Two regions of EpsL involved in species-specific protein-protein interactions with EpsE and EpsM of the general secretion pathway in Vibrio cholerae

Extracellular secretion of proteins via the type II or general secretion pathway in gram-negative bacteria requires the assistance of at least 12 gene products that are thought to form a complex apparatus through which secreted proteins are translocated. Although this apparatus is specifically required only for the outer membrane translocation step during transport across the bacterial cell envelope, it is believed to span both membranes. The EpsE, EpsL, and EpsM proteins of the type II apparatus in Vibrio cholerae are thought to form a trimolecular complex that is required to either control the opening and closing of the secretion pore or to transduce energy to the site of outer membrane translocation. EpsL is likely to play an important role in this relay by interacting with both the cytoplasmic EpsE protein and the cytoplasmic membrane protein EpsM, which is predominantly exposed on the periplasmic side of the membrane. We have now extended this model and mapped the separate regions within EpsL that contain the EpsE and EpsM binding domains. By taking advantage of the species specificity of the type II pathway, we have used chimeric proteins composed of EpsL and its homologue, ExeL, from Aeromonas hydrophila together with either EpsE or its Aeromonas homologue, ExeE, to complement the secretion defect in both epsL and exeL mutant strains. These studies have mapped the species-specific EpsE binding site to the N-terminal cytoplasmic region between residues 57 and 216 of EpsL. In addition, the species-specific EpsM binding site was mapped to the C-terminal half of EpsL by coimmunoprecipitation of EpsM with different EpsL-ExeL chimeras. This site is present in the region between amino acids 216 and 296, which contains the predicted membrane-spanning segment of EpsL.     

Type V protein secretion: simplicity gone awry?

Since its discovery in the late 1980's, the family of secreted proteins termed the autotransporters has been expanding continuously to become the largest group of secreted proteins in Gram-negative bacteria. The type V secretion pathway, which includes the autotransporters (type Va) together with the two-partner secretion system (type Vb) and the Oca family (type Vc), can be defined by secreted proteins that are (i) translocated across the outer membrane via a transmembrane pore formed by a beta-barrel and (ii) contain all the information required for translocation through the cell envelope. In the light of new discoveries and controversies in this research field, the secretion process of autotransporters, or the type Va secretion system, will be discussed here and placed in the context of the more general field of bacterial protein translocation.     

A TonB-like protein and a novel membrane protein containing an ATP-binding cassette function together in exotoxin secretion

Protein secretion by many Gram-negative bacteria occurs via the type II pathway involving translocation across the cytoplasmic and outer membranes in separate steps. The mechanism by which metabolic energy is supplied to the translocation across the outer membrane is unknown. Here we show that two Aeromonas hydrophila inner membrane proteins, ExeA and ExeB, are required for this process. ExeB bears sequence as well as topological similarity to TonB, a protein which opens gated ports for the inward translocation of ligands across the outer membrane. ExeA is a novel membrane protein which contains a consensus ATP-binding site. Mutations in this site dramatically decreased the rate of secretion of the toxin aerolysin from the cell. ExeB was stable when overproduced in the presence of ExeA, but was degraded when synthesized in its absence, indicating that the two proteins form a complex. These results suggest that ExeA and ExeB may act together to transduce metabolic energy to the opening of a secretion port in the outer membrane.     

Type II secretion: from structure to function

Gram-negative bacteria use the type II secretion system to transport a large number of secreted proteins from the periplasmic space into the extracellular environment. Many of the secreted proteins are major virulence factors in plants and animals. The components of the type II secretion system are located in both the inner and outer membranes where they assemble into a multi-protein, cell-envelope spanning, complex. This review discusses recent progress, particularly newly published structures obtained by X-ray crystallography and electron microscopy that have increased our understanding of how the type II secretion apparatus functions and the role that individual proteins play in this complex system.     

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.     

EscI: a crucial component of the type III secretion system forms the inner rod structure in enteropathogenic Escherichia coli

The T3SS (type III secretion system) is a multi-protein complex that plays a central role in the virulence of many gram-negative bacterial pathogens. This apparatus spans both bacterial membranes and transports virulence factors from the bacterial cytoplasm into eukaryotic host cells. The T3SS exports substrates in a hierarchical and temporal manner. The first secreted substrates are the rod/needle proteins which are incorporated into the T3SS apparatus and are required for the secretion of later substrates, the translocators and effectors. In the present study, we provide evidence that rOrf8/EscI, a poorly characterized locus of enterocyte effacement-encoded protein, functions as the inner rod protein of the T3SS of EPEC (enteropathogenic Escherichia coli). We demonstrate that EscI is essential for type III secretion and is also secreted as an early substrate of the T3SS. We found that EscI interacts with EscU, the integral membrane protein that is linked to substrate specificity switching, implicating EscI in the substrate-switching event. Furthermore, we showed that EscI self-associates and interacts with the outer membrane secretin EscC, further supporting its function as an inner rod protein. Overall, the results of the present study suggest that EscI is the YscI/PrgJ/MxiI homologue in the T3SS of attaching and effacing pathogens.     

Protein secretion in Pseudomonas aeruginosa.

The Gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into the extracellular medium. At least two distinct secretion pathways can be discerned. The majority of the exoproteins are secreted via a two-step mechanism. These proteins are first translocated across the inner membrane in a signal sequence-dependent fashion. The subsequent translocation across the outer membrane requires the products of at least 12 distinct xcp genes. The exact role of one of these proteins, the XcpA protein, has been resolved. It is a peptidase that is required for the processing of the precursors of four other Xcp proteins, thus allowing their assembly into the secretion apparatus. This peptidase is also required for the processing of the precursors of type IV pili subunits. Two other Xcp proteins, XcpR and XcpS, display extensive homology to proteins involved in pili biogenesis, which suggests that the assembly of the secretion apparatus and the biogenesis of type IV pili are related processes. The secretion of alkaline protease does not require the xcp gene products. This enzyme, which is encoded by the aprA gene, is not synthesized in a precursor form with an N-terminal signal sequence. Secretion across the two membranes probably takes place in one step at adhesion zones that may be constituted by three accessory proteins, designated AprD, AprE and AprF. The two secretion pathways found in P. aeruginosa appear to have disseminated widely among Gram-negative bacteria.     

Protein secretion in Pseudomonas aeruginosa

Abstract The Gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into the extracellular medium. At least two distinct secretion pathways can be discerned. The majority of the exoproteins are secreted via a two-step mechanism. These proteins are first translocated across the inner membrane in a signal sequence-dependent fashion. The subsequent translocation across the outer membrane requires the products of at least 12 distinct xcp genes. The exact role of one of these proteins, the XcpA protein, has been resolved. It is a peptidase that is required for the processing of the precursors of four other Xcp proteins, thus allowing their assembly into the secretion apparatus. This peptidase is also required for the processing of the precursors of type IV pili subunits. Two other Xcp proteins, XcpR and XcpS, display extensive homology to proteins involved in pili biogenesis, which suggests that the assembly of the secretion apparatus and the biogenesis of type IV pili are related processes. The secretion of alkaline protease does not require the xcp gene products. This enzyme, which is encoded by the aprA gene, is not synthesized in a precursor form with an N-terminal signal sequence. Secretion across the two membranes probably takes place in one step at adhesion zones that may be constituted by three accessory proteins, designated AprD, AprE and AprF. The two secretion pathways found in P. aeruginosa appear to habe disseminate widely among Gram-negative bacteria.     

On the path to uncover the bacterial type II secretion system

Gram-negative bacteria have evolved several secretory pathways to release enzymes or toxins into the surrounding environment or into the target cells. The type II secretion system (T2SS) is conserved in Gram-negative bacteria and involves a set of 12 to 16 different proteins. Components of the T2SS are located in both the inner and outer membranes where they assemble into a supramolecular complex spanning the bacterial envelope, also called the secreton. The T2SS substrates transiently go through the periplasm before they are translocated across the outer membrane and exposed to the extracellular milieu. The T2SS is unique in its ability to promote secretion of large and sometimes multimeric proteins that are folded in the periplasm. The present review describes recently identified protein-protein interactions together with structural and functional advances in the field that have contributed to improve our understanding on how the type II secretion apparatus assembles and on the role played by individual proteins of this highly sophisticated system.     

Protein secretion by hybrid bacterial ABC-transporters: specific functions of the membrane ATPase and the membrane fusion protein.

The Erwinia chrysanthemi metalloprotease C and the Serratia marcescens haem acquisition protein HasA are both secreted from Gram-negative bacteria by a signal peptide-independent pathway which requires a C-terminal secretion signal and a specific ABC-transporter made up of three proteins: a membrane ATPase (the ABC-protein), a second inner membrane component belonging to the membrane fusion protein family and an outer membrane polypeptide. HasA and protease C transporters are homologous although the secreted polypeptides share no sequence homology. Whereas protease C can use both translocators, HasA is secreted only by its specific transporter. Functional analysis of protease C and HasA secretion through hybrid transporters obtained by combining components from each system demonstrates that the ABC-protein is responsible for the substrate specificity and that inhibition of protease C secretion in the presence of HasA results from a defective interaction between HasA and the ABC-protein. We also show that the outer membrane protein, TolC, can combine with the membrane fusion protein HasE in the presence of either ABC-protein to form a functional transporter but not with the membrane fusion protein, PrtE. This indicates a specific interaction between the outer membrane component and the membrane fusion protein.     

Assembly of XcpR in the Cytoplasmic Membrane Is Required for Extracellular Protein Secretion in Pseudomonas aeruginosa

A broad range of extracellular proteins secreted by Pseudomonas aeruginosa use the type II or general secretory pathway (GSP) to reach the medium. This pathway requires the expression of at least 12 xcp gene products. XcpR, a putative nucleotide-binding protein, is essential for the secretion process across the outer membrane even though the protein contains no hydrophobic sequence that could target or anchor it to the bacterial envelope. For a better understanding of the relationship between XcpR and the other Xcp proteins which are located in the envelope, we have studied its subcellular localization. In a wild-type P. aeruginosa strain, XcpR was found associated with the cytoplasmic membrane. This association depends on the presence of the XcpY protein, which also appears to be necessary for XcpR stability. Functional complementation of an xcpY mutant required the XcpY protein to be expressed at a low level. Higher expression precluded the complementing activity of XcpY, although membrane association of XcpR was restored. This behavior suggested that an excess of free XcpY might interfere with the secretion by formation of inactive XcpR-XcpY complexes which cannot properly interact with their natural partners in the secretion machinery. These data show that a precise stoichiometric ratio between several components may be crucial for the functioning of the GSP.     

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.     

Insertion of the bacterial type III translocon: not your average needle stick

Bacterial type III secretion systems are thought to translocate virulence proteins directly from the bacterial cytoplasm into host cells through a continuous molecular channel. Little is known about how the apparatus itself interacts with membranes and whether insertion of this structure into the host membrane has consequences for the bacteria apart from its beneficial role in delivering virulence proteins. New evidence suggests that membrane insertion of the bacterial type III apparatus might turn on a calcium-dependent signaling pathway resulting in phagolysosomal fusion.     

Identification of two components of the Serratia marcescens metalloprotease transporter: protease SM secretion in Escherichia coli is TolC dependent.

The Serratia marcescens metalloprotease (protease SM) belongs to a family of proteins secreted from gram-negative bacteria by a signal peptide-independent pathway which requires a specific transporter consisting of three proteins: two in the inner membrane and one in the outer membrane. The prtDSM and prtESM genes encoding the two S. marcescens inner membrane components were cloned and expressed in Escherichia coli. Their nucleotide sequence revealed high overall homology with the two analogous inner membrane components of the Erwinia chrysanthemi protease secretion apparatus and lower, but still significant, homology with the two analogous inner membrane components of the E. coli hemolysin transporter. When expressed in E. coli, these two proteins, PrtDSM and PrtESM, allowed the secretion of protease SM only in the presence of TolC protein, the outer membrane component of the hemolysin transporter.     

Involvement of the twin-arginine translocation system in protein secretion via the type II pathway.

The general secretory pathway (GSP) is a two-step process for the secretion of proteins by Gram-negative bacteria. The translocation across the outer membrane is carried out by the type II system, which involves machinery called the secreton. This step is considered to be an extension of the general export pathway, i.e. the export of proteins across the inner membrane by the Sec machinery. Here, we demonstrate that two substrates for the Pseudomonas aeruginosa secreton, both phospholipases, use the twin-arginine translocation (Tat) system, instead of the Sec system, for the first step of translocation across the inner membrane. These results challenge the previous vision of the GSP and suggest for the first time a mosaic model in which both the Sec and the Tat systems feed substrates into the secreton. Moreover, since P.aeruginosa phospholipases are secreted virulence factors, the Tat system appears to be a novel determinant of bacterial virulence.     

External Loops at the C Terminus of Erwinia chrysanthemi Pectate Lyase C Are Required for Species-Specific Secretion through the Out Type II Pathway

The type II secretion system (main terminal branch of the general secretion pathway) is used by diverse gram-negative bacteria to secrete extracellular proteins. Proteins secreted by this pathway are synthesized with an N-terminal signal peptide which is removed upon translocation across the inner membrane, but the signals which target the mature proteins for secretion across the outer membrane are unknown. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete several isozymes of pectate lyase (Pel) by the out-encoded type II pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed pel genes from the other species, suggesting the existence of species-specific secretion signals within these proteins. The functional cluster of E. chrysanthemi out genes carried on cosmid pCPP2006 enables Escherichia coli to secrete E. chrysanthemi, but not E. carotovora, Pels. We exploited the high sequence similarity between E. chrysanthemi PelC and E. carotovora Pel1 to construct 15 hybrid proteins in which different regions of PelC were replaced with homologous sequences from Pel1. The differential secretion of these hybrid proteins by E. coli(pCPP2006) revealed M118 to D175 and V215 to C329 as regions required for species-specific secretion of PelC. We propose that the primary targeting signal is contained within the external loops formed by G274 to C329 but is dependent on residues in M118 to D170 and V215 to G274 for proper positioning.     

Evidence that a globular conformation is not compatible with FhaC-mediated secretion of the Bordetella pertussis filamentous haemagglutinin

The 220 kDa Bordetella pertussis filamentous haemagglutinin (FHA) is the major extracellular protein of this organism. It is exported using a signal peptide-dependent pathway, and its secretion depends on one specific outer membrane accessory protein, FhaC. In this work, we have investigated the influence of conformation on the FhaC-mediated secretion of FHA using an 80 kDa N-terminal FHA derivative, Fha44. In contrast to many signal peptide-dependent secretory proteins, no soluble periplasmic intermediate of Fha44 could be isolated. In addition, cell-associated Fha44 synthesized in the absence of FhaC did not remain competent for extracellular secretion upon delayed expression of FhaC, indicating that the translocation steps across the cytoplasmic and the outer membrane might be coupled. A chimeric protein, in which the globular B subunit of the cholera toxin, CtxB, was fused at the C-terminus of Fha44, was not secreted in B. pertussis or in Escherichia coli expressing FhaC. The hybrid protein was only secreted when both disulphide bond-forming cysteines of CtxB were replaced by serines or when it was produced in DsbA^?E. coli. The Fha44 portion of the secretion-incompetent hybrid protein was partly exposed on the cell surface. These results argue that the Fha44–CtxB hybrid protein transited through the periplasmic space, where disulphide bond formation is specifically catalysed, and that secretion across the outer membrane was initiated. The folded CtxB portion prevented extracellular release of the hybrid, in contrast to the more flexible CtxB domain devoid of cysteines. We propose a secretion model whereby Fha44 transits through the periplasmic space on its way to the cell surface and initiates its translocation through the outer membrane before being released from the cytoplasmic membrane. Coupling of Fha44 translocation across both membranes would delay the acquisition of its folded structure until the protein emerges from the outer membrane. Such a model would be consistent with the extensive intracellular proteolysis of FHA derivatives in B. pertussis.     

Identification of an additional member of the secretin superfamily of proteins in Pseudomonas aeruginosa that is able to function in type II protein secretion

Pseudomonas aeruginosa is a prolific exporter of virulence factors and contains three of the four protein secretion systems that have been described in Gram-negative bacteria. The P. aeruginosa type II general secretory pathway (GSP) is used to export the largest number of proteins from this organism, including lipase, phospholipase C, alkaline phosphatase, exotoxin A, elastase and LasA. Although these exoproteins contain no sequence similarity, they are specifically and efficiently transported by the secretion apparatus. Bacterial homologues of XcpQ (GspD), the only outer membrane component of this system, have been proposed to play the role of gatekeeper, by presumably interacting and recognizing the exported substrates to allow their passage through the outer membrane. While determining the phenotype of non-polar deletions in each of the xcp genes, we have shown that a deletion of the P. aeruginosa strain K xcpQ does not completely abolish protein secretion. As the proposed function of XcpQ should be requisite for secretion, we searched for additional factors that could carry out this role. A cosmid DNA library from a PAK strain deleted for xcpP-Z was tested for its ability to increase protein secretion by screening for enhanced growth on lipid agar, a medium that selects for the secretion of lipase. In this manner, we have identified an XcpQ homologue, XqhA, that is solely responsible for the residual export observed in a Δ xcpQ strain, although it is not required for efficient secretion in wild-type P. aeruginosa . We have also demonstrated that this protein is capable of recognizing all of the exoproteins of P. aeruginosa , arguing against the proposed role of members of the secretin family as determinants of specificity.     

Secretion and assembly of regular surface structures in Gram-negative bacteria

Bacteria synthesize large-sized surface structures through the ordered polymerization of protein subunits. This results in planar or tubular regular structures that have evolved to accomplish specific functions related to the particular environment in which these bacteria are found. Tubular assemblies known as flagella are the most complex structures known in bacteria and consist of a helical rigid filament, a torsion adapter or hook and a proton-fueled rotator known as the basal body. Pili or fimbriae are less complicated helical filaments, which consist of a major subunit and 3-5 minor subunits or pilins, whose main function is the attachment to specific surfaces. Planar structures known as S-layers are the simplest of these regular assemblies and are generally made up of a single subunit packed as a bidimensional crystal around the whole cell surface. Most of the components of these structures have to be secreted through the inner membrane (IM), the periplasm and the outer membrane (OM) before reaching their final destination. The so called general secretory pathway (GSP), or type II secretion system, appears to be implicated in this process to varying degrees, depending on the structure considered. A few S-layers and pili require GSP components but also need specific terminal branches, such as the well known chaperone-usher pathway. On the other hand, only two of the nearly 40 proteins involved in flagellar assembly are dependent on the GSP, while the external components are secreted through a specific pathway similar to the type III systems identified in some pathogens. Moreover, secretion of subunits of S-layers using dedicated type I machinery, without the involvement of any GSP component, has also been observed.