Comparative Genomics Reveal That Host-Innate Immune Responses Influence the Clinical Prevalence of Legionella pneumophila Serogroups

Abstract

Legionella pneumophila is the primary etiologic agent of legionellosis, a potentially fatal respiratory illness. Amongst the sixteen described L. pneumophila serogroups, a majority of the clinical infections diagnosed using standard methods are serogroup 1 (Sg1). This high clinical prevalence of Sg1 is hypothesized to be linked to environmental specific advantages and/or to increased virulence of strains belonging to Sg1. The genetic determinants for this prevalence remain unknown primarily due to the limited genomic information available for non-Sg1 clinical strains. Through a systematic attempt to culture Legionella from patient respiratory samples, we have previously reported that 34% of all culture confirmed legionellosis cases in Ontario (n = 351) are caused by non-Sg1 Legionella. Phylogenetic analysis combining multiple-locus variable number tandem repeat analysis and sequence based typing profiles of all non-Sg1 identified that L. pneumophila clinical strains (n = 73) belonging to the two most prevalent molecular types were Sg6. We conducted whole genome sequencing of two strains representative of these sequence types and one distant neighbour. Comparative genomics of the three L. pneumophila Sg6 genomes reported here with published L. pneumophila serogroup 1 genomes identified genetic differences in the O-antigen biosynthetic cluster. Comparative optical mapping analysis between Sg6 and Sg1 further corroborated this finding. We confirmed an altered O-antigen profile of Sg6, and tested its possible effects on growth and replication in in vitro biological models and experimental murine infections. Our data indicates that while clinical Sg1 might not be better suited than Sg6 in colonizing environmental niches, increased bloodstream dissemination through resistance to the alternative pathway of complement mediated killing in the human host may explain its higher prevalence.

Figure 1. Hybrid SBT-MLVA typing of a population based clinical repository of L. pneumophila.

Phylogenetic clusters were formed by UPGMA analysis of the combined typing data. Cluster 1 is identified in blue, while cluster 2 and 3 are shown in green and red, respectively.

Figure 2. Optical Maps and genome based comparisons of L. pneumophila strains.

(A) Optical map of L. pneumophilla Sg6 str. Thunder Bay (Middle) compared to Sg1 strains Philadelphia (Top) and Sg12 570-CO-H (Bottom). The regions in white indicate unique gene clusters, while areas in blue depict high similarity to Sg6. Regions in red are conserved between all three genome sequences. (B) UPGMA based cluster analysis of optical maps of sequenced L. pneumophila.

Figure 3. Intracellular growth of L. pneumophila.

(A) Biofilm production by Sg1 and Sg6 (crystal violet staining, OD_{600 nm}). (B) Binding and invasion of Sg6 str. Thunder Bay compared to Sg1 str. Philadelphia to/within human NCI-H292 lung epithelial cells. (C) Intracellular replication of L. pneumophilla in A. castellani. The magnitude of replication is reported in log10 CFU/ml. (D) Intracellular growth of Sg6 str. Thunder Bay (square) compared to Sg1 str. Philadelphia (diamond) within U937 derived Human Macrophage cells. Each data point is an average of three independent experiments. For each experiment data was collected from 3 wells and a mean value was determined. *denotes statistical significance as determined by a two-tailed student’s t-test with a P-value <0.05.

Figure 4. Pangenome comparison of L. pneumophila.

(A) BLAST map of L. pneumophila Sg6 str. Thunder Bay against several Sg1 strains. (B) Number of conserved (>65% identity) and divergent (<65% identity) proteins between Sg6 and a subset of core 5proteins of Sg1. Numbers showed in italics represent number of proteins that were considered homologous. (C) Conserved (>65% identity) and divergent (<65% identity) proteins among the unique Sg6 genes from panel B and all known Sg1 genes.

Figure 5. Optical Maps and genome based comparisons of L. pneumophila strains.

(A) Optical map of L. pneumophilla Sg6 str. Thunder Bay (Middle) compared to Sg1 strains Philadelphia (Top) and Sg12 570-CO-H (Bottom). The regions in white indicate unique gene clusters, while areas in blue depict high similarity to Sg6. Regions in red are conserved between all three genome sequences. (B) UPGMA based cluster analysis of optical maps of sequenced L. pneumophila. Percent difference at each node is indicated.

Figure 6. The O-antigen locus of L. pneumophila.

(A) Layout of O-antigen region in Sg1 str. Philadelphia and Sg6 str. Thunder Bay in the area of highest dissimilarity, corresponding to the LPS gene cluster. (B) 16% SDS gel analysis of purified LPS from Sg1 str. Philadelphia and Sg6 str. Thunder Bay. (C) Comparative serum resistance of (Sg1) Sg1 str. Philadelphia and (Sg6) Sg6 str. Thunder Bay in Non-immune Human Serum. Presented data are an average of three independent experiments. *denotes statistical significance as determined by a two-tailed student’s t-test with a P-value <0.05.

Figure 7. Serum Resistance of non-Sg1 L. pneumophila.

(A) Percentage survival of Sg1 str. Philadelphia vs Sg6 str. Thunder Bay in the presence of no Serum (NS), serum with Sg1 antibodies (S1), S1 heated for 30 min at 56°C (S1ΔH), serum with Sg6 antibodies (S6) and S6 heated for 30 min at 56°C (S6ΔH). (B) Percentage survival of L. pneumophila serogroups after incubation in 90% non-immune human serum for 1 hour at 37°C. Both panels A and B are an average of three independent experiments. (C) Relative prevalence of non-SG1 in the clinical isolates database maintained at Public Health Ontario Laboratories. (D) Immunodetection of complement proteins C₃ and (E) C₉ at the surface of NHS treated Sg1 str. Philadelphia vs Sg6 str. Thunder Bay. Panels on the left are fluorescent captures of the differential interference contrast images on the right. The bacteria are seen as individual rods in these images. Images were acquired using Quorum Optigrid microscope with a 63× oil immersion objective (Leica DMI6000B stand with a Hamamatsu EM-1K, EMCCD camera). Image acquisition and post-acquisition processing was performed using Volocity 4.3 software (Improvision).

Figure 8. Sg1 and Sg6 infections in A/J mice.

CFU counts of Sg1 and Sg6 in different organs and blood 48 h post-intratracheal inoculation. Both Sg1 and Sg6 groups consisted of 5 mice each.