A translocated bacterial protein protects vascular endothelial cells from apoptosis - PubMed (original) (raw)

A translocated bacterial protein protects vascular endothelial cells from apoptosis

Michael C Schmid et al. PLoS Pathog. 2006 Nov.

Abstract

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.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Bh BepA Meditates Inhibition of Apoptosis in ECs

(A and B) HUVECs were infected for 24 h with the indicated Bh strains (MOI = 300) or left uninfected for this period (control). If not indicated differently, apoptosis was then triggered by the addition of actinomycin D. Then, 12 h later, (A) morphological changes were visualized by recording phase contrast images (bar = 40 μm), and (B) caspase-3/-7 activities were determined with a specific fluorogenic peptide substrate. Arrowheads (A) indicate apoptotic cells displaying membrane blebbing. (C) Twenty-four hours after induction of apoptosis, the loss of membrane asymmetry was quantified by flow cytometric analysis of AlexaFluor488 Annexin V– and PI-stained cells, allowing us to quantify the rate of apoptotic cells (Annexin V–positive and PI-negative). Mean and standard deviation (SD) are shown for one representative out of three independent replica experiments. All strains were tested a minimum of three times in triplicate samples.

Figure 2

Figure 2. Bh BepA Is a Genuine VirB/VirD4 T4S Substrate That Is Translocated into ECs

(A) The bars indicate the parts of Bh BepA or Bh 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. (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_virB4_) 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. (C) Steady-state FLAG-Cya fusion protein levels of the indicated Bh strains grown on IPTG-containing medium.

Figure 3

Figure 3. Delineation and Subcellular Localization of the Region of Bh BepA Required for Inhibition of Apoptosis

(A) Schematic presentation of N-terminal GFP fusions to parts of Bh BepA. (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 _p_-values were determined by using an unpaired Student's t_-test. (C) The GFP–_Bh 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 _xy_-plane (upper image, overlay both channels, bar = 10 μm), and also in the xz_-plane at the dashed stroke line (lower images, single channels and overlay channels). (D) Fractionation of GFP–_Bh BepA fusion proteins into membrane and cytosolic fractions by ultracentrifugation of post-nuclear extracts harvested from transfected HEK293T cells.

Figure 4

Figure 4. Comparison of the Anti-Apoptotic Activities of BepA Homologs

(A) Anti-apoptotic activities of wild-type and isogenic Δ_virB4_ mutant strains of Bh in comparison with Bq and Bt. (B) Domain structure of Bh BepA, its paralogs Bh BepB and Bh BepC, and the orthologs Bt BepA and Bq BepA1/Bq BepA2. These homologs contain conserved FIC and BID domains in their N-terminal and C-terminal regions, respectively, except for Bq, where the orthologous locus is split between these domains into two separate open reading frames by an internal stop codon. (C) Anti-apoptotic activity of BepA homologs. HUVECs were infected with the indicated Bh 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.

Figure 5

Figure 5. Anti-Apoptotic BepA Homologs Mediate an Increase in Intracellular cAMP and an Upregulation of cAMP Response Genes

HUVECs were infected with the indicated Bh strains. The means and SD of one out of three independent replica experiments performed in triplicate samples are presented. (A) IL-8 was determined in culture supernatants after infection for 54 h with MOI = 300. (B) Expression of the cAMP-responsive genes pde4B and crem was determined by quantitative real-time PCR after infection for 54 h with MOI = 300. (C) Intracellular cAMP levels were determined after infection for 30 h with MOI = 150. In (B) and (C), samples marked with an asterisk (p < 0.05) differ statistically significantly from Δ_bepA–G_ using an unpaired Student's _t_-test.

Figure 6

Figure 6. A Pharmacologically Increased cAMP Level in ECs Mimics the Anti-Apoptotic Effect of Bh BepA

HUVECs were infected for 24 h with the indicated Bh 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.

Figure 7

Figure 7. Bh BepA Protects ECs against CTL-Mediated Cell Death

Confluent HUVEC monolayers fluorescently labeled with the cell-tracking dye CFSE were infected with the indicated bacterial strains for 24 h or left uninfected (control). Then cells were incubated in the presence (+ SMCY) or absence (− SMCY) of SMCY peptides, followed by washing. CTLs were added in an effector-to-target cell ratio of 5:1 for the indicated period, and after washing and Annexin V– and PI-staining, the apoptotic cell population of HUVECs was determined by flow cytometry analysis (CFSE-positive, Annexin V–positive, and PI-negative). (A) Dot plots are shown for representative samples. (B) Summary graph representing HUVEC apoptosis during CTL co-culture. The mean and SD of one of two independent experiments performed in duplicates is shown.

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