Specificity of the type II secretion systems of enterotoxigenic Escherichia coli and Vibrio cholerae for heat-labile enterotoxin and cholera toxin

Abstract

The Gram-negative type II secretion (T2S) system is a multiprotein complex mediating the release of virulence factors from a number of pathogens. While an understanding of the function of T2S components is emerging, little is known about what identifies substrates for export. To investigate T2S substrate recognition, we compared mutations affecting the secretion of two highly homologous substrates: heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) from Vibrio cholerae. Each toxin consists of one enzymatic A subunit and a ring of five B subunits mediating the toxin's secretion. Here, we report two mutations in LT's B subunit (LTB) that reduce its secretion from ETEC without global effects on the toxin. The Q3K mutation reduced levels of secreted LT by half, and as with CT (T. D. Connell, D. J. Metzger, M. Wang, M. G. Jobling, and R. K. Holmes, Infect. Immun. 63:4091-4098, 1995), the E11K mutation impaired LT secretion. Results in vitro and in vivo show that these mutants are not degraded more readily than wild-type LT. The Q3K mutation did not significantly affect CT B subunit (CTB) secretion from V. cholerae, and the E11A mutation altered LT and CTB secretion to various extents, indicating that these toxins are identified as secretion substrates in different ways. The levels of mutant LTB expressed in V. cholerae were low or undetectable, but each CTB mutant expressed and secreted at wild-type levels in ETEC. Therefore, ETEC's T2S system seems to accommodate mutations in CTB that impair the secretion of LTB. Our results highlight the exquisitely fine-tuned relationship between T2S substrates and their coordinate secretion machineries in different bacterial species.

FIG. 1.

Three mutations impair the secretion of LT from ETEC. (A) Representative immunoblots of TCA-precipitated total culture samples, adjusted for CFU, showing induced expression of wild-type LT (WT), LT[L25E], LT[Q3K], and LT[E11K] in strain jf570. Blots were probed with cross-reactive anti-CT antibody. (B) Strains expressing wild-type (WT) and the indicated mutant toxins were fractionated to isolate cell-free supernatant (Sup't) and periplasm (Pp). Each fraction was tested for toxin levels by G_M1 ELISA, with wild-type levels set to 100%. Supernatant levels were normalized to CFU, and periplasm levels were normalized to alkaline phosphatase activity. *, P < 0.01; **, P < 0.005; ***, P < 10⁻⁶ compared to wild-type (n ≥ 3); n.d., not determined. For some experiments, cultures were grown in the presence of a protease inhibitor cocktail (+ prot. inh.) (n ≥ 2). Inset: representative immunoblot showing levels of wild-type LT (WT) and LT[L25E] secreted from jf570. Cell-free supernatant fractions were precipitated with TCA, and samples were adjusted for CFU. Blots were probed with cross-reactive anti-CT antibody.

FIG. 2.

LT secretion mutants show WT protease sensitivity and pentamer formation. (A) Representative protein gels showing degradation of WT or the indicated mutant toxin. Purified toxin (500 ng) was incubated with pronase at the indicated final concentration for 1 h at 37°C. Following the incubation, samples were boiled and separated by 15% SDS-PAGE, and the gel was stained with Ruby Red. (B) Densitometric measurements of the intensities of the bands corresponding to LTB on the protein gels described in the legend for panel A. The intensity of the band corresponding to untreated WT LTB or the indicated mutant was set to 100%. *, P < 0.05 compared to WT at the same concentration (n ≥ 2) (C) Purified WT LTB and the indicated mutants were dissociated into monomers by treatment with acid, and then neutralized and allowed to reassemble into pentamers for the indicated number of minutes or overnight (o/n). Pentamer reformation was assessed by G_M1 ELISA, with values normalized to levels of each pentamer without acid treatment (no acid). *, P < 0.01, compared to WT at the same time point (n ≥ 3).

FIG. 3.

Mutation of Glu-11 impairs CTB secretion from V. cholerae. (A) Representative immunoblot of TCA-precipitated total culture samples, adjusted for CFU, showing induced expression of WT CTB, CTB[Q3K], CTB[E11K], and CTB[E11A] in strain P4. Blots were probed with anti-CT antibody. (B) Strains expressing WT CTB and the indicated CTB mutants were fractionated to isolate cell-free supernatant (Sup't) and periplasm (Pp). Each fraction was tested for pentamer levels by G_M1 ELISA, with WT levels set to 100%. Supernatant levels were normalized to CFU, and periplasm levels were normalized to alkaline phosphatase activity. *, P < 0.05; **, P < 0.0005 compared to wild-type (n ≥ 3). For some experiments, cultures were grown in the presence of a protease inhibitor cocktail (+ prot. inh.). §, P < 0.05 compared to E11K supernatant levels without protease inhibitor (n ≥ 2). (C) Representative protein gels showing degradation of WT CTB or the indicated mutant pentamer. Purified pentamer (500 ng) was incubated with pronase at the indicated final concentration for 1 h at 37°C. Following the incubation, samples were boiled and separated by 15% SDS-PAGE, and the gel was stained with Ruby Red. (D) Densitometric measurements of the intensities of the CTB bands on the protein gels described in the legend for panel C. The intensity of the band corresponding to untreated WT or the indicated mutant CTB was set to 100% (n ≥ 2). (E) Zones of clearance formed by V. cholerae on skim milk agar. Strains expressing WT CTB and the indicated mutant pentamers were plated on skim milk agar and incubated for 36 h at 37°C.

FIG. 4.

Secretion of CTB mutants is not impaired in ETEC. (A) Representative immunoblot of TCA-precipitated total culture samples, adjusted for CFU, showing induced expression of WT CTB, CTB[Q3K], CTB[E11K], and CTB[E11A] in strain jf570. The blot was probed with anti-CT antibody. (B) Strains expressing WT CTB and the indicated mutant pentamers were fractionated to isolate cell-free supernatant (Sup't) and periplasm (Pp). Each fraction was tested for pentamer levels by G_M1 ELISA, with WT levels set to 100%. Supernatant levels were normalized to CFU (n ≥ 3), and periplasm levels were normalized to alkaline phosphatase activity (n ≥ 2).

FIG. 5.

LTB mutants express poorly in V. cholerae. Representative Ruby Red protein stain (top) and immunoblot (bottom) of TCA-precipitated total culture samples, adjusted for CFU, showing induced expression of a vector control (Vec), WT LTB, LTB[Q3K], LTB[Q3A], LTB[E11K], and LTB[L25E] in strain P4. The blot was probed with cross-reactive anti-CT antibody.