The Gram-negative bacterium Bartonella henselae (Bh) is an emerging zoonotic pathogen that has been associated with a variety of human diseases including bacillary angiomatosis which is characterized by vasoproliferative tumor-like lesions on the skin and internal organs of some immunosuppressed individuals. Several virulence factors associated with Bartonella-induced pathogenesis have been characterized. However, the study of those virulence factors has been limited to in vitro cell culture systems due to the lack of a practical animal model. Therefore, we wanted to investigate whether the zebrafish embryo (Danio rerio) could be used to model human infection with Bh. We investigated if Bh can mount an infection in zebrafish embryos during their early stage of development. Our data showed that Tg(fli1:egfp)y1 zebrafish embryos supported a sustained Bh infection for 7 days with >10-fold bacterial replication when inoculated in the yolk sac. This was evident by plating of zebrafish homogenates, quantitative PCR, and confocal microscopy analysis. We assessed the interaction of Bh with EC and the phagocytic cells in live embryos by microscopy. Our data showed that aggregates of Bh interact with the endothelium of the embryo vasculature. Evidence showed that Bh recruited phagocytes to the site of infection in the Tg(mpx:GFP)uwm1 embryos. We also wanted to determine the response to infection with Bh. Infected embryos showed evidence of a Bh-induced angiogenic phenotype as well as an increase in expression of genes encoding pro-inflammatory factors and pro-angiogenic markers. A deletion mutant for the entire VirB type IV secretion system (ΔvirB2-11 supported bacterial replication although to a lesser degree compared to the wild type control. However, infection of zebrafish embryos with a deletion mutant in the major adhesin (BadA) resulted in little or no bacterial replication and a diminished pro-angiogenic and pro-inflammatory host response compared to wild type Bh, providing the first evidence that BadA is critical for in vivo infection. Thus, the zebrafish embryo provides the first practical animal model of Bh infection that will facilitate efforts to identify virulence factors and define molecular mechanisms of Bh pathogenesis.

Bacillary angiomatosis (BA), one of the clinical manifestations resulting from infection with the facultative intracellular bacterium Bartonella henselae, is characterized by angiogenic lesions. Endothelial cells have been identified as host cells for this pathogen and are presumed important for pathogenesis as lesions contain bacteria in direct contact with the endothelium. Lesions also contain infiltrating macrophages, which contribute to the angiogenic process during B. henselae infection by secreting vascular endothelial growth factor (VEGF). While virulence factors have been characterized, and the role for macrophages in B. henselae infection has been established, endothelial cell mediators of angiogenesis have not been well defined. We investigated three potentially important pathways that are triggered by the bacterial interactions with endothelial cells. We examined the ability of endothelial cells to upregulate the chemokines monocyte-macrophage chemoattracta nt protein-1 (MCP-1) and CXCL8 and the mechanism by which B. henselae secreted proteins (BHSP) induce endothelial cell proliferation. We determined that MCP-1 production is upregulated in response to B. henselae infection, which very likely contributes to angiogenic lesion formation by recruiting the VEGF-secreting macrophage. The chemokine CXCL8 is an important mediator of angiogenesis which can cause endothelial cell survival, proliferation, and capillary tube formation. We determined that CXCL8 is secreted from B. henselae-infected cells and contributes to B. henselae-induced angiogenesis in an autocrine manner. We also investigated the role of Ca2+ signaling during B. henselae infection. We determined that BHSP induce a robust intracellular Ca2+ response in HUVEC which originates from intracellular Ca2+ pools. Additionally, endothelial cell proliferation in response to BHSP required Ca2+ activity, indicating a role for intracellular Ca2+ pools during B. henselae-induced angio genesis. Endothelial cell proliferation during B. henselae infection possibly indicates a mechanism by which a pathogen induces proliferation of its host cell in order to propagate its own survival. Numerous factors culminate in the vascular lesions that are characteristic of BA. B. henselae infection represents an important and unique model for pathogen-triggered angiogenesis, and studies into the specific mechanisms of this process may elucidate host cell-pathogen interactions as well as pathways of pathogenic angiogenesis.