Permissible peptide insertions surrounding the signal peptide-mature protein junction of the ClpG prepilin: CS31A fimbriae of Escherichia coli as carriers of foreign sequences

Vartanian, Maurice Der; Méchin, Marie-Claire; Jaffeux, Bernard; Bertin, Yolande; Félix, Isabelle; Gaillard-Martinie, Brigitte

doi:10.1016/0378-1119(94)90229-1

Cited by 18 publications

(28 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The explanation for the slight effect of ClpG may be that it improved secretion by acting on the three-dimensional structure of its cognate leader peptide for correct inner membrane presentation, allowing optimal signal peptidase recognition. Conversely, ClpG alone was previously shown to be extracellularly undetectable in the absence of the CS31A fimbrial helper proteins (20). Altogether, these data suggest that mature STh might be able to gain access to the extracellular milieu upon its entry into the E. coli periplasm, once guided into this compartment by the ClpG leader peptide.…”

Section: Discussionsupporting

confidence: 52%

See 1 more Smart Citation

Extracellular DsbA-insensitive Folding of Escherichia coli Heat-stable Enterotoxin STa in Vitro

Batisson

Vartanian²

2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

To study the folding of human Escherichia coli heatstable enterotoxin STh, we used the major protein subunit of CS31A fimbriae (ClpG) as a marker of STh secretion and a provider of a signal peptide. We established that STh genetically fused to the N or C terminus of ClpG was able to mobilize ClpG to the culture supernatant while still retaining full enterotoxicity. These features indicate that the STh activity was not altered by the chimeric structure and suggest that spatial conformation of STh in the fusion is close to that of the native toxin, thus permitting recognition and activation of the intestinal STh receptor in vivo. In contrast to other studies, we showed that disulfide bond formation did not occur in the periplasm through the DsbA pathway and that there was no correlation between DsbA and secretion, folding, or activity. This discrepancy was not attributable to the chimeric nature of STh since there was no effect of dsbA or dsbB mutations on secretion and activity of recombinant STh from which ClpG had been deleted. Periplasmic and lysate fractions of dsbA ؉ and dsbA ؊ cells did not have any STh activity. In addition, the STh chimera was exclusively found in an inactive reduced form intracellularly and in an active oxidized form extracellularly, irrespective of the dsbA background. Subsequently, a time course experiment in regard to the secretion of STh from both dsbA ؉ and dsbA ؊ cells indicated that the enterotoxin activity (proper folding) in the extracellular milieu increased with time. Overall, these findings provide evidence that STa toxins can be cell-released in an unfolded state before being completely disulfide-bonded outside the cell.Escherichia coli heat-stable enterotoxin STa (also referred to as STI) is responsible for serious and sometimes fatal diarrheal disease in both humans and domestic animals (1). STa causes intestinal fluid secretion by binding to its cellular receptor, transmembrane guanylyl cyclase C (2), thus activating guanylyl cyclase C and leading to an increase in intracellular mucosal cGMP. This increase in cGMP adversely affects electrolyte flux in the intestine; sodium absorption is inhibited, and chloride secretion is stimulated. These ion flux changes result in the secretory diarrhea characteristic of enterotoxigenic E. coli infection (3, 4). The receptor binding and enterotoxic properties of STa have been mapped to a highly conserved 13-amino acid C-terminal core sequence essential for toxicity and the heatstable nature of the toxin. Six cysteine residues are present within this domain, and the three intramolecular disulfide bonds formed between the cysteine residues are necessary for toxicity of the molecule (5). The two species of STa, STp and STh, from porcine (or bovine) and human strains of enterotoxigenic E. coli, respectively, are typical extracellular peptides consisting of 18 (STp) or 19 (STh) amino acid residues (6, 7). STa is synthesized in the cytoplasm as a 72-amino acid precursor consisting of pre-, pro-, and mature STa regions (8, 9). The precursor i...

show abstract

Section: Discussionsupporting

confidence: 52%

“…1). Plasmid pHPCO838 was constructed from pDEV41155 (20) by in vitro site-directed mutagenesis (23) using mutagenic and selection primers. The former primer (5Ј-CGTGGCAGTAACTTATGTTAACTA-ATTGGCTTGA-3Ј) created a unique HpaI site and added a valine between the penultimate tyrosine and the last C-terminal residue (asparagine) of ClpG.…”

Section: Methodsmentioning

confidence: 99%

Extracellular DsbA-insensitive Folding of Escherichia coli Heat-stable Enterotoxin STa in Vitro

Batisson

Vartanian²

2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…One of the potential advantages of such a system is that repeated exposure of one or more epitopes along the length of the pilus should increase immunogenicity. The possible use of pili for such purposes is not a new idea (11,39,41,51), but many previous studies failed because insertions prevented polymerization of the pilus filament because of the unpredictable structural constraints. It will be interesting to determine the length and number of the foreign peptides that can be tagged onto PulG, whether there are other permissive sites for epitope insertion, and whether inserted peptides are immunogenic when delivered either on whole bacteria or in purified pili.…”

Section: Pseudopili or Real Pili?mentioning

confidence: 99%

Type IV-Like Pili Formed by the Type II Secreton: Specificity, Composition, Bundling, Polar Localization, and Surface Presentation of Peptides

Vignon

Köhler

Larquet³

et al. 2003

J Bacteriol

118

148

View full text Add to dashboard Cite

The secreton or type II secretion machinery of gram-negative bacteria includes several type IV pilin-like proteins (the pseudopilins) that are absolutely required for secretion. We previously reported the presence of a bundled pilus composed of the pseudopilin PulG on the surface of agar-grown Escherichia coli K-12 cells expressing the Klebsiella oxytoca pullulanase (Pul) secreton genes at high levels (N. Sauvonnet, G. Vignon, A. P. Pugsley, and P. Gounon, EMBO J. 19:2221-2228, 2000). We show here that PulG is the only pseudopilin in purified pili and that the phenomenon is not restricted to the Pul secreton reconstituted in E. coli or to PulG. For example, high-level expression of the endogenous E. coli gsp secreton genes caused production of bundled pili composed of the pseudopilin GspG, and the Pul secreton was able to form pili composed of PulG-like proteins from secreton systems of other bacteria. PulG derivatives in which the C terminus was extended by the addition of eight different peptides were also assembled into pili and functioned in secretion. Three of the C-terminal peptides were shown to be exposed along the entire length of the assembled pili. Hence, the C terminus of PulG may represent a permissive site for the insertion of immunogenic epitopes or other peptide sequences. One of these PulG variants, with a six-histidine tag at its C terminus, formed nonpolar, nonbundled pili, suggesting that bundle formation and polar localization are not correlated with the ability of PulG to function in secretion. We propose that the PulG pilus is an artifactual manifestation of a periplasmic "pseudopilus" and that cycles of pseudopilus extension and retraction within the periplasm propel pullulanase through secretin channels in the outer membrane. Abnormally long pili that extend beyond the outer membrane are produced only when pilus length control and retraction are deregulated by overproduction of the major pseudopilus subunit (PulG).The secreton or type II secretion (T2S) system permits the energy-dependent secretion of a limited number of specific proteins from the periplasm in gram-negative bacteria (33). Many of the 12 or more secreton components share extensive sequence similarity with components of the type IV piliation (T4P) system in gram-negative bacteria (24, 33). In the T2S system, these "shared" components include the pilins themselves (called pseudopilins in the secreton system) and prepilin peptidase, the enzyme that removes a short N-terminal peptide and then N-methylates type IV pilin precursors (25,29,35,37) and type IV pilin-like proteins. The N-terminal prepeptide and approximately 20-residue hydrophobic domains of the type IV pilins and pseudopilins are highly conserved (24, 33). In the pullulanase (PulA)-specific secreton from Klebsiella oxytoca, there are five type IV pseudopilins (PulG, PulH, Pull, PulJ, and PulK) (31,36,40).In the absence of any direct evidence for the assembly of pseudopilins into a pilus (34, 38), they were proposed to form a small, intraperiplasmic structure (the ...

show abstract

“…Recently, ClpG, the major subunit of E. coli CS31A fimbriae, has been shown to accept various virus epitopes without affecting CS31A formation (6,10,11,27). In this study, we exploited ClpG as a provider of a signal peptide and an extracellular export carrier for the active cysteine-containing passenger peptide, the small human heat-stable enterotoxin STh of E. coli.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, formation of CS31A-STh fimbriae at the cell surface of recombinant E. coli DH5␣ strains was examined by adhesion assay on monolayers of Intestine-407 cells and by electron microscopy after immunogold labeling with primary rabbit anti-ClpG antibodies and secondary goat anti-rabbit colloidal gold-labeled IgG (Sigma). The adherence and immunolabeling assays were performed as previously described by Di Martino et al (13) and Der Vartanian et al (10), respectively. Strains DH5␣(pEH524) and DH5␣(pDSPH524) were used as positive and negative controls, respectively.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2000

Self Cite

View full text Add to dashboard Cite

We have successfully used the major subunit ClpG of Escherichia 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 (disulfidebonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH 2 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 of E. 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.

show abstract

Permissible peptide insertions surrounding the signal peptide-mature protein junction of the ClpG prepilin: CS31A fimbriae of Escherichia coli as carriers of foreign sequences

Cited by 18 publications

References 32 publications

Extracellular DsbA-insensitive Folding of Escherichia coli Heat-stable Enterotoxin STa in Vitro

Extracellular DsbA-insensitive Folding of Escherichia coli Heat-stable Enterotoxin STa in Vitro

Type IV-Like Pili Formed by the Type II Secreton: Specificity, Composition, Bundling, Polar Localization, and Surface Presentation of Peptides

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

Contact Info

Product

Resources

About