A Translocated Bacterial Protein Protects Vascular Endothelial Cells from Apoptosis

The modulation of host cell apoptosis by bacterial pathogens is of critical importance for the outcome of the infection process. The capacity of Bartonella henselae and B. quintana to cause vascular tumor formation in immunocompromised patients is linked to the inhibition of vascular endothelial cell (EC) apoptosis. Here, we show that translocation of BepA, a type IV secretion (T4S) substrate, is necessary and sufficient to inhibit EC apoptosis. Ectopic expression in ECs allowed mapping of the anti-apoptotic activity of BepA to the Bep intracellular delivery domain, which, as part of the signal for T4S, is conserved in other T4S substrates. The anti-apoptotic activity appeared to be limited to BepA orthologs of B. henselae and B. quintana and correlated with (i) protein localization to the host cell plasma membrane, (ii) elevated levels of intracellular cyclic adenosine monophosphate (cAMP), and (iii) increased expression of cAMP-responsive genes. The pharmacological elevation of cAMP levels protected ECs from apoptosis, indicating that BepA mediates anti-apoptosis by heightening cAMP levels by a plasma membrane–associated mechanism. Finally, we demonstrate that BepA mediates protection of ECs against apoptosis triggered by cytotoxic T lymphocytes, suggesting a physiological context in which the anti-apoptotic activity of BepA contributes to tumor formation in the chronically infected vascular endothelium

Paleo-Immunology: Evidence Consistent with Insertion of a Primordial Herpes Virus-Like Element in the Origins of Acquired Immunity

BACKGROUND:The RAG encoded proteins, RAG-1 and RAG-2 regulate site-specific recombination events in somatic immune B- and T-lymphocytes to generate the acquired immune repertoire. Catalytic activities of the RAG proteins are related to the recombinase functions of a pre-existing mobile DNA element in the DDE recombinase/RNAse H family, sometimes termed the "RAG transposon". METHODOLOGY/PRINCIPAL FINDINGS:Novel to this work is the suggestion that the DDE recombinase responsible for the origins of acquired immunity was encoded by a primordial herpes virus, rather than a "RAG transposon." A subsequent "arms race" between immunity to herpes infection and the immune system obscured primary amino acid similarities between herpes and immune system proteins but preserved regulatory, structural and functional similarities between the respective recombinase proteins. In support of this hypothesis, evidence is reviewed from previous published data that a modern herpes virus protein family with properties of a viral recombinase is co-regulated with both RAG-1 and RAG-2 by closely linked cis-acting co-regulatory sequences. Structural and functional similarity is also reviewed between the putative herpes recombinase and both DDE site of the RAG-1 protein and another DDE/RNAse H family nuclease, the Argonaute protein component of RISC (RNA induced silencing complex). CONCLUSIONS/SIGNIFICANCE:A "co-regulatory" model of the origins of V(D)J recombination and the acquired immune system can account for the observed linked genomic structure of RAG-1 and RAG-2 in non-vertebrate organisms such as the sea urchin that lack an acquired immune system and V(D)J recombination. Initially the regulated expression of a viral recombinase in immune cells may have been positively selected by its ability to stimulate innate immunity to herpes virus infection rather than V(D)J recombination Unlike the "RAG-transposon" hypothesis, the proposed model can be readily tested by comparative functional analysis of herpes virus replication and V(D)J recombination

Comparison of the Anti-Apoptotic Activities of BepA Homologs

<div><p>(A) Anti-apoptotic activities of wild-type and isogenic Δ<i>virB4</i> mutant strains of <i>Bh</i> in comparison with <i>Bq</i> and <i>Bt</i>.</p><p>(B) Domain structure of <i>Bh</i> BepA, its paralogs <i>Bh</i> BepB and <i>Bh</i> BepC, and the orthologs <i>Bt</i> BepA and <i>Bq</i> BepA1/<i>Bq</i> BepA2. These homologs contain conserved FIC and BID domains in their N-terminal and C-terminal regions, respectively, except for <i>Bq,</i> where the orthologous locus is split between these domains into two separate open reading frames by an internal stop codon.</p><p>(C) Anti-apoptotic activity of BepA homologs. HUVECs were infected with the indicated <i>Bh</i> strains for 24 h, followed by apoptotic induction with actinomycin D for 12 h. Caspase-3/-7 activities were then determined with a specific fluorogenic peptide substrate. Mean and SD are illustrated for one representative out of three independent experiments.</p></div

Delineation and Subcellular Localization of the Region of <i>Bh</i> BepA Required for Inhibition of Apoptosis

<div><p>(A) Schematic presentation of N-terminal GFP fusions to parts of <i>Bh</i> BepA.</p><p>(B) Determination of apoptosis following ectopic expression of the constructs illustrated in (A). GFP-BepA fusion proteins were ectopically expressed in HUVECs for 24 h, followed by 12 h incubation in the presence or absence of actinomycin D as indicated. The loss of membrane asymmetry in transfected cells (GFP-positive) was then quantified by flow cytometric analysis of APC-Annexin V– and PI-stained cells, allowing us to quantify the rate of apoptotic cells (Annexin V–positive and PI-negative). The means and SD of three independent replica experiments are shown. The <i>p</i>-values were determined by using an unpaired Student's <i>t</i>-test.</p><p>(C) The GFP–<i>Bh</i> BepA fusion proteins illustrated in (A) were ectopically expressed for 24 h in HEK293T cells. Cells were immunochemically stained to label the cell surface with Texas Red–conjugated WGA. Confocal pictures were taken for GFP (green channel) and WGA (red channel) in the <i>xy</i>-plane (upper image, overlay both channels, bar = 10 μm), and also in the <i>xz</i>-plane at the dashed stroke line (lower images, single channels and overlay channels).</p><p>(D) Fractionation of GFP–<i>Bh</i> BepA fusion proteins into membrane and cytosolic fractions by ultracentrifugation of post-nuclear extracts harvested from transfected HEK293T cells.</p></div