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 [13
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 [13
], as well as by using immunoelectron microscopy (data not shown). As shown in , 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.
B.pertussis organisms and OMV in human pathology specimens
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 (). 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 (). B.pertussis native OMV () and enriched OMV () exhibit similar morphologies. Importantly, as shown in , 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.
Transmission EM images of B.pertussis and OMV
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
]. 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 , 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 (). 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 , 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.
Distribution of periplasmic and cytoplasmic markers in B.pertussis and OMV
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 [23
]. 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 , 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 (). 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 [24
]. 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
]. As expected, intoxication by purified ACT is reduced >96% (compared to the untreated cells) by either antibody, but unaffected by isotype controls (). In contrast, intoxication of J774.A1 cells by OMV-ACT is unaffected by either antibody.
Effect of antibodies and cytochalasin-D on intoxication by ACT and OMV-ACT
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 [26
]. 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 (). 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 (). 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 [28
]. VacA-containing vesicles are also endocytosed into vacuoles as is soluble VacA, suggesting that the recognition and processing may be the same [29
]. In contrast, A.actinomycetemcomitans
OMV, like those of B.pertussis
, interact with target-cell membranes independent of the OMV-associated leukotoxin or microfilament function [30
]. 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.