Bacterial surface association of heat-labile enterotoxin through lipopolysaccharide after secretion via the general secretory pathway

Abstract

Heat-labile enterotoxin (LT) is an important virulence factor expressed by enterotoxigenic Escherichia coli. The route of LT secretion through the outer membrane and the cellular and extracellular localization of secreted LT were examined. Using a fluorescently labeled receptor, LT was found to be specifically secreted onto the surface of wild type enterotoxigenic Escherichia coli. The main terminal branch of the general secretory pathway (GSP) was necessary and sufficient to localize LT to the bacterial surface in a K-12 strain. LT is a heteromeric toxin, and we determined that its cell surface localization was mediated by the its B subunit independent of an intact G(M1) ganglioside binding site and that LT binds lipopolysaccharide and G(M1) concurrently. The majority of LT secreted into the culture supernatant by the GSP in E. coli associated with vesicles. Only a mutation in hns, not overexpression of the GSP or LT, caused an increase in vesicle yield, supporting a specific vesicle formation machinery regulated by the nucleoid-associated protein HNS. We propose a model in which LT is secreted by the GSP across the outer membrane, secreted LT binds lipopolysaccharide via a G(M1)-independent binding region on its B subunit, and LT on the surface of released outer membrane vesicles interacts with host cell receptors, leading to intoxication. These data explain a novel mechanism of vesicle-mediated receptor-dependent delivery of a bacterial toxin into a host cell.

Fig. 1. Detection of cell surface-associated LT

A, wild type ETEC and mutant cells grown to log phase were incubated with BODIPY-G_M1. Fluorescence was determined, and values were normalized to cell number (RFU/CFU). Data shown are from one reproducible representative experiment. n ≥ 3. B, as indicated, HNS and Δhns K-12-expressing GSP, ΔGSP, LT, LTB, and/or LTB_G33D from plasmids were incubated with BODIPY-G_M1, and RFU/CFU was determined as in A. Error bars = mean ± S.E.; n ≥ 3. C, immunoprecipitation of cells because of surface-associated LTB. Values shown are fold over nonspecific settling seen in the background strain (Δhns/GSP). Error bars = ± S.E.; n = 7.

Fig. 2. Binding of exogenous LT to the surface of LT-deficient ETEC

A, immunoblot of LT bound to untreated or pretreated E9034P. Incubation with LT and cell pretreatments with Pronase or inhibitor-inactivated Pronase are indicated. B, densitometric analysis of immunoblots of E9034P incubated with buffer (no LT), LT and buffer (B), or LT preincubated with the following compounds: galactose (Gal); phosphatidylglycerol (PG); phosphatidylethanolamine (PE); O55 LPS (O55); Ra LPS (Ra); Lipid A (A); and monosialoganglioside (G_M1). Error bars = mean ± S.E.; n ≥ 3. *, p < 0.005 by Student’s t test.

Fig. 3. Binding to LPS does not affect LT toxicity

LT preincubated with buffer or an excess of LPS or G_M1 in the amounts indicated was applied to Y1 cells and scored for toxicity. Black bars, 6.0 nmol LT; gray bars, 600 pmol LT; hatched bars, 60 pmol LT. Error bars = mean ± S.E.; n = 3.

Fig. 4. GSP-secreted LT is associated with vesicles and vesicle production is HNS-regulated

A, toxicity of total and size-fractionated (100-kDa filtrate) cell-free supernatants from cultures of Δhns/GSP/LT (n = 16) (black bars) and Δhns/GSP (n = 8) (gray bars). Error bars = mean ± S.E. Inset, standard curve of LT toxicity on Y1 cells. Error bars = mean ± S.E.; n ≥ 6. B, vesicle yields per CFU of indicated strains compared with Δhns. Error bars = mean ± S.E.; n = 6.

Fig. 5. Model of LT secretion across the outer membrane of ETEC and interaction between vesicles carrying LT and host cells

After secretion by the GSP, LT binds to LPS on the cell surface via LTB. Consequently, vesicles released from the cell have LT on their surface that can act as a tether between G_M1 on the host cell and LPS on the vesicle. Internalization of vesicles and their associated LT cause toxicity in the host cell. Steps are outlined under “Discussion.”