Two-partner secretion in Gram-negative bacteria: a thrifty, specific pathway for large virulence proteins

A collection of large virulence exoproteins, including Ca2+-independent cytolysins, an iron acquisition protein and several adhesins, are secreted by the two-partner secretion (TPS) pathway in various Gram-negative bacteria. The hallmarks of the TPS pathway are the presence of an N-proximal module called the 'secretion domain' in the exoproteins that we have named the TpsA family, and the channel-forming beta-barrel transporter proteins we refer to as the TpsB family. The genes for cognate exoprotein and transporter protein are usually organized in an operon. Specific secretion signals are present in a highly conserved region of the secretion domain of TpsAs. TpsBs probably serve as specific receptors of the TpsA secretion signals and as channels for the translocation of the exoproteins across the outer membrane. A subfamily of transporters also mediates activation of their cognate cytolysins upon secretion. The exoproteins are synthesized as precursors with an N-terminal cleavable signal peptide, and a subset of them carries an extended signal peptide of unknown function. According to our current model, the exoproteins are probably translocated across the cytoplasmic membrane in a Sec-dependent fashion, and their signal peptide is probably processed by a LepB-type signal peptidase. The N-proximal secretion domain directs the exoproteins towards their transporters early, so that translocation across both membranes is coupled. The exoproteins transit through the periplasm in an extended conformation and fold progressively at the cell surface before eventually being released into the extracellular milieu. Several adhesins also undergo extensive proteolytic processing upon secretion. The genes of many new TpsAs and TpsBs are found in recently sequenced genomes, suggesting that the TPS pathway is widespread.     

The Polypeptide Transport-associated (POTRA) Domains of TpsB Transporters Determine the System Specificity of Two-partner Secretion Systems

The two-partner secretion (TPS) systems of Gram-negative bacteria secrete large TpsA exoproteins by a dedicated TpsB transporter in the outer membrane. TpsBs contain an N-terminal module located in the periplasm that includes two polypeptide transport-associated (POTRA) domains. These are thought to initiate secretion of a TpsA by binding its N-terminal secretion signal, called the TPS domain. Neisseria meningitidis encodes up to five TpsA proteins that are secreted via only two TpsB transporters: TpsB1 and TpsB2. Of these two, the TpsB2 recognizes the TPS domains of all TpsAs, despite their sequence diversity. By contrast, the TpsB1 shows a limited recognition of a TPS domain that is shared by two TpsAs. The difference in substrate specificity of the TpsBs enabled us to investigate the role of the POTRA domains in the selection of TPS domains. We tested secretion of TPS domains or full-length TpsAs by TpsB mutants with deleted, duplicated, and exchanged POTRA domains. Exchanging the two POTRA domains of a TpsB resulted in a switch in specificity. Furthermore, exchanging a single POTRA domain showed that each of the two domains contributed to the cargo selection. Remarkably, the order of the POTRA domains could be reversed without affecting substrate selection, but this aberrant order did result in an alternatively processed secretion product. Our results suggest that secretion of a TpsA is initiated by engaging both POTRA domains of a TpsB transporter and that these select the cognate TpsAs for secretion.     

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 (TPS) systems use β‐barrel proteins of the Omp85‐TpsB superfamily to transport large exoproteins across the outer membranes of Gram‐negative bacteria. The Bordetella FHA/FhaC proteins are prototypical of TPS systems in which the exoprotein contains a large C‐terminal prodomain that is removed during translocation. Although it is known that the FhaB prodomain is required for FHA function in vivo, its role in FHA maturation has remained mysterious. We show here that the FhaB prodomain is required for the extracellularly located mature C‐terminal domain (MCD) of FHA to achieve its proper conformation. We show that the C‐terminus of the prodomain is retained intracellularly and that sequences within the N‐terminus of the prodomain are required for this intracellular localization. We also identify sequences at the C‐terminus of the MCD that are required for release of mature FHA from the cell surface. Our data support a model in which the intracellularly located prodomain affects the final conformation of the extracellularly located MCD. We hypothesize that maturation triggers cleavage and degradation of the prodomain.     

Type V secretion: From biogenesis to biotechnology

The two membranes of Gram-negative bacteria contain protein machines that have a general function in their assembly. To interact with the extra-cellular milieu, Gram-negatives target proteins to their cell surface and beyond. Many specialized secretion systems have evolved with dedicated translocation machines that either span the entire cell envelope or localize to the outer membrane. The latter act in concert with inner-membrane transport systems (i.e. Sec or Tat). Secretion via the Type V secretion system follows a two-step mechanism that appears relatively simple. Proteins secreted via this pathway are important for the Gram-negative life-style, either as virulence factors for pathogens or by contributing to the survival of non-invasive environmental species. Furthermore, this system appears well suited for the secretion of biotechnologically relevant proteins. In this review we focus on the biogenesis and application of two Type V subtypes, the autotransporters and two-partner secretion (TPS) systems. For translocation across the outer membrane the autotransporters require the assistance of the Bam complex that also plays a generic role in the assembly of outer membrane proteins. The TPS systems do use a dedicated translocator, but this protein shows resemblance to BamA, the major component of the Bam complex. Interestingly, both the mechanistic and more applied studies on these systems have provided a better understanding of the secretion mechanism and the biogenesis of outer membrane proteins. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Topology and maturation of filamentous haemagglutinin suggest a new model for two-partner secretion

Two-partner secretion (TPS) is the most widely distributed secretion pathway known. These systems export large exoproteins through highly conserved channel-forming beta-barrel proteins. Filamentous haemagglutinin (FHA), expressed by Bordetella species, is the prototypical TPS family member. Here we show that the C-terminus of mature FHA, as opposed to the N-terminus as previously proposed, is exposed on the cell surface and is required for mediating adherence to cultured epithelial cells. We show that the C-terminus of the FHA pro-protein (FhaB) is required for FHA function in vitro and in vivo and we show that cleavage of FhaB to form FHA is not the mechanism by which FHA is released from the cell. Based on these data, we propose a new model for TPS. This model provides an explanation for the energetics of export of globular protein domains across membranes in the absence of ATP and it suggests a new mechanism for the control of protein folding.     

Protein secretion in the absence of ATP: the autotransporter,two-partner secretion and chaperone/usher pathways of Gram-negative bacteria (Review)

Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In Gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these ‘self-sufficient’ pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.     

The underlying mechanisms of type II protein secretion

The cell envelope of Gram-negative bacteria is composed of two membranes, which are separated by the peptidoglycan-containing periplasm. Whereas the envelope forms an essential barrier against harmful substances, it is nevertheless a compartment of intense traffic for large proteins such as enzymes and toxins. Numerous studies dealing with the molecular mechanism of protein secretion have revealed that Gram-negative bacteria evolved different strategies to achieve this process. Among them, the type II secretion mechanism is part of a two-step process. Exoproteins following this pathway are synthesized as signal peptide-containing precursors. After cleavage of the signal peptide, the mature exoproteins are released into the periplasm, where they fold. The type II machinery, also known as the secreton, is responsible for the translocation of the periplasmic intermediates across the OM. The type II system is broadly conserved in Gram-negative bacteria and involves a set of 12-16 different proteins named GspC-M, GspAB, GspN, GspO, and GspS. The type II secretion system is highly reminiscent of the type IV piliation assembly system. Based on findings about the subcellular localisation of the Gsp components, protein-protein interactions between Gsps and their multimerisation status, structural data and electron microscopy observation, it could be proposed a working model that strikingly runs both systems in parallel.     

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.     

Protein secretion in the absence of ATP: the autotransporter, two-partner secretion and chaperone/usher pathways of gram-negative bacteria (review)

Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these 'self-sufficient' pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.     

Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase

The xcp genes are required for the secretion of most extracellular proteins by Pseudomonas aeruginosa. The products of these genes are essential for the transport of exoproteins across the outer membrane after they have reached the periplasm via a signal sequence-dependent pathway. To date, analysis of three xcp genes has suggested the conservation of this secretion pathway in many Gram-negative bacteria. Furthermore, the xcpA gene was shown to be identical to pilD, which encodes a peptidase involved in the processing of fimbrial (pili) subunits, suggesting a connection between pili biogenesis and protein secretion. Here the nucleotide sequences of seven other xcp genes, designated xcpR to -X, are presented. The N-termini of four of the encoded Xcp proteins display similarity to the N-termini of type IV pili, suggesting that XcpA is involved in the processing of these Xcp proteins. This could indeed be demonstrated in vivo. Furthermore, two other proteins, XcpR and XcpS, show similarity to the PilB and PilC proteins required for fimbriae assembly. Since XcpR and PilB display a canonical nucleotide-binding site, ATP hydrolysis may provide energy for both systems.     

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 β-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 β-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°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.     

Secretins of Pseudomonas aeruginosa: large holes in the outer membrane

Pseudomonas aeruginosa produces a large number of exoproteins, ranging from the ADP-ribosyltransferases exotoxin A and ExoS to degradative enzymes, such as elastase and chitinase. As it is a gram-negative bacterium, P. aeruginosa must be able to transport these exoproteins across both membranes of the cell envelope. In addition, also proteins that are part of cellular appendages, such as type IV pili and flagella, have to cross the cell envelope. Whereas the majority of the proteins transported across the inner membrane are dependent on the Sec channel, the systems for translocation across the outer membrane seem to be more diverse. Gram-negative bacteria have invented a number of different strategies during the course of evolution to achieve this goal. Although these transport machineries seem to be radically different, many of them actually depend on a member of the secretin protein family for their function. Recent results show that secretins form a large complex in the outer membrane, which constitutes the actual translocation channel. Understanding the working mechanism of this protein translocation channel could open up new strategies to target molecular machineries at the heart of many important virulence factors.     

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.     

Colicin A hybrids: a genetic tool for selection of type II secretion-proficient Pseudomonas strains

The Gram-negative bacterium Pseudomonas aeruginosa secretes the majority of its extracellular proteins by the type II secretion mechanism, a two-step process initiated by translocation of signal peptide-bearing exoproteins across the inner membrane. The periplasmic forms are transferred across the outer membrane by a machinery consisting of 12 xcp gene products. Although the type II secretion machinery is conserved among Gram-negative bacteria, interactions between the secreted proteins and the machinery are specific. The lack of a selectable phenotype has hampered the development of genetic strategies for studying type II secretion. We report a novel strategy to identify rare events, such as those that allow heterologous secretion or identification of extragenic suppressors correcting xcp defects. This is based on creating a host-vector system where the non-secretory phenotype is lethal. The original tool we designed is a hybrid protein containing elastase and the pore-forming domain of colicin A.     

Secretion signal of the filamentous haemagglutinin, a model two-partner secretion substrate

The sorting of proteins to their proper subcellular compartment requires specific addressing signals that mediate interactions with ad hoc transport machineries. In Gram-negative bacteria, the widespread two-partner secretion (TPS) pathway is dedicated to the secretion of large, mostly virulence-related proteins. The secreted TpsA proteins carry a characteristic 250-residue-long N-terminal 'TPS domain' essential for secretion, while their TpsB transporters are pore-forming proteins that specifically recognize their respective TpsA partners and mediate their translocation across the outer membrane. However, the nature of the secretion signal has not been elucidated yet. The whooping cough agent Bordetella pertussis secretes its major adhesin filamentous haemagglutinin (FHA) via the TpsB transporter FhaC. In this work, we show specific interactions between an N-terminal fragment of FHA containing the TPS domain and FhaC by using two different techniques, an overlay assay and a pull-down of the complex. FhaC recognizes only non-native conformations of the TPS domain, corroborating the model that in vivo, periplasmic FHA is not yet folded. By generating single amino acid substitutions, we have identified interaction determinants forming the secretion signal. They are found unexpectedly far into the TPS domain and include both conserved and variable residues, which most likely explains the specificity of the TpsA-TpsB interaction. The N-terminal domain of FhaC is involved in the FHA-FhaC interaction, in agreement with its proposed function and periplasmic localization.     

Protein secretion systems of Pseudomonas aeruginosa and P fluorescens

Gram-negative bacteria have evolved numerous systems for the export of proteins across their dual-membrane envelopes. Three of these systems (types I, III and IV) secrete proteins across both membranes in a single energy-coupled step. Four systems (Sec, Tat, MscL and Holins) secrete only across the inner membrane, and four systems [the main terminal branch (MTB), fimbrial usher porin (FUP), autotransporter (AT) and two-partner secretion families (TPS)] secrete only across the outer membrane. We have examined the genome sequences of Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens Pf0-1 for these systems. All systems except type IV were found in P. aeruginosa, and all except types III and IV were found in P. fluorescens. The numbers of each such system were variable depending on the system and species examined. Biochemical and physiological functions were assigned to these systems when possible, and the structural constituents were analyzed. Available information regarding the mechanisms of transport and energy coupling as well as physiological functions is summarized. This report serves to identify and characterize protein secretion systems in two divergent pseudomonads, one an opportunistic human pathogen, the other a plant symbiont.     

Substrate recognition by the POTRA domains of TpsB transporter FhaC

Widespread in Gram-negative bacteria, the two-partner secretion (TPS) pathway mediates the secretion of large, β-helical 'TpsA' proteins with various functions. TpsA proteins harbour a conserved, N-proximal TPS domain essential for secretion. TpsB transporters specifically recognize their TpsA partners in the periplasm and mediate their translocation across the outer membrane through a hydrophilic channel. The FHA/FhaC pair of Bordetella pertussis represents a model TPS system. FhaC is composed of a β barrel preceded by two periplasmic POTRA domains in tandem. Here we show that both POTRAs are involved in FHA recognition. Surface plasmon resonance analyses indicated an interaction of micromolar affinity between the POTRAs and the TPS domain with fast association and dissociation steps, consistent with the transient character of this interaction in vivo. Major interaction sites in POTRAs correspond to hydrophobic grooves formed by a β sheet edge and the flanking α helix, well-suited to accommodate extended, amphipathic strands of the substrate and consistent with β augmentation. The initial recruitment of the TPS domain to POTRAs appears to be facilitated by electrostatic attractions. A domain corresponding to the first part of the repeat-rich central region of FHA is also recognized by the POTRAs, suggesting successive interactions in the course of secretion.     

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.     

System Specificity of the TpsB Transporters of Coexpressed Two-Partner Secretion Systems of Neisseria meningitidis

The two-partner secretion (TPS) systems of Gram-negative bacteria consist of a large secreted exoprotein (TpsA) and a transporter protein (TpsB) located in the outer membrane. TpsA targets TpsB for transport across the membrane via its ∼30-kDa TPS domain located at its N terminus, and this domain is also the minimal secretory unit. Neisseria meningitidis genomes encode up to five TpsAs and two TpsBs. Sequence alignments of TPS domains suggested that these are organized into three systems, while there are two TpsBs, which raised questions on their system specificity. We show here that the TpsB2 transporter of Neisseria meningitidis is able to secrete all types of TPS domains encoded in N. meningitidis and the related species Neisseria lactamica but not domains of Haemophilus influenzae and Pseudomonas aeruginosa. In contrast, the TpsB1 transporter seemed to be specific for its cognate N. meningitidis system and did not secrete the TPS domains of other meningococcal systems. However, TpsB1 did secrete the TPS2b domain of N. lactamica, which is related to the meningococcal TPS2 domains. Apparently, the secretion depends on specific sequences within the TPS domain rather than the overall TPS domain structure.