key: cord-0943365-i3mebqtq
authors: Der Vartanian, Maurice; Méchin, Marie-Claire; Jaffeux, Bernard; Bertin, Yolande; Félix, Isabelle; Gaillard-Martinie, Brigitte
title: Permissible peptide insertions surrounding the signal peptide-mature protein junction of the ClpG prepilin: CS31A fimbriae of Escherichia coli as carriers of foreign sequences
date: 1994-10-11
journal: Gene
DOI: 10.1016/0378-1119(94)90229-1
sha: 95349ecdf1b234c3a53dfd1c8dc9364a340aa0fe
doc_id: 943365
cord_uid: i3mebqtq

Abstract The clpG gene, expressing the Escherichia coli major CS31A fimbrial subunit ClpG, was subjected to random mutagenesis by insertion of an EcoRI linker and a kanamycin-resistance (KmR) cassette into the multiple newly generated EcoRI sites. The KmR gene was then excised by PstI, which left a 48-bp linker representing the heterologous sequence. The same procedure was followed to introduce a synthetic oligodeoxyribonucleotide (oligo) corresponding to epitope C from the spike protein S from the porcine transmissible gastroenteritis coronavirus (TGEV). Nine insertion/deletion mutants (indels) that contained long foreign peptides variously located around the ClpG signal peptide (SP) processing site were characterized. A striking feature of this study is the variety of amino acid (aa) insertions in the ClpG prepilin that have little or no effect on CS31A fimbria biogenesis. These ‘permissive’ sites tolerate inserts of 18 or 19 aa and accept sequences of different natures in view of their aa composition, charge and hydrophobicity. The results obtained here are also interesting in light of the high level of aa sequence conservation seen in the SP and N-terminal domains of the ClpG-related subunits. The structure-function relationship of the ClpG SP is discussed. The TGEV-C epitope fused to the N-terminal end of the mature ClpG protein was cell-surface exposed, as observed on immuno-electron microscopy. Therefore, the CS31A fimbria seems to be a potent tool for the presentation of foreign antigenic determinants or the production of heterologous polypeptides in E. coli.

the heterologous sequence. The same procedure was followed to introduce a synthetic oligodeoxyribonucleotide (oligo) corresponding to epitope C from the spike protein S from the porcine transmissible gastroenteritis coronavirus (TGEV). Nine insertion/deletion mutants (indels) that contained long foreign peptides variously located around the ClpG signal peptide (SP) processing site were characterized.

A striking feature of this study is the variety of amino acid (aa) insertions in the ClpG prepilin that have little or no effect on CS31A fimbria biogenesis. These 'permissive' sites tolerate inserts of 18 or 19 aa and accept sequences of different natures in view of their aa composition, charge and hydrophobicity. The results obtained here are also interesting in light of the high level of aa sequence conservation seen in the SP and N-terminal domains of the ClpG-related subunits. The structure-function relationship of the ClpG SP is discussed. The TGEV-C epitope fused to the N-terminal end of the mature ClpG protein was cell-surface exposed, as observed on immuno-electron microscopy. Therefore, the CS31A fimbria seems to be a potent tool for the presentation of foreign antigenic determinants or the production of heterologous polypeptides in E. coli. INTRODUCTION Polymer CS31A is a plasmid-encoded fibrillar protein associated with animal and human pathogenic isolates of Escherichia coli (Contrepois et al., 1989; Cherifi et al., CS3 I A biogcncsis ( Martin ct al., I99 I ). They include the ma_jor ClpG monomer and several accessory proteins involvcd in the stabilization, transport and assembly of CIpG. Extensive nt sequence homology throughout the accessory protein-encoding genes of CS31 A. KHH and F41 operons has been demonstrated by Southern hybridization analysis (Casey et al., 1990 : Martin ct al.. 1991 Korth ct al., 1991 ) . The accessory systems mediating cxpression of these operons may be functionally interchangeable, indicating that CS3lA. K88 and F41

fimbriae arc members of a closely related family ( Korth et al.. 1992) . The c<lpG gent codes for the ClpG precursor containing a typical Gram-bacterial 2l-XI SP, whose processing results in a mature polypeptide of 257 aa with a deduced hrl, of 26 777, and migrating as a 29-kDa protein in SDS-PAGE. The nt sequence of clpG has a homology of 60 and 30%. and its corresponding aa sequence an identity of 46 and 24"/0 with those of,firrG and f'41, which encode the major subunits FaeG and F4l composing the K88 and F41 fimbriae. respectively (Girardcau et al., 1991) . By analogy with the known K8X timbria biogenesis (Bakker, 1991) , it seems likely that the ClpG precursor is processed by signal peptidase 1. and exported across the cytoplasmic membrane through the Secdependent part of the general secretory pathway (see, for a review, Pugsley, 1993) . In contrast to the general divcrsity found in fimbrial signal sequences, a highly conserved (80%) SP was observed for ClpG, FaeG and F4l prepilins, with an identical cleavage site for ClpG and FaeG.

of the SP in concert with high homology in N-terminal domains of the ClpG. FaeG and F41 subunits suggests that extreme functional pressure is applied to conserve this sequence (Girardeau et al.. 1991) .

The aims of this study were to show that in spite of the extensive modifications engineered in ClpG SP. the resulting mutated prepilins are normally processed, and that a viral antigenic determinant inserted into ClpG prepilin between the residues -I and + I is presented at the E. co/i cell surface. RESULTS of the epitope C-oligo insert, and then complemented by pDSPH524 in E. co/i DHSa. Colonies with :I TGEV-C cpitope insertion were screened and analysed by in situ immunoblotting for CS3lA fimbria production and C epitope antigenicity.

All of the colonies reacted with anti-ClpG pAb but only 2"/;) with the neutralizing site-Cspecific 3b.5 mAb. The sequencing of plasmid-DNAs from clones immunoreactive against mAb 3b.5 indicated that all TGEV-C epitope insertions were located precisely between SP and mature ClpG. One of the clones, ClpG 21/22.3C. was selected for Western blot analysis with an anti-ClpG pAb and the mAb 3b.5, using PAGE both (Fig. 4C ). The method involved the epitope recognition by the 3b.5 mAb and its indirect labeling with goat anti-mouse immunoglobulin G (IgG) conjugated with gold particles. Taken together, these findings demonstrate that epitope C is properly exposed on the monomeric and oligomeric forms of the native hybrid protein at the E. coli cell-surface.

Of the remaining 98% of clones that reacted only with anti-ClpG pAb, some were analysed by plasmid-DNA sequencing. In all cases, the TGEV-C epitope-encoding oligo was inserted in-phase in the incorrect orientation and gave mutants ClpG 9/10. 

The examination of the aa sequence immediately upstream from the experimentally determined processing site (Girardeau et al., 1988; Fig. 5) shows that the 21-aa SP of the preClpG has a perfect similarity to typical Holegona, 1980). The ClpG SP is considerably different from the SP of type-IV pilins or homologues that are transported through a specialized Set-independent export machinery and processed by a cognate prepilin peptidase (see, for a review, Hobbs and Mattick, 1993).

We located by DNA sequencing nine mutations generated by random oligo-insertion mutagenesis which specifically altered regions surrounding the signal sequence of clpG without drastically affecting the CS31A fimbria biogenesis on the basis of colony-and Western immunoblotting (data not shown) or immuno-EM (only for ClpG 21/22.3C mutant; Fig. 4C ). In all cases, depending on the reading frame and surrounding nt, 18 or 19 aa were inserted in-phase into the different sites at positions -13/-12, -7/+8, -6/-l and -l/+1 (Fig.5) Fig. 5 and Table I ). The final modifications are insertions with or without deletions, which are referred to as indels (Betton et al., 1993) . Overall, these heterologous insertions introduced 447 charged aa residues, with an average net charge of -3 to + 1, and several hydrophilic aa, with an average hydrophobicity of -0.25 to -3.55 (Table I) .

The indels ClpG 9/10.1 and ClpG 9/10.2, which contain a large disrupting aa sequence in the central hydrophobic core of SP, produced an anti-ClpG pAb-reacting 29-kDa protein comparable to the wt mature ClpG. In contrast, the hybrid ClpG monomers extracted from mutants teins whose sizes, as expected, were slightly greater than that of wt ClpG (Table I) .

Since in-frame substitutions in the wt cleavage site do not appear to affect the capacity of some indel pilins to reach the bacterial surface, processing must occur at some (*) r---TGEV C Epitope (12 aa) with either the murine mAh 3h.5 or a rabbit anti-ClpG pAh, as primary Ah, and either a goat anti-mouse or a goat anti-rabbit IgG conjugated to peroxidase, as secondary Ah. Sheet, were stained wjith 4chloronaphtol.

Lanes: I, hybrid ClpG proteins; 2. wt ClpC proteins: (+) and (-) refer to the presence or the absence of TGEV-C epitope. respectively.

The 29 unknown second site. probably in or upstream from the foreign sequences of modified preClpG, as supported by the finding that in some mutants the mature species clearly migrated slower on SDS-PAGE than did the wt mature ClpG. As suggested by Fikes et al. ( 1990) . there may be circumstances in which the presence of alternate cleavage sites ensures processing in the event of a mutational alteration elsewhere in the signal which shifts the proximity of the core to the normal cleavage site.

To investigate whether ClpG SP specificity is essential for CS31A biogenesis. we fused in-frame the ornpA signal sequence to the structural C/PC gene devoid of its signal sequence (Fig. 7A ). The choice of the E. r~~/i OmpA outer membrane protein was based on the high sequence homology between its SP and those of ClpG, FaeG and F41 prepilins (Fig. 6A ). This new construct, carried by pOPA31, expressed a hybrid polypeptide detectable only after total lysis of the cells, suggesting an incorrect fimbrial assembly of hybrid proteins across the outer membrane. Western blot analysis with anti-ClpG pAb (Fig. 7B ) revealed that cells harboring complemented pDEV41155 produced only the wt mature ClpG, whereas cells bearing complemented pOPA significantly XCCLImulated only the hybrid precursor protein in view of its expected size of about 3 I -kDa. The conclusion that the pended in PBS (pH 7.2). Cells wcrc then labclcd on EM grids with mAh 3h.5 and IO-nm colloidal gold-labeled goat anti-mouse Ah. Gold labeling of intact cells was carried out essentially as described by Girardeau et al. (198X) . The grids were EM examined with a Phrhps EM400. OmpA SP did not mediate its proper translocation across the cytoplasmic membrane was strengthened by Western blot analysis with the anti-FLAG peptide MlmAb. This mAb specifically binds to the eight aa FLAG peptide (DYKDDDDK; Fig. 7A ) only when it is located at the free N terminus of a FLAG fusion protein, but does not bind to an unprocessed N-terminal FLAG fusion protein (Prickett et al., 1989) . Thus, given that the fusion ( Fig. 7B) , and from the data reported above, we conclude that the OmpA SP was unprocessed. Alignment and comparison of the SP aa sequences of ClpG, FaeG and F41 prepilins with that of OmpA (Fig. 6A) showed a cryptic consensus sequence, namely S-A-(A)-V/A-S, only at the C-terminal end of the ClpG, FaeG and F41 hydrophobic cores. It contains polar Ser, hydrophobic Ala and Val residues, which form an a-helix. Previous studies have pointed out the importance of an a-helical conformation in the hydrophobic region for SP function (Pugsley, 1993) . In particular, the controlling factor seems to be the overall propensity for helix forma- In-frame fusion between onlpA signal sequence and c/pG gene is shown. tion rather than the length of the helical region (Lehnhardt et al., 1987) . That this phenomena is not applicable to 0mpA::ClpG fusion, since it was unprocessed in spite of a normal cc-helix-structured

OmpA SP, implies that conformation of the SP may be influenced by the mature region. An earlier study (Girardeau et al., 1991 ) showed a substantial scqucncc similarit! both bctwccn the mature regions of ClpG. (Bertin et al.. 1993 ). appears as a good candidate capable of interacting with both ClpG SP and mature ClpG. Indeed. Hultgren et al. ( 1989) reported that processing of the PapG adhesin by signal peptidase I is cnhanccd by its interaction with the PapD pilin chapcronc. and that the last 13 aa at the C-terminal part of the mature PapG were directly involved in the PapG-PapD complex formation.

Fimbriae are usually composed of scberal different subunits. all of which arc recognized by the same molecular chaperone.

If our second hypothesis is true. the specific mechanism by which the membrane translocation of the major fimbrial subunits occurs through the Ser-flanked sequence recognition by ClpE. must be also applicable to other exported proteins members of the fimbrial assembly machinery. In agreement with this rcasoning. most SP :I;I scqucnccs of nine CS31 A and KXX accessory proteins revealed a Ser-flanked segment of 4 7 aa residues near the peptidase cleavage site ( Fig. hB) Indeed. despite the extensive modifications in the signal sequence region, the mutant ClpG pilin was processed in every case. In particular, the normal peptidase recognition sequence is dispensable since major substitutions of rcsiducs across this processing junction were permissive for CS31A fimbria production. Although this is the first time these findings have been reported for an E. coli pilin, similar observations were made with Pseudomonas aeruginosa type-IV pilin leader-peptides mutants (Strom and Lory, 199 1) (2) In agreement with the conclusions of Hemila et al.

(1992), fusions containing intact SP, which are common when producing heterologous proteins, are not necessarily the most suitable.

(3) The work reported here suggests a specific functional interrelationship of the ClpG SP with its associated mature region. A possibility is that the mature region and the SP interact either directly through definite aa zones of recognition, or indirectly in association with other components of the CS31A fimbria assembly machinery.

(4) It is possible to insert a viral antigenic determinant into CS31A fimbriae in such a way that it is cell-surface exposed and that intact recombinant cells are recognized by a mAb directed against the viral epitope. This opens up the possibility of creating diagnostic reagents based on whole cells, of developing new live vaccins and of producing foreign polypeptides.

Studies on the K88 Fimbriae of Enteropathogenic Escherichia co/i. Thesis

The ClpE protein involved in biogenesis of the CS3lA capsule-like antigen is a member of a periplasmic chaperone family in Gram-negative bacteria

Location of tolerated insertions/deletions in the structure of the maltose binding protein

Rapid alkaline extraction procedure for screening recombinant plasmid DNA

Characterization of bovine septicemic, bovine diarrheal, and human enteroinvasive Escherichiu co/i that hybridize with K88 and F41 accessory gene probes but do not express these adhesins

Factors and marker of virulence in Escherichia coli from human septicemia

Analysis of membrane and surface protein sequences with hydrophobic moment plot

Maturation of Escherichiu co/i maltose-binding protein by signal peptidase I in vivo

Further developments of pro

Sequence analysis of the clpG gene, which codes for surface antigen CS3lA subunit: evidence of an evolutionary relationship between CS3lA, KS8 and F41 subunit genes

CS31A, a new K88 related fimbrial antigen on bovine enterotoxigenie and septicemic Escherichiu coli strains

We are grateful to Dr H. Laude (INRA, Laboratoire de Virologie et Immunologie Moleculaires, France) for kindly providing the sequence of the antigenic TGEV-C epitope and the mAb 3b.5. This work was supported by grants AGRE-0008-C from European Economic Community (ECLAIR program).