The role of the VirB/VirD4/Bep system in "Bartonella henselae"-triggered vascular proliferation

Bartonella henselae (Bh) rarely shows any symptoms in its feline reservoir host but is responsible for a number of clinical outcomes during infection of the human incidental host. Bh shares with B. quintana (Bq) and B. bacilliformis (Bb) the remarkable capacity to induce tumour-like vascular proliferations. The study of Bartonella-host cell interactions might not only provide insides into the pathogenesis of Bh but might also serve as a model to investigate general mechanisms involved pathological angiogenesis, which is a hallmark of tumour growth. The Bh VirB/VirD4 type IV secretion system (T4SS) and the thereby translocated Bartonella-effector proteins (Beps) have been shown to be responsible for the subversion of a number of endothelial cell (EC) functions upon infection. This thesis aimed to elucidate the role of the VirB/VirD4/Bep system in Bh-triggered angioproliferation. The first chapter characterizes the Bh VirB T4SS translocated protein BepA as an effector mediating the inhibition of apoptosis in human umbilical vein endothelial cells (HUVECs). Either overexpressed in the effector-less ∆bepA-G mutant or ectopically expressed, BepA was sufficient to promote the anti-apoptotic activity. Delineation of BepA revealed that this activity is confined to the BID (Bep intracellular delivery) domain of BepA. Interestingly, only the homologue BepA2 (corresponding to the BID domain plus C-terminus of BepA) from Bq inhibited apoptosis as well. In contrast to Bh and Bq, B. tribocorum is not associated to vascular proliferation and its BepA-homologue did not show any anti-apoptotic activity. Upon translocation into the host cell BepA was targeted to the membrane. BepA-mediated anti-apoptosis correlated with an increase in cAMP and increased expression of cAMP-responsive genes, pointing to a mechanism involving the regulation of this second messenger. BepA not only inhibited chemically ActD-induced but also CTL-triggered apoptosis. Thus, BepA has the potential to play a role in vasoproliferation in an indirect way by enhancing cell survival. Chapter 2 presents a spheroid-based three-dimensional in vitro sprouting assay established to address the angiogenic potential of Bh. Compared to spheroids from uninfected HUVECs, spheroids from HUVECs pre-infected with Bh wild-type showed an increased sprouting activity, albeit sprout morphology was distinct from the sprouts induced by vascular endothelial growth factor (VEGF). Formation of those sprouts was in part VirB/Bep-dependent. Overexpressed BepA in the ∆bepA-G mutant strongly promoted sprout formation. Delineation of the domain required for this stimulation indicates parallels to the anti-apoptotic activity. BepD showed a moderate sprout-promoting effect. In contrary, BepG, involved in cytoskeletal rearrangement, displayed a potent interference with sprout formation. This novel in vitro model of Bh-triggered sprouting angiogenesis revealed distinct activities of the Beps in modulating sprout formation and contributing to the regulation of the Bh angiogenic activity in the course of the chronic vascular infection. In chapter 3 the effect of exogenous VEGF on Bh-infected ECs is addressed. VEGF is thought to be involved in Bh-induced vascular tumour formation by promoting EC proliferation in a paracrine manner. Assessing the biological activity of VEGF in assays such as proliferation, wound assay and capillary-like sprout formation revealed an intriguing interference of the VirB/VirD4/Bep system with responsiveness of infected ECs to stimulation with VEGF. Analysis of the VEGF receptor 2 pathways showed that Bh inhibited phosphorylation of tyrosine 1175. In Bh-infected ECs stimulated with VEGF, PLCγ1 was less recruited and phosphorylated and consequently downstream calcium flux and ERK1/2 activation were blocked. These data rather challenge the idea of a VEGF-driven paracrine loop in the presence of an active VirB/VirD4/Bep system and emphasize the modulatory role of the VirB T4SS balancing the angiogenic activity of Bh. The last chapter reports the role of the VirB/VirD4/Bep system in the paracrine-loop model of VEGF production. HeLa cells produced VEGF upon exposure to Bh in a VirB/Bep-dependent manner. BepD elicited a strong stimulation of VEGF secretion in HeLa cells when overexpressed in the ∆bepB-G mutant. A shift in colour of the cell culture supernatant, depending on the cell culture media used, was associated to VirB T4SS translocation activity but not particularly to VEGF production. The shift probably is due to acidification of the supernatant. Hence, in addition to the previously reported BadA also the VirB/VirD4/Bep system, especially BepD, independently is able to trigger the production of an essential regulator of angiogenesis.