Evolution and diversification of secreted protein effectors in the order Legionellales

Abstract: The evolution of a large, diverse group of intracellular bacteria was previously very difficult to study. Recent advancements in both metagenomic methods and bioinformatics has made it possible. This thesis investigates the evolution of the order Legionellales. The study concentrates on a group of proteins essential for pathogenesis and host manipulation in the order, called effector proteins. The role of effectors in host adaptation, evolutionary history and the diversification of the order were investigated using a multitude of bioinformatics methods. First, the abundance and distribution of the known effector proteins in the orderwas found to cover newly discovered clades. There was a clear distinction between the proteins present in Legionellales and the outgoup, indicating the important role of the effectors in the order. Further, the effectors with known functions found in the new clades, particularly in Berkiella, revealed potential modes of host manipulation of this group. Secondly, the evolution of the effector gene content in the order shed light on theevolution of the order, as well as on the potential evolutionary differences between Legionellaceae and Coxiellaceae. In general, most of the effectors were gained early in the last common ancestor of Legionellales and Legionellaceae, as further indication of their role in the diversification of the order. New effector genes were acquired in the Legionellaceae even up to recent speciation events, whereas Coxiellacea have lost more protein coding genes with time. These differences may be due to horizontal gene transfer in the case of gene gains in Legionellaceae and loss of selection in the case of gene losses in Coxiellaceae. Third, the early evolution of core gained effector proteins for the order was studied.Two of the eight investigated core effectors seem to have a connection to eukaryotes, the rest to other bacteria, indicating both inter-domain and within bacteria horizontal gene transfer. In particular, one effector protein with eukaryotic motif gained at the last common ancestor of Legionellales, was found in all the clades and is therefore an important evolutionary link that may have allowed Legionellales to utilize eukaryotic hosts.