Analysis of the Legionella longbeachae genome and transcriptome uncovers unique strategies to cause Legionnaires' disease

Abstract

Legionella pneumophila and L. longbeachae are two species of a large genus of bacteria that are ubiquitous in nature. L. pneumophila is mainly found in natural and artificial water circuits while L. longbeachae is mainly present in soil. Under the appropriate conditions both species are human pathogens, capable of causing a severe form of pneumonia termed Legionnaires' disease. Here we report the sequencing and analysis of four L. longbeachae genomes, one complete genome sequence of L. longbeachae strain NSW150 serogroup (Sg) 1, and three draft genome sequences another belonging to Sg1 and two to Sg2. The genome organization and gene content of the four L. longbeachae genomes are highly conserved, indicating strong pressure for niche adaptation. Analysis and comparison of L. longbeachae strain NSW150 with L. pneumophila revealed common but also unexpected features specific to this pathogen. The interaction with host cells shows distinct features from L. pneumophila, as L. longbeachae possesses a unique repertoire of putative Dot/Icm type IV secretion system substrates, eukaryotic-like and eukaryotic domain proteins, and encodes additional secretion systems. However, analysis of the ability of a dotA mutant of L. longbeachae NSW150 to replicate in the Acanthamoeba castellanii and in a mouse lung infection model showed that the Dot/Icm type IV secretion system is also essential for the virulence of L. longbeachae. In contrast to L. pneumophila, L. longbeachae does not encode flagella, thereby providing a possible explanation for differences in mouse susceptibility to infection between the two pathogens. Furthermore, transcriptome analysis revealed that L. longbeachae has a less pronounced biphasic life cycle as compared to L. pneumophila, and genome analysis and electron microscopy suggested that L. longbeachae is encapsulated. These species-specific differences may account for the different environmental niches and disease epidemiology of these two Legionella species.

Figure 1. Whole-genome synteny map of L.longbeachae strain NSW150 and L. pneumophila strain Paris.

The linearized chromosomes were aligned and visualized by Lineplot in MAGE. Syntenic relationships comprising at least 8 genes are indicated by green and red lines for genes found on the same strand or on opposite strands, respectively. IS elements (pink), ribosomal operons (blue) and tRNAs (green) are also indicated.

Figure 2. Intracellular growth of the wild-type and the dotA mutant strain in mouse and amoeba infection.

(A) Intracellular replication of L. longbeachae in Acanthamoeba castellanii. Blue, wild-type L. longbeachae strain NSW150; Red, dotA::Km mutant. Results are expressed as log₁₀ CFU. Each time point (in hours, x-axis) represents the mean ± SD of two independent experiments. Infections were performed at 37°C. (B) CI values from mixed infections of A/J mice. Mice were inoculated with approximately 10⁶ CFU of each strain under investigation and were sacrificed at 24 h or 72 h after infection to examine the bacterial content of their lungs. Competition experiment between L. longbeachae and the dotA::Km mutant representative of 2 independent experiments. (C) Single infections of A/J mice with L. longbeachae wt and the dotA::Km mutant strain. Results are expressed as log₁₀ CFU. Note: to maintain numbers in the lung L. longbeachae must be replicating Non-replicating bacteria are cleared in this infection model over 72 h (eg. dotA mutant) .

Figure 3. Putative capsule and LPS encoding loci in the genome of L. longbeachae.

(A) 48 kb chromosomal region highly conserved in the four L. longbeachae genomes sequenced putatively encoding the capsular biosynthesis genes. (B) 24 kb chromosomal region differing between Sg1 and Sg2 isolates putatively encoding the lippolysaccaride biosynthesis genes of L. longebachae. Colors indicate different classes of genes: magenta, synthesis pathway of nucleoside sugar precursors; blue, glycosyltranferase; yellow transportation; grey, genes of unknown.

Figure 4. Electron microscopy showing the presence of capsule like structures.

Transmission electron micrographs of L. longbeache cells cultured in BYE broth to post exponential growth phase (OD600 3.8). Black arrows, puative capsule structures, red Arrow, putative pili.

Figure 5. Alignment of the chromosomal regions of L. pneumophila and L. longbeachae coding the flagella biosynthesis genes.

The comparison shows that all except the regulatory genes are missing in L. longbeachae. Red, conserved regulator encoding genes, grey arrows orthologous genes among the genomes, white arrows, non orthologues genes.