Crystal Structure of a Full-Length Autotransporter

The autotransporter (AT) secretion mechanism is the most common mechanism for the secretion of virulence factors across the outer membrane (OM) from pathogenic Gram-negative bacteria. In addition, ATs have attracted biotechnological and biomedical interest for protein display on bacterial cell surfaces. Despite their importance, the mechanism by which passenger domains of ATs pass the OM is still unclear. The classical view is that the β-barrel domain provides the conduit through which the unfolded passenger moves, with the energy provided by vectorial folding of the β-strand-rich passenger on the extracellular side of the OM. We present here the first structure of a full-length AT, the esterase EstA from Pseudomonas aeruginosa, at a resolution of 2.5 Å. EstA has a relatively narrow, 12-stranded β-barrel that is covalently attached to the passenger domain via a long, curved helix that occupies the lumen of the β-barrel. The passenger has a structure that is dramatically different from that of other known passengers, with a globular fold that is dominated by α-helices and loops. The arrangement of secondary-structure elements suggests that the passenger can fold sequentially, providing the driving force for passenger translocation. The esterase active-site residues are located at the apical surface of the passenger, at the entrance of a large hydrophobic pocket that contains a bound detergent molecule that likely mimics substrate. The EstA structure provides insight into AT mechanism and will facilitate the design of fusion proteins for cell surface display.     

Vectorial transport and folding of an autotransporter virulence protein during outer membrane secretion

Autotransporter (AT) proteins are a large and diverse family of extracellular virulence proteins from Gram-negative bacteria, characterized by a central β-helix domain within the mature virulence protein. It is not clear how these proteins cross the outer membrane (OM) quickly and efficiently, without assistance from an external energy source such as ATP or a proton gradient. Conflicting results in the literature have led to several proposed mechanisms for AT OM secretion, including a concerted process, or vectorial secretion with different directionalities. We introduced pairs of cysteine residues into the passenger sequence of pertactin, an AT virulence protein from Bordetella pertussis , and show that OM secretion of the passenger domain stalls due to the formation of a disulphide bond. We further show that the C-terminus of the pertactin passenger domain β-helix crosses the OM first, followed by the N-terminal portions of the virulence protein. In vivo proteolytic digestion shows that the C-terminus is exposed to the extracellular milieu during stalling, and forms stable structure. These AT secretion and folding features can potentially facilitate efficient secretion.     

A conserved aromatic residue in the autochaperone domain of the autotransporter Hbp is critical for initiation of outer membrane translocation

Autotransporters are bacterial virulence factors that share a common mechanism by which they are transported to the cell surface. They consist of an N-terminal passenger domain and a C-terminal β-barrel, which has been implicated in translocation of the passenger across the outer membrane (OM). The mechanism of passenger translocation and folding is still unclear but involves a conserved region at the C terminus of the passenger domain, the so-called autochaperone domain. This domain functions in the stepwise translocation process and in the folding of the passenger domain after translocation. In the autotransporter hemoglobin protease (Hbp), the autochaperone domain consists of the last rung of the β-helix and a capping domain. To examine the role of this region, we have mutated several conserved aromatic residues that are oriented toward the core of the β-helix. We found that non-conservative mutations affected secretion with Trp(1015) in the cap region as the most critical residue. Substitution at this position yielded a DegP-sensitive intermediate that is located at the periplasmic side of the OM. Further analysis revealed that Trp(1015) is most likely required for initiation of processive folding of the β-helix at the cell surface, which drives sequential translocation of the Hbp passenger across the OM.     

Autotransporter β-domains have a specific function in protein secretion beyond outer-membrane targeting

Autotransporters (ATs) of Gram-negative bacteria contain an N-proximal passenger domain that is transported to the extracellular milieu and a C-terminal β-domain that inserts into the outer membrane (OM) in a β-barrel conformation. This β-domain facilitates translocation of the passenger domain across the OM and has long been considered to be the translocation pore. However, available crystal structures of β-domains show that the β-barrel pore is too narrow for the observed transport of folded elements within the passenger domains. ATs have recently been shown to interact with the β-barrel assembly machinery. These findings questioned a direct involvement of the β-domain in passenger translocation and suggested that it may only target the passenger to the β-barrel assembly machinery pore. To address the function of the β-domain in more detail, we have replaced the β-domain of the Escherichia coli AT hemoglobin protease by β-domains originating from other OM proteins. Furthermore, we have modified the diameter of the β-domain pore. The mutant proteins were analyzed for their capacity to insert into the OM and for surface display of the passenger. Our results show that efficient passenger secretion requires a specific β-domain that not only functions as a targeting device but also is directly involved in the translocation of the passenger to the cell surface.     

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.     

Of linkers and autochaperones: an unambiguous nomenclature to identify common and uncommon themes for autotransporter secretion

Autotransporter (AT) proteins provide a diverse array of important virulence functions to Gram‐negative bacterial pathogens, and have also been adapted for protein surface display applications. The ‘autotransporter’ moniker refers to early models that depicted these proteins facilitating their own translocation across the bacterial outer membrane. Although translocation is less autonomous than originally proposed, AT protein segments upstream of the C‐terminal transmembrane β‐barrel have nevertheless consistently been found to contribute to efficient translocation and/or folding of the N‐terminal virulence region (the ‘passenger’). However, defining the precise secretion functions of these AT regions has been complicated by the use of multiple overlapping and ambiguous terms to define AT sequence, structural, and functional features, including ‘autochaperone’, ‘linker’ and ‘junction’. Moreover, the precise definitions and boundaries of these features vary among ATs and even among research groups, leading to an overall murky picture of the contributions of specific features to translocation. Here we propose a unified, unambiguous nomenclature for AT structural, functional and conserved sequence features, based on explicit criteria. Applied to 16 well‐studied AT proteins, this nomenclature reveals new commonalities for translocation but also highlights that the autochaperone function is less closely associated with a conserved sequence element than previously believed.     

An alternative outer membrane secretion mechanism for an autotransporter protein lacking a C‐terminal stable core

Autotransporter (AT) proteins are a broad class of virulence factors from Gram‐negative pathogens. AT outer membrane (OM) secretion appears simple in many regards, yet the mechanism that enables transport of the central AT ‘passenger’ across the OM remains unclear. OM secretion efficiency for two AT passengers is enhanced by a ～ 20 kDa stable core at the C‐terminus of the passenger, but studies on a broader range of AT proteins are needed in order to determine whether a stability difference between the passenger N‐ and C‐terminus represents a truly common mechanistic feature. Yersinia pestis YapV is homologous to Shigella flexneri IcsA, and like IcsA, YapV recruits mammalian neural Wiskott–Aldrich syndrome protein (N‐WASP). In vitro, the purified YapV passenger is functional and rich in β‐sheet structure, but lacks a ～ 20 kDa C‐terminal stable core. However, the N‐terminal 49 residues of the YapV passenger globally destabilize the entire YapV passenger, enhancing its OM secretion efficiency. These results indicate that the contributions of AT passenger sequences to OM secretion efficiency extend beyond a C‐terminal stable core, and highlight a role of the passenger N‐terminus in reducing passenger stability in order to facilitate OM secretion of some AT proteins.     

A conserved stable core structure in the passenger domain beta-helix of autotransporter virulence proteins

In Gram-negative bacteria, a wide variety of virulence factors are secreted via the autotransporter (AT) pathway. Intriguingly, there is no significant concentration of ATP in the periplasm, nor a proton gradient across the OM, so the energetic origin of efficient secretion of AT proteins is unknown. More than 97% of AT proteins are predicted to contain right-handed parallel beta-helical structure, and the three crystal structures available for AT passenger domains each contain a long right-handed parallel beta-helix. Previous studies have shown that pertactin, an AT from Bordetella pertussis, exhibits three-state folding and has a C-terminal stable core structure. Here, we show that Pet, an unrelated AT from Escherichia coli, also exhibits three-state unfolding and also has a stable core structure. Deletion mutants, mass spectrometry, and N-terminal sequencing demonstrate that the Pet stable core is also located near the C-terminus of the passenger domain. Moreover, sequence analysis suggests that three-state folding and a C-terminal stable core structure could be important general features of the biogenesis of AT proteins in vivo.     

Limited tolerance towards folded elements during secretion of the autotransporter Hbp

Many virulence factors secreted by pathogenic Gram-negative bacteria belong to the autotransporter (AT) family. ATs consist of a passenger domain, which is the actual secreted moiety, and a beta-domain that facilitates the transfer of the passenger domain across the outer membrane. Here, we analysed folding and translocation of the AT passenger, using Escherichia coli haemoglobin protease (Hbp) as a model protein. Dual cysteine mutagenesis, instigated by the unique crystal structure of the Hbp passenger, resulted in intramolecular disulphide bond formation dependent on the periplasmic enzyme DsbA. A small loop tied off by a disulphide bond did not interfere with secretion of Hbp. In contrast, a bond between different domains of the Hbp passenger completely blocked secretion resulting in degradation by the periplasmic protease DegP. In the absence of DegP, a translocation intermediate accumulated in the outer membrane. A similar jammed intermediate was formed upon insertion of a calmodulin folding moiety into Hbp. The data suggest that Hbp can fold in the periplasm but must retain a certain degree of flexibility and/or modest width to allow translocation across the outer membrane.     

Comparative Analysis of the Biochemical and Functional Properties of C-Terminal Domains of Autotransporters

Autotransporters (ATs) are the largest group of proteins secreted by Gram-negative bacteria and include many virulence factors from human pathogens. ATs are synthesized as large precursors with a C-terminal domain that is inserted in the outer membrane (OM) and is essential for the translocation of an N-terminal passenger domain to the extracellular milieu. Several mechanisms have been proposed for AT secretion. Self-translocation models suggest transport across a hydrophilic channel formed by an internal pore of the β-barrel or by the oligomerization of C-terminal domains. Alternatively, an assisted-translocation model suggests that transport employs a conserved machinery of the bacterial OM such as the Bam complex. In this work we have investigated AT secretion by carrying out a comparative study to analyze the conserved biochemical and functional features of different C-terminal domains selected from ATs of gammaproteobacteria, betaproteobacteria, alphaproteobacteria, and epsilonproteobacteria. Our results indicate that C-terminal domains having an N-terminal α-helix and a β-barrel constitute functional transport units for the translocation of peptides and immunoglobulin domains with disulfide bonds. In vivo and in vitro analyses show that multimerization is not a conserved feature in AT C-terminal domains. Furthermore, we demonstrate that the deletion of the conserved α-helix severely impairs β-barrel folding and OM insertion and thereby blocks passenger domain secretion. These observations suggest that the AT β-barrel without its α-helix cannot form a stable hydrophilic channel in the OM for protein translocation. The implications of our data for an understanding of AT secretion are discussed.The classical autotransporter (AT) family, also known as the type Va protein secretion system, represents the largest group of proteins secreted by Gram-negative bacteria and includes many virulence factors from important human pathogens (10, 17). Bacteria produce AT proteins as large polypeptide precursors, with their virulence activity (e.g., cytotoxins, adhesins, and proteases, etc.) present in a passenger domain flanked by an N-terminal signal peptide (sp) for Sec-dependent translocation across the bacterial inner membrane (IM) and a C-terminal domain of ∼30 to 40 kDa for insertion into the bacterial outer membrane (OM) (see Fig. 2A). A self-translocation model was originally proposed to explain the secretion mechanism of AT proteins across the OM, based mostly on data obtained with the IgA protease (IgAP) from Neisseria gonorrhoeae (43). In this model the C-terminal domain of ATs was supposed to fold in the OM as a β-barrel protein with an internal hydrophilic pore that could be used for the translocation of the passenger domain. The finding that the B subunit of cholera toxin (CtxB) should not have disulfide bonds for its secretion when fused as a heterologous passenger to the C-terminal domain of IgAP (30, 31) indirectly suggests passenger translocation in an unfolded conformation through a narrow channel expected for a β-barrel. Similar observations with the C-terminal domains of IcsA from Shigella flexneri (56) and AIDA-I from Escherichia coli (36) supported this model.Previous work done by our group challenged the original self-translocation model, since a 45-kDa C-terminal fragment of IgAP was shown to form oligomeric ring-shaped complexes with a central hydrophilic pore of ∼2 nm (63). In addition, this C-terminal fragment of IgAP was found to translocate folded immunoglobulin (Ig) domains with disulfide bonds to the bacterial surface, indicating that at least a ∼2-nm pore was being used for passenger secretion (61, 62). These data led us to propose a “multimeric” version of the self-translocation model in which the secretion of the passenger may occur through the central channel assembled by the oligomerization of the C-terminal domains in the OM. Studies with IcsA from S. flexneri (7, 46, 47, 64) and EspP from E. coli (53) also provided evidence indicating that native and heterologous passengers adopt folded or at least partially folded conformations in the periplasm before OM translocation. Conversely, a limited capacity for the translocation of folded native passengers with engineered disulfide bonds has been reported by studies with Hbp from E. coli (23) and pertactin from Bordetella pertussis (24). Crystallographic structures of the C-terminal domains of NalP from Neisseria meningitidis (41) and EspP from E. coli (2) revealed distinct β-barrel folding with 12 amphipathic β-strands and one N-terminal α-helix filling the central hydrophilic pore of the β-barrel. No indication of oligomerization was obtained with the crystallographic data. In addition, the putative protein-conducting channels of the EspP and NalP β-barrels (of ∼1 nm in diameter) were found to be closed due to the presence of the internal α-helix, which would impede the transport of passenger polypeptides (either folded or unfolded) through the reported structures. Thus, an alternative model was proposed for the assisted translocation of ATs (3, 41), in which the protein-conducting channel for secretion across the OM would be provided by the conserved Bam complex. The Bam complex is required for the insertion of β-barrel proteins (32), and the depletion of its essential component BamA (formerly YaeT in E. coli and Omp85 in Neisseria) prevents the insertion of several ATs in the OM (i.e., IcsA and SepA from S. flexneri, AIDA-I and Hbp from E. coli, and BrkA from B. pertussis) (21, 50). BamA was reported to form hydrophilic pores in lipid membranes in vitro (54) and to cross-link in vivo with the passenger domain of a slow-secretion mutant of EspP (19), which supports a role for BamA in translocation.Despite the above-described progress made in our understanding of ATs, their actual molecular mechanism of secretion remains uncertain. This is partially because the reported information is based on studies with different model AT proteins and nonhomogenous experimental approaches used by different laboratories, which sometimes produce data that are difficult to compare or may be conflicting. Here, we report a comparative study to determine conserved biochemical and functional properties found in AT C-terminal domains. Following a uniform experimental approach for six AT C-terminal domains selected from the gammaproteobacteria, betaproteobacteria, alphaproteobacteria, and epsilonproteobacteria, we have investigated their capacities for the secretion of peptides and globular domains, their pore formation and oligomerization properties, and their requirement for an N-terminal α-helix for AT function and C-terminal domain stability. Our results shed light on the secretion mechanism of ATs from the conserved structural features found in their C-terminal domains.     

Stepwise Folding of an Autotransporter Passenger Domain Is Not Essential for Its Secretion

Autotransporters are a superfamily of virulence proteins produced by Gram-negative bacteria. They consist of an N-terminal β-helical domain (“passenger domain”) that is secreted into the extracellular space and a C-terminal β-barrel domain (“β-domain”) that anchors the protein to the outer membrane. Because the periplasm lacks ATP, vectorial folding of the passenger domain in a C-to-N-terminal direction has been proposed to drive the secretion reaction. Consistent with this hypothesis, mutations that disrupt the folding of the C terminus of the passenger domain of the Escherichia coli O157:H7 autotransporter EspP have been shown to cause strong secretion defects. Here, we show that point mutations introduced at specific locations near the middle or N terminus of the EspP β-helix that perturb folding also impair secretion, but to a lesser degree. Surprisingly, we found that even multiple mutations that potentially abolish the stability of several consecutive rungs of the β-helix only moderately reduce secretion efficiency. Although these results provide evidence that the free energy derived from passenger domain folding contributes to secretion efficiency, they also suggest that a significant fraction of the energy required for secretion is derived from another source.     

Protein secretion through autotransporter and two-partner pathways

Two distinct protein secretion pathways, the autotransporter (AT) and the two-partner secretion (TPS) pathways are characterized by their apparent simplicity. Both are devoted to the translocation across the outer membrane of mostly large proteins or protein domains. As implied by their name, AT proteins contain their own transporter domain, covalently attached to the C-terminal extremity of the secreted passenger domain, while TPS systems are composed of two separate proteins, with TpsA being the secreted protein and TpsB its specific transporter. In both pathways, the secreted proteins are exported in a Sec-dependent manner across the inner membrane, after which they cross the outer membrane with the help of their cognate transporters. The AT translocator domains and the TpsB proteins constitute distinct families of protein-translocating, outer membrane porins of Gram-negative bacteria. Both types of transporters insert into the outer membrane as beta-barrel proteins possibly forming oligomeric pores in the case of AT and serve as conduits for their cognate secreted proteins or domains across the outer membrane. Translocation appears to be folding-sensitive in both pathways, indicating that AT passenger domains and TpsA proteins cross the periplasm and the outer membrane in non-native conformations and fold progressively at the cell surface. A major difference between AT and TPS pathways arises from the manner by which specificity is established between the secreted protein and its transporter. In AT, the covalent link between the passenger and the translocator domains ensures the translocation of the former without the need for a specific molecular recognition between the two modules. In contrast, the TPS pathway has solved the question of specific recognition between the TpsA proteins and their transporters by the addition to the TpsA proteins of an N-proximal module, the conserved TPS domain, which represents a hallmark of the TPS pathway.     

A conserved region within the Bordetella pertussis autotransporter BrkA is necessary for folding of its passenger domain

Autotransporter secretion represents a unique mechanism that Gram-negative bacteria employ to deliver proteins to their cell surface. BrkA is a Bordetella pertussis autotransporter protein that mediates serum resistance and contributes to adherence of the bacterium to host cells. BrkA is a 103 kDa protein that is cleaved to form a 73 kDa alpha-domain and a 30 kDa beta domain. The alpha domain, also referred to as the passenger domain, is responsible for the effector functions of the protein, whereas the beta domain serves as a transporter. In an effort to characterize BrkA secretion, we have shown that BrkA has a 42 amino acid signal peptide for transit across the cytoplasmic membrane, and a translocation unit made up of a short linker region fused to the beta-domain to ferry the passenger domain to the bacterial surface through a channel formed by the beta-domain. In this report, we provide genetic, biochemical and structural evidence demonstrating that a region within the BrkA passenger (Glu601-Ala692) is necessary for folding the passenger. This region is not required for surface display in the outer membrane protease OmpT-deficient Escherichia coli strain UT5600. However, a BrkA mutant protein bearing a deletion in this region is susceptible to digestion when expressed in E. coli strains expressing OmpT suggesting that the region is required to maintain a stable structure. The instability of the deletion mutant can be rescued by surface expressing Glu601-Ala692in trans suggesting that this region is acting as an intramolecular chaperone to effect folding of the passenger concurrent with or following translocation across the outer membrane.     

Size and conformation limits to secretion of disulfide-bonded loops in autotransporter proteins

Autotransporters are a superfamily of virulence factors typified by a channel-forming C terminus that facilitates translocation of the functional N-terminal passenger domain across the outer membrane of Gram-negative bacteria. This final step in the secretion of autotransporters requires a translocation-competent conformation for the passenger domain that differs markedly from the structure of the fully folded secreted protein. The nature of the translocation-competent conformation remains controversial, in particular whether the passenger domain can adopt secondary structural motifs, such as disulfide-bonded segments, while maintaining a secretion-competent state. Here, we used the endogenous and closely spaced cysteine residues of the plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli to investigate the effect of disulfide bond-induced folding on translocation of an autotransporter passenger domain. We reveal that rigid structural elements within disulfide-bonded segments are resistant to autotransporter-mediated secretion. We define the size limit of disulfide-bonded segments tolerated by the autotransporter system demonstrating that, when present, cysteine pairs are intrinsically closely spaced to prevent congestion of the translocator pore by large disulfide-bonded regions. These latter data strongly support the hairpin mode of autotransporter biogenesis.     

Biogenesis of β-barrel integral proteins of bacterial outer membrane

Gram-negative bacteria are enveloped by two membranes, the inner (cytoplasmic) (CM) and the outer (OM). The majority of integral outer membrane proteins are arranged in β-barrels of cylindrical shape composed of amphipathic antiparallel β-strands. In bacteria, β-barrel proteins function as water-filled pores, active transporters, enzymes, receptors, and structural proteins. Proteins of bacterial OM are synthesized in the cytoplasm as unfolded polypeptides with an N-terminal sequence that marks them for transport across the CM. Precursors of membrane proteins move through the aqueous medium of the cytosol and periplasm under the protection of chaperones (SecB, Skp, SurA, and DegP), then cross the CM via the Sec system composed of a polypeptide-conducting channel (SecYEG) and ATPase (SecA), the latter providing the energy for the translocation of the pre-protein. Pre-protein folding and incorporation in the OM require the participation of the Bam-complex, probably without the use of energy. This review summarizes current data on the biogenesis of the β-barrel proteins of bacterial OM. Data on the structure of the proteins included in the multicomponent system for delivery of the OM proteins to their destination in the cell and on their complexes with partners, including pre-proteins, are pre-sented. Molecular models constructed on the basis of structural, genetic, and biochemical studies that describe the mechanisms of β-barrel protein assembly by this molecular transport machinery are also considered.     

Roles of Periplasmic Chaperone Proteins in the Biogenesis of Serine Protease Autotransporters of Enterobacteriaceae

The serine protease autotransporters of Enterobacteriaceae (SPATEs) represent a large family of virulence factors. The prevailing model for autotransporter secretion comprises entry to the periplasm via the Sec apparatus, followed by an obscure series of steps in which the C terminus of the periplasmic species inserts into the outer membrane as a β-barrel protein, accompanied by translocation of the passenger domain to the bacterial cell surface. Little is known about the fate of the autotransporter proteins in the periplasm, including whether accessory periplasmic proteins are involved in translocation to the external milieu. Here we studied the role of the major periplasmic chaperones in the biogenesis of EspP, a prototype SPATE protein produced by Escherichia coli O157:H7. The yeast two-hybrid approach, secretion analysis of chaperone mutant strains, and surface plasmon resonance analysis (SPR) revealed direct protein-protein interactions between the periplasmic SurA and DegP chaperones and either the EspP-β or EspP passenger domains. The secretion of EspP was moderately reduced in the surA and skp mutant strains but severely impaired in the degP background. Site-directed mutagenesis of highly conserved aromatic amino acid residues in the SPATE family resulted in ∼80% reduction of EspP secretion. Synthetic peptides containing aromatic residues derived from the EspP passenger domain blocked DegP and SurA binding to the passenger domain. SPR suggested direct protein-protein interaction between periplasmic chaperones and the unfolded EspP passenger domain. Our data suggest that translocation of AT proteins may require accessory factors, calling into question the moniker “autotransporter.”Secretion of proteins to the surface of gram-negative bacteria requires passage through the inner membrane (IM), the periplasm, and the outer membrane (OM). This formidable series of obstacles can be overcome only by complex biological processes. The autotransporter (AT) system, probably the most common gram-negative secretion mechanism (13), is characterized by formation of an OM β-barrel comprised of the C terminus of the periplasmic species. The precise events required for AT translocation across the OM, however, are controversial. The original model for OM translocation comprised targeting to the periplasm via the Sec apparatus, followed by formation of an OM β-barrel, which mediates passage of an unfolded or partially folded N-terminal passenger domain to the extracellular milieu (30). Three models of AT translocation have gained some acceptance (3, 16). According to the hairpin model, translocation of the passenger domain is initiated with the C-terminal end of the passenger forming a hairpin structure inside the AT β-barrel, followed by movement of the rest of the passenger through the barrel''s pore in a C-to-N direction. Under the Omp85 model, the pore-forming Omp85 (YaeT in Escherichia coli) OM protein (OMP) facilitates insertion of the AT translocator domain into the OM, whereupon the AT passenger domain translocates through the Omp85 pore. A third model entails the combination of the hairpin and Omp85 models, including concerted insertion and translocation. All models must reconcile observations seemingly in conflict. Bernstein and colleagues reported cleavage of the mature passenger by a protease located inside the C-terminal AT barrel (10); yet, the dimensions of the folded AT barrel channel are by most accounts too narrow to accommodate even a partially folded passenger species, which is suggested from experimental periplasmic disulfide bond formation within the passenger domain (7, 19, 21).The term “autotransporter” was initially proposed on the assumption that the translocated species contained all necessary information for movement to the extracellular space. We and others have challenged that assumption (11, 14). Recently, several periplasmic proteins have been implicated in the targeting and assembly of extracytoplasmic proteins, principally OMPs (27). Three biological functions have been recognized for these periplasmic proteins: (i) molecular chaperones such as DegP, SurA, Skp, FkpA, PpiA, and PpiD (1, 5, 8, 9, 23, 26) stabilize nonnative conformations of target proteins and facilitate their folding; (ii) peptidyl-prolyl cis-trans isomerases, such as SurA, PpiD, and FkpA (9, 33, 36), catalyze the rate-limiting steps of isomerization during folding; and (iii) proteases, such as DegP and DegQ (22), degrade unproductive or misfolded proteins. Recent reports have suggested the involvement of chaperones during the passage of the AT through the periplasm (31, 43), although the mechanisms have not been defined.Here we demonstrate further the requirement for periplasmic chaperones in the biogenesis of the serine protease ATs of Enterobacteriaceae (SPATEs). Our data suggest a requirement for these periplasmic factors in translocation and suggest direct binding of the chaperone proteins to specific highly conserved motifs in the AT passenger and β-domains.     

The Protein-disulfide Isomerase DsbC Cooperates with SurA and DsbA in the Assembly of the Essential β-Barrel Protein LptD

The assembly of the β-barrel proteins present in the outer membrane (OM) of Gram-negative bacteria is poorly characterized. After translocation across the inner membrane, unfolded β-barrel proteins are escorted across the periplasm by chaperones that reside within this compartment. Two partially redundant chaperones, SurA and Skp, are considered to transport the bulk mass of β-barrel proteins. We found that the periplasmic disulfide isomerase DsbC cooperates with SurA and the thiol oxidase DsbA in the folding of the essential β-barrel protein LptD. LptD inserts lipopolysaccharides in the OM. It is also the only β-barrel protein with more than two cysteine residues. We found that surAdsbC mutants, but not skpdsbC mutants, exhibit a synthetic phenotype. They have a decreased OM integrity, which is due to the lack of the isomerase activity of DsbC. We also isolated DsbC in a mixed disulfide complex with LptD. As such, LptD is identified as the first substrate of DsbC that is localized in the OM. Thus, electrons flowing from the cytoplasmic thioredoxin system maintain the integrity of the OM by assisting the folding of one of the most important β-barrel proteins.     

A structurally informed autotransporter platform for efficient heterologous protein secretion and display

ABSTRACT: BACKGROUND: The self-sufficient Autotransporter (AT) pathway, ubiquitous in Gram-negative bacteria, combines a relatively simple protein secretion mechanism with a high transport capacity. ATs consist of a secreted passenger domain and a beta-domain that facilitates transfer of the passenger across the cell-envelope. They have a great potential for the extracellular expression of recombinant proteins but their exploitation has suffered from the limited structural knowledge of carrier ATs. Capitalizing on its crystal structure, we have engineered the Escherichia coli AT Hemoglobin protease (Hbp) into a platform for the secretion and surface display of heterologous proteins, using the Mycobacterium tuberculosis vaccine target ESAT6 as a model protein. RESULTS: Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a beta-helical core structure (beta-stem) was left intact. The resulting Hbp-ESAT6 chimeras were efficiently and stably secreted into the culture medium of E. coli. On the other hand, Hbp-ESAT6 fusions containing a truncated beta-stem appeared unstable after translocation, demonstrating the importance of an intact beta-stem. By interrupting the cleavage site between passenger and beta-domain, Hbp-ESAT6 display variants were constructed that remain cell associated and facilitate efficient surface exposure of ESAT6 as judged by proteinase K accessibility and whole cell immuno-EM analysis. Upon replacement of the passenger side domain of an alternative AT, EspC, ESAT6 was also efficiently secreted, showing the approach is more generally applicable to ATs. Furthermore, Hbp-ESAT6 was efficiently displayed in an attenuated Salmonella typhimurium strain upon chromosomal integration of a single encoding gene copy, demonstrating the potential of the Hbp platform for live vaccine development. CONCLUSIONS: We developed the first structurally informed AT platform for efficient secretion and surface display of heterologous proteins. The platform has potential with regard to the development of recombinant live vaccines and may be useful for other biotechnological applications that require high-level secretion or display of recombinant proteins by bacteria.     

Intimin-mediated export of passenger proteins requires maintenance of a translocation-competent conformation

Intimins from pathogenic bacteria promote intimate bacterial adhesion to epithelial cells. Several structurally similar domains form on the bacterial cell surface an extended rigid rod that exposes the carboxy-terminal domain, which interacts with the translocated intimin receptor. We constructed a series of intimin-derived fusion proteins consisting of carboxy-terminally truncated intimin and the immunoglobulin light-chain variable domain REIv, ubiquitin, calmodulin, beta-lactamase inhibitor protein, or beta-lactamase. By systematically investigating the intimin-mediated cell surface exposure of these passenger domains in the presence or absence of compounds that interfere with outer membrane stability or passenger domain folding, we acquired experimental evidence that intimin-mediated protein export across the outer membrane requires, prior to export, the maintenance of a translocation-competent conformation that may be distinct from the final protein structure. We propose that, during export, competition exists between productive translocation and folding of the passenger domain in the periplasm into a stable conformation that is not compatible with translocation through the bacterial outer membrane. These results may expand understanding of the mechanism by which intimins are inserted into the outer membrane and expose extracellular domains on the cell surface.     

Looks can be deceiving: recent insights into the mechanism of protein secretion by the autotransporter pathway

Autotransporters are a large superfamily of cell surface proteins produced by Gram‐negative bacteria that consist of an N‐terminal extracellular domain (‘passenger domain’) and a C‐terminal β‐barrel domain that resides in the outer membrane (OM). Although it was originally proposed that the passenger domain is translocated across the OM through a channel formed exclusively by the covalently linked β‐barrel domain, this idea has been strongly challenged by a variety of observations. Recent experimental results have suggested a new model in which both the translocation of the passenger domain and the membrane integration of the β‐barrel domain are facilitated by the Bam complex, a highly conserved heteroligomer that plays a general role in OM protein assembly. Other factors, including periplasmic chaperones and inner membrane proteins, have also recently been implicated in the biogenesis of at least some members of the autotransporter superfamily. New results have raised intriguing questions about the energetics of the secretion reaction and the relationship between the assembly of autotransporters and the assembly of other classes of OM proteins. Concomitantly, new mechanistic and structural insights have expanded the utility of the autotransporter pathway for the surface display of heterologous peptides and proteins of interest.