Luis Ángel Fernández

Engineering a mouse metallothionein on the cell surface of Ralstonia eutropha CH34 for immobilization of heavy metals in soil.

Here we describe targeting of the mouse metallothionein I (MT) protein to the cell surface of the heavy metal-tolerant Ralstonia eutropha (formerly Alcaligenes eutrophus) CH34 strain, which is adapted to thrive in soils highly polluted with metal ions. DNA sequences encoding MT were fused to the autotransporter β-domain of the IgA protease of Neisseria gonorrhoeae, which targeted the hybrid protein toward the bacterial outer membrane. The translocation, surface display, and functionality of the chimeric MTβ protein was initially demonstrated in Escherichia coli before the transfer of its encoding gene (mtb) to R. eutropha. The resulting bacterial strain, named R. eutropha MTB, was found to have an enhanced ability for immobilizing Cd2+ ions from the external media. Furthermore, the inoculation of Cd2+-polluted soil with R. eutropha MTB decreased significantly the toxic effects of the heavy metal on the growth of tobacco plants (Nicotiana bentamiana).

Production of Functional Single-Chain Fv Antibodies in the Cytoplasm of Escherichia coli

Production of intracellular antibodies in Escherichia coli has been thought unlikely owing to an inability to form stable disulfide bonds in the cytoplasm, a necessary step in the folding of most immunoglobulin (Ig) domains. This work investigates whether E. coli strains carrying mutations in the major intracellular disulfide bond-reduction systems (i.e. the thioredoxin and the glutathione/glutaredoxin pathways) allow the oxidation and folding of single chain variable fragment (scFv) antibodies in the cytoplasm. The effect of the co-expression of disulfide bond chaperones in these cells was also examined. An scFv that recognizes the alternative sigma factor sigma(54) was used as a model to investigate disulfide bond formation and the folding of Ig domains in E. coli. The results demonstrate that functional intrabodies, with oxidized disulfide bonds in their Ig domains, are produced efficiently in E. coli cells carrying mutations in the glutathione oxidoreductase (gor) and the thioredoxin reductase (trxB) genes and co-expressing a signal-sequence-less derivative of the disulfide-bond isomerase DsbC ((Delta)ssDsbC). We obtained evidence indicating that (Delta)ssDsbC acts as a chaperone promoting the correct folding and oxidation of scFvs.

Export of autotransported proteins proceeds through an oligomeric ring shaped by C‐terminal domains

An investigation was made into the oligomerization, the ability to form pores and the secretion‐related properties of the 45 kDa C‐terminal domain of the IgA protease (C‐IgAP) from Neisseria gonorrhoeae. This protease is the best studied example of the autotransporters (ATs), a large family of exoproteins from Gram‐negative bacteria that includes numerous virulence factors from human pathogens. These proteins contain an N‐terminal passenger domain that em bodies the secreted polypeptide, while the C‐domain inserts into the outer membrane (OM) and trans locates the linked N‐module into the extracellular medium. Here we report that purified C‐IgAP forms an oligomeric complex of ∼500 kDa with a ring‐like structure containing a central cavity of ∼2 nm diameter that is the conduit for the export of the N‐domains. These data overcome the previous model for ATs, which postulated the passage of the N‐module through the hydrophilic channel of the β‐barrel of each monomeric C‐domain. Our results advocate a secretion mechanism not unlike other bacterial export systems, such as the secretins or fimbrial ushers, which rely on multimeric complexes assembled in the OM.

Probing secretion and translocation of a beta-autotransporter using a reporter single-chain Fv as a cognate passenger domain.

The mechanism of protein secretion mediated by the β‐domain of the Neisseria gonorrhoeae IgA protease, a paradigm of a family of secreted polypeptides of Gram‐negative bacteria called autotransporters, has been examined using a single‐chain antibody (scFv) as a reporter passenger domain to monitor the translocation process. Fusion of a scFv to the β‐module of the IgA protease allowed us to investigate the passage of the chimeric protein through the periplasm, its insertion into the outer membrane and the movement of the N‐terminal moiety towards the cell surface. As the binding activity of the scFv to its target antigen is entirely dependent on the formation of disulphide bonds, the relationship between secretion, folding and formation of S–S bridges could be analysed in detail. In contrast to the current notion that only an unfolded N‐passenger domain can be translocated through the β‐domain, our results show that the scFv is able to pass through the outer membrane, albeit at a threefold reduced level, in an active conformation with its disulphide bonds preformed in the periplasm through the action of the DsbA product. These data call for a re‐evaluation of the prevailing model for secretion of the N‐domain of autotransporters.

Structural tolerance of bacterial autotransporters for folded passenger protein domains

In this report we investigate the capacity of bacterial autotransporters (AT) to translocate folded protein domains across the outer membrane (OM). Polypeptides belonging to the AT family contain a C‐terminal domain that supports the secretion of the N‐domain (the passenger) across the OM of Gram‐negative bacteria. Despite some controversial data, it has been widely accepted that N‐passenger domains of AT must be unfolded and devoid of disulphide bonds for efficient translocation. To address whether or not AT are able to translocate folded protein domains across the OM, we employed several types of recombinant antibodies as heterologous N‐passengers of the transporter C‐domain of IgA protease (C‐IgAP) of Neisseria gonorroheae. The N‐domains used were single chain Fv fragments (scFv) and variable mono‐domains derived from camel antibodies (VHH) selected on the basis of their distinct and defined folding properties (i.e. enhanced solubility, stability and presence or not of disulphide bonds). Expression of these hybrids in Escherichia coli shows that stable scFv and VHH domains are efficiently (>99%) translocated towards the bacterial surface regardless of the presence or not of disulphide bonds on their structure. Antigen‐binding assays demonstrate that surface‐exposed scFv and VHH domains are correctly folded and thus able to bind their cognate antigens. Expression of scFv‐ or VHH‐C‐IgAP hybrids in E. coli dsbA or fkpA mutant cells reveals that these periplasmic protein chaperones fold these N‐domains before their translocation across the OM. Furthermore, large N‐passengers composed of strings of VHH domains were secreted in a folded state by AT with no loss of efficacy (>99%) despite having multiple disulphide bonds. Thus AT can efficiently translocate toward the cell surface folded N‐passengers composed of one, two or three immunoglobulin (Ig) domains, each with a folded diameter between ∼2 nm and having disulphide bonds. This tolerance for folded protein domains of ∼2 nm fits with the diameter of the central hydrophilic channel proposed for the ring‐like oligomeric complex assembled by C‐IgAP in the OM.

Specific Secretion of Active Single-Chain Fv Antibodies into the Supernatants of Escherichia coli Cultures by Use of the Hemolysin System

ABSTRACT A simple method for the nontoxic, specific, and efficient secretion of active single-chain Fv antibodies (scFvs) into the supernatants ofEscherichia coli cultures is reported. The method is based on the well-characterized hemolysin transport system (Hly) of E. coli that specifically secretes the target protein from the bacterial cytoplasm into the extracellular medium without a periplasmic intermediate. The culture media that accumulate these Hly-secreted scFvs can be used in a variety of immunoassays without purification. In addition, these culture supernatants are stable over long periods of time and can be handled basically as immune sera.

Role of Periplasmic Chaperones and BamA (YaeT/Omp85) in Folding and Secretion of Intimin from Enteropathogenic Escherichia coli Strains

Intimin is a bacterial adhesin located on the surface of enteropathogenic Escherichia coli and other related bacteria that is believed to self-translocate across the outer membrane (OM), and therefore it has been regarded as a member of the type V secretion system (T5SS), which includes classical autotransporters (ATs). However, intimin has few structural similarities to classical ATs and an opposite topology with an OM-embedded N region and a secreted C region. Since the actual secretion mechanism of intimin is unknown, we investigated intimin biogenesis by analyzing its requirement of periplasmic chaperones (DsbA, SurA, Skp, and DegP) and of OM protein BamA (YaeT/Omp85) for folding, OM insertion, and translocation. Using full-length and truncated intimin polypeptides, we demonstrate that DsbA catalyzes the formation of a disulfide bond in the D3 lectin-like domain of intimin in the periplasm, indicating that this secreted C-terminal domain is at least partially folded prior to its translocation across the OM. We also show that SurA chaperone plays the major role for periplasmic transport and folding of the N region of intimin, whereas the parallel pathway made by Skp and DegP chaperones plays a secondary role in this process. Further, we demonstrate that BamA is essential for the insertion of the N region of intimin in the OM and that the protease activity of DegP participates in the degradation of misfolded intimin. The significance of these findings for a BamA-dependent secretion mechanism of intimin is discussed in the context of T5SSs.

Prokaryotic expression of antibodies and affibodies

Abstract Recent advances have been made in the development of systems for the display and expression of recombinant antibodies and affibodies in filamentous phages, Escherichia coli and other prokaryotic cells. Emphasis has been placed on improving phage and phagemid vectors, alternative systems for expression in different cellular compartments (e.g. the outer membrane, periplasm, cytoplasm and extracellular secretion) and novel multimerization systems for generating bivalent or multivalent binding molecules.

Conjugative transfer can be inhibited by blocking relaxase activity within recipient cells with intrabodies

Horizontal transfer of antibiotic resistance genes carried by conjugative plasmids poses a serious health problem. As conjugative relaxases are transported to recipient cells during bacterial conjugation, we investigated whether blocking relaxase activity in the recipient cell might inhibit conjugation. For that purpose, we used an intrabody approach generating a single‐chain Fv antibody library against the relaxase TrwC of conjugative plasmid R388. Recombinant single‐chain Fv antibodies were engineered for cytoplasmic expression in Escherichia coli cells and either selected in vitro for their specific binding to TrwC, or in vivo by their ability to interfere with conjugation using a high‐throughput mating assay. Several intrabody clones were identified showing specific inhibition against R388 conjugation upon cytoplasmic expression in the recipient cell. The epitope recognized by one of these intrabodies was mapped to a region of TrwC containing Tyr‐26 and involved in the conjugative DNA‐processing termination reaction. These findings demonstrate that the transferred relaxase plays an important role in the recipient cell and open a new approach to identify specific inhibitors of bacterial conjugation.

Formation of disulphide bonds during secretion of proteins through the periplasmic‐independent type I pathway

In this work, we have investigated whether the bacterial type I secretion pathway, which does not have a periplasmic intermediate of the secreted protein, allows the formation of disulphide bridges. To this end, the formation of disulphide bonds has been studied in an antibody single‐chain Fv (scFv) fragment secreted by the Escherichia coli haemolysin (Hly) transporter (a paradigm of type I secretion). The scFv antibody fragment was used as a disulphide bond and protein‐folding reporter, as it contains two disulphide bridges that are required for its correct folding (i.e. to preserve its antigen‐binding activity). We show that an scFv–HlyA hybrid secreted by Hly type I transporter (TolC, HlyB, HlyD) is accumulated in the extracellular medium with the disulphide bonds correctly formed. Neither periplasmic and inner membrane‐bound Dsb enzymes (e.g. DsbC, DsbG, DsbB and DsbD) nor cytoplasmic thioredoxins (TrxA and TrxC) were required for scFv–HlyA oxidation. However, a mutation of the thioredoxin reductase gene (trxB), which leads to the cytoplasmic accumulation of the oxidized forms of thioredoxins, had a specific inhibitory effect on the Hly‐dependent secretion of disulphide‐containing proteins. These data suggest that premature cytoplasmic oxidation of the substrate may interfere with the secretion process. Taken together, these results indicate not only that the type I system tolerates secretion of disulphide‐containing proteins, but also that disulphide bonds are specifically formed during the passage of the polypeptide through the export conduit.