(319h) Association with Outer Membrane Vesicles Drastically Alters Bacterial Toxin Activity

Historically, most bacterial protein toxins have been purified and studied in their water-soluble, free form. The mechanisms of these toxins and their roles in disease have been well-established. However, it has recently been observed that many bacterial toxins can also be released in association with outer membrane vesicles (OMVs), which are produced from the outer membrane of Gram negative bacteria. Our lab has studied the activity of two bacterial toxins that are associated with OMVs and found significant differences in the delivery and activity of both when they are associated with OMVs compared to when they are free. Vibrio cholerae, the causative agent of cholera, secretes cholera toxin (CT), which binds to the ganglioside GM1 on host cells to initiate its toxic mechanism. We have discovered that most of the secreted CT is actually associated with OMVs, not in its free form, and in its OMV-associated form, the toxin does not bind to GM1. This finding highlights the need to understand delivery of this toxin in its OMV-associated form, as inhibitors of GM1 binding by CT are unlikely to be effective anti-virulence strategies. Similarly, the oral bacterium, Aggregatibacter actinomycetemcomitans, secretes a leukotoxin (LtxA) in a water-soluble form. LtxA recognizes cholesterol and an integrin receptor, LFA-1, on host cells. Because LFA-1 is only expressed by human immune cells, this toxin (in its water-soluble form) has been reported to be active only against these specific cells. However, OMV-associated LtxA is delivered to cells in an LFA-1-independent manner and is active in cells that do not express LFA-1. Importantly, this finding demonstrates that the activity of this toxin is much greater than has been suggested in the past. Together, our results indicate that bacteria package protein toxins in vesicles to enable long-distance delivery and to expand the range of activity of the toxin. In order to fully characterize the virulence of pathogenic organisms, it is essential to study these proteins in their vesicle-associated form, as well as their free form.