Abstract
Cholera toxin (CT), the major virulence factor of Vibrio cholerae, is an AB5 toxin secreted through the type II secretion system (T2SS). Upon secretion, the toxin initiates endocytosis through the interaction of the B pentamer with the GM1 ganglioside receptor on small intestinal cells. In addition to the release of CT in the free form, the bacteria secrete CT in association with outer membrane vesicles (OMVs). Previously, we demonstrated that strain 569B releases OMVs that encapsulate CT and which interact with host cells in a GM1-independent mechanism. Here, we have demonstrated that OMV-encapsulated CT, while biologically active, does not exist in an AB5 form; rather, the OMVs encapsulate two enzymatic A-subunit (CTA) polypeptides. We further investigated the assembly and secretion of the periplasmic CT and found that a major fraction of periplasmic CTA does not participate in the CT assembly process and instead is continuously encapsulated within the OMVs. Additionally, we found that the encapsulation of CTA fragments in OMVs is conserved among several Inaba O1 strains. We further found that under conditions in which the amount of extracellularly secreted CT increases, the concentration of OMV-encapsulated likewise CTA increases. These results point to a secondary mechanism for the secretion of biologically active CT that does not depend on the CTB-GM1 interaction for endocytosis.
Highlights
Cholera is an acute and often fatal diarrheal disease that is caused by effective colonization of Vibrio cholerae in the small intestine [1,2,3]
Our previous studies on Classical V. cholerae 569B demonstrated that cholera toxin (CT) is secreted under low-salt conditions; because a majority of the toxin is encapsulated within the outer membrane vesicles (OMVs) and is not located on the surface of the vesicles, it is not detectable with the GM1 ELISA
These results indicate that the encapsulation of composed of an enzymatic A-subunit (CTA) within the vesicles increases detected in the disrupted OMVs
Summary
Cholera is an acute and often fatal diarrheal disease that is caused by effective colonization of Vibrio cholerae in the small intestine [1,2,3]. The unfolded CTA1 triggers the ER-associated degradation (ERAD) machinery to retro-translocate to the cytosol, where it activates adenylate cyclase, which in turn increases the cyclic AMP (cAMP) level, causing activation of the cystic fibrosis transmembrane conductance regulator (CFTR) This leads to an enhanced efflux of chloride ions and water into the intestinal lumen, inducing watery diarrhea [13,14,15,16]. In our previous study on the V. cholerae 569B strain (Classical), we demonstrated that under a low-salt growth condition, the majority of the secreted CT is in an OMV-associated form and is located exclusively inside the vesicles. This location prevents the toxin from being detected by conventional CT-detection assays. We assessed the strain-dependence of our results by analyzing OMVs from two pandemic O1 El Tor strains, C6706 and N16961
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