Perinuclear Localization of Internalized Outer Membrane Vesicles Carrying Active Cytolethal Distending Toxin from Aggregatibacter actinomycetemcomitans

Abstract

Aggregatibacter actinomycetemcomitans is implicated in aggressive forms of periodontitis. Similarly to several other Gramnegative species, this organism produces and excretes a cytolethal distending toxin (CDT), a genotoxin associated with cell distention, G 2 cell cycle arrest, and/or apoptosis in many mammalian cell types. In this study, we have identified A. actinomycetemcomitans outer membrane vesicles (OMVs) as a vehicle for simultaneous delivery of multiple proteins, including CDT, into human cells. The OMV proteins were internalized in both HeLa cells and human gingival fibroblasts (HGF) via a mechanism of OMV fusion with lipid rafts in the plasma membrane. The active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. In accordance with a tight association of CdtB with OMVs, vesicles isolated from A. actinomycetemcomitans strain D7SS (serotype a), in contrast to OMVs from a D7SS cdtABC mutant, induced a cytolethal distending effect on HeLa and HGF cells, indicating that OMV-associated CDT was biologically active. Association of CDT with OMVs was also observed in A. actinomycetemcomitans isolates belonging to serotypes b and c, indicating that OMV-mediated release of CDT may be conserved in A. actinomycetemcomitans. Although the role of A. actinomycetemcomitans OMVs in periodontal disease has not yet been elucidated, our present data suggest that OMVs could deliver biologically active CDT and additional virulence factors into susceptible cells of the periodontium.

Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles Are Internalized in Human Host Cells and Trigger NOD1- and NOD2-Dependent NF-κB Activation

“…A possible explanation for such observations may be that endocytic pathways differ based on the size of the particle (65,66). Thus, as A. actinomycetemcomitans, similar to many additional bacterial species, disseminates vesicles of heterogenous sizes (approximately 10 to 300 nm in diameter) (3,7,19,48), differentially sized subpopulations of bacterial OMVs may be internalized via discrete pathways of endocytosis. A. actinomycetemcomitans OMV uptake was only partly reduced by filipin III, an inhibitor of caveola-dependent endocytosis (62,67,68), preventing OMV colocalization with caveolin protein (69).…”

“…Outer membrane vesicles (OMVs) released by this organism carry multiple proteins, and density gradient centrifugation supports the notion that there may be subpopulations of vesicles exhibiting slight variations in protein composition (7,19). Further to earlier reports revealing the OMV association of leukotoxin (LtxA) and a concomitant leukotoxic activity of A. actinomycetemcomitans OMVs (19,20), we have demonstrated that the OMVs could simultaneously deliver multiple proteins, including OmpA and biologically active cytolethal distending toxin (CDT), into HeLa cells and human gingival fibroblasts (HGF) (7). Hence, OMVs released by this species likely play a role in periodontal disease by delivering biologically active toxins and additional virulence factors into susceptible cells of the periodontium.…”

“…Control treatment corresponded to PBS which had been preincubated with PKH26 in the same manner as the OMVs. Where applicable, vesicles were incubated with cells in the presence of the inhibiting agent filipin III (Sigma-Aldrich) at a final concentration of 10 g/ml as in earlier studies (7,8,50). The inhibitor monensin (Sigma-Aldrich) was used at a final concentration of 2 M or 10 M. To estimate the effect of these inhibitors on cell viability, a neutral red uptake assay was carried out as described previously (51).…”

“…Via MVs, bacteria can expose host cells to relatively high concentrations of toxins and additional virulence factors without the requirement of a close contact between the bacterial and target mammalian cells (2)(3)(4)(5)(6)(7)(8)(9). MVs have also several defensive functions, including a role in antimicrobial peptide resistance (6,10).…”

Bacterial Outer Membrane Vesicles in Trafficking, Communication and the Host-Pathogen Interaction

“…These observations reinforced the intuitive prediction that OMVs likely form at regions of the OM that are only loosely connected to PG, an idea that remains an important consideration in developing models for OMV biogenesis. Integral OM transporters and porins are commonly identified in OMVs [Lee et al, 2008], including members of the OmpA family [Berlanda Scorza et al, 2008;Kesty et al, 2004;McMahon et al, 2012;Rompikuntal et al, 2012;Sidhu et al, 2008], which are interesting because they can function both as porins or as lipoprotein-like tethers between the OM and PG, depending upon their conformation [Nikaido, 2003]. OMVs have also been shown to contain many proteins of periplasmic origin, giving rise to the idea that OMV formation can serve as a secretory or excretory mechanism for select proteins [Chatterjee and Das, 1967;Kadurugamuwa and Beveridge, 1995].…”