The A Subunit of Type IIb Enterotoxin (LT-IIb) Suppresses the Proinflammatory Potential of the B Subunit and Its Ability to Recruit and Interact with TLR2

Abstract

The type IIb heat-labile enterotoxin of Escherichia coli (LT-IIb) and its nontoxic pentameric B subunit (LT-IIb-B5) display different immunomodulatory activities, the mechanisms of which are poorly understood. We investigated mechanisms whereby the absence of the catalytically active A subunit from LT-IIb-B5 renders this molecule immunostimulatory through TLR2. LT-IIb-B5, but not LT-IIb, induced TLR2-mediated NF-κB activation and TNF-α production. These LT-IIb-B5 activities were antagonized by LT-IIb; however, inhibitors of adenylate cyclase or protein kinase A reversed this antagonism. The LT-IIb antagonistic effect is thus likely dependent upon the catalytic activity of its A subunit, which causes elevation of intracellular cAMP and activates cAMP-dependent protein kinase A. Consistent with this, a membrane-permeable cAMP analog and a cAMP-elevating agonist, but not catalytically defective point mutants of LT-IIb, mimicked the antagonistic action of wild-type LT-IIb. The mutants moreover displayed increased proinflammatory activity compared with wild-type LT-IIb. Additional mechanisms for the divergent effects on TLR2 activation by LT-IIb and LT-IIb-B5 were suggested by findings that the latter was significantly stronger in inducing lipid raft recruitment of TLR2 and interacting with this receptor. The selective use of TLR2 by LT-IIb-B5 was confirmed in an assay for IL-10, which is inducible by both LT-IIb and LT-IIb-B5 at comparable levels; TLR2-deficient macrophages failed to induce IL-10 in response to LT-IIb-B5 but not in response to LT-IIb. These differential immunomodulatory effects by LT-IIb and LT-IIb-B5 have important implications for adjuvant development and, furthermore, suggest that enterotoxic E. coli may suppress TLR-mediated innate immunity through the action of the enterotoxin A subunit.

LT-IIc, a New Member of the Type II Heat-Labile Enterotoxin Family Encoded by an Escherichia coli Strain Obtained from a Nonmammalian Host

“…Alignments revealed that the 27.31-kDa polypeptide of OS-1 was homologous to the A polypeptides of type II HLTs and to the A polypeptides of type I HLTs (Table 2). Amino acids Arg7, Asp9, His44, Ser61, Val97, Tyr104, Pro106, His107, Glu110, and Glu112, which are crucial for the ADP-ribosylating activity of CT and LT-I (35) and LT-IIb (19), were identified in the 27.31-kDa polypeptide, albeit shifted by two amino acid positions (Fig. 4).…”

Pathogen induction of CXCR4/TLR2 cross-talk impairs host defense function

“…However, upon Ab-mediated neutralization of IL-10, we did not observe significant reversal of the inhibitory effects (not shown). We then turned our attention to cAMP because inhibition of NF-B and TNF-␣ and concomitant augmentation of IL-10 are reminiscent of the immunomodulatory action of cAMP-inducing enterotoxins, as we previously observed (17).…”

“…Levels of cAMP in activated cell extracts were measured by using a cAMP enzyme immunoassay kit (Cayman Chemical) (17). Induction of PKA activity was determined by using lysates of activated cells and the ProFluor PKA assay (Promega).…”

Toll-Like Receptor 2-Mediated Interleukin-8 Expression in Gingival Epithelial Cells by the Tannerella forsythia Leucine-Rich Repeat Protein BspA

“…BspA binding to TLR2. TLR2 binding was measured as previously described (18). Briefly, 96-well microtiter wells were coated overnight at 4°C with 20 g/ml of purified recombinant mouse TLR2 (R&D Systems, Minneapolis, MN).…”