Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles - PubMed (original) (raw)

Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles

Gina M Donato et al. FEBS Lett. 2012.

Abstract

Bordetella pertussis adenylate cyclase toxin (ACT) intoxicates cells by producing intracellular cAMP. B. pertussis outer membrane vesicles (OMV) contain ACT on their surface (OMV-ACT), but the properties of OMV-ACT were previously unknown. We found that B. pertussis in the lung from a fatal pertussis case contains OMV, suggesting an involvement in pathogenesis. OMV-ACT and ACT intoxicate cells with and without the toxin's receptor CD11b/CD18. Intoxication by ACT is blocked by antitoxin and anti-CD11b antibodies, but not by cytochalasin-D; in contrast, OMV-ACT is unaffected by either antibody and blocked by cytochalasin-D. Thus OMV-ACT can deliver ACT by processes distinct from those of ACT alone.

Copyright © 2012 Federation of European Biochemical Societies. All rights reserved.

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Figures

Figure 1

Figure 1. B.pertussis organisms and OMV in human pathology specimens

Electron microscopic images of outer membrane vesicles associated with B.pertussis are from lung tissue obtained at autopsy from a child with fatal B. pertussis infection. Arrows indicate OMV. Scale bar=0.1 μm; magnification=30,000X.

Figure 2

Figure 2. Transmission EM images of B.pertussis and OMV

A. B.pertussis GMT1 and associated OMV were negatively stained with 2% NanoVan. Arrows indicate OMV attached to the bacterial surface; bracket represents OMV released into the culture medium; scale bar=1 μm; magnification=15,000X. B. Ultrathin section of embedded bacteria and attached OMV stained with uranyl acetate and lead citrate. Scale bar=0.2 μm; magnification=40,000X. C. OMV prepared and purified from GMT1 by the method of Hozbor et al. [19] were negatively stained with 2% PTA pH 7.0. Scale bar=0.5 μm; magnification=30,000X. D. J774.A1 cells were intoxicated comparably by enriched and native OMV from GMT1. The comparison of intoxication was based on equalized AC enzymatic activities. Enriched OMV from the ACT-negative strain, BP348, did not cause intoxication. * p<0.0001 compared to GMT1 enriched OMV.

Figure 3

Figure 3. Distribution of periplasmic and cytoplasmic markers in B.pertussis and OMV

A. GMT1(pTH22), a B. pertussis strain expressing the periplasmic enzyme marker, AP, was fractionated and the periplasmic and cytoplasmic fractions assayed for AP activity. Data represent the blanked mean ± S.E.M. from 4 independent experiments. B. Enriched OMV from GMT1(pTH22) were lysed and assayed for AP enzyme activity. AP activity was detected in the OMV lysate and luminal fraction, but not the membrane fraction. Representative graph from 3 independent experiments. C. Enriched GMT1 OMV were lysed and assayed for MDH via the oxidation of β-NADH, which is reflected as a decrease in OD340. MDH activity of the B pertussis lysate is shown in inset. Representative graph from 3 independent experiments.

Figure 4

Figure 4. Trypsin-sensitivity assay

OMV-ACT and purified ACT were incubated with or without 40 μg/ml trypsin for 5 min at 37°C and then, 80 μg/ml trypsin inhibitor was added. As a control, samples were incubated with trypsin inhibitor prior to trypsin treatment. These samples were assayed for (A) adenylate cyclase enzymatic activity and (B) intoxication. Data reported as a percentage of the untreated controls, which were set at 100%. At equivalent ACT enzymatic activity, intoxication for purified ACT was 9215 ± 1241 pmol cAMP/mg J774A.1 protein and for OMV-ACT it was 9896 ± 270 pmol cAMP/mg J774A.1 protein. All data represent the mean ± S.E.M. with background subtracted of duplicate samples from at least 2 independent experiments. * p<0.007 compared to untreated controls.

Figure 5

Figure 5. Effect of antibodies and cytochalasin-D on intoxication by ACT and OMV-ACT

A. OMV-ACT and purified ACT were incubated with 20 μg/ml anti-ACT (3D1) or control antibody (mouse isotype IgG1) for 10 min at 4°C and then added directly to J774A.1 cells. Alternatively, J774A.1 macrophages were pre-treated with 10 μg/ml anti-CD11b or control antibody (rat isotype IgG2b) at 4°C for 1 hr before purified ACT or OMV-ACT was added. Cells were incubated at 37°C for 1 hr and then cAMP measured. Data reported as a percentage of the untreated controls, which were set at 100%. At equivalent ACT enzymatic activity, intoxication for ACT was 10280 ± 1786 pmol cAMP/mg J774A.1 protein and for OMV-ACT it was 5117 ± 442 pmol cAMP/mg J774A.1 protein. All data represent the mean ± S.E.M. with background subtracted of duplicate samples from at least 2 independent experiments. * p<0.0018 compared to untreated controls. B. J774.A1 macrophages were incubated with or without 10 μg/ml cytochalasin-D for 1 hr at 37°C before OMV-ACT or purified ACT was added and incubated an additional hour at 37°C. * p=0.0009 compared to untreated control. C. J774.A1 and CHO cells were each treated with cytochalasin-D as described in (5B), equal amounts of OMV-ACT were added and incubated an additional hour at 37°C. Buffer control was 121 ± 23 pmol cAMP/mg cell protein; DMSO-treated control cells was 37 ± 6 pmol cAMP/mg cell protein. All data represent the mean ± S.E.M. of duplicate samples from at least 2 independent experiments. * p<0.0045 compared to untreated controls.

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