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B.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.
Bordetella pertussis is the causative agent of whooping cough, which is increasing in incidence, despite high immunization rates . This Gram-negative organism produces a number of virulence factors, including adenylate cyclase toxin (ACT), filamentous hemagglutinin (FHA), pertactin (PRN) and pertussis toxin (PT) . ACT uses the αMβ2 integrin CD11b/CD18 as a receptor, but also intoxicates cells not expressing CD11b/CD18 [3-5]. Following binding, regardless of CD11b/CD18, the ACT catalytic domain is translocated into the cell and activated by calmodulin to convert ATP to cyclic AMP (cAMP), a process referred to in this study and elsewhere as “intoxication”.
Most Gram-negative bacteria produce outer membrane vesicles (OMV) containing outer-membrane proteins, carbohydrates and lipids. These structures have been extensively studied and recognized to have a role in pathogenesis of some bacterial infectious diseases [6;7]. Hozbor et al. showed that B.pertussis produce OMV containing ACT (OMV-ACT) and other virulence factors and proposed use of these OMV as an acellular pertussis vaccine [8;9]. Virtually nothing is known about the effects of OMV-ACT, as illustrated by the fact that neither the Hozbor publications nor two recent reviews on OMV contain information on virulence-associated activities of OMV-ACT. In our studies of intoxication by OMV-ACT, we find that OMV-ACT acts as a delivery system for ACT, but by a process that is different from that used by purified ACT.
B.pertussis strains (GMT1, GMT1(pTH22) and BP348) were grown on Bordet-Gengou (BG) agar (Difco) containing 10% defibrinated sheep blood (Cocalico) and then modified Stainer-Scholte liquid medium (SSM)  at 35.5°C. GMT1 is a wild-type B.pertussis strain  and BP348 contains a transposon insertion in cyaA , rendering it defective in the production of ACT. GMT1(pTH22) was created for this study, as described below.
OMV were isolated from culture supernatants and bacterial cells as described by Hozbor et al. . For OMV from culture supernatant (referred to as “native OMV”), GMT1 was grown in SSM, centrifuged and filtered to remove remaining bacteria. OMV were obtained by centrifugation at 150,000 × g for 1 hr at 4°C and washing in TE (20 mM Tris-HCl, 2 mM EDTA pH 8.5). Following the final centrifugation, OMV pellets were stored in TE at -20°C.
For OMV from treatment of bacteria (designated “enriched OMV”), strains were grown to logarithmic phase, centrifuged and resuspended in TE. OMV were released by sonication on ice in five, 1-min bursts. The bacteria were removed by centrifugation and the OMV-containing supernatants were centrifuged at 100,000 × g for 2 hr at 4°C. The pellets were resuspended in 2% deoxycholate (DOC) and further purified by centrifugation on a 60% sucrose cushion at 100,000 × g for 2 hr at 4°C. The OMV band was collected from the interface, washed and stored in TE at -20°C. OMV protein concentrations were determined by BCA protein assay (Pierce). The absence of viable bacteria in the OMV was established by lack of growth on BG plates.
For B.pertussis cultured in vitro, an aliquot of GMT1 or isolated OMV was added to formvar and carbon-coated, nickel-mesh grids for 1 min, excess liquid removed and samples negatively stained with either 2% NanoVan (Nanoprobes) or 2% phosphotungstic acid (PTA) pH 7.0 for 1 min. Bacteria and vesicles were visualized by TEM. For high resolution images, GMT1 was prepared for sectioning by fixing in 2.5% glutaraldehyde for 1 hr at room temperature (RT). Bacteria were centrifuged, washed in 0.1 M phosphate buffer pH 7.4, post-fixed for 1 hr in 1% osmium tetroxide, dehydrated and embedded in epoxy resin (EMBED 812; Electron Microscopy Sciences). After polymerization, ultrathin sections were collected, mounted, stained with uranyl acetate and lead citrate and examined according to routine procedures.
For autopsy samples, formalin-fixed, paraffin-embedded lung tissue was deparaffinized, post-fixed in 1% osmium tetroxide, dehydrated, and embedded in a mixture of Epon substitute and Araldite. Sections were stained with uranyl acetate and lead citrate and microscopy was performed at the CDC, as previously described .
AC enzymatic activity was measured by the conversion of [32P]-ATP to [32P]-cAMP, as previously described . Comparisons between native and enriched OMV and between OMV-ACT and purified ACT were made on the basis of equalized AC enzymatic activities.
ACT was expressed from E.coli XL-1 Blue and purified by ion exchange and affinity chromatographies, as described previously .
J774A.1 cells (murine macrophage cell line) and CHO-K1 cells (Chinese hamster ovary epithelial cell line) were grown in 96-well plates in Dulbecco’s modified Eagle’s medium with high glucose (DMEM; Gibco) or Ham’s F12 medium with L-glutamine (Gibco), respectively, plus 10% heat-inactivated fetal bovine serum (FBS; Gibco). Purified ACT or OMV-ACT was added to cells, which were then incubated 1 hr, washed twice and lysed for cAMP measurement using a chemiluminescence-based ELISA (cAMP-screen system; Applied Biosystems).
We introduced E.coli phoA (encoding alkaline phosphatase) into B.pertussis specifically for the purpose of validating enriched OMV. Since OMV arise by budding from the outer membrane independent of the cytoplasmic membrane and cytosol, they are expected to contain periplasmic proteins and to exclude cytosolic proteins. GMT1 was conjugated with an E. coli donor strain SM10(pTH22) (kindly provided by Drusilla Burns, FDA), which carries phoA . Ex-conjugates were selected on BG agar containing gentamicin and streptomycin. The resulting strain, which was designated GMT1(pTH22), was grown in SSM and fractionated to yield periplasmic and cytoplasmic compartments, as previously described . The bacterial pellet was resuspended in 0.2 M Tris pH 8.0 and spheroplast buffer (0.2 M Tris pH 8.0, 1 M sucrose, 0.5% Zwittergent-316, 0.1 mg/ml lysozyme) was added. The supernatant following centrifugation contained the periplasmic fraction and spheroplasts disrupted by osmotic lysis provided the cytoplasmic fraction.
Enriched OMV (20 μg) from GMT1- and GMT1(pTH22) were lysed by rotating at RT for 2 hr in spheroplast buffer (described above) to obtain total lysate. After an aliquot was removed, the remainder was centrifuged at 144,000 × g for 1 hr at 4°C to obtain lumen and membrane fractions. The membranes were resuspended in TE and both fractions stored at -20°C. Alkaline phosphatase (AP) activity was measured according to the method described by Brickman and Beckwith, with minor modifications . E. coli AP (MP Biomedicals) was the positive control. The assay for malate dehydrogenase (MDH) was performed as suggested by the manufacturer of the substrates and enzyme (Sigma). Porcine heart MDH (Sigma) was used as a positive control. Incubation of B.pertussis on ice for 10 min in a lysis buffer, followed by freeze-thaw cycles and sonication, yielded a bacterial whole cell lysate as a control.
OMV-ACT and purified ACT were treated with trypsin (40 μg/ml, 5 min, 37°C) and then 80 μg/ml trypsin inhibitor was added to stop the reaction. As controls, OMV-ACT and purified ACT were treated with trypsin inhibitor prior to the addition of trypsin, in order to mimic the experimental conditions. Enzyme activity and intoxication were measured, as described above.
OMV-ACT and purified ACT were incubated (10 min, 4°C) with 20 μg/ml 3D1 or control mouse IgG antibody (Sigma) and then added to macrophages. Alternatively, macrophages were treated (1 hr, 4°C) with 10 μg/ml anti-CD11b (M1/70) or control rat IgG2b antibody (BD Pharmingen) before ACT or OMV-ACT was added. Intoxication was measured after 1 hr, as described above. Intoxication in the absence of prior incubation with either antibody served as control.
J774A.1 and CHO cells were incubated with or without 10 μg/ml cytochalasin-D, then OMV-ACT or purified ACT was added to the cells, incubated an additional 1 hr at 37°C and cAMP was measured. As controls, cytochalasin-D (10 μg/ml) and DMSO (final concentration of 1%) were added separately to target cells and cAMP quantified.
In light of the observations of Hozbor et al. , we asked whether OMV are produced in vivo during infection with B.pertussis. Previously, autopsy airway specimens from children, who died of pertussis, were examined using electron microscopy . B.pertussis organisms were identified in the cilia of respiratory epithelium of a subsegemental bronchus in a section of lung tissue by immunohistochemical staining and molecular analysis , as well as by using immunoelectron microscopy (data not shown). As shown in Figure 1, these lung sections from a fatal case of pertussis reveal B.pertussis organisms with associated OMV, some in the process of forming and others already released. These images, which illustrate for the first time B.pertussis OMV in vivo, prompted investigation of a possible role for OMV in the delivery of ACT to host cells.
This investigation began with characterization of B.pertussis OMV and optimization of their preparation. Transmission electron microscopy (TEM) of negatively stained, unfixed B.pertussis (GMT1) from an overnight culture reveals vesicles attached to the cell surface and released into the medium (Figure 2A). Stained, ultrathin sections of fixed and embedded bacteria allow a higher resolution image of the native OMV, with characteristic membrane bilayer surrounding electron-dense material (Figure 2B). B.pertussis native OMV (Figure 2A) and enriched OMV (Figure 2C) exhibit similar morphologies. Importantly, as shown in Figure 2D, native OMV and enriched OMV at equal amounts of enzymatic activity are comparable in their ability to elicit cAMP production in J774.A1 cells. Enriched OMV from ACT-negative strain, BP348, serve as the negative control.
By virtue of the process by which OMV are formed, budding of outer membrane without disturbing the cytoplasmic membrane, they contain periplasmic components, but not material from the cytoplasm [20-22]. To further validate the procedure used by Hozbor et al., we characterized the luminal contents of enriched OMV. A B.pertussis strain (GMT1(pTH22)) expressing E.coli alkaline phosphatase (AP) was created specifically for this purpose, since AP is localized to the periplasmic space. As illustrated in Figure 3A, 94% of the AP activity from (GMT1(pTH22)) is periplasmic. In enriched OMV obtained from GMT1(pTH22), the AP activity was detected solely in the OMV lumen fraction (Figure 3B). We also tested OMV from (GMT1(pTH22)) for cytoplasmic contamination by assaying for the intracellular enzyme malate dehydrogenase (MDH). Oxidation of β-NADH by MDH results in a decrease in absorbance at 340 nm. As shown in Figure 3C, there is MDH activity from the B.pertussis whole cell lysate (inset panel) as well as the control enzyme from porcine heart, but none in OMV lysates or fractions thereof.
Together, these data demonstrate that enriched OMV from B.pertussis are comparable morphologically to native OMV and show the characteristic features of OMV, namely containing periplasmic AP, but no cytoplasmic MDH. Because the yield of native OMV produced by B.pertussis grown in vitro decreases by the filtration step necessary to remove residual bacteria from the culture medium, and the comparability of native and enriched OMV, we used enriched OMV for the subsequent studies of OMV-ACT.
It was recognized at the time of discovery of B.pertussis ACT that a large amount of toxin is associated with the bacterial surface and both cell-associated and purified ACT are very sensitive to trypsin . Since OMV are derived from the outer membrane of proliferating B.pertussis, we hypothesized that OMV-ACT would be exposed on the external surface and thus susceptible to added trypsin. As shown in Figure 4A, exposure of OMV-ACT to trypsin results in an 88% decrease in AC enzymatic activity (as compared to untreated OMV-ACT) and this loss of activity is accompanied by an equivalent (91%) reduction in their ability to intoxicate J774.A1 cells (Figure 4B). When purified ACT is treated with trypsin under the same conditions, there is a comparable loss of toxin activity. Together, these data confirm that OMV-ACT is in a location, most likely the external surface of the OMV that makes it susceptible to exogenously added trypsin.
In previous studies of the surface-exposed B.pertussis ACT, we hypothesized that this ACT could be transferred to target cells, resulting in intoxication. This hypothesis, however, was not correct; it is newly secreted ACT that is responsible for intoxicating cells . In order to determine the relative proportion of ACT released as OMV-associated rather than free toxin, we isolated OMV from culture supernatants and measured AC enzymatic activity. We found that native OMV-ACT represents 1.2 ± 0.06% of the total ACT released into the bacteria-cleared supernatant of B.pertussis cultures grown in vitro. Our discovery that OMV-ACT is able to deliver ACT and thus intoxicate target cells provides a potential alternative mechanism by which ACT on the bacterial surface may contribute to intoxication of host cells. To understand this process better, we investigated the mechanism by which eukaryotic cells are intoxicated by OMV-ACT.
Intoxication by purified ACT involves binding to CD11b/CD18, when present, and translocation into the cell. This intoxication is inhibited >95% by preventing binding of ACT to its receptor with anti-CD11b antibody or by interfering with translocation with anti-ACT monoclonal antibody 3D1 [3;25]. As expected, intoxication by purified ACT is reduced >96% (compared to the untreated cells) by either antibody, but unaffected by isotype controls (Figure 5A). In contrast, intoxication of J774.A1 cells by OMV-ACT is unaffected by either antibody.
Unlike other bacterial toxins whose entry into cells is mediated by a toxin-receptor interaction and a host cell-mediated process, the pathway by which isolated ACT enters host cells is not a microfilament-dependent process, such as endocytosis or phagocytosis . The differences between OMV-ACT and isolated ACT in response to anti-toxin and anti-receptor antibodies suggest that they enter by different mechanisms. To explore this hypothesis, we evaluated the effects of cytochalasin-D, an inhibitor of microfilament function, on intoxication by OMV-ACT. Consistent with previous findings, intoxication by ACT is not affected by cytochalasin-D (Figure 5B). In striking contrast, intoxication by OMV-ACT is prevented by treatment with cytochalasin-D. To test whether this response to OMV-ACT in J774.A1 cells is a general phenomenon, we compared ACT and OMV-ACT in non-phagocytic, CD11b/CD18-negative CHO cells. The potency of OMV-ACT is comparable between cell types and the addition of cytochalasin-D to CHO cells decreases OMV-mediated intoxication by 85% relative to untreated cells (Figure 5C). Overall these data suggest that the process by which OMV-ACT intoxicates both cell types is CD11b/CD18-independent, microfilament-dependent and mechanistically distinct from intoxication by isolated ACT. Thus, although OMV-ACT is unlikely the primary pathway for ACT delivery since approximately only 1% of the total released active ACT is secreted via native OMV, it may provide an important supplemental mechanism for B.pertussis intoxication of host cells. Since OMV-mediated delivery of ACT could presumably occur at a distance and in the absence of bacteria, OMV-ACT would circumvent the need for B.pertussis to be in close contact with the host cell in order to exert its effects, as is the requirement for newly synthesized ACT.
These results are particularly of interest in light of the recent observations by Chatterjee and Chaudhuri on OMV from V. cholerae . In studies of the delivery of cholera toxin (CT) to intestinal epithelial cells by OMV, they observed that intoxication is blocked by addition of the CT receptor, GM1 ganglioside, prior to incubation with target cells. These data are interpreted to indicate that the interaction of OMV-CT with the epithelial cells is mediated by an interaction of CT with GM1. Similarly, E.coli heat-labile toxin (LT) acts as an OMV adhesin, binding GM1 as does CT, and leads to endocytosis . VacA-containing vesicles are also endocytosed into vacuoles as is soluble VacA, suggesting that the recognition and processing may be the same . In contrast, A.actinomycetemcomitans OMV, like those of B.pertussis, interact with target-cell membranes independent of the OMV-associated leukotoxin or microfilament function . Thus, there are several mechanisms by which OMV deliver virulence factors to host cells. Our studies establish that OMV from B.pertussis can deliver ACT to target cells and that production of OMV occurs in vivo during clinical pertussis.
This work was supported by grant AI18000 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
We wish to thank the following individuals for providing reagents and assistance: Peggy Cotter for GMT1, Drusilla Burns and Scott Stibitz for pTH22 and Meta Kuehn for OMV discussions.
The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
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