Passenger Domain Research Articles

Influence of the passenger domain of a model autotransporter on the properties of its translocator domain

Autotransporters are a superfamily of proteins secreted by Gram-negative bacteria including many virulence factors. They are modular proteins composed of an N-terminal signal peptide, a surface-exposed 'passenger' domain carrying the activity of the protein, and a C-terminal 'translocator' domain composed of an alpha-helical linker region and a transmembrane beta-barrel. The translocator domain plays an essential role for the secretion of the passenger domain across the outer membrane; however, the mechanism of autotransport remains poorly understood. The whooping cough agent Bordetella pertussis produces an autotransporter serine-protease, SphB1, which is involved in the maturation of an adhesin at the bacterial surface. SphB1 also mediates the proteolytic maturation of its own precursor. We used SphB1 as a model autotransporter and performed the first comparisons of the biochemical and biophysical properties of an isolated translocator domain with those of the same domain preceded by the C-terminal moiety of its natural passenger. By using cross-linking and dynamic light scattering, we provide evidence that the passenger domain promotes the auto-association of SphB1, although these interactions appear rather labile. Electrophysiological studies revealed that the passenger domain of the autotransporter appears to maintain the translocator channel in a low-conductance conformation, most likely by stabilizing the alpha-helix inside the pore. That the passenger may significantly influence AT physicochemical properties is likely to be relevant for the in vivo maturation and stability of AT proteins.

Moraxella catarrhalisSynthesizes an Autotransporter That Is an Acid Phosphatase

Moraxella catarrhalis O35E was shown to synthesize a 105-kDa protein that has similarity to both acid phosphatases and autotransporters. The N-terminal portion of the M. catarrhalis acid phosphatase A (MapA) was most similar (the BLAST probability score was 10(-10)) to bacterial class A nonspecific acid phosphatases. The central region of the MapA protein had similarity to passenger domains of other autotransporter proteins, whereas the C-terminal portion of MapA resembled the translocation domain of conventional autotransporters. Cloning and expression of the M. catarrhalis mapA gene in Escherichia coli confirmed the presence of acid phosphatase activity in the MapA protein. The MapA protein was shown to be localized to the outer membrane of M. catarrhalis and was not detected either in the soluble cytoplasmic fraction from disrupted M. catarrhalis cells or in the spent culture supernatant fluid from M. catarrhalis. Use of the predicted MapA translocation domain in a fusion construct with the passenger domain from another predicted M. catarrhalis autotransporter confirmed the translocation ability of this MapA domain. Inactivation of the mapA gene in M. catarrhalis strain O35E reduced the acid phosphatase activity expressed by this organism, and this mutation could be complemented in trans with the wild-type mapA gene. Nucleotide sequence analysis of the mapA gene from six M. catarrhalis strains showed that this protein was highly conserved among strains of this pathogen. Site-directed mutagenesis of a critical histidine residue (H233A) in the predicted active site of the acid phosphatase domain in MapA eliminated acid phosphatase activity in the recombinant MapA protein. This is the first description of an autotransporter protein that expresses acid phosphatase activity.

Incorporation of a polypeptide segment into the β‐domain pore during the assembly of a bacterial autotransporter

Bacterial autotransporters consist of an N-terminal 'passenger domain' that is transported into the extracellular space by an unknown mechanism and a C-terminal 'beta-domain' that forms a beta-barrel in the outer membrane. Recent studies have revealed that fully assembled autotransporters have an unusual architecture in which a small passenger domain segment traverses the pore formed by the beta-domain. It is unclear, however, whether this configuration forms prior to passenger domain translocation or results from the translocation of the passenger domain through the beta-domain pore. By examining the accessibility of tobacco etch virus protease sites and single-cysteine residues in the passenger domain of the Escherichia coli O157:H7 autotransporter EspP at different stages of protein biogenesis, we identified a novel pre-translocation intermediate whose topology resembles that of the fully assembled protein. This intermediate was isolated in the periplasm in cell fractionation experiments. The data strongly suggest that the EspP beta-domain and an embedded polypeptide segment are integrated into the outer membrane as a single pre-formed unit. The data also provide indirect evidence that at least some outer membrane proteins acquire considerable tertiary structure prior to their membrane integration.

Autotransporter structure reveals intra-barrel cleavage followed by conformational changes.

Autotransporters are virulence factors produced by Gram-negative bacteria. They consist of two domains, an N-terminal 'passenger' domain and a C-terminal beta-domain. beta-domains form beta-barrel structures in the outer membrane while passenger domains are translocated into the extracellular space. In some autotransporters, the two domains are separated by proteolytic cleavage. Using X-ray crystallography, we solved the 2.7-A structure of the post-cleavage state of the beta-domain of EspP, an autotransporter produced by Escherichia coli strain O157:H7. The structure consists of a 12-stranded beta-barrel with the passenger domain-beta-domain cleavage junction located inside the barrel pore, approximately midway between the extracellular and periplasmic surfaces of the outer membrane. The structure reveals an unprecedented intra-barrel cleavage mechanism and suggests that two conformational changes occur in the beta-domain after cleavage, one conferring increased stability on the beta-domain and another restricting access to the barrel pore.

Highly Attenuated Bordetella pertussis Strain BPZE1 as a Potential Live Vehicle for Delivery of Heterologous Vaccine Candidates

Bordetella pertussis, the causative agent of whooping cough, is a promising and attractive candidate for vaccine delivery via the nasal route, provided that suitable attenuation of this pathogen has been obtained. Recently, the highly attenuated B. pertussis BPZE1 strain has been described as a potential live pertussis vaccine for humans. We investigated here the use of BPZE1 as a live vehicle for heterologous vaccine candidates. Previous studies have reported the filamentous hemagglutinin (FHA), a major B. pertussis adhesin, as a carrier to express foreign antigens in B. pertussis. In this study, we also examined the BrkA autotransporter as a surface display system. Three copies of the neutralizing peptide SP70 from enterovirus 71 (EV71) were fused to FHA or in the passenger domain of BrkA, and each chimera was expressed in BPZE1. The FHA-(SP70)3 and BrkA-(SP70)3 chimeras were successfully secreted and exposed at the bacterial surface, respectively. Nasal administration of the live recombinant strains triggered a strong and sustained systemic anti-SP70 antibody response in mice, although the titers and neutralizing activities against EV71 were significantly higher in the sera of mice immunized with the BrkA-(SP70)3-producing strain. These data indicate that the highly attenuated BPZE1 strain is a potential candidate for vaccine delivery via the nasal route with the BrkA autotransporter as an alternative to FHA for the presentation of the heterologous vaccine antigens.

Crystal structure of the Helicobacter pylori vacuolating toxin p55 domain

Helicobacter pylori VacA, a pore-forming toxin secreted by an autotransporter pathway, causes multiple alterations in human cells, contributes to the pathogenesis of peptic ulcer disease and gastric cancer, and is a candidate antigen for inclusion in an H. pylori vaccine. Here, we present a 2.4-A crystal structure of the VacA p55 domain, which has an important role in mediating VacA binding to host cells. The structure is predominantly a right-handed parallel beta-helix, a feature that is characteristic of autotransporter passenger domains but unique among known bacterial protein toxins. Notable features of VacA p55 include disruptions in beta-sheet contacts that result in five beta-helix subdomains and a C-terminal domain that contains a disulfide bond. Analysis of VacA protein sequences from unrelated H. pylori strains, including m1 and m2 forms of VacA, allows us to identify structural features of the VacA surface that may be important for interactions with host receptors. Docking of the p55 structure into a 19-A cryo-EM map of a VacA dodecamer allows us to propose a model for how VacA monomers assemble into oligomeric structures capable of membrane channel formation.

Are bacterial ‘autotransporters’ really transporters?

Autotransporters are bacterial outer membrane proteins that consist of a large N-terminal extracellular domain ('passenger domain') and a C-terminal beta-barrel domain ('beta domain'). The beta domain was originally proposed to function as a channel that transports its own passenger domain across the outer membrane. Results of recent structural, biochemical and molecular genetic studies, however, have challenged this idea. Here I describe an alternative model in which translocation of the passenger domain is mediated by an exogenous factor (possibly a newly identified factor necessary for assembly of outer membrane proteins called 'Omp85/YaeT'), whereas the beta domain only targets the protein to the outer membrane and serves as a membrane anchor.

Protein Secretion in Gram-Negative Bacteria via the Autotransporter Pathway

Autotransporters are a large and diverse superfamily of proteins produced by pathogenic gram-negative bacteria that are composed of an N-terminal passenger domain, which typically harbors a virulence function, and a C-terminal beta domain. It has long been known that the beta domain anchors the protein to the outer membrane and facilitates transport of the passenger domain into the extracellular space. Despite the apparent simplicity of the autotransporter pathway, several aspects of autotransporter biogenesis remain poorly understood, most notably the mechanism by which the passenger domain is translocated across the outer membrane. Here we review recent evidence that the enormous sequence diversity of both passenger and beta domains belies a remarkable conservation of structure. We also discuss insights into each stage of autotransporter biogenesis that have emerged from recent structural, biochemical, and imaging studies.

The Structure of the Haemophilus influenzae HMW1 Pro-piece Reveals a Structural Domain Essential for Bacterial Two-partner Secretion

In pathogenic Gram-negative bacteria, many virulence factors are secreted via the two-partner secretion pathway, which consists of an exoprotein called TpsA and a cognate outer membrane translocator called TpsB. The HMW1 and HMW2 adhesins are major virulence factors in nontypeable Haemophilus influenzae and are prototype two-partner secretion pathway exoproteins. A key step in the delivery of HMW1 and HMW2 to the bacterial surface involves targeting to the HMW1B and HMW2B outer membrane translocators by an N-terminal region called the secretion domain. Here we present the crystal structure at 1.92 A of the HMW1 pro-piece (HMW1-PP), a region that contains the HMW1 secretion domain and is cleaved and released during HMW1 secretion. Structural analysis of HMW1-PP revealed a right-handed beta-helix fold containing 12 complete parallel coils and one large extra-helical domain. Comparison of HMW1-PP and the Bordetella pertussis FHA secretion domain (Fha30) reveals limited amino acid homology but shared structural features, suggesting that diverse TpsA proteins have a common structural domain required for targeting to cognate TpsB proteins. Further comparison of HMW1-PP and Fha30 structures may provide insights into the keen specificity of TpsA-TpsB interactions.

Characterization of an Immunogenic Outer Membrane Autotransporter Protein, Arp, of Bartonella henselae

Bartonella henselae is a recently recognized pathogenic bacterium associated with cat scratch disease, bacillary angiomatosis, and bacillary peliosis. This study describes the cloning, sequencing, and characterization of an antigenic autotransporter gene from B. henselae. A cloned 6.0-kb BclI-EcoRI DNA fragment expresses a 120-kDa B. henselae protein immunoreactive with 21.2% of sera from patients positive for B. henselae immunoglobulin G antibodies by indirect immunofluorescence, with 97.3% specificity and no cross-reactivity with antibodies against various other organisms. DNA sequencing of the clone revealed one open reading frame of 4,320 bp with a deduced amino acid sequence that shows homology to the family of autotransporters. The autotransporters are a group of proteins that mediate their own export through the outer membrane and consist of a passenger region, the alpha-domain, and an outer membrane transporter region, the beta-domain. The passenger domain shows homology to a family of pertactin-like adhesion proteins and contains seven, nearly identical 48-amino-acid repeats not found in any other bacterial or Bartonella DNA sequences. The passenger alpha-domain has a calculated molecular mass of 117 kDa, and the transporter beta-domain has a calculated molecular mass of 36 kDa. The clone expresses a 120-kDa protein and a protein that migrates at approximately 38 kDa exclusively in the outer membrane protein fraction, suggesting that the 120-kDa passenger protein remains associated with the outer membrane after cleavage from the 36-kDa transporter.

Autotransporter proteins: novel targets at the bacterial cell surface

Autotransporter proteins constitute a family of outer membrane/secreted proteins that possess unique structural properties that facilitate their independent transport across the bacterial membrane system and final routing to the cell surface. Autotransporter proteins have been identified in a wide range of Gram-negative bacteria and are often associated with virulence functions such as adhesion, aggregation, invasion, biofilm formation and toxicity. The importance of autotransporter proteins is exemplified by the fact that they constitute an essential component of some human vaccines. Autotransporter proteins contain three structural motifs: a signal sequence, a passenger domain and a translocator domain. Here, the structural properties of the passenger and translocator domains of three type Va autotransporter proteins are compared and contrasted, namely pertactin from Bordetella pertussis, the adhesion and penetration protein (Hap) from Haemophilus influenzae and Antigen 43 (Ag43) from Escherichia coli. The Ag43 protein is described in detail to examine how its structure relates to functional properties such as cell adhesion, aggregation and biofilm formation. The widespread occurrence of autotransporter-encoding genes, their apparent uniform role in virulence and their ability to interact with host cells suggest that they may represent rational targets for the design of novel vaccines directed against Gram-negative pathogens.

Expression, Processing, and Localization of PmpD of Chlamydia trachomatis Serovar L2 during the Chlamydial Developmental Cycle

BackgroundWhile families of polymorphic membrane protein (pmp) genes have been identified in several Chlamydia species, their function remains mostly unknown. These proteins are of great interest, however, because of their location in the outer membrane and possible role in chlamydial virulence.Methodology/Principal FindingWe analyzed the relative transcription of the pmpD gene, a member of the pmp gene family in C. trachomatis serovar L2, and its protein product translation and processing during the chlamydial developmental cycle. By real-time reverse transcription polymerase chain reaction, the pmpD gene was found to be upregulated at 16 to 24 four hours after infection. Using polyclonal antibodies generated against the predicted passenger domain of PmpD, we demonstrated that it is initially localized on the surface of reticulate bodies, followed by its secretion outside Chlamydia starting at 24 hours after infection. In elementary bodies, we found a ≈157 kDa PmpD only inside the cell. Both events, the upregulation of pmpD gene transcription and PmpD protein processing and secretion, are coincidental with the period of replication and differentiation of RBs into EBs. We also demonstrated that, in the presence of penicillin, the cleavage and secretion of the putative passenger domain was suppressed.Conclusion/SignificanceOur results are in agreement with the general concept that PmpD is an autotransporter protein which is post-translationally processed and secreted in the form of the putative passenger domain outside Chlamydia at mid- to- late point after infection, coinciding with the development of RBs into EBs.

Crystallization and preliminary X-ray diffraction analysis of P30, the transmembrane domain of pertactin, an autotransporter fromBordetella pertussis

P30, the 32 kDa transmembrane C-terminal domain of pertactin from Bordetella pertussis, is supposed to form a beta-barrel inserted into the outer membrane for the translocation of the passenger domain. P30 was cloned and expressed in inclusion bodies in Escherichia coli. After refolding and purification, the protein was crystallized using the sitting-drop vapour-diffusion method at 292 K. The crystals diffract to a resolution limit of 3.5 A using synchrotron radiation and belong to the hexagonal space group P6(1)22, with unit-cell parameters a = b = 123.27, c = 134.43 A.

Cleavage of a bacterial autotransporter by an evolutionarily convergent autocatalytic mechanism

Bacterial autotransporters are comprised of an N-terminal 'passenger domain' and a C-terminal beta barrel ('beta domain') that facilitates transport of the passenger domain across the outer membrane. Following translocation, the passenger domains of some autotransporters are cleaved by an unknown mechanism. Here we show that the passenger domain of the Escherichia coli O157:H7 autotransporter EspP is released in a novel autoproteolytic reaction. After purification, the uncleaved EspP precursor underwent proteolytic processing in vitro. An analysis of protein topology together with mutational studies strongly suggested that the reaction occurs inside the beta barrel and revealed that two conserved residues, an aspartate within the beta domain (Asp(1120)) and an asparagine (Asn(1023)) at the P1 position of the cleavage junction, are essential for passenger domain cleavage. Interestingly, these residues were also essential for the proteolytic processing of two distantly related autotransporters. The data strongly suggest that Asp(1120) and Asn(1023) form an unusual catalytic dyad that mediates self-cleavage through the cyclization of the asparagine. Remarkably, a very similar mechanism has been proposed for the maturation of eukaryotic viral capsids.

Mutations affecting the biogenesis of the AIDA-I autotransporter

Autotransporters are simple systems that Gram-negative bacteria employ to secrete proteins to their surfaces or into the extracellular milieu. They consist of an N-terminal passenger domain and a C-terminal domain that is thought to insert into the outer membrane and mediate the secretion of the passenger domain. Despite the apparent simplicity of these secretion systems, their mechanism of translocation is still not completely understood. To study this mechanism, we used the AIDA-I autotransporter adhesin of Escherichia coli. We introduced mutations at several sites in a junction region of the passenger domain, close to the membrane-embedded domain. We observed that the mutations dramatically affected the biogenesis of AIDA-I. The same mutations, however, did not affect the translocation of a chimeric construct where MalE, the E. coli periplasmic maltose binding protein, replaced most of the passenger domain of AIDA-I. Our results emphasize the function of this region in the biogenesis of AIDA-I and suggest that it plays its role by interacting with and/or promoting folding of native passenger domains.

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.

Comparison of a fimbrial versus an autotransporter display system for viral epitopes on an attenuated Salmonella vaccine vector

Attenuated Salmonella have been used as vectors to deliver foreign antigens as live vaccines. We have previously developed an efficient surface-display system by genetically engineering 987P fimbriae to present transmissible gastroenteritis virus (TGEV) C and A epitopes for the induction of anti-TGEV antibodies with a Salmonella vaccine vector. Here, this system was compared with an autotransporter protein surface display system. The TGEV C and A epitopes were fused to the passenger domain of the MisL autotransporter of Salmonella. Expression of both the MisL- and 987P subunit FasA-fusions to the TGEV epitopes were under the control of in vivo-induced promoters. Expression of the TGEV epitopes from the Salmonella typhimurium CS4552 ( crp cya asd pgtE) vaccine strain was greater when the epitopes were fused to MisL than when they were fused to the 987P FasA subunit. However, when BALB/c mice were orally immunized with the Salmonella vector expressing the TGEV epitopes from either one of the fusion constructs or both together, the highest level of anti-TGEV antibody was obtained with the 987P-TGEV immunogen-displaying vector. This result suggested that better immune responses towards specific epitopes could be obtained by using a polymeric display system such as fimbriae.

TheMoraxella catarrhalisAutotransporter McaP Is a Conserved Surface Protein That Mediates Adherence to Human Epithelial Cells through Its N-Terminal Passenger Domain

The protein McaP was previously shown to be an adhesin expressed by the Moraxella catarrhalis strain O35E, which also displays esterase and phospholipase B activities (J. M. Timpe et al., Infect. Immun. 71:4341-4350, 2003). In the present study, sequence analysis suggests that McaP is a conventional autotransporter protein that contains a 12-stranded beta-barrel transporter module (amino acids [aa] 383 to 650) linked to a surface-exposed passenger domain exhibiting lipolytic activity (aa 62 to 330). An in-frame deletion removing most of this predicted N-terminal passenger domain was engineered, and Escherichia coli expressing the truncated McaP protein exhibited greatly reduced adherence to A549 human lung epithelial cells compared to E. coli expressing wild-type McaP. Site-directed mutagenesis of a serine residue at position 62 of McaP, predicted to be important for the lipolytic activity of the protein, resulted in loss of hydrolysis of p-nitrophenyl ester of caproate. E. coli expressing this mutated McaP, however, adhered to A549 monolayers at levels greater than recombinant bacteria expressing the wild-type adhesin. These results indicate that the predicted passenger domain of McaP is involved in both the binding and the lipolytic activity of the molecule and demonstrate that the adhesive properties of McaP do not require its lipolytic activity. Sequence analysis of mcaP from eight Moraxella catarrhalis strains revealed that the gene product is highly conserved at the amino acid level (98 to 100% identity), and Western blot analysis demonstrated that a panel of 16 isolates all express McaP. Flow cytometry experiments using antibodies raised against various portions of McaP indicated that its predicted passenger domain as well as transporter module contain surface-exposed epitopes. In addition to binding to the surface of intact bacteria, these antibodies were found to decrease adherence of M. catarrhalis to A549 human lung cells by up to 47% and to reduce binding of recombinant E. coli expressing McaP by 98%. These results suggest that McaP should be considered as a potential vaccine antigen.

Protease activity, secretion, cell entry, cytotoxicity, and cellular targets of secreted autotransporter toxin of uropathogenic Escherichia coli.

The secreted autotransporter toxin (Sat), found predominantly in uropathogenic Escherichia coli, is a member of the SPATE (serine protease autotransporters of Enterobacteriaceae) family and, as such, has serine protease activity and causes cytopathic effects on various cell types. To assess the contribution of the serine protease active site to the mechanism of action of Sat, mutations were made in the first (S256I), in the second (S258A), or in both (S256I/S258A) serine residues within the active site motif. Mutations in the first or both serines reduced protease activity to background levels (P<0.001); a single mutation in the second serine reduced activity by 60% compared to wild type (P<0.001). After reversion of the S256I mutation to wild type (I256S), we confirmed S256 as the catalytically active serine. None of these mutations affected secretion of the mature passenger domain or release into the supernatant. The S256I mutation, however, abrogated the cytotoxicity of Sat on human bladder (UM-UC-3) and kidney (HEK 293) epithelial cells, characterized by rounding and elongation, respectively, and a high level of cell detachment. Moreover, S256 is essential for Sat to mediate cytoskeletal contraction and actin loss in host cells as well as to degrade specific membrane/cytoskeletal (fodrin and leukocyte function-associated molecule 1) and nuclear [microtubule-associated proteins, LIM domain-only protein 7, Rap GTPase-activating protein, poly(ADP-ribose) polymerase] proteins in vitro. Lastly, Sat was internalized by host cells and localized to the cytoskeletal fraction where membrane/cytoskeletal target proteins reside.

Role of the α-Helical Linker of the C-Terminal Translocator in the Biogenesis of the Serine Protease Subfamily of Autotransporters

Autotransporters are secreted virulence factors that comprise three domains: an N-terminal signal peptide, an internal passenger domain, and a C-terminal beta-domain. The mechanism of passenger translocation across the outer membrane remains undefined, with four models having been proposed: the "hairpin," the "threading," the "multimeric," and the "Omp85 (YaeT)" models. In an attempt to understand autotransporter biogenesis, we screened the sequences of the serine protease subfamily of autotransporters (SPATEs) for conserved features indicative of a common secretion mechanism. Our analyses revealed a strictly conserved 14-amino-acid motif within the predicted alpha-helical linker region, upstream of the beta-domain of SPATEs. We investigated the function of this motif through a mutagenesis approach using Tsh as a model. Our studies demonstrate that mutations throughout the conserved motif do not block insertion of the beta-domain into the outer membrane. However, nonconservative mutations of four hydrophobic (V1099, L1102, G1107, and L1109) and three polar (N1100, K1104, and R1105) residues of the motif severely decrease or even abolish Tsh biogenesis. Further studies showed that these mutations interfere with passenger transport across the outer membrane. Bioinformatical analyses suggest that the critical polar and hydrophobic amino acids localize on opposite sides of the helix that runs through the beta-barrel pore. Our data indicate that the conserved motif is important for passenger secretion across the outer membrane and that mutations in certain residues severely affect the secretion process. We discuss how these results fit with the four proposed models for autotransporter secretion and potential applications in antimicrobial and vaccine development.