Separation of heterogeneous Aggregatibacter actinomycetemcomitans outer membrane vesicles reveals that toxin sorting is driven by surface-associated DNA

Abstract

Aggregatibacter actinomycetemcomitans is a Gram-negative oral bacterium associated with localized aggressive periodontitis as well as some systemic diseases. The strains of A. actinomycetemcomitans most closely associated with periodontal disease tend to produce more of a secreted leukotoxin (LtxA) than isolates from healthy carriers, suggesting a key role for this toxin in disease progression. LtxA is secreted via a type 1 secretion system, from the bacterial cytosol across both the inner and outer membranes in a single step into the supernatant, where it is able to interact with host cells. Upon secretion, some of the toxin associates with the bacterial cell membrane, enabling its release in association with the surface of outer membrane vesicles (OMVs), small, spherical vesicles derived from the outer membrane. We have previously observed that the highly leukotoxic A. actinomycetemcomitans strain JP2 produces two populations of OMVs, a highly abundant population of small (<100 nm in diameter) OMVs, and a less abundant population of large (>300 nm in diameter) OMVs. Here, we have investigated the association of LtxA with the two populations of OMVs varying in size. Our results indicate that surface-associated DNA drives the selective sorting of LtxA to large OMVs.

Heterogeneity of Size and Toxin Distribution in Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles

“…Our observations that OMVs produced during the late exponential phase contain more LtxA and consist of a greater fraction of large OMVs are consistent with our prior finding that LtxA is more abundant on large OMVs [ 39 , 40 , 41 ]. Knowing that LtxA is located on the surface of OMVs [ 12 ] and that it is a membrane-active protein that is able to induce curvature in model membrane systems [ 42 ], we hypothesized that the preferential association of LtxA with large OMVs might be mediated by the production of LtxA during the exponential phase.…”

“…In prior work, we found that LtxA is located predominately on the larger OMVs produced by the A. actinomycetemcomitans strain JP2 [ 39 , 40 ], consistent with a previous study of a different strain of A. actinomycetemcomitans , strain D7SS, in which it was found that LtxA was associated with OMVs with reduced density, that is, larger OMVs, compared to another exotoxin, cytolethal distending toxin (Cdt) [ 35 ]. The D7SS strain is a nonfimbriated smooth colony mutant of a clinical isolate of A. actinomycetemcomitans , suggesting that the heterogeneous sorting of LtxA on OMVs is a common and clinically relevant phenomenon.…”

Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles

“…Moreover, this finding provides evidence for the exposure of the outer leaflet of the bacterial membrane since the dye is membrane impermeable therefore it cannot detect cytoplasmic DNA. This observation aligns with reports suggesting that DNA is situated on the exterior of these vesicles and is reinforced by the impermeability of YO-PRO-1 to the membrane 47,57,58 .…”

“…Consequently, we conducted tests to detect the presence of bacterial OMV components within the surface. Our primary goal was to detect the presence of surface-associated DNA, which is present on OMVs 47,57,58 . To accomplish this, we first created POPC/PEG-PE SLBs and OM-SBs labeled with the DiI membrane dye.…”

“…However, there have been numerous reports of its resistance to commonly used antibiotics for treatment 43,44 . As part of its virulence, A. actinomycetemcomitans produces leukotoxin (LtxA), which is secreted into the extracellular milieu as a free protein and is also released in association with OMVs, where it resides on the surface along with surface-associated DNA [45][46][47] . This highlights the critical role of OMVs in pathogenesis, which is why we seek to utilize them, given their relevance in the disease process.…”