LT-IIc, a new member of the type II heat-labile enterotoxin family encoded by an Escherichia coli strain obtained from a nonmammalian host

Abstract

Two families of bacterial heat-labile enterotoxins (HLTs) have been described: the type I HLTs are comprised of cholera toxin (CT) of Vibrio cholerae, LT-I of Escherichia coli, and several related HLTs; the type II HLTs are comprised of LT-IIa and LT-IIb. Herein, we report LT-IIc, a new type II HLT encoded from an enterotoxigenic E. coli (ETEC) strain isolated from an avian host. Using a mouse Y1 adrenal cell bioassay, LT-IIc was shown to be less cytotoxic than CT, LT-IIa, or LT-IIb. Cytotoxicity of LT-IIc was partially neutralized by antisera recognizing LT-IIa or LT-IIb but not by anti-CT antiserum. Genes encoding putative A polypeptide and B polypeptides of LT-IIc were arranged in an operon which was flanked by potential prophage sequences. Analysis of the nucleotide and predicted amino acid sequences demonstrated that the A polypeptide of LT-IIc has moderate homology to the A polypeptides of CT and LT-I and high homology to the A polypeptides of LT-IIa and LT-IIb. The B polypeptide of LT-IIc exhibited no significant homology to the B polypeptides of CT and LT-I and only moderate homology to the B polypeptides of LT-IIa and LT-IIb. The binding pattern of LT-IIc for gangliosides was distinctive from that of either LT-IIa or LT-IIb. The data suggest that other types of the type II HLT subfamily are circulating in the environment and that host specificity of type II HLT is likely governed by changes in the B polypeptide which mediate binding to receptors.

FIG. 1.

Southern hybridizations of potential ETEC strains isolated from ostriches. HindIII-digested genomic DNAs isolated from four ETEC ostrich isolates (OS-1, OS-2, OS-3, and OS-4), E. coli SA53 (LT-IIa), E. coli Ec41 (LT-IIb), and E. coli DH5α (control strain) were hybridized with nucleotide probes homologous to genes encoding LT-IIa holotoxin (A), LT-IIb holotoxin (B), B polypeptide of LT-IIa (C), or B polypeptide of LT-IIb (D). The gene encoding the A polypeptide of LT-IIa contains a HindIII restriction site. Thus, digestion of SA53 genomic DNA with HindIII produced two hybridizing fragments. Southern hybridization conditions were chosen for moderately high stringency to detect DNA fragments having closely related nucleotide sequences.

FIG. 2.

SDS-PAGE of LT-IIc and related HLT. Purified holotoxins were resolved by sodium dodecyl sulface-polyacrylamide gel electrophoresis and stained with Coomassie blue dye. Lane 1, protein molecular mass standard (M); lane 2, CT; lane 3, LT-IIa-His₆; lane 4, LT-IIb-His₆; lane 5, LT-IIc-His₆. Molecular masses are indicated in kDa.

FIG. 3.

Schematic of the LT-IIc locus. An open reading frame encoding a potential bacteriophage lysozyme (33) is located 5′ of the LT-IIc locus. An open reading frame encoding a potential bacteriophage protein containing a ParB-like nucleotide binding domain (4) is positioned 3′ of the LT-IIc locus.

FIG. 4.

Multiple-sequence alignment of the predicted amino acid sequences of the A polypeptides of the type I and type II HLTs. Amino acid sequences were aligned by using Clustal 2.0.12 (http://www.ebi.ac.uk/Tools/clustalw2/index.html). pLT-IA, porcine LT-IA; hLT-IA, human LT-IA.

FIG. 5.

Multiple-sequence alignment of the predicted amino acid sequences of the B polypeptides of the type I and type II HLTs. Amino acid sequences were aligned by using Clustal 2.0.12 (http://www.ebi.ac.uk/Tools/clustalw2/index.html). Conserved Thr13, Thr14, Thr34, and Trp92 amino acids that are required for binding of the B pentamers of the type II HLT to gangliosides are underlined (5, 6, 35). The TLR2 interaction motifs [N₂-AMAA(I/V)LS-COOH] of the type II HLT are highlighted in gray.

FIG. 6.

Accumulation of cAMP. Peritoneal macrophages from naïve mice were treated with LT-IIa, LT-IIb, or LT-IIc. Data (arithmetic means ± standard errors of the means; n = 3) are reported as amounts of cAMP in intoxicated cells. *, statistical difference from the untreated group (P < 0.001).

FIG. 7.

Binding specificity of LT-IIc, LT-IIa, and LT-IIb for gangliosides. Polyvinyl microtiter plates were coated with 10 ng of purified ganglioside or with a mixture of gangliosides. Enterotoxins were incubated in the wells of the ganglioside-coated plates, after which the wells were probed with rabbit anti-LT-IIa, anti-LT-IIb, or anti-LT-IIc polyclonal antiserum. Microtiter plates were developed with alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibody and nitrophenyl phosphate and analyzed with a Versamax microplate reader at 405 nm. Results (absorbance at 405 nm) are reported as the means of three replicate experiments ± 1 standard error of the mean (SEM).