Analysis of Intracellular Trafficking of Cholera Toxin B Subunits Depending on Ganglioside GM1 in Tissue-Resident Macrophages.

Cholera toxin (CT) is a bacterial exotoxin secreted by Vibrio cholerae. CT is comprised of one toxic A subunit (CTA) and five nontoxic B subunits (CTB). CTB is required for internalization of CT through binding with a membrane glycolipid ganglioside GM1. Previously, we clarified that CTB could induce production of proinflammatory cytokine interleukin-1β (IL-1β) from murine resident peritoneal macrophages (RPMs) in synergy with lipopolysaccharide (LPS) through a ganglioside GM1-dependent manner. Then, CTB was incorporated into endoplasmic reticulum (ER) depending on GM1 and activate ER stress sensor, inositol-requiring enzyme 1 alpha (IRE1α). IRE1α was required for both ER stress responses and IL-1β production in CTB- plus LPS-stimulated RPMs. These findings indicate that ganglioside GM1 functions not only for cell-surface receptor for CTB but also for cross talk with the ER stress sensor, IRE1α, in RPMs. Here, we describe the protocols for analysis of intracellular trafficking of CTB depending on ganglioside GM1 in RPMs.