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

PrimeArray: genome-scale primer design for DNA-microarray construction

Summary: PrimeArray is a Windows program that computes oligonuceotide primer pairs for genome-scale gene amplification by the Polymerase Chain Reaction (PCR). The program supports the automated extraction of coding sequences (CDS) from various input-file formats and allows highly automated primer pair-optimization. Availability: The program is freely available for non-profit use via request from the authors. Contact: [email protected]

Characterization of the Genome Composition of Bartonella koehlerae by Microarray Comparative Genomic Hybridization Profiling

Bartonella henselae is present in a wide range of wild and domestic feline hosts and causes cat-scratch disease and bacillary angiomatosis in humans. We have estimated here the gene content of Bartonella koehlerae, a novel species isolated from cats that was recently identified as an agent of human endocarditis. The investigation was accomplished by comparative genomic hybridization (CGH) to a microarray constructed from the sequenced 1.93-Mb genome of B. henselae. Control hybridizations of labeled DNA from the human pathogen Bartonella quintana with a reduced genome of 1.58 Mb were performed to evaluate the accuracy of the array for genes with known levels of sequence divergence. Genome size estimates of B. koehlerae by pulsed-field gel electrophoresis matched that calculated by the CGH, indicating a genome of 1.7 to 1.8 Mb with few unique genes. As in B. quintana, sequences in the prophage and the genomic islands were reported absent in B. koehlerae. In addition, sequence variability was recorded in the chromosome II-like region, where B. koehlerae showed an intermediate retention pattern of both coding and noncoding sequences. Although most of the genes missing in B. koehlerae are also absent from B. quintana, its phylogenetic placement near B. henselae suggests independent deletion events, indicating that host specificity is not solely attributed to genes in the genomic islands. Rather, the results underscore the instability of the genomic islands even within bacterial populations adapted to the same host-vector system, as in the case of B. henselae and B. koehlerae

The BatR/BatS Two-Component Regulatory System Controls the Adaptive Response of Bartonella henselae during Human Endothelial Cell Infection ▿ † ‡

Here, we report the first comprehensive study of Bartonella henselae gene expression during infection of human endothelial cells. Expression of the main cluster of upregulated genes, comprising the VirB type IV secretion system and its secreted protein substrates, is shown to be under the positive control of the transcriptional regulator BatR. We demonstrate binding of BatR to the promoters of the virB operon and a substrate-encoding gene and provide biochemical evidence that BatR and BatS constitute a functional two-component regulatory system. Moreover, in contrast to the acid-inducible (pH 5.5) homologs ChvG/ChvI of Agrobacterium tumefaciens, BatR/BatS are optimally activated at the physiological pH of blood (pH 7.4). By conservation analysis of the BatR regulon, we show that BatR/BatS are uniquely adapted to upregulate a genus-specific virulence regulon during hemotropic infection in mammals. Thus, we propose that BatR/BatS two-component system homologs represent vertically inherited pH sensors that control the expression of horizontally transmitted gene sets critical for the diverse host-associated life styles of the alphaproteobacteria

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

A Pharmacologically Increased cAMP Level in ECs Mimics the Anti-Apoptotic Effect of <i>Bh</i> BepA

<p>HUVECs were infected for 24 h with the indicated <i>Bh</i> strains or left uninfected (control) in the absence or presence of either (A) forskolin (1 μM) and IBMX (10 μM) or (B) dibutyryl cAMP (1 mM). If indicated, apoptosis was then induced with actinomycin D. Caspase-3/-7 activities were determined 9 h later. All strains were tested in triplicates a minimum of three times.</p

<i>Bh</i> BepA Is a Genuine VirB/VirD4 T4S Substrate That Is Translocated into ECs

<div><p>(A) The bars indicate the parts of <i>Bh</i> BepA or <i>Bh</i> BepD that were fused to Cya. These reporter fusions were used to monitor translocation via the VirB/VirD4 system. All constructs contain an N-terminal FLAG epitope for immunological detection of the encoded fusion protein.</p><p>(B) Quantification of the amount of intracellular cAMP in HUVECs infected for 20 h with the indicated bacterial strains (MOI = 300). Isogenic strains with a functional (wild-type) or non-functional (Δ<i>virB4</i>) VirB/VirD4 T4S system were used to express the different Cya reporter constructs. Mean and SD are shown for one representative out of three independent replica experiments.</p><p>(C) Steady-state FLAG-Cya fusion protein levels of the indicated <i>Bh</i> strains grown on IPTG-containing medium.</p></div

Anti-Apoptotic BepA Homologs Mediate an Increase in Intracellular cAMP and an Upregulation of cAMP Response Genes

<div><p>HUVECs were infected with the indicated <i>Bh</i> strains. The means and SD of one out of three independent replica experiments performed in triplicate samples are presented.</p><p>(A) IL-8 was determined in culture supernatants after infection for 54 h with MOI = 300.</p><p>(B) Expression of the cAMP-responsive genes <i>pde4B</i> and <i>crem</i> was determined by quantitative real-time PCR after infection for 54 h with MOI = 300.</p><p>(C) Intracellular cAMP levels were determined after infection for 30 h with MOI = 150.</p><p>In (B) and (C), samples marked with an asterisk (<i>p</i> < 0.05) differ statistically significantly from Δ<i>bepA–G</i> using an unpaired Student's <i>t</i>-test.</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