The aim of our studies is to gain a molecular understanding of the function of type IV secretion (T4S) systems in establishing persistent bacterial infection in the host. T4S systems are ancestrally related to bacterial conjugation systems that mediate interbacterial DNA transfer. Bacterial pathogens targeting eukaryotic host cells have adopted these supramolecular protein assemblies for the intracellular delivery of virulence factors from the bacterial cytoplasm directly into the host cell cytoplasm. Our longstanding research on the vascular tumor-inducing pathogens of the genus Bartonella revealed crucial roles of two distinct T4S systems, VirB and Trw, in the ability of these bacteria to colonize, invade and persist within vascular endothelial cells and erythrocytes, respectively (see Figure 1, reviewed in Dehio, 2008, Cell. Microbiol.; and Harms and Dehio, 2012, Clin. Microbiol. Rev.).

More recently, we have initiated a new project to study the role of the T4S system VirB in intracellular persistence of the closely related bacterial pathogens of the genus Brucella that represent the etiological agents of brucellosis - the worldwide most important bacterial zoonosis. We are using a multi-disciplinary research approach including genetics, genomics, biochemistry, structural biology, cell biology, animal experimentation and bioinformatics in order to reveal the cellular, molecular and evolutionary basis of T4S in bacterial persistence of Bartonella and Brucella. Moreover, we employ a systems biology approach to reveal the host cell signaling network underlying cell entry and intracellular persistence of these pathogens in order to define novel targets for the development of innovative anti-infectiva.

T4S systems play diverse roles in Bartonella-host interaction: They are essential for establishing persistent infection and contribute to host adaptability
A functional and comparative genomics approach allowed us to demonstrate that both the VirB and Trw T4S systems of Bartonella represent essential virulence factors for establishing chronic infection in mammals. Further, these virulence devices must have played major roles during evolution in facilitating adaptation of these pathogens to their specific mammalian reservoirs (Saenz et al., 2007, Nat. Genet.; Engel et al., 2011, PLoS Genetics). Genetic and cell biological analysis of Trw has shown that this T4S system mediates the host-restricted adhesion to erythrocytes (Vayssier et al., 2010). Important to note, during adoption of this dedicated role in host interaction this T4S system has lost its ancestral substrate transfer capability. In contrast, we have shown that the VirB T4S is capable of translocating DNA into endothelial target host cells in a process similar to the interbacterial DNA transfer mediated by the ancestral conjugation systems (Schroeder et al., 2011, PNAS; reviewed in Llosa et al., 2012, Trends Microbiol.). However, the physiological role of the VirB T4S system is to translocate a cocktail of Bartonella effector proteins (Beps) into vascular endothelial cells that subvert cellular functions to the benefit of the pathogen (Schulein et al., 2005, PNAS). An evolutionary genomics study allowed us to propose that the horizontally acquired VirB T4S system and its translocated Bep effectors facilitated adaptations to novel hosts via two parallel adaptive radiations (Engel et al., 2011, PLoS Genet.). We showed that the functional versatility and adaptive potential of the VirB T4S system evolved convergently – prior to the radiations – by consecutive rounds of lineage-specific gene duplication events followed by functional diversification. This resulted in two diverse arrays of Bep effector proteins in the two radiating lineages of Bartonella. Together, we established Bartonella as a bacterial paradigm of adaptive radiation, allowing for the first time to study the molecular and evolutionary basis of this fundamental evolutionary process for the generation of organismic diversity in bacteria.

Structure/function analysis of VirB-translocated Bep effector proteins of Bartonella
The cocktail of Bep effectors translocated by the VirB T4S system into vascular endothelial cells mediates multiple cellular effects, including anti-apoptosis, internalization of bacterial aggregates via the F-actin-dependent invasome structure and proinflammatory activation (Schulein et al., 2005, PNAS). Defining the cellular targets and molecular mechanisms of how these Beps interfere with eukaryotic signaling processes have become a focus of our recent studies. The C-terminal parts of the Beps carry the Bep intracellular delivery (BID) domain that serves as T4S signal, but has in several instances adopted additional effector function within host cells. A prominent example is the BID domain of BepA that binds adenylate cyclase to potentiate Gαs-dependent cAMP production, which leads to inhibition of apoptosis in vascular endothelial cells (Pulliainen et al., 2012, PNAS). The N-terminal parts of the Beps carry diverse domains or peptide motifs considered to mediate effector functions within host cells. For instance, upon translocation the effectors BepD, BepE and BepF become tyrosine-phosphorylated on short N-terminal repeat motifs, thereby interfering with eukaryotic signal transduction processes (Selbach et al., 2009, Cell Host & Microbe). Together with the Schirmer group (Biozentrum) we study the structure/function relationship of the Fic domains that are present in the N-terminus of multiple Beps and their ancestors and mediate post-translational modifications of specific host target proteins via covalent transfer of AMP (AMPylation) (Palanivelu et al., 2011, Protein Sci.; Pieles et al., 2014, Proteomics). A particular focus of these studies is the identification of target proteins (Harms et al., 2015, Cell Rep.) and the regulation of the AMPylation activity, i.e. via binding of the Fic domain to an inhibitory protein termed antitoxin (Engel et al., 2012, Nature; Stanger et al., 2016, PNAS). Structure analysis of the BID domain serving as T4S signal in all Beps revealed a novel fold characterized by four-helix bundle topped by a hook (Stanger et al., 2016, Structure). Importantly, beyond the broadly conserved role as T4S signal, individual BID domains have also evolved secondary effector functions within host cells, such as mediating anti-apoptosis (Pulliainen et al., 2012, PNAS) or by safeguarding cells from deleterious effects caused by other Beps (Okujava et al., 2014, PLoS Patho.).

A systems biology approach to Bartonella and Brucella entry and intracellular persistence in human cells
The goal of the former InfectX (2010-2013) and present TargetInfectX (2014-2017) research and development project (RTD) of the Swiss-wide systems biology initiative SystemsX.ch is to comprehensively identify components of the human infectome for a set of important bacterial and viral pathogens and to develop new mathematical and computational methods with predictive power to reconstruct key signaling pathways controlling pathogen entry into human cells. We are using a systems biology approach to reconstruct host signaling processes underlying Bartonella and Brucella entry into the human model cell line HeLa that lead to the establishment of a persisting intracellular infection. For Bartonella henselae, the VirB T4S effector BepG or the combined activity of the effectors BepC/BepF was found to inhibit endocytic uptake of individual bacteria, thereby redirecting bacterial uptake to the invasome-mediated pathway facilitating the uptake of large bacterial aggregates (reviewed in Eicher and Dehio, 2012, Cell. Microbiol.). This unique cell entry process is mediated by massive F-actin rearrangements that depend on the small GTPases Rac1, the Rac1-effector Scar1, and the F-actin organizing complex Arp2/3 (Rhomberg et al., 2009, Cell Microbiol.; Truttmann et al., 2011, Cell Microbiol.) and bi-directional signaling via the integrin pathway (Truttmann et al., 2011, J. Cell Sci.). The uptake process triggered by Brucella abortus is less well defined but considered to depend on lipid rafts and the small GTPase Cdc42. Genome-wide RNA interference screens and related modeling approaches currently performed on the basis of high-content fluorescence microscopy assays for pathogen entry and intracellular replication should facilitate the comprehensive identification of the human infectomes involved in establishing persistent intracellular infection of these pathogens as a first step towards the identification of human targets suitable for the development of a new class of anti-infectives that interfere with the function of host proteins essential for pathogen infection.