Two-partner Secretion Pathway Research Articles

Large-Scale Conformational Changes of FhaC Provide Insights Into the Two-Partner Secretion Mechanism.

The Two-Partner secretion pathway mediates protein transport across the outer membrane of Gram-negative bacteria. TpsB transporters belong to the Omp85 superfamily, whose members catalyze protein insertion into, or translocation across membranes without external energy sources. They are composed of a transmembrane β barrel preceded by two periplasmic POTRA domains that bind the incoming protein substrate. Here we used an integrative approach combining in vivo assays, mass spectrometry, nuclear magnetic resonance and electron paramagnetic resonance techniques suitable to detect minor states in heterogeneous populations, to explore transient conformers of the TpsB transporter FhaC. This revealed substantial, spontaneous conformational changes on a slow time scale, with parts of the POTRA2 domain approaching the lipid bilayer and the protein’s surface loops. Specifically, our data indicate that an amphipathic POTRA2 β hairpin can insert into the β barrel. We propose that these motions enlarge the channel and initiate substrate secretion. Our data propose a solution to the conundrum how TpsB transporters mediate protein secretion without the need for cofactors, by utilizing intrinsic protein dynamics.

Bordetella Filamentous Hemagglutinin, a Model for the Two-Partner Secretion Pathway.

Bacteria use a variety of mechanisms to translocate proteins from the cytoplasm, where they are synthesized, to the cell surface or extracellular environment or directly into other cells, where they perform their ultimate functions. Type V secretion systems (T5SS) use β-barrel transporter domains to export passenger domains across the outer membranes of Gram-negative bacteria. Distinct among T5SS are type Vb or two-partner secretion (TPS) systems in which the transporter and passenger are separate proteins, necessitating a mechanism for passenger-translocator recognition in the periplasm and providing the potential for reuse of the translocator. This review describes current knowledge of the TPS translocation mechanism, using Bordetella filamentous hemagglutinin (FHA) and its transporter FhaC as a model. We present the hypothesis that the TPS pathway may be a general mechanism for contact-dependent delivery of toxins to target cells.

Systematic Disruption of the Nonpolar β‐Helix Core of Hemolysin A and its Site‐Specific Effect on Protein Structure, Function, and Secretion

Gram‐negative bacteria utilize a two‐partner secretion (TPS) pathway to export disease causing virulence factors. This pathway is a member of the Type Vb secretion systems which are thought to utilize the energy of protein folding to accomplish secretion across the outer membrane. In the TPS pathway, the secreted protein (TpsA) is produced along with a specific outer membrane protein (TpsB), which uses POTRA domains to recognize nonpolar segments on the TpsA. In our current model, this recognition leads to transport and folding of the TpsA protein via a Brownian ratchet mechanism. Utilizing this TPS pathway, Hemolysin A (HpmA), a hemolytic protein from Proteus mirabilis, is able to cross the bacterial outer membrane. A version of this protein, comprising a conserved TPS domain and truncated at position 265 (HpmA265), serves as a model for secretion and folding. HpmA265 adopts a β‐helical fold that encompasses three subdomains. Folded HpmA265 can function in trans as a template to fold and activate unfolded full‐length HpmA into its hemolytically active form (Template Assisted Hemolytic Activity – TAHA). This templating is thought to mimic the Brownian ratchet mechanism of secretion‐coupled folding of the full‐length protein. Previous work suggests that destabilization of the lowest stability subdomain enhances templating while destabilization of the second subdomain reduces templating ability. To identify the positional dependence of this effect, we have systematically replaced nonpolar amino acids with polar amino acids in the nonpolar core of the protein. Using CD spectroscopy to quantify the destabilization caused by each of the introduced variants, we have correlated the folding free energies of each variant with measured values of templating ability determined via TAHA assays. These measurements have allowed us to refine our model for how HpmA265 templates the folding of full‐length HpmA. In vivo, the nonpolar amino acids that were targeted may also play a role in the POTRA domain recognition. Previous data has shown that introduction of polar residues can increase secretion, suggesting that any destabilization of the folded form is offset by changes to POTRA domain interactions. To more fully map this effect, we have quantitatively determined the levels of secretion for each variant protein. Together, these results have allowed us to refine our model of the driving mechanisms in the TPS pathway, down to an amino acid level.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Site‐selective alterations within the hemolysin A non‐polar core

The secretion of virulence factors often aids bacterial pathogenesis. The two‐partner secretion (TPS) pathway, harboring both A (TpsA) and B‐components (TpsB), is the most commonly used gram‐negative virulence factor secretion system. In fact, whooping cough, meningitis, and certain food‐borne illnesses have been attributed to TPS pathway containing gram‐negative bacterial species. Systematically, TpsA members are activated concomitant with Tps‐dependent secretion across the outer membrane. Upon secretion, TpsA members elicit a variety of functions including cytolysis, adhesion, contact‐growth inhibition, and iron sequestration. Collectively, these TpsA functions provide advantageous invasion and proliferation strategies within host cells. Structurally, TpsA members can be divided into a two‐partner secretion (TPS) domain and a functional domain. All TPS domains are constructed from a 300‐residue right‐handed, parallel β‐helix structure, and recognize their cognate TpsB partner. In order to further understand the relationship between TPS domains and TpsA structure and function, we have implemented a truncated form of hemolysin A (HpmA265), a TpsA member from Proteus mirabilis. In previous studies, HpmA265 was structurally separated into three sequential folding subdomains: polar core, non‐polar core, and carboxy‐terminus. Specifically, these research investigations targeted valine 158 (V158) and phenylalanine 215 (F215) located within the first and last parallel β‐strands of the non‐polar core subdomain. A series of site‐selective variations were established at both V158 and F215. These variant forms of HpmA265 were characterized structurally via protease sensitivity and protein folding techniques, while functionality was ascertained within a template‐assisted hemolysis assay. Structurally, the V158 variants have destabilized the unfolding transitions associated with both the polar and non‐polar core subdomains, while leaving functionality unaffected. Site‐selective variants at F215 have selectively destabilized the non‐polar core subdomain, while leaving the unfolding transition attributed to the polar core subdomain unaffected. Additionally, the F215 variants do not affect template‐assisted hemolysis. Therefore, our results have been able to dissect the structural stability within the non‐polar core subdomain from template‐assisted function. These results have expanded the understanding for the implementation of TPS domains within gram‐negative bacteria.Support or Funding InformationFunding for this research was provided by: University Wisconsin – La Crosse Faculty Research Grant Program (TMW) and University Wisconsin – La Crosse Undergraduate Research and Creativity Grant Program (JDG).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural determinants of the interaction between the TpsA and TpsB proteins in the Haemophilus influenzae HMW1 two-partner secretion system.

The two-partner secretion (TPS) pathway in Gram-negative bacteria consists of a TpsA exoprotein and a cognate TpsB outer membrane pore-forming translocator protein. Previous work has demonstrated that the TpsA protein contains an N-terminal TPS domain that plays an important role in targeting the TpsB protein and is required for secretion. The nontypeable Haemophilus influenzae HMW1 and HMW2 adhesins are homologous proteins that are prototype TpsA proteins and are secreted by the HMW1B and HMW2B TpsB proteins. In the present study, we sought to define the structural determinants of HMW1 interaction with HMW1B during the transport process and while anchored to the bacterial surface. Modeling of HMW1B revealed an N-terminal periplasmic region that contains two polypeptide transport-associated (POTRA) domains and a C-terminal membrane-localized region that forms a pore. Biochemical studies demonstrated that HMW1 engages HMW1B via interaction between the HMW1 TPS domain and the HMW1B periplasmic region, specifically, the predicted POTRA1 and POTRA2 domains. Subsequently, HMW1 is shuttled to the HMW1B pore, facilitated by the N-terminal region, the middle region, and the NPNG motif in the HMW1 TPS domain. Additional analysis revealed that the interaction between HMW1 and HMW1B is highly specific and is dependent upon the POTRA domains and the pore-forming domain of HMW1B. Further studies established that tethering of HMW1 to the surface-exposed region of HMW1B is dependent upon the external loops of HMW1B formed by residues 267 to 283 and residues 324 to 330. These observations may have broad relevance to proteins secreted by the TPS pathway. Secretion of HMW1 involves a recognition event between the extended form of the HMW1 propiece and the HMW1B POTRA domains. Our results identify specific interactions between the HMW1 propiece and the periplasmic HMW1B POTRA domains. The results also suggest that the process of HMW1 translocation involves at least two discrete steps, including initial interaction between the HMW1 propiece and the HMW1B POTRA domains and then a separate translocation event. We have also discovered that the HMW1B pore itself appears to influence the translocation process. These observations extend our knowledge of the two-partner secretion system and may be broadly relevant to other proteins secreted by the TPS pathway.

FhaC takes a bow to FHA in the two-partner do-si-do.

FhaC is an outer membrane transporter from Bordetella pertussis belonging to the two-partner secretion (TPS) pathway with its primary role being the secretion of the virulence factor filamentous haemagglutinin (FHA). FhaC serves as a model transporter of the TPS pathway and significant work has been done to characterize the role of FhaC in FHA secretion. Recent studies characterized interactions between FHA and the POTRA domains of FhaC, suggesting that secretion may involve a successive translocation mechanism mediated by β-augmentation and/or electrostatic interactions. Moreover, it was also shown that reconstituted FhaC is necessary and sufficient to transport FHA into proteoliposomes. While the crystal structure of FhaC clearly suggests a role in transport, the putative transport pore is plugged by an N-terminal α-helix (H1 helix) that occludes access by FHA. Therefore, it has been proposed that the H1 helix must be expelled from the pore in order for secretion of FHA to occur. However, this has yet to be shown experimentally. Guérin et al. (2014) report the first direct experimental evidence to show that the FhaC H1 helix is quite dynamic and exchanges between closed and open states upon interaction with FHA.

Two-partner secretion: as simple as it sounds?

The two-partner secretion (TPS) pathway is a branch of type V secretion. TPS systems are dedicated to the secretion across the outer membrane of long proteins that form extended β-helices. They are composed of a ‘TpsA’ cargo protein and a ‘TpsB’ transporter, which belongs to the Omp85 superfamily. This basic design can be supplemented by additional components in some TPS systems. X-ray structures are available for the conserved TPS domain of several TpsA proteins and for one TpsB transporter. However, the molecular mechanisms of two-partner secretion remain to be deciphered, and in particular, the specific role(s) of the TPS domain and the conformational dynamics of the TpsB transporter. Deciphering the TPS pathway may reveal functional features of other transporters of the Omp85 superfamily.

The Prodomain of the Bordetella Two-Partner Secretion Pathway Protein FhaB Remains Intracellular yet Affects the Conformation of the Mature C-terminal Domain

The Prodomain of the Bordetella Two-Partner Secretion Pathway Protein FhaB Remains Intracellular yet Affects the Conformation of the Mature C-terminal Domain

Two-partner Secretion of Gram-negative Bacteria: A SINGLE β-BARREL PROTEIN ENABLES TRANSPORT ACROSS THE OUTER MEMBRANE

The mechanisms of protein secretion by pathogenic bacteria remain poorly understood. In gram-negative bacteria, the two-partner secretion pathway exports large, mostly virulence-related "TpsA" proteins across the outer membrane via their dedicated "TpsB" transporters. TpsB transporters belong to the ubiquitous Omp85 superfamily, whose members are involved in protein translocation across, or integration into, cellular membranes. The filamentous hemagglutinin/FhaC pair of Bordetella pertussis is a model two-partner secretion system. We have reconstituted the TpsB transporter FhaC into proteoliposomes and demonstrate that FhaC is the sole outer membrane protein required for translocation of its cognate TpsA protein. This is the first in vitro system for analyzing protein secretion across the outer membrane of gram-negative bacteria. Our data also provide clear evidence for the protein translocation function of Omp85 transporters.

Direct Observation of Multistate Folding in a Single Beta-Helical Protein

Filamentous haemagglutinin (FHA) is the major adhesin of B. pertussis, the bacterium that causes whooping cough. It is the prototypical member of the Two-Partner Secretion pathway family, a class of proteins associated with virulence in Gram-negative bacteria. Such proteins are large yet efficiently exported across the bacterial outer membrane without an obvious energy source, suggesting the hypothesis that translocation is driven by folding. Here, we use magnetic tweezers to apply stable and constant forces to single molecules corresponding to the N-terminal 480 amino acids of FHA (which initiate outer membrane translocation) and observe equilibrium unfolding and refolding in multiple discrete steps. This distributed (rather than cooperative) folding of isolated FHA provides evidence for processive, vectorial folding in vivo.

Type V Secretion: the Autotransporter and Two-Partner Secretion Pathways

The autotransporter and two-partner secretion (TPS) pathways are used by E. coli and many other Gram-negative bacteria to delivervirulence factors into the extracellular milieu.Autotransporters arecomprised of an N-terminal extracellular ("passenger") domain and a C-terminal β barrel domain ("β domain") that anchors the protein to the outer membrane and facilitates passenger domain secretion. In the TPS pathway, a secreted polypeptide ("exoprotein") is coordinately expressed with an outer membrane protein that serves as a dedicated transporter. Bothpathways are often grouped together under the heading "type V secretion" because they have many features in common and are used for the secretion of structurally related polypeptides, but it is likely that theyhave distinct evolutionary origins. Although it was proposed many years ago that autotransporterpassenger domains are transported across the outer membrane through a channel formed by the covalently linked β domain, there is increasing evidence that additional factors are involved in the translocation reaction. Furthermore, details of the mechanism of protein secretion through the TPS pathway are only beginning to emerge. In this chapter I discussour current understanding ofboth early and late steps in the biogenesis of polypeptides secreted through type V pathways and current modelsofthe mechanism of secretion.

Functional importance of a conserved sequence motif in FhaC, a prototypic member of the TpsB/Omp85 superfamily

In Gram-negative bacteria, the two-partner secretion pathway mediates the secretion of TpsA proteins with various functions. TpsB transporters specifically recognize their TpsA partners in the periplasm and mediate their transport through a hydrophilic channel. The filamentous haemagglutinin adhesin (FHA)/FhaC pair represents a model two-partner secretion system, with the structure of the TpsB transporter FhaC providing the bases to decipher the mechanism of action of these proteins. FhaC is composed of a β-barrel preceded by two periplasmic polypeptide-transport-associated (POTRA) domains in tandem. The barrel is occluded by an N-terminal helix and an extracellular loop, L6, folded back into the FhaC channel. In this article, we describe a functionally important motif of FhaC. The VRGY tetrad is highly conserved in the TpsB family and, in FhaC, it is located at the tip of L6 reaching the periplasm. Replacement by Ala of the invariant Arg dramatically affects the secretion efficiency, although the structure of FhaC and its channel properties remain unaffected. This substitution affects the secretion mechanism at a step beyond the initial TpsA-TpsB interaction. Replacement of the conserved Tyr affects the channel properties, but not the secretion activity, suggesting that this residue stabilizes the loop in the resting conformation of FhaC. Thus, the conserved motif at the tip of L6 represents an important piece of two-partner secretion machinery. This motif is conserved in a predicted loop between two β-barrel strands in more distant relatives of FhaC involved in protein transport across or assembly into the outer membranes of bacteria and organelles, suggesting a conserved function in the molecular mechanism of transport.

The Haemophilus influenzae HMW1C Protein Is a Glycosyltransferase That Transfers Hexose Residues to Asparagine Sites in the HMW1 Adhesin

The Haemophilus influenzae HMW1 adhesin is a high-molecular weight protein that is secreted by the bacterial two-partner secretion pathway and mediates adherence to respiratory epithelium, an essential early step in the pathogenesis of H. influenzae disease. In recent work, we discovered that HMW1 is a glycoprotein and undergoes N-linked glycosylation at multiple asparagine residues with simple hexose units rather than N-acetylated hexose units, revealing an unusual N-glycosidic linkage and suggesting a new glycosyltransferase activity. Glycosylation protects HMW1 against premature degradation during the process of secretion and facilitates HMW1 tethering to the bacterial surface, a prerequisite for HMW1-mediated adherence. In the current study, we establish that the enzyme responsible for glycosylation of HMW1 is a protein called HMW1C, which is encoded by the hmw1 gene cluster and shares homology with a group of bacterial proteins that are generally associated with two-partner secretion systems. In addition, we demonstrate that HMW1C is capable of transferring glucose and galactose to HMW1 and is also able to generate hexose-hexose bonds. Our results define a new family of bacterial glycosyltransferases.

Reversible negative design destabilization of hemolysin A

Hemolysin A (HpmA) from Proteus mirabilis is a member of the two‐partner secretion (TPS) pathway found in Gram‐negative bacteria. The TPS pathway provides an energy independent mechanism for secretion of large virulence factors, like adhesions and hemolysins. A truncated form of HpmA, termed HpmA265, containing the first 265 residues, facilitates biphasic, cooperative, heat stable, and template‐assisted hemolytic activity. The crystal structure of HpmA265 was recently solved, revealing a right‐handed beta‐helix fold, and a dry dimeric interface formed between C‐terminal adjacent HpmA265 monomers. To investigate the role of the C‐terminal dry dimeric interface during template‐assisted hemolysis, a negative design concept was employed. A positively charged lysine residue was used to replace Leu263 located within the dry, dimeric interface. The resultant mutant has been termed L263K. Template‐assisted hemolytic assays confirmed a decreased ability to activate full length HpmA, while circular dichroism (CD) confirmed structural destabilization within L263K. Also, SDS‐PAGE, mass spectrometry, N‐terminal sequencing, and surface plasmon resonance confirmed a degradation event at the C‐terminus of L263K. Re‐analysis via template‐assisted hemolytic assays and CD confirmed the restoration of template‐assisted hemolysis and beta‐signal within degraded L263K. These data demonstrate the C‐terminal dry dimeric interface is the important site of interaction between HpmA265 and full length HpmA. The research was supported by the UW‐La Crosse Dean's Distinguished Undergraduate Research Fellowship to AD, along with an NSF grant (MCB0744754) to TW.

Role of DegP for two‐partner secretion in Bordetella

Sorting of proteins destined to the surface or the extracellular milieu is mediated by specific machineries, which guide the protein substrates towards the proper route of secretion and determine the compartment in which folding occurs. In gram-negative bacteria, the two-partner secretion (TPS) pathway is dedicated to the secretion of large proteins rich in beta-helical structure. The secretion of the filamentous haemagglutinin (FHA), a 230 kDa adhesin of Bordetella pertussis, represents a model TPS system. FHA is exported by the Sec machinery and transits through the periplasm in an extended conformation. From there it is translocated across the outer membrane by its dedicated transporter FhaC to finally fold into a long beta-helix at the cell surface in a progressive manner. In this work, we show that B. pertussis lacking the periplasmic chaperone/protease DegP has a strong growth defect at 37 degrees C, and the integrity of its outer membrane is compromised. While both phenotypes are significantly aggravated by the presence of FHA, the chaperone activity of DegP markedly alleviates the periplasmic stress. In vitro, DegP binds to non-native FHA with high affinity. We propose that DegP chaperones the extended FHA polypeptide in the periplasm and is thus involved in the TPS pathway.

Structural and Functional Studies of Truncated Hemolysin A from Proteus mirabilis

In this study we analyzed the structure and function of a truncated form of hemolysin A (HpmA265) from Proteus mirabilis using a series of functional and structural studies. Hemolysin A belongs to the two-partner secretion pathway. The two-partner secretion pathway has been identified as the most common protein secretion pathway among Gram-negative bacteria. Currently, the mechanism of action for the two-partner hemolysin members is not fully understood. In this study, hemolysis experiments revealed a unidirectional, cooperative, biphasic activity profile after full-length, inactive hemolysin A was seeded with truncated hemolysin A. We also solved the first x-ray structure of a TpsA hemolysin. The truncated hemolysin A formed a right-handed parallel beta-helix with three adjoining segments of anti-parallel beta-sheet. A CXXC disulfide bond, four buried solvent molecules, and a carboxyamide ladder were all located at the third complete beta-helix coil. Replacement of the CXXC motif led to decreased activity and stability according to hemolysis and CD studies. Furthermore, the crystal structure revealed a sterically compatible, dry dimeric interface formed via anti-parallel beta-sheet interactions between neighboring beta-helix monomers. Laser scanning confocal microscopy further supported the unidirectional interconversion of full-length hemolysin A. From these results, a model has been proposed, where cooperative, beta-strand interactions between HpmA265 and neighboring full-length hemolysin A molecules, facilitated in part by the highly conserved CXXC pattern, account for the template-assisted hemolysis.

Template‐assisted hemolytic activity of carboxy‐terminal mutants of truncated hemolysin A

Proteus species, a common urinary tract infectious gram‐negative bacterium, contains the virulence factor hemolysin A. Hemolysin A is one member of the two‐partner secretion (TPS) pathway. In the TPS pathway hemolysin B (HpmB) secretes and activates hemolysin A from the periplasm into the external environment. Additional studies have reported the activation of inactive hemolysin A after mixing with an exogenous pool of truncated and post‐HpmB secreted hemolysin A. This so‐called template‐assisted hemolytic activity has been monitored under a number of different conditions. More recently, a 3‐dimensional structure of truncated hemolysin A, dubbed HpmA265, has been determined. In this crystal structure one of the important interactions occurs between the carboxy‐terminal ends of two beta‐helix monomers, thereby forming a cross beta‐stranded, dry dimer interface. In an effort to understand the template‐assisted activity of HpmA265, two types of mutants were constructed. First, a series of carboxy‐terminal mutants were constructed where a lysine residue was substituted within the normally hydrophobic core. Additionally, a truncated form of hemolysin A was constructed to contain only the first 141 residues and has been termed HpmA141. Each of these mutants were purified and analyzed for template‐assisted activity and temperature stability. The lysine replacements engineered at the carboxy‐terminus of HpmA265 did not inhibit activity. However, temperature stability differences have been observed between these carboxy‐terminal mutants and HpmA265. Furthermore, HpmA141 showed some residual template‐assisted activity even though it only harbors the first 141 residues. The later results are supportive of a model whereby the carboxy‐terminal end of truncated HpmA is an important interface during template‐assisted activity. Research supported by NSF‐RUI (MCB 0744754)

A Filamentous Hemagglutinin-Like Protein of Xanthomonas axonopodis pv. citri, the Phytopathogen Responsible for Citrus Canker, Is Involved in Bacterial Virulence

Xanthomonas axonopodis pv. citri, the phytopathogen responsible for citrus canker has a number of protein secretion systems and among them, at least one type V protein secretion system belonging to the two-partner secretion pathway. This system is mainly associated to the translocation of large proteins such as adhesins to the outer membrane of several pathogens. Xanthomonas axonopodis pv. citri possess a filamentous hemagglutinin-like protein in close vicinity to its putative transporter protein, XacFhaB and XacFhaC, respectively. Expression analysis indicated that XacFhaB was induced in planta during plant-pathogen interaction. By mutation analysis of XacFhaB and XacFhaC genes we determined that XacFhaB is involved in virulence both in epiphytic and wound inoculations, displaying more dispersed and fewer canker lesions. Unexpectedly, the XacFhaC mutant in the transporter protein produced an intermediate virulence phenotype resembling wild type infection, suggesting that XacFhaB could be secreted by another partner different from XacFhaC. Moreover, XacFhaB mutants showed a general lack of adhesion and were affected in leaf surface attachment and biofilm formation. In agreement with the in planta phenotype, adhesin lacking cells moved faster in swarming plates. Since no hyperflagellation phenotype was observed in this bacteria, the faster movement may be attributed to the lack of cell-to-cell aggregation. Moreover, XacFhaB mutants secreted more exopolysaccharide that in turn may facilitate its motility. Our results suggest that this hemagglutinin-like protein is required for tissue colonization being mainly involved in surface attachment and biofilm formation, and that plant tissue attachment and cell-to-cell aggregation are dependent on the coordinated action of adhesin molecules and exopolysaccharides.

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 a Novel Two-Partner Secretion System in Escherichia coli O157:H7

Gram-negative bacteria contain multiple secretion pathways that facilitate the translocation of proteins across the outer membrane. The two-partner secretion (TPS) system is composed of two essential components, a secreted exoprotein and a pore-forming beta barrel protein that is thought to transport the exoprotein across the outer membrane. A putative TPS system was previously described in the annotation of the genome of Escherichia coli O157:H7 strain EDL933. We found that the two components of this system, which we designate OtpA and OtpB, are not predicted to belong to either of the two major subtypes of TPS systems (hemolysins and adhesins) based on their sequences. Nevertheless, we obtained direct evidence that OtpA and OtpB constitute a bona fide TPS system. We found that secretion of OtpA into the extracellular environment in E. coli O157:H7 requires OtpB and that when OtpA was produced in an E. coli K-12 strain, its secretion was strictly dependent on the production of OtpB. Furthermore, using OtpA/OtpB as a model system, we show that protein secretion via the TPS pathway is extremely rapid.