From FIC to BID : target identification and functional characterization of "Bartonella" effector proteins

Pathogens belonging to the genus Bartonella employ a unique stealth infection strategy that involves evasion from the host immune system, replication in the endothelium and persistence in erythrocytes. A key factor in colonization of the replicative niche is the manipulation of nucleated cells to the benefit of bacterial uptake, survival, proliferation or spreading. To this end, Bartonella spp. translocate a set of bacterial effectors via a VirB/VirD4 type IV secretion system (T4SS) into the host cell. Upon translocation, several Bartonella effector proteins (Beps) hijack host cell signaling cascades, thus, subverting host cellular functions to promote pathogenicity, yet their underlying mechanism remains largely elusive. Although pathogenicity factors evolved independently in radiating lineages of Bartonellae, Beps share a common domain architecture. The C-terminal part of all Beps consists of a Bartonella intracellular delivery domain (BID) and a positively charged tail region that primarily serve as a bi-partite secretion signal. Apart from translocation, some BID-domains acquired additional functions and interfere with host cell signaling resulting in cytoskeletal rearrangements during pathogen entry. The N-terminal part is less conserved and can harbor phospho-tyrosine motifs, additional BID-domains or share the ancestral domain architecture with a filamentation induced by cAMP (FIC) domain. This domain was recently shown to catalyze the transfer of an AMPmoiety onto target proteins, a process called AMPylation or adenylylation. Although the FICdomain is widely distributed and can be found in all kingdoms of life, the only identified targets are small GTPases of the Ras superfamily. In this study, we aimed to identify target proteins of different Beps and to gain insights into their molecular function. In Research Article I, we describe that BepA of B. henselae elevates intracellular cAMP-levels by activating eukaryotic adenylyl cyclase (AC) synergistically with the a-subunit of stimulating heterotrimeric G-protein (Gas). Further we could show that BepA is a conditional activator of AC and directly interacts with at least one of the catalytically active cytosolic AC domains. Furthermore, we established a mass spectrometry based strategy to identify targets of post translational modifications on the example of AMPylation that is presented in Research Article II. To this end, we used stable isotope-labeled ATP in in vitro AMPylation assays on crude cell lysates which results in the formation of reporter ion clusters in subsequent LC-MS analysis. Applying this strategy on an exemplary Fic protein, Bep2 of B. rochalimae, we identified vimentin as a target protein. As vimentin is not structurally related to small GTPases, we exhibit cytoskeletal components as a new target class of Fic protein-mediated AMPylation. Taken together, Bartonella effector proteins target a plethora of host cell proteins and are thereby manipulating key elements of host cell signaling. Therefore, they developed a high level of versatility in their target proteins and molecular mechanisms ranging from complex formation to posttranslational modifications. We hypothesize that both of these attributes play fundamental roles in the establishment of chronic infections. Furthermore, the understanding of these basic functionalities will be useful in the development of cell biology tools or of innovative therapeutics