The rate of folding dictates substrate secretion by the Escherichia coli hemolysin type 1 secretion system

Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the "folding" mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function.     

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.     

Mutations in HlyD, part of the type 1 translocator for hemolysin secretion, affect the folding of the secreted toxin

HlyD, a member of the membrane fusion protein family, is essential for the secretion of the RTX hemolytic toxin HlyA from Escherichia coli. Random point mutations affecting HlyA secretion were obtained, distributed in most periplasmic regions of the HlyD molecule. Analysis of the secretion phenotypes of different mutants allowed the identification of regions in HlyD involved in different steps of HlyA translocation. Four mutants, V349-I, T85-I, V334-I and L165-Q, were conditionally defective, a phenotype shown to be linked to the presence of inhibitory concentrations of Ca2+ in extracellular medium. Hly mutant T85-I was defective at an early stage in secretion, while mutants V334-I and L165-Q appeared to accumulate HlyA in the cell envelope, indicating a block at an intermediate step. Mutants V349-I, V334-I, and L165-Q were only partially defective in secretion, allowing significant levels of HlyA to be transported, but in the case of V349-I and L165-Q the HlyA molecules secreted showed greatly reduced hemolytic activity. Hemolysin molecules secreted from V349-I and V334-I are defective in normal folding and can be reactivated in vitro to the same levels as HlyA secreted from the wild-type translocator. Both V349-I and V334-I mutations mapped to the C-terminal lipoyl repeat motif, involved in the switching from the helical hairpin to the extended form of HlyD during assembly of the functional transport channel. These results suggest that HlyD is an integral component of the transport pathway, whose integrity is essential for the final folding of secreted HlyA into its active form.     

Using an E. coli Type 1 secretion system to secrete the mammalian, intracellular protein IFABP in its active form

A biotechnological production of proteins through protein secretion systems might be superior to the conventional cytoplasmic production, because of the absence of large amounts of proteases present in the extracellular space and the ease of purification or downstream processing. However, secretion of proteins is still a trial-and-error approach and many proteins fail to be secreted. Recently, a study of a Type 1 secretion system revealed that the folding rate of the passenger protein dictates secretion efficiency. Here, the well-known MalE failed to be secreted when fused to a C-terminal fragment of the natural substrate haemolysin A. In contrast, slow-folding mutants of MalE were secreted in high yields. However, MalE is a bacterial protein that is targeted to the periplasmic space of E. coli and possesses the intrinsic capability to cross a membrane. Therefore, we applied the same approach for another eukaryotic protein that resides in the cytoplasm. As an example, we chose the intestinal fatty acid binding protein (IFABP) and highlight the universal potential of this Type 1 secretion system to secrete proteins with slow-folding kinetics (here the G121V mutant). Finally, a one-step purification protocol was established yielding 1mg of pure IFABP G121V per liter culture supernatant. Moreover, secreted IFABP G121V was shown to reach a folded state, which is biologically active.     

Structural requirements for dimerization,glycosylation, secretion,and biological function of VPF/VEGF

Vascular permeability factor (VPF) also known as vascular endothelial growth factor (VEGF), is a dimeric protein that affects endothelial cell (EC) and vascular functions including enhancement of microvascular permeability and stimulation of EC growth. To investigate the structural features of VPF/VEGF necessary for efficient dimerization, secretion, and biological activities, we employed site-directed mutagenesis with a Cos-1 cell expression system. Several cysteine residues essential for VPF dimerization were identified by mutation analysis of the Cys-25, Cys-56, and Cys-67 residues. Mutant VPF isoforms lacking either of these cysteines were secreted as monomers and were completely inactive in both vascular permeability and endothelial cell mitotic assays. VPF Cys-145 mutant protein was efficiently secreted as a glycosyaated, dimeric polypeptide, but had a reduction in biological activities. The site of N-linked glycosylation was directly identified as Asn-74, which, when mutated produced an inefficiently secreted dimeric protein without post-translational glycosylation, yet maintained full vascular permeability activity. Finally, we found that one VPF mutant isoform Cys-101 was not secreted and this mutant functioned as a dominant-negative suppressor of wild-type VPF secretion as demonstrated by co-expression assays in Cos-1 cells.     

Secretion and degradation of mutant leucine-specific binding protein molecules containing C-terminal deletions

The leucine-specific binding protein (LS-BP), a periplasmic component of the Escherichia coli high-affinity leucine transport system, is initially synthesized in a precursor form with a 23 amino acid N-terminal leader sequence that is removed during secretion of the protein into the periplasm. Using in vitro mutagenesis, deletion mutants of the LS-BP gene have been constructed with altered or missing amino acid sequences in the C-terminal portion of the protein. These altered binding proteins exhibited normal processing and secretion but were rapidly degraded in the periplasmic space. In the presence of an uncoupler of the transmembrane potential (CCCP) the precursor forms accumulated in the membrane and were protected from degradation. The altered binding proteins also were secreted by spheroplasts of E coli, after which they were easily detected.     

Secretion of the Rhizobium leguminosarum nodulation protein NodO by haemolysin-type systems

The Rhizobium leguminosarum biovar viciae nodulation protein NodO is partially homologous to haemolysin of Escherichia coli and, like haemolysin, is secreted into the growth medium. The NodO protein can be secreted by a strain of E. coli carrying the cloned nodO gene plus the haemolysin secretion genes hlyBD, in a process that also requires the outer membrane protein encoded by tolC. The related protease secretion genes, prtDEF, from Erwinia chrysanthemi also enable E. coli to secrete NodO. The Rhizobium genes encoding the proteins required for NodO secretion are unlinked to nodO and are unlike other nod genes, since they do not require flavonoids or NodO for their expression. Although proteins similar to NodO were not found in rhizobia other than R. leguminosarum bv. viciae, several rhizobia and an Agrobacterium strain containing the cloned nodO gene were found to have the ability to secrete NodO. These observations indicate that a wide range of the Rhizobiaceae have a protein secretion mechanism analogous to that which secretes haemolysin and related toxins and proteases in the ENterobacteriaceae.     

Isolation of the Actinobacillus pleuropneumoniae haemolysin gene and the activation and secretion of the prohaemolysin by the HlyC, HlyB and HlyD proteins of Escherichia coli

The gene encoding the c. 105 kD secreted haemolysin protein of the porcine pathogen Actinobacillus pleuropneumoniae serotype 1 has been isolated by screening a lambda gt11 expression library in Escherichia coli with antiserum raised against the wild-type protein. A derivative recombinant DNA pJFF702 expressed the hlylA haemolysin gene from the pUC19 lac promoter but the resulting haemolysin I protein remained within the E. coli cell and was haemolytically inactive. Export of the intracellular A. pleuropneumoniae prohaemolysin out into the medium was achieved by the presence in trans of the E. coli haemolysin secretion genes hlyB and hlyD, and high levels of intracellular haemolytic activity were attained similarly by the E. coli post-translational haemolysin activator gene, hlyC. Southern hybridization of A. pleuropneumoniae parental DNA nevertheless indicated only a low degree of nucleotide sequence identity to the haemolysin structural and secretion genes hlyA and hlyB of E. coli. The data show that despite substantial nucleotide sequence divergence the A. pleuropneumoniae serotype 1 haemolysin determinant is closely related to that which is dispersed throughout other Gram-negative human and animal pathogens.     

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 (V(HH)) 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 V(HH) 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 V(HH) domains are correctly folded and thus able to bind their cognate antigens. Expression of scFv- or V(HH)-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 V(HH) 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 approximately 2 nm and having disulphide bonds. This tolerance for folded protein domains of approximately 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.     

Serratia marcescens S-layer protein is secreted extracellularly via an ATP-binding cassette exporter, the Lip system

The Serratia marcescens Lip exporter belonging to the ATP-binding cassette (ABC) exporter is known to be involved in signal peptide-independent extracellular secretion of a lipase and a metalloprotease. Although the genes of secretory proteins and their ABC exporters are usually all reported to be linked in several Gram-negative bacteria, neither the lipase nor the protease gene is located close to the Lip exporter genes, lipBCD . A gene ( slaA ) located upstream of the lipBCD genes was cloned, revealing that it encodes a polypeptide of 100 kDa and is partially similar to the Caulobacter crescentus paracrystalline cell surface layer (S-layer) protein. The Lip exporter-deficient mutants of S . marcescens failed to secrete the SlaA protein. Electron micrography demonstrated the cell surface layer of S . marcescens . The S-layer protein was secreted to the cultured media in Escherichia coli cells carrying the Lip exporter. Three ABC exporters, Prt, Has and Hly systems, could not allow the S-layer secretion, indicating that the S . marcescens S-layer protein is strictly recognized by the Lip system. This is the first report concerning secretion of an S-layer protein via its own secretion system.     

The toxic role of alpha-haemolysin in the pathogenesis of experimental Escherichia coli infection in mice

Filtrates from strains of Escherichia coli possessing plasmid-cloned haemolysin (Hly) genes and from strains possessing 'wild' Hly plasmids were lethal for mice on intravenous inoculation; similar doses of preparations from derivatives of these strains in which the Hly genes had been rendered non-functional or which did not possess the 'wild' plasmids were not. Live cultures of both kinds of Hly+ strain usually had a lower lethal dose for mice on intraperitoneal inoculation than the corresponding Hly- forms. Mice that had been inoculated with Hly+ forms had shorter survival times and lower numbers of organisms in peritoneal washings, lungs and blood at point of death than mice that had been inoculated with the corresponding Hly- forms; this was also so for mice pre-treated with FeSO4, a procedure which rendered mice equally susceptible to the lethal effects of the Hly+ and Hly- forms of a strain. In FeSO4-treated mice the numbers of organisms in the tissues of those dying from infection with Hly+ organisms were no higher than they were at the same time after inoculation in others given the corresponding Hly- forms; before mice of the latter category died the numbers of organisms in their tissues increased greatly. The clinical and pathological signs exhibited by mice inoculated with Hly+ organisms, but not with Hly- organisms, resembled those exhibited by mice inoculated with bacteria-free haemolysin preparations. These results suggest that haemolysin played a significant role in the pathogenesis of the disease produced by the Hly+ organisms by having a direct toxic action on the host.     

Functional analysis of the enteropathogenic Escherichia coli type III secretion system chaperone CesT identifies domains that mediate substrate interactions

In many Gram-negative bacteria, a key indicator of pathogenic potential is the possession of a specialized type III secretion system, which is utilized to deliver virulence effector proteins directly into the host cell cytosol. Many of the proteins secreted from such systems require small cytosolic chaperones to maintain the secreted substrates in a secretion-competent state. One such protein, CesT, serves a chaperone function for the enteropathogenic Escherichia coli (EPEC) translocated intimin receptor (Tir) protein, which confers upon EPEC the ability to alter host cell morphology following intimate bacterial attachment. Using a combination of complementary biochemical approaches, functional domains of CesT that mediate intermolecular interactions, involved in both chaperone-chaperone and chaperone-substrate associations, were determined. The CesT N-terminal is implicated in chaperone dimerization, whereas the amphipathic alpha-helical region of the C-terminal, is intimately involved in substrate binding. By functional complementation of chaperone domains using the Salmonella SicA chaperone to generate chaperone chimeras, we show that CesT-Tir interaction proceeds by a mechanism potentially common to other type III secretion system chaperones.     

Sucrase is an intramolecular chaperone located at the C-terminal end of the sucrase-isomaltase enzyme complex

The sucrase-isomaltase enzyme complex (pro-SI) is a type II integral membrane glycoprotein of the intestinal brush border membrane. Its synthesis commences with the isomaltase (IM) subunit and ends with sucrase (SUC). Both domains reveal striking structural similarities, suggesting a pseudo-dimeric assembly of a correctly folded and an enzymatically active pro-SI. The impact of each domain on the folding and function of pro-SI has been analyzed by individual expression and coexpression of the individual subunits. SUC acquires correct folding, enzymatic activity and transport competence and is secreted into the external milieu independent of the presence of IM. By contrast, IM persists as a mannose-rich polypeptide that interacts with the endoplasmic reticulum resident molecular chaperone calnexin. This interaction is disrupted when SUC is coexpressed with IM, indicating that SUC competes with calnexin for binding of IM. The interaction between SUC and the membrane-anchored IM leads to maturation of IM and blocks the secretion of SUC into the external milieu. We conclude that SUC plays a role as an intramolecular chaperone in the context of the pro-SI protein. To our knowledge all intramolecular chaperones so far identified are located at the N-terminal end. SUC is therefore the first C-terminally located intramolecular chaperone in mammalian cells.     

Secretion of active β-lactamase to the medium mediated by the Escherichia coli haemolysin transport pathway

An in frame gene fusion containing the coding region for mature β-lactamase and the 3′-end of hylA encoding the haemolysin secretion signal, was constructed under the control of a lac promoter. The resulting 53 kDa hybrid protein was specifically secreted to the external medium in the presence of the haemolysin translocator proteins, HlyB and HlyD. The specific activity of the β-lactamase portion of the secreted protein (measured by the hydrolysis of penicillin G), approximately 1 U/μg protein, was close to that of authentic, purified TEM-β-lactamase. This is an important example of a hybrid protein that is enzymatically active, and secreted via the haemolysin pathway. Previous studies have indicated that haemolysin is secreted directly into the medium, bypassing the periplasm, to which β-lactamase is normally targeted. This study indicated, therefore, that normal folding of an active β-lactamase, can occur, at least when fused to the HlyA C-terminus, without the necessity of entering the periplasm. Despite the secretion of approximately 5 μg/ml levels of the active β-lactamase fusion into the medium, there was maximally only a 50% detectable increase in the LD₅₀ for resistance to ampicillin at the individual cell level. This result suggests that, normally, resistance to ampicillin requires a high concentration of the enzyme close to killing targets, i.e. in the periplasm, in order to achieve significant levels of protection.     

Analysis of the haemolysin transport process through the secretion from Escherichia coli of PCM, CAT or β-galactosidase fused to the Hly C-terminal signal domain

Secretion of haemolysin (HlyA) is secA independent, but depends upon two accessory membrane proteins, HlyB and HlyD, encoded by the hly determinant. A fourth (cytoplasmic) protein, HlyC, is required to activate HlyA post-translationally, but has no role in export. Deletion studies have previously shown that the HlyA molecule contains a targeting signal close to the C-terminus which specifically directs its secretion to the medium. This targeting signal has been variously located within the terminal 27, 53, 60 or 113 amino acids. In this paper, we have sought to confirm the presence of a C-terminal targeting signal and to analyse the specificity of the Hly transport system through fusion of C-terminal fragments of HlyA to heterologous polypeptides. A C-terminal fragment (23 kDa) of HlyA, when fused at the C-terminus, efficiently promoted the secretion of the eukaryotic protein prochymosin (PCM) to the medium via HlyB and HlyD. This result is in contrast to previous findings that prochymosin, preceded by the alkaline phosphatase signal sequence, cannot be translocated across the Escherichia coli inner membrane. The HlyA targeting domain was also used to secrete to the medium varying portions of chloramphenicol acetyltransferase (CAT) and 98 per cent of the beta-galactosidase (LacZ) molecule (both E. coli cytoplasmic proteins). In the case of the PCM and CAT fusions the efficiency of secretion was reduced as the proportion of the PCM and CAT molecule increased. This result is consistent with inhibition of secretion through the irreversible folding of the larger passenger protein fragments, or the occlusion of the HlyA targeting signal by upstream sequences. Analysis of the nature of the C-terminal domain promoting secretion of prochymosin, demonstrated that shortening the signal domain from 218 to 113 amino acids significantly reduced the efficiency of secretion. This result may also reflect the importance of maintaining an independently folded signal motif well separated from a passenger domain.     

Analysis of signals for secretion in the staphylococcal protein A gene.

Different constructs of the gene encoding staphylococcal protein A were introduced in Staphylococcus aureus and S. xylosus as well as Escherichia coli. The product of the gene without the cell wall anchoring domain was efficiently secreted in all three hosts. N-terminal sequencing of the affinity-purified mature protein revealed a common processing site after the alanine residue at position 36. In contrast, when an internal IgG-binding fragment of protein A (region B) was inserted after the protein A signal sequence, the product was poorly secreted and N-terminal sequencing revealed no processing at the normal site. This demonstrates that the structure of the polypeptide chain beyond the signal peptide cleavage site can affect cleavage. Another construct, containing the N-terminal IgG-binding part of the mature protein A (region E) followed by region B, gave correct processing and efficient secretion. Unexpectedly, the gene product, EB, was not only secreted and correctly processed, but was also excreted to the culture medium of E. coli. Secretion vectors containing the protein A signal sequence were constructed to facilitate secretion of foreign gene products. Insertion of the E. coli gene phoA, lacking its own promoter and signal sequence, led to efficient secretion of alkaline phosphatase both in E. coli and S. aureus.