Structure and function of Bartonella effector protein 1: target and interdomain interactions

Bartonella are gram-negative facultative intracellular pathogens that follow a stealth infection strategy, to persist in erythrocytes and thereby be ingested by bloodsucking arthropods that serve as vectors. To achieve this persistent infection, most Bartonella translocate effector proteins via the VirB4/D4 type IV secretion system into host cells, to modulate responses during their infection cycle. The majority of Bartonella effector proteins (BEPs) is made up by a common domain arrangement, consisting of an essential C-terminal BID domain, carrying the type IV secretion signal, and an N-terminal FIC domain, that usually catalyzes post translational modifications of target proteins, most prominently the transfer of AMP, called AMPylation. While knowledge about the Fic protein family has grown rapidly in the last decade, only targets for a few of its members could be identified so far. In this study, I used structural and biophysical methods to characterize Bep1, that consists of the most abundant FIC and BID domain arrangement. First, I analysed Bep1 from B. rochalimae and studied its exquisite target selectivity towards the Rac subfamily of Rho GTPases. For this purpose, I set up a new method for the quantification of AMPylation reactions, called online Ion Exchange Chromatography assay, and developed a python pipeline for automated data processing. I used kinetic studies in combination with mutagenesis to narrow interactions down to two crucial salt-bridges between Bep1 and its targets. Second, I crystallized the full-length effector Bep1 from B. clarridgeiea and could identify a fold at the C-terminus, that was previously described as unstructured tail. I was able to combine the structural analysis with hot-spots of sequence conservation, and found interactions that are critical for the shape of Bartonella effectors containing a Fic domain. These results might hint at a mechanism for an unfolding process necessary for translocation of these effectors through the T4SS.