Evolution and host-specific adaptations of Legionella pneumophila

Sammanfattning: How bacteria evolve pathogenic traits is shaped by their communities and environments. Legionella pneumophila is ubiquitous in aquatic habitats, where it persists by replicating within a broad range of protozoan hosts. Using the same mechanisms, L. pneumophila may also accidentally infect humans, causing a severe pneumonia known as Legionnaires’ disease. As hosts, humans are evolutionary dead-ends, resulting in the loss of human-specific adaptations after infection. This thesis aims to identify and characterise these host adaptations.In Paper I, we study the in-patient evolution of L. pneumophila. We collected a large set of strains from sporadic infections and outbreaks, pairing clinical isolates with their respective environmental sources. Using comparative genomic analyses, we identified two genes individually mutated in three independent infections. One gene encoded an outer membrane protein, a homolog from the OmpP1/FadL family, and the other an EAL domain-containing protein. These results suggest that convergent evolution may be at play and that these mutations are potential candidates for human-specific host adaptations.In Paper II, we investigate host adaptation and the selective pressures that drive it using a long-term experimental evolution approach. We passaged L. pneumophila in Acanthamoeba castellanii and U937 macrophages, separately and in alternation, for over 800 generations. We found 49 fixed mutations across the 18 evolved populations: two distinct mutations in RpsL, which confers streptomycin resistance, as well as two additional mutations, each consistently associated with one of the former, in the chaperonin GroES or in RpsD, a known compensatory mutation. Mutations in the lipopolysaccharide synthesis operon were observed only in lineages passaged in A. castellanii, whilst mutations in LerC were fixed in six lineages passaged in U937, making these candidate mutations for host-specific adaptations.In Paper III, we shift focus to A. castellanii, a natural host of L. pneumophila. We describe a novel method for high-efficiency transfection of this amoeba with a cationic polymer. Using a systematic approach to test different parameters, we found that widely available and inexpensive polyethylenimines can be used to transfect A. castellanii at a much greater efficiency than the currently used reagents.In conclusion, these studies suggest that although L. pneumophila can infect humans, it is sub-optimally adapted for it, and offer potential determinants of host-specificity in L. pneumophila.