M. Contrepois

Full Capacity of Recombinant Escherichia coli Heat-Stable Enterotoxin Fusion Proteins for Extracellular Secretion, Antigenicity, Disulfide Bond Formation, and Activity

ABSTRACT We have successfully used the major subunit ClpG ofEscherichia coli CS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length. However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported. To address this possibility, we genetically fused peptides containing all or part of the E. coli human heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG. Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the E. coli cells in a heat-stable active oxidized (disulfide-bonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH2 or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity. Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner. These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition). Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the E. coli cell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis. None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface ofE. coli, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins. In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway. In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of E. coli for culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin.

An Escherichia coli CS31A fibrillum chimera capable of inducing memory antibodies in outbred mice following booster immunization with the entero-pathogenic coronavirus transmissible gastroenteritis virus

Abstract CS31A fibrillae are thin, flexible, heteropolymeric proteinaceous appendages exposed as a capsule-like material around the cell surface of certain Escherichia coli strains. Two antigenic peptides of the S spike glycoprotein (TGEV-S) amino acids (aa) 363–371 and 521–531 of the transmissible gastroenteritis virus (TGEV) were tandemly introduced in the loop-structured, variable region aa 202–218 of the major ClpG subunit protein composing the bulk of CS31A. The resulting hybrid fibrillae with a 25 aa heterologous peptide were produced at the cell surface. Using a monoclonal antibody (Mab) specific for the TGEV epitopes, purified hybrid fibrillae were analysed in Western blotting under native conditions, which showed that the two viral epitopes were recognized immunologically as an integral part of the hybrid fibrillae, and therefore that they were antigenically active. The immunogenicity of the fusion construct was evaluated with live recombinant bacteria, purified hybrid ClpG monomers, and purified chimeric CS31A polymers. Whatever the form of hybrid used as antigen, intraperitoneally immunized outbred mice elicited serum anti-TGEV peptides antibodies (Abs) with significant titres and capable of recognizing native TGEV particles, indicating that the epitopes are exposed in an immunogenic conformation in all cases. However, virus neutralization titres were only obtained after immunization with either purified polymers or monomers. Furthermore, 4 months after an ultimate immunization with 20 μg of hybrid fibrillae mice developed a strong anamnestic Ab response against the two TGEV peptides following booster inoculation with virions. We conclude that CS31A fibrillae carrying a combination of TGEV epitopes as insert can induce an immunological memory in outbred animals infected with TGEV, and therefore that hybrid CS31A fibrillae may prove efficient as components of a subunit vaccine.

A study of relationships among F17 a producing enterotoxigenic and non-enterotoxigenic Escherichia coli strains isolated from diarrheic calves

We investigated the clonal relationships among 41 enterotoxigenic (ETEC) or non-enterotoxigenic (NETEC) Escherichia coli strains producing the F17 a fimbriae isolated from diarrheic calves in France or Belgium in the early 1980s. Twenty-three of the 26 ETEC strains were highly clonally related, most of them with a O101:K32:H9-serotype. The NETEC strains were also divided in clonal subgroups, most of them with O101:H-serotype. The F17 a positive ETEC strains are no longer isolated from diarrheic calves in these countries. It is postulated that the use of a vaccine including O101, K32 and H9 antigens in addition to K99 (F5) explains the strongly reduced isolation of the O101:K32:H9, K99 (F5) E. coli clone.

Correlation between esterase electrophoretic polymorphism and virulence–associated traits in extra–intestinal invasive strains of Escherichia coli

The electrophoretic variations of carboxylesterase B and of esterases A, C and I, the presence of mannose resistant haemagglutinin, alpha-haemolysin, cytotoxic necrotizing factor type 1 (CNF1) and certain O antigens were compared in 150 strains of Escherichia coli responsible for extra-intestinal infections. Electrophoretic mobilities of outer membrane proteins (OMP) were also studied for strains belonging to O4, O6, O7, O8 and O75 serogroups. Fast migrating allozymes of carboxylesterase B (pattern B1) were correlated with slow migrating allozymes of esterase C, serogroups O7 and O8, lack of virulence factor, and particular OMP patterns, whereas slow migrating allozymes of carboxylesterase B (pattern B2) were correlated with fast migrating allozymes of esterase C, serogroups O2, O4, O6, O18 and O75, virulence factor production, and distinct OMP patterns. Allozymes of esterases A and I were not clearly correlated with the distribution of virulence factors. The pattern B2 was more strongly associated with CNF1 than with alpha-haemolysin and mannose resistant haemagglutinin. These results substantiate the view that the electrophoretic pattern B2 of carboxylesterase B identified most of the highly pathogenic strains implicated in extra-intestinal infection of humans.

Light and electronic microscopic studies of the changes in the intestinal mucosa of gnotobiotic calves infected with a wild rotavirus

Summary Experimental infection of four gnotobiotic calves by a rotavirus isolated from the faeces of diarrheic calves caused non-fatal diarrhoea without dehydration. Lesions of the alimentary mucosa appear well before the onset of diarrhoea and are characterized by a release of mucus in the anterior part of the intestine. By the time diarrhoea begin, different types of rotavirus particules are present in the enterocytes of the mucosa, and mucus contents of distal intestine are affected. At this stage of the disease, the virus causes epithelial cells in the small intestine to desquamate and changes the secretion of mucus in the small as well as the large intestine. The very intense virus multiplication is completed within 18 h. Intracellular rotavirus disappear very quickly after the onset of diarrhoea, and cannot be detected even during the acute phase of the disease. Regeneration of the mucosa takes place slowly, and the cellular and structural lesions are still visible more than 4 days after the diarrhoea stopped.