A Translocated Effector Required for <i>Bartonella</i> Dissemination from Derma to Blood Safeguards Migratory Host Cells from Damage by Co-translocated Effectors

Abstract

<div><p>Numerous bacterial pathogens secrete multiple effectors to modulate host cellular functions. These effectors may interfere with each other to efficiently control the infection process. <i>Bartonellae</i> are Gram-negative, facultative intracellular bacteria using a VirB type IV secretion system to translocate a cocktail of <i><u>B</u>artonella</i><u>e</u>ffector <u>p</u>roteins (Beps) into host cells. Based on <i>in vitro</i> infection models we demonstrate here that BepE protects infected migratory cells from injurious effects triggered by BepC and is required for <i>in vivo</i> dissemination of bacteria from the dermal site of inoculation to blood. Human endothelial cells (HUVECs) infected with a Δ<i>bepE</i> mutant of <i>B. henselae</i> (<i>Bhe</i>) displayed a cell fragmentation phenotype resulting from Bep-dependent disturbance of rear edge detachment during migration. A Δ<i>bepCE</i> mutant did not show cell fragmentation, indicating that BepC is critical for triggering this deleterious phenotype. Complementation of Δ<i>bepE</i> with BepE<i><sub>Bhe</sub></i> or its homologues from other <i>Bartonella</i> species abolished cell fragmentation. This cyto-protective activity is confined to the C-terminal <i><u>B</u>artonella</i><u>i</u>ntracellular <u>d</u>elivery (BID) domain of BepE<i><sub>Bhe</sub></i> (BID2.E<i><sub>Bhe</sub></i>). Ectopic expression of BID2.E<i><sub>Bhe</sub></i> impeded the disruption of actin stress fibers by Rho Inhibitor 1, indicating that BepE restores normal cell migration via the RhoA signaling pathway, a major regulator of rear edge retraction. An <i>intradermal</i> (<i>i.d.</i>) model for <i>B. tribocorum (Btr)</i> infection in the rat reservoir host mimicking the natural route of infection by blood sucking arthropods allowed demonstrating a vital role for BepE in bacterial dissemination from derma to blood. While the <i>Btr</i> mutant Δ<i>bepDE</i> was abacteremic following <i>i.d.</i> inoculation, complementation with BepE<i><sub>Btr</sub></i>, BepE<i><sub>Bhe</sub></i> or BIDs.E<i><sub>Bhe</sub></i> restored bacteremia. Given that we observed a similar protective effect of BepE<i><sub>Bhe</sub></i> on infected bone marrow-derived dendritic cells migrating through a monolayer of lymphatic endothelial cells we propose that infected dermal dendritic cells may be involved in disseminating <i>Bartonella</i> towards the blood stream in a BepE-dependent manner.</p></div>