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.