Adhesin degradation accelerates delivery of heat-labile toxin by enterotoxigenic Escherichia coli

Abstract

Many enteric pathogens, including enterotoxigenic Escherichia coli (ETEC), produce one or more serine proteases that are secreted via the autotransporter (or type V) bacterial secretion pathway. These molecules have collectively been referred to as SPATE proteins (serine protease autotransporter of the Enterobacteriaceae). EatA, an autotransporter previously identified in ETEC, possesses a functional serine protease motif within its secreted amino-terminal passenger domain. Although this protein is expressed by many ETEC strains and is highly immunogenic, its precise function is unknown. Here, we demonstrate that EatA degrades a recently characterized adhesin, EtpA, resulting in modulation of bacterial adhesion and accelerated delivery of the heat-labile toxin, a principal ETEC virulence determinant. Antibodies raised against the passenger domain of EatA impair ETEC delivery of labile toxin to epithelial cells suggesting that EatA may be an effective target for vaccine development.

FIGURE 1.

EatA modulates epithelial cell adhesion. a, schematic of EatA protein structure showing from left to right the signal peptide (black), the passenger domain (open) with the site of serine protease catalytic triad (His-134, Asp-162, and Ser-267), and the β-barrel transport domain (gray). b, Caco-2 cell adherence assays showing adherence by ETEC wild type strain H10407 (wt) or the eatA mutant (jf904) complemented with empty vector plasmid (pSB001), plasmids expressing either rEatA (pSP014), or mutant protein bearing a mutation in the putative serine protease motif (pSP019). Shown below each strain are immunoblots of corresponding TCA-precipitated culture supernatants demonstrating production of EatA protein. c, addition of exogenous recombinant EatA passenger domain (rEatAp), but not the mutant protein rEatAp(H134R) restores adherence to wild type levels (ø = no protein added). d, antibodies against the EatA passenger domain alter adherence of H10407 to target cells. Shown are total cell-associated bacteria shown in presence (+) or absence (−) of affinity-purified antibody (α) directed against the EatA passenger. * denote p values determined by two-tailed t test [unpaired]; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).

FIGURE 2.

EatA regulates colonization of murine small intestine. Intestinal colonization at 24 h (a) or 72 h (b) following infection. c, competition assay between jf876 (lacZ::KmR) and jf904 (eatA::CmR). The competitive index = ((mutant (CmR)/wild type (KmR))output cfu/(mutant/wild type) input cfu). Fecal shedding of organisms at 48 h (d),and 72 h (e) following infection. a, b, d, and e involved administration of 1.5 × 10⁷ cfu of either jf876 an H10407 derivative (lacZ::KmR) or strain jf904 (eatA::CmR). Competition assay in c involved co-administration of ≈1 × 10⁴ cfu of both strains.

FIGURE 3.

EatA binds and degrades the EtpA adhesin molecule. a, fluorescently stained (SyproRuby) SDS-polyacrylamide gel of supernatant proteins concentrated from WT bacteria (left) and the eatA mutant (right). Arrowhead indicates migration of EatA passenger protein in the parent, and brace shows accumulated EtpA glycoprotein in the mutant following growth in Luria broth for 7 h. b, EtpA immunoblots demonstrating accumulation of EtpA exoprotein in (TCA-precipitated) culture supernatants of the eatA jf904 mutant (ΔeatA) relative to the wild type ETEC H10407 strain (wt) or the mutant complemented with a plasmid expressing the WT eatA gene in trans (pSP014). ø = no antibody added, c, growth curves of eatA⁺ (WT) and eatA⁻ (ΔeatA) strains. d, far Western blot showing binding of mutant (H134R) EatA passenger domain to EtpA-myc-His. Addition of serine protease inhibitor, 4-amidinophenylmethanesulfonyl fluoride hydrochloride (APMSF) (e) or affinity-purified polyclonal antibodies directed against the EatA passenger domain (αEatAp) (f) prevents degradation of EtpA by the EatA protease. Mutation of the putative serine protease motif (H134R) prevents degradation of EtpA when compared with equimolar amounts of WT EatA protein over time (g) or over a range of concentrations (h).

FIGURE 4.

EatA accelerates delivery of heat-labile toxin. a, ganglioside-binding ELISA demonstrating production of heat-labile toxin by H10407 (wt), the eatA mutant, and negative control (eltA) strain, which contains a mutation in the genes for LT. b, activation of cAMP in target Caco-2 intestinal epithelial cells by WT ETEC, eatA strain, and eatA mutant complemented with plasmid expressing either the recombinant EatA protein (pSP014) or the mutant serine protease-deficient protein pSP019 (c) accelerated epithelial cell (Caco-2) cAMP activation induced by the WT strain relative to the mutant or mutant complemented with plasmid expressing the serine protease-deficient protein. The eltA mutant (jf571) is shown as a negative control. d, antibody against EatA passenger domain inhibits cAMP activation in target epithelial cells. Data in b and d were obtained at t = 2 h post-infection. (ø = no antibody added; α-EatAp refers to purified polyclonal rabbit antibodies raised against the EatA passenger domain; α-control refers to antibody purified from preimmune sera obtained from the same rabbit.

FIGURE 5.

EatA does not directly activate host cAMP or enhance toxin processing. a, effect of exogenously added LT, rEatAp, or the combination of the two molecules together on cAMP activation in target Caco-2 cells. b, rEatAp does not nick LT to promote toxicity. Shown is a Coomassie-stained gel of SDS-PAGE-separated proteins following incubation of LT with EatA passenger domain or trypsin. Approximate molecular weights are shown at left and the predicted proteins are shown at right. The 1st lane at left contains LT incubated with trypsin as a positive control for proteolytic cleavage of A into A1 and A2 domains.

FIGURE 6.

eatA mutants exhibit altered adherence architecture in vitro. In confocal microscopy images, wild type ETEC H10407 adheres to Caco-2 intestinal cells as single organisms or small groups of a few organisms/cell (a–d), whereas eatA mutants form large clusters loosely attached to the cell surface (e–h), ×40 oil. Shown in i and j are individual frames from z-stack images acquired at ×100 corresponding to center of fields shown in e–h. DIC, differential interference contrast.