Comparative analyses of Legionella species identifies genetic features of strains causing Legionnaires' disease

Abstract

Background: The genus Legionella comprises over 60 species. However, L. pneumophila and L. longbeachae alone cause over 95% of Legionnaires’ disease. To identify the genetic bases underlying the different capacities to cause disease we sequenced and compared the genomes of L. micdadei, L. hackeliae and L. fallonii (LLAP10), which are all rarely isolated from humans.

Results: We show that these Legionella species possess different virulence capacities in amoeba and macrophages, correlating with their occurrence in humans. Our comparative analysis of 11 Legionella genomes belonging to five species reveals highly heterogeneous genome content with over 60% representing species-specific genes; these comprise a complete prophage in L. micdadei, the first ever identified in a Legionella genome. Mobile elements are abundant in Legionella genomes; many encode type IV secretion systems for conjugative transfer, pointing to their importance for adaptation of the genus. The Dot/Icm secretion system is conserved, although the core set of substrates is small, as only 24 out of over 300 described Dot/Icm effector genes are present in all Legionella species. We also identified new eukaryotic motifs including thaumatin, synaptobrevin or clathrin/coatomer adaptine like domains.

Conclusions: Legionella genomes are highly dynamic due to a large mobilome mainly comprising type IV secretion systems, while a minority of core substrates is shared among the diverse species. Eukaryotic like proteins and motifs remain a hallmark of the genus Legionella. Key factors such as proteins involved in oxygen binding, iron storage, host membrane transport and certain Dot/Icm substrates are specific features of disease-related strains.

Figure 1

Intracellular replication of L. hackeliae , L. micdadei and L. fallonii (LLAP10). (A) THP-1 derived macrophages at 37°C. (B) A. castellanii culture at 20°C. (C) A. castellanii plate test at 37°C and 30°C. L. pneumophila strain Paris wild type (wt) and ∆dotA were used as positive and negative controls, respectively. Intracellular replication for each strain was determined by recording the number of colony-forming units (CFU) through plating on BCYE agar. Blue, L. pneumophila strain Paris; red, ∆dotA; orange, L. micdadei; violet, L. hackeliae; green, L. fallonii (LLAP10). Results are expressed as Log10 ratio CFU Tn/T0 and each point represents the mean ± standard deviation of two or three independent experiments. The error bars represent the standard deviation, but some were too small to clearly appear in the figure.

Figure 2

Shared and specific content of the different Legionella species/strains analyzed in this study. Each petal and color represents one genome. The number in the center of the diagram represents the orthologous genes shared by all the genomes. The number inside of each individual petal corresponds to the specific genes of each genome with non-orthologous genes in any of the other genomes. (A) Core genome of five Legionella species including seven L. pneumophila genomes. (B) Core genome when one representative of each Legionella species is taken into account.

Figure 3

Phylogenetic tree of six Legionella species and seven L. pneumophila strains and their shared Dot/Icm substrates. Neighbor-joining tree based on the concatenation of 816 protein-coding genes from 11 Legionella genomes. C. burnetii was used as out-group. The tree was constructed using MEGA and JTT as model of evolution. The values above nodes indicate the bootstrap values. The values in blue circles represent the number of Dot/Icm substrates shared by the species in the corresponding cluster, suggesting that they were present in the common ancestor. The values inside blues squares are the number of Dot/Icm substrates shared between L. pneumophila strains and the remaining species (for example, the species L. micdadei and L. pneumophila share 33 Dot/Icm substrates).

Figure 4

L. fallonii synthesizes cellulose. (A) Genomic organization and Blastx comparison of the regions encoding the cellulose synthesis machinery in E. coli, L. fallonii, L. dumofii and L. anisa. The gray color code represents the Blast matches; the darker the gray the better the blast match. (B) Growth of L. fallonii on calcofluor agar plates that shows cellulose synthesis as visualized under long-wave UV light. L. fallonii is fluorescent due to the binding of calcofluor to cellulose. In contrast L. pneumophila that was used as negative control is not.

Figure 5

The L. micdadei and L. fallonii genomes contain specific flagellar-encoding regions . Genomic organization and Blastx comparison of the specific flagellar gene clusters in L. micdadei and L. fallonii. The gray color code represents the Blast matches; the darker the gray the better the blast match. Pink arrows point to tRNA genes. Protein names and their predicted function in L. micdadei are indicated below.

Figure 6

Genome comparison of two L. micdadei strains . The complete genome sequences of the two L. micdadei strains included in this study were aligned using the software Mauve. The two strains align perfectly with the exception of three mobile genetic elements specifically present in strain L. micdadei ATCC33218 and one specifically present in the Victorian isolate. The specific regions of each genome are indicated. The 'Lvh region' is indicated, as this region is, with a high number of SNPs, quite divergent between the two isolates.

Figure 7

Phylogenetic analysis shows the eukaryotic origin of the carboxypeptidase S28 family protein (Llo0042/Lfa0022). The species belonging to bacteria and eukaryotes are shown in red and green, respectively. Numbers next to tree nodes correspond to bootstrap values. The bar at the bottom represents the estimated evolutionary distance.