genome. Circles from outside in: gene distribution on the forward (1) and reverse (2) strands (fig. S6), color-coded according to function (fig. S2). (3) tRNA (black) and rRNA (red) genes and the chromosomal plasmid-like pLP45 region (green). (4) G+C content, lower (blue) and higher (red) than genome average. (5) GC-skew. oriC was positioned in the rpmH-dnaA intergenic region (figs. S3 and S4). Other regions of interest are shown in fig. S5.

As expected for an intracellular pathogen, examination of its genome indicates that L. pneumophila has undergone horizontal gene transfer. Several homologs of eukaryotic genes were identified (11), including the previously described ralF gene, encoding a guanosine triphosphatase (GTPase) modifier required for recruiting the ADP ribosylation factor (ARF) GTPase to the Legionella-containing vacuole (8). Some phage-derived and insertion sequences, constituting ∼2.4% of the genome, are dispersed throughout the genome; others form nine clusters in addition to two loci containing the icm/dot genes encoding the type IVB secretion system (12, 13), the lvh/lvr cluster encoding a typical type IV secretion system (14), and a region containing F plasmid–derived tra/trb genes (15).

The Philadelphia 1 strain possesses a plasmid-like element of 45 kbp (pLP45) that exists in a circular episomal form or within the chromosome. The lvh/lvr gene region, one of the few extensive loci with an elevated G+C content (fig. S5), is found within this element, along with genes potentially involved in DNA recombination. We identified a 100-kb region (fig. S5) containing several genes encoding efflux transporters for heavy metals and other toxic substances. The presence of tRNA, phage-related genes, and transposase genes near the extremities of this region suggest that it was acquired via horizontal transfer. This “efflux island” may contribute to Legionella's ability to flourish in plumbing systems and persist in the presence of toxic biocides.

The fully sequenced species most closely related to L. pneumophila is the obligate intracellular pathogen Coxiella burnettii, also belonging to the order Legionellales. Its proteome has a high number of basic proteins, which may explain its ability to replicate within phagolysosomes (16). L. pneumophila replicates in a vacuole resembling the endoplasmic reticulum; its proteome has a lower average isoelectric point (9). Altogether, Legionella shares ∼42% of its genes with Coxiella despite differences in genome size (3.4 and 1.9 Mbp, respectively). To date the icm/dot genes have been found only in Legionella spp. and C. burnettii. Out of several identified L. pneumophila effectors delivered by the Icm/Dot system, only the recently described sid genes (6) have counterparts in C. burnetii. Very few other genes are shared exclusively by L. pneumophila and C. burnettii [the only named genes are the “enhanced entry” genes enhA and enhB (17)], implying that specific regulatory circuits or transport capabilities determine the features that distinguish them from other bacteria (table S2).

Of Legionella's gene complement, 60% of the genes have homologs among phylogenetically diverse intracellular bacteria (Coxiella, Salmonella, Chlamydia, Rickettsia, Brucella, and Mycobacterium species), comparable to the 63% found in seven fully sequenced related γ-proteobacteria, suggesting that the species' similar life-styles and common origin may equally affect gene complement similarity.

Paralogous gene family expansions may be associated with adaptation to specific environments, development of novel life strategies, or other species-specific adaptations. In a search for Legionella gene family expansions relative to other bacterial genomes (table S3), we identified several examples including β-lactamase, factor for inversion stimulation, and some transporters (9), even though overall Legionella does not display a high genome redundancy, as judged by its modest average gene family expansion rate. Among intracellular pathogens, Legionella groups with Brucella melitensis and Salmonella species (table S4), whereas reduced genome organisms (e.g., Rickettsia and Coxiella) exhibit low expansion values, and M. tuberculosis contains many expanded gene families including several related to lipid synthesis.

Legionella has multiple family members for genes encoding enhanced entry proteins, peptide methionine sulfoxide reductase, zinc metalloproteases, polyhydroxyalkanoic acid synthase, transposases, and effectors. The Sid effectors, identified on the basis of their ability to be transferred to another cell via the Icm/Dot type IVB system (6), form three families in L. pneumophila.

Legionella survives and replicates in axenic cultures, fresh water, soil, and in biofilms with other organisms, as well as within intracellular vacuoles of amoebae, ciliates, and human cells. To best utilize the nutrients within these diverse environments, the bacteria would need a broad range of membrane transporters. We identified more than 350 binding proteins and permeases in the Legionella genome, representing 62 separate substrate classes.

Among its many multidrug transporters, there is an expanded 11-member major facilitator family in Legionella. Nine similar proteins occur in C. burnetii, which has an unusually high overall density of multidrug transporters in its genome (18), but no clearcut representatives of this particular family exist in other intracellular pathogens or in the substantially larger genomes of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa. L. pneumophila has more members of not only this particular gene family, but of multidrug transporters as a general class. It has an unusually high number of genes encoding putative effluxers for toxic compounds and heavy metals relative to other γ-proteobacteria, perhaps because the natural protozoan hosts for Legionella accumulate heavy metals, including lead and cadmium, from the environment (19).

A family of four glutamate/γ-aminobutyrate–specific amino acid antiporters is apparently represented by two members in Coxiella and only one member in 10 other species examined (GadC in E. coli). This antiporter is involved in bacterial survival within acidic environments (20); its possible functional redundancy may account for the suggested ability of L. pneumophila to survive in a putative acidic compartment late in its life cycle (16).

Because L. pneumophila utilizes amino acids as carbon and energy sources and cannot ferment or oxidize carbohydrates (21), we were surprised to find an intact glycolytic chain, pyruvate dehydrogenase complex, tricarboxylic acid cycle and respiratory chain, and a glucose-6-phosphate transporter. Perhaps these pathways are used when bacteria are exposed to the complex molecular content of intracellular organelles of host cells or potentially nutrient-rich biofilms. Legionella is reportedly auxotrophic for several amino acids, so it was surprising to find potential genes for their synthetic pathways: cysteine from pyruvate or serine, methionine from cysteine, and both phenylalanine and tyrosine from phosphoenolpyruvate. Even if some of these genes are not expressed under laboratory growth conditions, their presence presumably relates to the organism's ability to persist in diverse environments.

In addition to previously recognized virulence factors (table S5), we identified new candidates (table S6), including homologs of genes encoding virulence functions in other bacteria. Moreover, ∼145 apparent secreted or membrane proteases and other hydrolases, some of which may function as virulence factors, exist in L. pneumophila. Among fully sequenced organisms, this is exceeded only by the predatory Bdellovibrio (22). L. pneumophila has been proposed to utilize bacterial-induced apoptotic (early) and/or necrotic pore-forming (late) events to exit infected hosts (23); its putative hydrolases may be involved in these processes.

The genome sequence of L. pneumophila offers the opportunity to explain its broad host range and extraordinary ability to resist eradication in water supplies. Having lists of genes unique to Legionella or shared with unrelated bacteria with similar life-styles, it should now be possible to determine experimentally which of them distinguish Legionella species displaying different host preferences or pathogenicity.

Supporting Online Material

www.sciencemag.org/cgi/content/full/305/5692/1966/DC1

Materials and Methods

SOM Text

Tables S1 to S6

Figs. S1 to S6

References and Notes

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