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 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 Salmonella typhimurium InvH protein is an outer membrane lipoprotein required for the proper localization of InvG

The secretion of pathogenicity factors by Salmonella typhimurium is mediated by a type III secretion system that includes an outer membrane protein of the secretin family. Related secretins are also required for f1 phage assembly and type II secretion. When the C-terminal 43 amino acids of the S. typhimurium secretin InvG are added to f1 pIV, the chimeric f1 pIV-'InvG₄₃ protein becomes dependent on the co-expression of another gene, invH , for function in phage assembly. [³H]-palmitic acid labelling, globomycin sensitivity and density gradient flotation were used to demonstrate that InvH is an outer membrane lipoprotein that is processed by signal peptidase II. A complex between chimeric f1 pIV-'InvG₄₃ and InvH was demonstrated in vivo. InvH was shown to be required for the proper localization of InvG in the outer membrane and for the secretion of the virulence factor SipC. These results suggest that InvH and InvG are part of the functional outer membrane translocation complex in type III secretion systems.     

Channel-tunnels: outer membrane components of type I secretion systems and multidrug efflux pumps of Gram-negative bacteria

For translocation across the cell envelope of Gram-negative bacteria, substances have to overcome two permeability barriers, the inner and outer membrane. Channel-tunnels are outer membrane proteins, which are central to two distinct export systems: the type I secretion system exporting proteins such as toxins or proteases, and efflux pumps discharging antibiotics, dyes, or heavy metals and thus mediating drug resistance. Protein secretion is driven by an inner membrane ATP-binding cassette (ABC) transporter while drug efflux occurs via an inner membrane proton antiporter. Both inner membrane transporters are associated with a periplasmic accessory protein that recruits an outer membrane channel-tunnel to form a functional export complex. Prototypes of these export systems are the hemolysin secretion system and the AcrAB/TolC drug efflux pump of Escherichia coli, which both employ TolC as an outer membrane component. Its remarkable conduit-like structure, protruding 100 ? into the periplasmic space, reveals how both systems are capable of transporting substrates across both membranes directly from the cytosol into the external environment. Proteins of the channel-tunnel family are widespread within Gram-negative bacteria. Their involvement in drug resistance and in secretion of pathogenic factors makes them an interesting system for further studies. Understanding the mechanism of the different export apparatus could help to develop new drugs, which block the efflux pumps or the secretion system. Electronic Publication     

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.     

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.     

Salmonella InvG forms a ring-like multimer that requires the InvH lipoprotein for outer membrane localization

Salmonella species translocate virulence effector proteins from the bacterial cytoplasm into mammalian host cells by means of a type III secretion apparatus, encoded by the pathogenicity island-1 (SPI-1). Little is known about the assembly and structure of this secretion apparatus, but the InvG protein is essential and could be an outer membrane secretion channel for the effector proteins. We observed that in recombinant Escherichia coli , the yield of InvG was enhanced by co-expression of InvH, and showed that mutation of invH decreased the level of InvG in wild-type Salmonella typhimurium . In E. coli , InvG alone was able to form an SDS-resistant multimer, but InvG localization to the outer membrane was dependent upon InvH, a lipoprotein itself located in the outer membrane, and no other SPI-1 specific protein. InvG targeted to the outer membrane by InvH became accessible to extracellular protease. InvG and InvH did not, however, appear to form a stable complex. Electron microscopy of InvG membrane protein purified from E. coli revealed that it forms an oligomeric ring-like structure with inner and outer diameters, 7 nm and 15 nm respectively.     

CsgE is a curli secretion specificity factor that prevents amyloid fibre aggregation

Curli are extracellular amyloid fibres produced by Escherichia coli that are critical for biofilm formation and adhesion to biotic and abiotic surfaces. CsgA and CsgB are the major and minor curli subunits, respectively, while CsgE, CsgF and CsgG direct the extracellular localization and assembly of curli subunits into fibres. The secretion and stability of CsgA and CsgB are dependent on the outer membrane lipoprotein CsgG. Here, we identified functional interactions between CsgG and CsgE during curli secretion. We discovered that CsgG overexpression restored curli production to a csgE strain under curli-inducing conditions. In antibiotic sensitivity and protein secretion assays, CsgG expression alone allowed translocation of erythromycin and small periplasmic proteins across the outer membrane. Coexpression of CsgE with CsgG blocked non-specific protein and antibiotic passage across the outer membrane. However, CsgE did not block secretion of proteins containing a 22-amino-acid putative outer membrane secretion signal of CsgA (A22). Finally, using purified proteins, we found that CsgE prohibited the self-assembly of CsgA into amyloid fibres. Collectively, these data indicate that CsgE provides substrate specificity to the curli secretion pore CsgG, and acts directly on the secretion substrate CsgA to prevent premature subunit assembly.     

Outer membrane translocons: structural insights into channel formation

Gram-negative bacteria need to maintain the integrity of their outer membrane while also regulating the secretion of toxins and other macromolecules. A variety of dedicated outer membrane proteins (OMPs) facilitate this process. Recent structural work has shown that some of these proteins adopt classical β-barrel transmembrane structures and rely on structural changes within the barrel lumen to allow passage of substrate proteins. Other secretion systems have OMP components which use transmembrane α-helices and appear to function in a different way. Here we review a selection of recent structural studies which have major ramifications for our understanding of the passage of macromolecules across the outer membrane.     

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.     

Protein import and export across the bacterial outer membrane

The bacterial outer membrane forms an impermeable barrier to the environment, but a wide variety of substances must cross it without compromising the membrane. Perhaps, the most fascinating transport phenomenon is the import and export of very large protein toxins using relatively small β-barrel proteins residing in the outer membrane. Progress has been made on three systems in recent years that shed light on this process. In this review, we summarize bacteriocin (toxin) import using TonB-dependent transporters and protein secretion by autotransporters and two partner secretion systems.     

Hydrophobic residues of the autotransporter EspP linker domain are important for outer membrane translocation of its passenger

The autotransporter family of proteins is an important class of Gram-negative secreted virulence factors. Their secretion mechanism comprises entry to the periplasm via the Sec apparatus, followed by formation of an outer membrane beta barrel, which allows the N-terminal passenger domain to pass to the extracellular space. Several groups have identified a region immediately upstream of the beta domain that is important for outer membrane translocation, the so-called linker region. Here we characterize this region in EspP, a prototype of the serine protease autotransporters of enterobacteriaceae. We hypothesized that the folding of this region would be important in the outer membrane translocation process. We tested this hypothesis using a mutagenesis approach in conjunction with a series of nested deletions and found that in the absence of a complete passenger, mutations to the C-terminal helix, but not the upstream linker, significantly decrease secretion efficiency. However, in the presence of the passenger mutations to the amino-terminal region of the linker decrease secretion efficiency. Moreover, amino acids of hydrophobic character play a crucial role in linker function, suggesting the existence of a hydrophobic core or hydrophobic interaction necessary for outer membrane translocation of autotransporter proteins.     

The mechanism of secretion of hemolysin and other polypeptides from Gram-negative bacteria

In the secretion of polypeptides from Gram-negative bacteria, the outer membrane constitutes a specific barrier which has to be circumvented. In the majority of systems, secretion is two-step process, with initial export to the periplasm involving an N-terminal signal sequence. Transport across the outer membrane then involves a variable number of ancillary polypeptides including both periplasmic and outer membrane. While such ancillary proteins are probably specific for each secreted protein, the mechanism of movement across the outer membrane is unknown. In contrast to these systems, secretion of theE. coli hemolysin (HlyA) has several distinctive features. These include a novel targeting signal located within the last 50 or so C-terminal amino acids, the absence of any periplasmic intermediates in transfer, and a specific membrane-bound translocator, HlyB, with important mammalian homologues such as P-glycoprotein (Mdr) and the cystic fibrosis protein. In this review we discuss the nature of the HlyA targeting signal, the structure and function of HlyB, and the probability that HlyA is secreted directly to the medium through a trans-envelope complex composed of HlyB and HlyD.     

Secretion of the Serratia marcescens HasA protein by an ABC transporter.

We previously identified a Serratia marcescens extracellular protein, HasA, able to bind heme and required for iron acquisition from heme and hemoglobin by the bacterium. This novel type of extracellular protein does not have a signal peptide and does not show sequence similarities to other proteins. HasA secretion was reconstituted in Escherichia coli, and we show here that like many proteins lacking a signal peptide, HasA has a C-terminal targeting sequence and is secreted by a specific ATP binding cassette (ABC) transporter consisting of three proteins, one inner membrane protein with a conserved ATP binding domain, called the ABC; a second inner membrane protein; and a third, outer membrane component. Since the three S. marcescens components of the HasA transporter have not yet been identified, the reconstituted HasA secretion system is a hybrid. It consists of the two S. marcescens inner membrane-specific components, HasD and HasE, associated with an outer membrane component coming from another bacterial ABC transporter, such as the E. coli TolC protein, the outer membrane component of the hemolysin transporter, or the Erwinia chrysanthemi PrtF protein, the outer membrane component of the protease transporter. This hybrid transporter was first shown to allow the secretion of the S. marcescens metalloprotease and the E. chrysanthemi metalloproteases B and C. On account of that, the two S. marcescens components HasD and HasE were previously named PrtDSM and PrtESM, respectively. However, HasA is secreted neither by the PrtD-PrtE-PrtF transporter (the genuine E. chrysanthemi protease transporter) nor by the HlyB-HlhD-TolC transporter (the hemolysin transporter). Moreover, HasA, coexpressed in the same cell, strongly inhibits the secretion of proteases B and C by their own transporter, indicating that the E. chrysanthemi transporter recognizes HasA. Since PrtF could replace TolC in the constitution of the HasA transporter, this indicates that the secretion block does not take place at the level of the outer membrane component but rather at an earlier step of interaction between HasA and the inner membrane components.     

Secretion of pectate lyase and protein composition of the outer membrane of Erwinia chrysanthemi ENA49 cells

The combined changes (specters of isoenzymes of intracellular and extracellular pectatelyase, protein composition of periplasm and outer membrane) in the cells of E. chrysanthemi ENA49 from the periodical culture growing on the inducer containing medium have been studied. The beginning of active pectatelyase synthesis was established to be accompanied by the temporal intracellular accumulation of the enzyme. The cellular capability of pectatelyase secretion to the outer medium correlates with the presence of the definite protein in the outer membrane. The different pathways for secretion of pectatelyase isoenzymes PLb, PLc, PLd and PLe are suggested.     

Subcellular location and unique secretion of the hemolysin of Serratia marcescens

It is shown that Serratia marcescens exports a hemolysin to the cell surface and secretes it to the extracellular space. Escherichia coli containing the cloned hemolysin genes shlA and shlB exported and secreted the S. marcescens hemolysin. A nonhemolytic secretion-incompetent precursor of the hemolysin, designated ShlA*, was synthesized in a shlB deletion mutant and accumulated in the periplasmic space of E. coli. Immunogold-labeled ultrathin sections revealed ShlA* bound to the outer face of the cytoplasmic membrane and to the inner face of the outer membrane. A number of mutants carrying 3' deletions in the shlA gene secreted truncated polypeptides, the smallest of which contained only 261 of the 1578 amino acids of the mature ShlA hemolysin, showing that the information for export to the cell surface of E. coli and secretion into the culture medium is located in the NH2-terminal segment of the hemolysin. We propose a secretion pathway in which ShlA and ShlB are exported across the cytoplasmic membrane via a signal sequence-dependent mechanism. ShlB is integrated into the outer membrane. ShlA is translocated across the outer membrane with the help of ShlB. During the latter export process or at the cell surface, ShlA acquires the hemolytically active conformation and is released to the extracellular space. The hemolysin secretion pathway appears to be different from any other secretion system hitherto reported and involves only a single specific export protein.     

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.     

Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway

The energy requirement for the second step in pullulanase secretion by the general secretory pathway was studied in Escherichia coli . In order to uncouple the two steps in the secretion pathway (across the cytoplasmic and outer membranes, respectively) and to facilitate kinetic analysis of secretion, a variant form of pullulanase lacking its N-terminal fatty acid membrane anchor was used. The transport of the periplasmic secretion intermediate form of this protein across the outer membrane was not inhibited by concentrations of sodium arsenate in excess of those required to reduce ATP levels to ≤10% of their normal value. Pullulanase secretion was inhibited by the protonophore carbonyl cyanide m -chlorophenyl hydrazone at concentrations which were similar to those reported by others to be required to prevent solute uptake or the export and processing of preproteins across the cytoplasmic membrane, but which were in excess of those required to fully dissipate the proton-motive force and to reduce lactose uptake to a significant extent.     

Biology of type II secretion

The type II secretion pathway or the main terminal branch of the general secretion pathway, as it has also been referred to, is widely distributed among Proteobacteria, in which it is responsible for the extracellular secretion of toxins and hydrolytic enzymes, many of which contribute to pathogenesis in both plants and animals. Secretion through this pathway differs from most other membrane transport systems, in that its substrates consist of folded proteins. The type II secretion apparatus is composed of at least 12 different gene products that are thought to form a multiprotein complex, which spans the periplasmic compartment and is specifically required for translocation of the secreted proteins across the outer membrane. This pathway shares many features with the type IV pilus biogenesis system, including the ability to assemble a pilus-like structure. This review discusses recent findings on the organization of the secretion apparatus and the role of its various components in secretion. Different models for pilus-mediated secretion through the gated pore in the outer membrane are also presented, as are the possible properties that determine whether a protein is recognized and secreted by the type II pathway.     

Cellular locations of Pseudomonas syringae pv. syringae HrcC and HrcJ proteins, required for harpin secretion via the type III pathway

The complete hrp-hrc-hrmA cluster of Pseudomonas syringae pv. syringae 61 encodes 28 polypeptides. A saprophytic bacterium carrying this cluster is capable of secreting HrpZ-a harpin encoded by hrpZ-in an hrp-dependent manner, which suggests that this cluster contains sufficient components to assemble functional type III secretion machinery. Sequence data show that HrcJ and HrcC are putative outer membrane proteins, and nonpolar mutagenesis demonstrates they are all required for HrpZ secretion. In this study, we investigated the cellular localization of the HrcC and HrcJ proteins by Triton solubilization, sucrose-gradient isopycnic centrifugation, and immunogold labeling of the bacterial cell surface. Our results indicate that HrcC is indeed an outer membrane protein and that HrcJ is located between both membranes. Their membrane localization suggests that they might be involved in the formation of a supramolecular structure for protein secretion.