Legionella pneumophila pangenome reveals strain-specific virulence factors

Abstract

Background: Legionella pneumophila subsp. pneumophila is a gram-negative gamma-Proteobacterium and the causative agent of Legionnaires' disease, a form of epidemic pneumonia. It has a water-related life cycle. In industrialized cities L. pneumophila is commonly encountered in refrigeration towers and water pipes. Infection is always via infected aerosols to humans. Although many efforts have been made to eradicate Legionella from buildings, it still contaminates the water systems. The town of Alcoy (Valencian Region, Spain) has had recurrent outbreaks since 1999. The strain "Alcoy 2300/99" is a particularly persistent and recurrent strain that was isolated during one of the most significant outbreaks between the years 1999-2000.

Results: We have sequenced the genome of the particularly persistent L. pneumophila strain Alcoy 2300/99 and have compared it with four previously sequenced strains known as Philadelphia (USA), Lens (France), Paris (France) and Corby (England).Pangenome analysis facilitated the identification of strain-specific features, as well as some that are shared by two or more strains. We identified: (1) three islands related to anti-drug resistance systems; (2) a system for transport and secretion of heavy metals; (3) three systems related to DNA transfer; (4) two CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems, known to provide resistance against phage infections, one similar in the Lens and Alcoy strains, and another specific to the Paris strain; and (5) seven islands of phage-related proteins, five of which seem to be strain-specific and two shared.

Conclusions: The dispensable genome disclosed by the pangenomic analysis seems to be a reservoir of new traits that have mainly been acquired by horizontal gene transfer and could confer evolutionary advantages over strains lacking them.

Figure 1

Pangenome overview. The Venn diagram showing the core genome and the genes specific of the strains L. pneumophila Alcoy, Philadelphia, Lens, Paris and Corby. Genes overlapping at least 70% length and 80% of similarity were considered orthologs.

Figure 2

Pangenome state. Rarefaction curves applied to different strains of L. pneumophila (5 genomes), E. coli (8 genomes), Streptococcus pyogenes (8 genomes), S. agalactiae (8 genomes), and Staphilococcus aureus (9 genomes). See Additional file 6 for accession numbers of used genomes.

Figure 3

Functional analysis. Functional analysis. COGs distribution within the core and dispensable compartments of L. pneumophila pangenome.

Figure 4

Strain related orthology maps. Strain related orthology maps. Each map shows comparative analysis based on each genome. From outside to inside: ring 1, gene positions according with COG categories color code; ring 2, GC content; ring 3, GC Skew; ring 4, island positions (color code according with islands legend); rings 5 to 8 show the BLAST homology for each gene versus the other four genomes (color code according with genomes legend).

Figure 5

Islands distribution on L. pneumophila MLST tree topology from D'Auria et al. (2008) [9]. The left side (a) shows the islands appearance according to the tree topology obtained by the phylogenetic analysis. Each color is related to island type (see legend). On the right side (b) is reported the multiple genome alignment obtained by Mauve software applying default parameters [59]: homolog blocks are drawn with identical colors in forward and reverse strands; diagonal lines connect homologous blocks from each genome; colored blocks (according to island-type) on the light pink area between strands indicate the position of each island.