Outer Membrane Vesicles of Vibrio cholerae Protect and Deliver Active Cholera Toxin to Host Cells via Porin-Dependent Uptake

Abstract

Outer membrane vesicles (OMVs) are an emerging research field due to their multifactorial composition and involvement in interspecies and intraspecies communication. Recent studies indicate that vesicle release by Gram-negative bacterial pathogens is increased during in vivo colonization, as exemplified by the facultative human pathogen Vibrio cholerae upon oral ingestion by the host. In this study, we investigate the fate of OMVs produced by the Gram-negative facultative pathogen V. cholerae. We show that vesicles produced by the clinically relevant El Tor biotype are readily taken up by human intestinal cell lines. We identify outer membrane porins of V. cholerae, i.e., OmpU and OmpT, as the required surface effectors on OMVs for cellular uptake, and we pinpoint the uptake mechanism as caveolin-mediated endocytosis. Furthermore, we show that OMVs derived from V. cholerae grown under virulence-inducing conditions act as potent vehicles for delivery of bioactive cholera toxin to intestinal epithelial cells. In contrast to free cholera toxin secreted via the type II secretion system, OMV-associated cholera toxin is protected from degradation by intestinal proteases. Taken together, these data show that OMV-associated cholera toxin can sustain longer periods in the intestinal tract and preserve toxin effects, as indicated by a prolonged increase of cAMP levels in the intestinal tissue. IMPORTANCE Cholera is still a massive global health burden because it causes large outbreaks with millions of infections and thousands of deaths every year. Several studies have contributed to the knowledge of this pathogen, although key parts are still missing. We aim to broaden our understanding of Vibrio cholerae infections, virulence, and toxicity by drawing attention to the involvement of OMVs in these core processes. Upon host entry, V. cholerae increases secretion of OMVs, which can carry the main virulence factor, cholera toxin, to distant host intestinal cells. We show that specific outer membrane porins on the vesicle surface mediate endocytosis of the vesicles into intestinal cells. With protection by the vesicles, cholera toxin activity endures even in the presence of intestinal proteases. It is tempting to hypothesize that the extended half-life of vesicle-associated cholera toxin allows it to target host cells distant from the primary colonization sites.

Extracellular vesicles of the probiotic E. coli O83 activate innate immunity and prevent allergy in mice

“…The human lung epithelial cell line A549 was used for the uptake assay. Cells were cultured according to the published protocols (36)(37)(38) using the appropriate culture media (Table S1). Culture conditions, stimulation conditions, and methods for analysis, as well as the methodology for the internalisation assay are given in the Appendix.…”

Extracellular Vesicles: Recent Insights Into the Interaction Between Host and Pathogenic Bacteria

“…Bacteria exploit bEVs as an alternative secretory pathway to cover the shortage of other secretory systems, and the mechanisms by which bEV-associated toxins enter host cells may be completely different ( 45 ). Compared with free toxin, bEVs provide protection against digestion by intestinal proteases, thus allowing the bacteria to target host cells distant from the primary colonization sites ( 46 ). BEVs are able to travel through barriers.…”

In vitro and ex vivo modeling of enteric bacterial infections

“… 112 , 113 Alternatively, cholera toxin can also be delivered to intestinal epithelial cells in outer membrane vesicles that protect it from intestinal proteases. 114 Upon entry, the catalytic subunit of cholera toxin ADP-ribosylates the α subunit of GTP-binding proteins (G s ) to irreversibly inhibit its GTPase activity, resulting in constitutive activation of adenylate cyclase. Excessive cyclic AMP concentrations then activate protein kinase A (PKA), which phosphorylates its target, the chloride channel CFTR, leading to Cl − , HCO3 − and water efflux.…”