Rapid adaptations of Legionella pneumophila to the human host

Abstract

Legionella pneumophila are host-adapted bacteria that infect and reproduce primarily in amoeboid protists. Using similar infection mechanisms, they infect human macrophages, and cause Legionnaires' disease, an atypical pneumonia, and the milder Pontiac fever. We hypothesized that, despite the similarities in infection mechanisms, the hosts are different enough that there exist high-selective value mutations that would dramatically increase the fitness of Legionella inside the human host. By comparing a large number of isolates from independent infections, we identified two genes, mutated in three unrelated patients, despite the short duration of the incubation period (2-14 days). One is a gene coding for an outer membrane protein (OMP) belonging to the OmpP1/FadL family. The other is a gene coding for an EAL-domain-containing protein involved in cyclic-di-GMP regulation, which in turn modulates flagellar activity. The clinical strain, carrying the mutated EAL-domain-containing homologue, grows faster in macrophages than the wild-type strain, and thus appears to be better adapted to the human host. As human-to-human transmission is very rare, fixation of these mutations into the population and spread into the environment is unlikely. Therefore, parallel evolution - here mutations in the same genes observed in independent human infections - could point to adaptations to the accidental human host. These results suggest that despite the ability of L. pneumophila to infect, replicate in and exit from macrophages, its human-specific adaptations are unlikely to be fixed in the population.

Keywords: Legionella pneumophila; Legionnaires’ disease; comparative genomics; host-specific adaptations; molecular evolution.

Fig. 1.

Unrooted maximum-likelihood phylogenetic tree of L. pneumophila . All samples in this study, as well as a few reference ones (strains Paris, Philadelphia, Lorraine, Lens, Corby and Alcoy) are included. Strains from the Murcia outbreak are collapsed for readability and shown in an inset. Environmental isolates are shown in blue, and clinical isolates in orange. Comparisons from single cases are shown with brackets. Samples containing mutations in the EAL-containing protein and samples containing mutations in the OmpP1/FadL protein are indicated by arrows. The tree is based on a Legionella -specific cgMLST scheme, resulting in an alignment of 2 253 410 nt positions. Circles on branches represent the percentage of bootstrap trees supporting the node. Bootstrap support values <60 are not shown. Bar, average number of substitutions per site.

Fig. 2.

Number of mutations per gene in this study compared to 1000 simulations based on random sampling. The left panel is a subset of the right panel. The numbers of genes mutated once or more in comparisons between an environmental and clinical isolate observed in this study are displayed with green diamonds (103 genes mutated once, seven mutated twice, two mutated three times). The distribution of the number of genes mutated the same number of times in 1000 random samplings of 123 SNPs among an estimated 2445 non-essential genes of L. pneumophila strain Paris is displayed with violin plots. Individual data points are overlaid as a cloud of points.

Fig. 3.

Intracellular replication of L. pneumophila in A. castellanii (Acas) and human macrophage-derived U937 cells at an m.o.i. of 0.1. DA38626 (clinical isolate, pale blue) carries the mutated OmpP1/FadL homologe (lpg0707), while DA38627 (environmental, dark blue) has the wild-type gene. DA48501 (clinical isolate, pale green) carries a mutated EAL-containing protein (lpg0891), while DA48502 (environmental, dark green) has the wild-type gene. Two controls are shown: L. pneumophila strain Paris wild-type (Lp. Paris, orange) and a DotA mutant of the same strain, deficient for intracellular growth (ΔDotA, red). (a) Growth, as measured by the c.f.u. count ratio relative to T₀, over time (x-axis, in days). Each curve shows the average of three replicates. Error bars show standard deviation. (b) Ratios of c.f.u. counts after 2 days (T_48h) compared to c.f.u. counts at T₀. Each dot represents a replicate, and box-and-whiskers plots summarize the data. P-values of two-sample t-tests are shown for the comparison between the mutant and wild-type alleles, for both the OmpP1/FadL gene and the EAL-containing protein. Top panels show growth in A. castellanii (Acas), while bottom panels show growth in human macrophage-like U937 cells.

Fig. 4.

Predicted structure of Pseudomonas aeruginosa FadL homologue (PDB: 3DWO), with the corresponding location of the variants highlighted in blue (left: strain BrisbaneLP47, G26*, aligned to S28 in 3DWO; middle: strain PHHL01023034, W245*, aligned to L300 in 3DWO; right: DA38626, G311D, aligned to T362 in 3DWO). Red indicates the part of the protein that would not be translated after the introduced stop codons.

Fig. 5.

Predicted structure of Pseudomonas aeruginosa MorA homologue (PDB: 4RNH), with variants highlighted in blue. Top: G662D mutation in strain PHHL01023035, aligned to G1315. Bottom: I711T in strain DA48501, aligned to I1365. The G157R mutation in strain PHH072360604 does not align to the crystallized part of MorA and is thus not shown here.