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Abstract
In this study, we report how the cholera toxin (CT) A subunit (CTA), the enzyme moiety responsible for signaling alteration in host cells, enters the exosomal pathway, secretes extracellularly, transmits itself to a cell population. The first evidence for long-term transmission of CT’s toxic effect via extracellular vesicles was obtained in Chinese hamster ovary (CHO) cells. To follow the CT intracellular route towards exosome secretion, we used a novel strategy for generating metabolically-labeled fluorescent exosomes that can be counted by flow cytometry assay (FACS) and characterized. Our results clearly show the association of CT with exosomes, together with the heat shock protein 90 (HSP90) and Protein Disulfide Isomerase (PDI) molecules, proteins required for translocation of CTA across the ER membrane into the cytoplasm. Confocal microscopy showed direct internalization of CT containing fluorescent exo into CHO cells coupled with morphological changes in the recipient cells that are characteristic of CT action. Moreover, Me665 cells treated with CT-containing exosomes showed an increase in Adenosine 3’,5’-Cyclic Monophosphate (cAMP) level, reaching levels comparable to those seen in cells exposed directly to CT. Our results prompt the idea that CT can exploit an exosome-mediated cell communication pathway to extend its pathophysiological action beyond an initial host cell, into a multitude of cells. This finding could have implications for cholera disease pathogenesis and epidemiology.
Highlights
In the 21st century, cholera remains an epidemic or endemic disease in many parts of the world
Our results clearly show the association of cholera toxin (CT) with exosomes, together with the heat shock protein 90 (HSP90) and Protein Disulfide Isomerase (PDI) molecules, proteins required for translocation of CTA across the ER membrane into the cytoplasm
We previously reported that Cav-1, a structural component of caveolae formation, is highly expressed in human metastatic melanoma cell lines, and is retrieved in isolated fractions of extracellular vesicles (EV) [16]
Summary
In the 21st century, cholera remains an epidemic or endemic disease in many parts of the world. CT is made up of two major subunits, A and B [2], similar to other members of the AB5 family of toxins, and, once secreted by bacteria as a holotoxin, enters host cells by hijacking endogenous internalization and intracellular trafficking pathways, culminating in the induction of toxicity [3]. Exosomes (exo) are vesicles of 30–150 nm diameter that are secreted by cells into their environment They are generated by inward budding of endosomal membranes to form multivesicular bodies (MVBs). The lethal factor (LF) of Anthrax toxin, a major Bacillus anthracis virulence factor, is translocated into the lumen of endosomal intraluminal vesicles (ILVs). It persists in them for days, and can be transmitted to neighboring cells via exosomes [13]. To follow the transport of CT along the MVB/exosome route, we take advantage of a new methodology based on the fluorescent labeling of the phospholipid bilayer of exosomes that enabled us to trace and quantify exosome secretion [15]
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