Autotransporter Pathway Research Articles

BamA and BamD Are Essential for the Secretion of Trimeric Autotransporter Adhesins.

The BAM complex in Escherichia coli is composed of five proteins, BamA-E. BamA and BamD are essential for cell viability and are required for the assembly of β-barrel outer membrane proteins. Consequently, BamA and BamD are indispensable for secretion via the classical autotransporter pathway (Type 5a secretion). In contrast, BamB, BamC, and BamE are not required for the biogenesis of classical autotransporters. Recently, we demonstrated that TamA, a homologue of BamA, and its partner protein TamB, were required for efficient secretion of proteins via the classical autotransporter pathway. The trimeric autotransporters are a subset of the Type 5-secreted proteins. Unlike the classical autotransporters, they are composed of three identical polypeptide chains which must be assembled together to allow secretion of their cognate passenger domains. In contrast to the classical autotransporters, the role of the Bam and Tam complex components in the biogenesis of the trimeric autotransporters has not been investigated fully. Here, using the Salmonella enterica trimeric autotransporter SadA and the structurally similar YadA protein of Yersinia spp., we identify the importance of BamA and BamD in the biogenesis of the trimeric autotransporters and reveal that BamB, BamC, BamE, TamA and TamB are not required for secretion of functional passenger domain on the cell surface.ImportanceThe secretion of trimeric autotransporters (TAA’s) has yet to be fully understood. Here we show that efficient secretion of TAAs requires the BamA and D proteins, but does not require BamB, C or E. In contrast to classical autotransporter secretion, neither trimeric autotransporter tested required TamA or B proteins to be functionally secreted.

Sequential Translocation of Polypeptides across the Bacterial Outer Membrane through the Trimeric Autotransporter Pathway.

Trimeric autotransporter adhesins (TAAs) are a family of bacterial outer membrane (OM) proteins that are comprised of three identical subunits. Each subunit contains an N-terminal extracellular ("passenger") domain and a short C-terminal segment that contributes four β strands to a single 12-stranded β barrel. The mechanism by which the passenger domains are translocated across the OM and the energetics of the translocation reaction are poorly understood. To address these issues, we examined the secretion of modified versions of the passenger domain of UpaG, a TAA produced by Escherichia coli CFT073. Using the SpyTag-SpyCatcher system to probe passenger domain localization, we found that both intrinsically disordered polypeptides fused to the UpaG passenger domain and artificially disulfide-bonded polypeptides were secreted effectively but relatively slowly. Surprisingly, we also found that in some cases, the three nonnative passenger domain segments associated with a single trimer were secreted sequentially. Photo-cross-linking experiments indicated that incompletely assembled UpaG derivatives remained bound to the barrel assembly machinery (Bam) complex until all three passenger domains were fully secreted. Taken together, our results strongly suggest that the secretion of polypeptides through the TAA pathway is coordinated with the assembly of the β barrel domain and that the folding of passenger domains in the extracellular space maximizes the rate of secretion. Furthermore, our work provides evidence for an unprecedented sequential mode of protein translocation, at least under specific experimental conditions.IMPORTANCE Trimeric autotransporter adhesins (TAAs) are specialized bacterial outer membrane proteins consisting of three identical subunits. TAAs contain large extracellular domains that trimerize and promote virulence, but the mechanism by which they are secreted is poorly understood. We found that the extracellular domains of a native TAA were secreted rapidly but that disordered and artificially folded polypeptides fused to native passenger domains were secreted in a slow, sequential fashion. Our results strongly suggest that the efficient secretion of native extracellular domains is driven by their trimerization following export but that alternative energy sources can be harnessed to secrete nonnative polypeptides. Furthermore, we obtained evidence that TAA extracellular domains are secreted before the assembly of the linked membrane spanning domain is completed.

Type V Secretion in Gram-Negative Bacteria.

Type V, or "autotransporter," secretion is a term used to refer to several simple protein export pathways that are found in a wide range of Gram-negative bacteria. Autotransporters are generally single polypeptides that consist of an extracellular ("passenger") domain and a β barrel domain that anchors the protein to the outer membrane (OM). Although it was originally proposed that the passenger domain is secreted through a channel formed solely by the covalently linked β barrel domain, experiments performed primarily on the type Va, or "classical," autotransporter pathway have challenged this hypothesis. Several lines of evidence strongly suggest that both the secretion of the passenger domain and the membrane integration of the β barrel domain are catalyzed by the barrel assembly machinery (Bam) complex, a conserved hetero-oligomer that plays an essential role in the assembly of most integral OM proteins. The secretion reaction appears to be driven at least in part by the folding of the passenger domain in the extracellular space. Although many aspects of autotransporter biogenesis remain to be elucidated, it will be especially interesting to determine whether the different classes of proteins that fall under the type V rubric-most of which have not been examined in detail-are assembled by the same basic mechanism as classical autotransporters.

Inhibition of autotransporter biogenesis by small molecules.

SummaryDisarming pathogens by targeting virulence factors is a promising alternative to classic antibiotics. Many virulence factors in Gram‐negative bacteria are secreted via the autotransporter (AT) pathway, also known as Type 5 secretion. These factors are secreted with the assistance of two membrane‐based protein complexes: Sec and Bam. To identify inhibitors of the AT pathway, we used transcriptomics analysis to develop a fluorescence‐based high‐throughput assay that reports on the stress induced by the model AT hemoglobin protease (Hbp) when its secretion across the outer membrane is inhibited. Screening a library of 1600 fragments yielded the compound VUF15259 that provokes cell envelope stress and secretion inhibition of the ATs Hbp and Antigen‐43. VUF15259 also impairs β‐barrel folding activity of various outer membrane proteins. Furthermore, we found that mutants that are compromised in outer membrane protein biogenesis are more susceptible to VUF15259. Finally, VUF15259 induces the release of vesicles that appear to assemble in short chains. Taken together, VUF15259 is the first reported compound that inhibits AT secretion and our data are mostly consistent with VUF15259 interfering with the Bam‐complex as potential mode of action. The validation of the presented assay incites its use to screen larger compound libraries with drug‐like compounds.

The secreted autotransporter toxin (Sat) does not act as a virulence factor in the probiotic Escherichia coli strain Nissle 1917.

BackgroundEscherichia coli Nissle 1917 (EcN) is a probiotic used in the treatment of intestinal diseases. Although it is considered safe, EcN is closely related to the uropathogenic E. coli strain CFT073 and contains many of its predicted virulence elements. Thus, it is relevant to assess whether virulence-associated genes are functional in EcN. One of these genes encodes the secreted autotransporter toxin (Sat), a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs) that are secreted following the type V autotransporter pathway. Sat is highly prevalent in certain E. coli pathogenic groups responsible for urinary and intestinal infections. In these pathogens Sat promotes cytotoxic effects in several lines of undifferentiated epithelial cells, but not in differentiated Caco-2 cells.ResultsHere we provide evidence that sat is expressed by EcN during the colonization of mouse intestine. The EcN protein is secreted as an active serine protease, with its 107 kDa-passenger domain released into the medium as a soluble protein. Expression of recombinant EcN Sat protein in strain HB101 increases paracellular permeability to mannitol in polarized Caco-2 monolayers. This effect, also reported for the Sat protein of diffusely adherent E. coli, is not observed when this protein is expressed in the EcN background. In addition, we show that EcN supernatants confer protection against Sat-mediated effects on paracellular permeability, thus indicating that other secreted EcN factors are able to prevent barrier disruption caused by pathogen-related factors. Sat is not required for intestinal colonization, but the EcNsat::cat mutant outcompetes wild-type EcN in the streptomycin-treated mouse model. Analysis of the presence of sat in 29 strains of the ECOR collection isolated from stools of healthy humans shows 34.8 % positives, with high prevalence of strains of the phylogenetic groups D and B2, related with extra-intestinal infections.ConclusionsSat does not act as a virulence factor in EcN. The role of Sat in intestinal pathogenesis relies on other genetic determinants responsible for the bacterial pathotype.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0591-5) contains supplementary material, which is available to authorized users.

Looks can be deceiving: recent insights into the mechanism of protein secretion by the autotransporter pathway.

Autotransporters are a large superfamily of cell surface proteins produced by Gram-negative bacteria that consist of an N-terminal extracellular domain ('passenger domain') and a C-terminal β-barrel domain that resides in the outer membrane (OM). Although it was originally proposed that the passenger domain is translocated across the OM through a channel formed exclusively by the covalently linked β-barrel domain, this idea has been strongly challenged by a variety of observations. Recent experimental results have suggested a new model in which both the translocation of the passenger domain and the membrane integration of the β-barrel domain are facilitated by the Bam complex, a highly conserved heteroligomer that plays a general role in OM protein assembly. Other factors, including periplasmic chaperones and inner membrane proteins, have also recently been implicated in the biogenesis of at least some members of the autotransporter superfamily. New results have raised intriguing questions about the energetics of the secretion reaction and the relationship between the assembly of autotransporters and the assembly of other classes of OM proteins. Concomitantly, new mechanistic and structural insights have expanded the utility of the autotransporter pathway for the surface display of heterologous peptides and proteins of interest.

An autotransporter display platform for the development of multivalent recombinant bacterial vector vaccines.

BackgroundThe Autotransporter pathway, ubiquitous in Gram-negative bacteria, allows the efficient secretion of large passenger proteins via a relatively simple mechanism. Capitalizing on its crystal structure, we have engineered the Escherichia coli autotransporter Hemoglobin protease (Hbp) into a versatile platform for secretion and surface display of multiple heterologous proteins in one carrier molecule.ResultsAs proof-of-concept, we demonstrate efficient secretion and high-density display of the sizeable Mycobacterium tuberculosis antigens ESAT6, Ag85B and Rv2660c in E. coli simultaneously. Furthermore, we show stable multivalent display of these antigens in an attenuated Salmonella Typhimurium strain upon chromosomal integration. To emphasize the versatility of the Hbp platform, we also demonstrate efficient expression of multiple sizeable antigenic fragments from Chlamydia trachomatis and the influenza A virus at the Salmonella cell surface.ConclusionsThe successful efficient cell surface display of multiple antigens from various pathogenic organisms highlights the potential of Hbp as a universal platform for the development of multivalent recombinant bacterial vector vaccines.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0162-8) contains supplementary material, which is available to authorized users.

Analysis of surface-exposed outer membrane proteins in Helicobacter pylori.

More than 50 Helicobacter pylori genes are predicted to encode outer membrane proteins (OMPs), but there has been relatively little experimental investigation of the H. pylori cell surface proteome. In this study, we used selective biotinylation to label proteins localized to the surface of H. pylori, along with differential detergent extraction procedures to isolate proteins localized to the outer membrane. Proteins that met multiple criteria for surface-exposed outer membrane localization included known adhesins, as well as Cag proteins required for activity of the cag type IV secretion system, putative lipoproteins, and other proteins not previously recognized as cell surface components. We identified sites of nontryptic cleavage consistent with signal sequence cleavage, as well as C-terminal motifs that may be important for protein localization. A subset of surface-exposed proteins were highly susceptible to proteolysis when intact bacteria were treated with proteinase K. Most Hop and Hom OMPs were susceptible to proteolysis, whereas Hor and Hof proteins were relatively resistant. Most of the protease-susceptible OMPs contain a large protease-susceptible extracellular domain exported beyond the outer membrane and a protease-resistant domain at the C terminus with a predicted β-barrel structure. These features suggest that, similar to the secretion of the VacA passenger domain, the N-terminal domains of protease-susceptible OMPs are exported through an autotransporter pathway. Collectively, these results provide new insights into the repertoire of surface-exposed H. pylori proteins that may mediate bacterium-host interactions, as well as the cell surface topology of these proteins.

Charge-dependent secretion of an intrinsically disordered protein via the autotransporter pathway

Autotransporters are a large class of virulence proteins produced by Gram-negative bacteria. They contain an N-terminal extracellular ("passenger") domain that folds into a β-helical structure and a C-terminal β-barrel ("β") domain that anchors the protein to the outer membrane. Because the periplasm lacks ATP, the source of energy that drives passenger domain secretion is unknown. The prevailing model postulates that vectorial folding of the β-helix in the extracellular space facilitates unidirectional secretion of the passenger domain. In this study we used a chimeric protein composed of the 675-residue receptor-binding domain (RD) of the Bordetella pertussis adenylate cyclase toxin CyaA fused to the C terminus of the Escherichia coli O157:H7 autotransporter EspP to test this hypothesis. The RD is a highly acidic, repetitive polypeptide that is intrinsically disordered in the absence of calcium. Surprisingly, we found that the RD moiety was efficiently secreted when it remained in an unfolded conformation. Furthermore, we found that neutralizing or reversing the charge of acidic amino acid clusters stalled translocation in the vicinity of the altered residues. These results challenge the vectorial folding model and, together with the finding that naturally occurring passenger domains are predominantly acidic, provide evidence that a net negative charge plays a significant role in driving the translocation reaction.

Bacterial serine proteases secreted by the autotransporter pathway: classification, specificity, and role in virulence

Serine proteases exist in eukaryotic and prokaryotic organisms and have emerged during evolution as the most abundant and functionally diverse group. In Gram-negative bacteria, there is a growing family of high molecular weight serine proteases secreted to the external milieu by a fascinating and widely employed bacterial secretion mechanism, known as the autotransporter pathway. They were initially found in Neisseria, Shigella, and pathogenic Escherichia coli, but have now also been identified in Citrobacter rodentium, Salmonella, and Edwardsiella species. Here, we focus on proteins belonging to the serine protease autotransporter of Enterobacteriaceae (SPATEs) family. Recent findings regarding the predilection of serine proteases to host intracellular or extracellular protein-substrates involved in numerous biological functions, such as those implicated in cytoskeleton stability, autophagy or innate and adaptive immunity, have helped provide a better understanding of SPATEs' contributions in pathogenesis. Here, we discuss their classification, substrate specificity, and potential roles in pathogenesis.

Flagellar Localization of a Helicobacter pylori Autotransporter Protein

ABSTRACTHelicobacter pylori contains four genes that are predicted to encode proteins secreted by the autotransporter (type V) pathway. One of these, the pore-forming toxin VacA, has been studied in great detail, but thus far there has been very little investigation of three VacA-like proteins. We show here that all three VacA-like proteins are >250 kDa in mass and localized on the surface of H. pylori. The expression of the three vacA-like genes is upregulated during H. pylori colonization of the mouse stomach compared to H. pylori growth in vitro, and a wild-type H. pylori strain outcompeted each of the three corresponding isogenic mutant strains in its ability to colonize the mouse stomach. One of the VacA-like proteins localizes to a sheath that overlies the flagellar filament and bulb, and therefore, we designate it FaaA (flagella-associated autotransporter A). In comparison to a wild-type H. pylori strain, an isogenic faaA mutant strain exhibits decreased motility, decreased flagellar stability, and an increased proportion of flagella in a nonpolar site. The flagellar localization of FaaA differs markedly from the localization of other known autotransporters, and the current results reveal an important role of FaaA in flagellar localization and motility.

A structurally informed autotransporter platform for efficient heterologous protein secretion and display

BackgroundThe self-sufficient autotransporter (AT) pathway, ubiquitous in Gram-negative bacteria, combines a relatively simple protein secretion mechanism with a high transport capacity. ATs consist of a secreted passenger domain and a β-domain that facilitates transfer of the passenger across the cell-envelope. They have a great potential for the extracellular expression of recombinant proteins but their exploitation has suffered from the limited structural knowledge of carrier ATs. Capitalizing on its crystal structure, we have engineered the Escherichia coli AT Hemoglobin protease (Hbp) into a platform for the secretion and surface display of heterologous proteins, using the Mycobacterium tuberculosis vaccine target ESAT6 as a model protein.ResultsBased on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a β-helical core structure (β-stem) was left intact. The resulting Hbp-ESAT6 chimeras were efficiently and stably secreted into the culture medium of E. coli. On the other hand, Hbp-ESAT6 fusions containing a truncated β-stem appeared unstable after translocation, demonstrating the importance of an intact β-stem. By interrupting the cleavage site between passenger and β-domain, Hbp-ESAT6 display variants were constructed that remain cell associated and facilitate efficient surface exposure of ESAT6 as judged by proteinase K accessibility and whole cell immuno-EM analysis. Upon replacement of the passenger side domain of an alternative AT, EspC, ESAT6 was also efficiently secreted, showing the approach is more generally applicable to ATs. Furthermore, Hbp-ESAT6 was efficiently displayed in an attenuated Salmonella typhimurium strain upon chromosomal integration of a single encoding gene copy, demonstrating the potential of the Hbp platform for live vaccine development.ConclusionsWe developed the first structurally informed AT platform for efficient secretion and surface display of heterologous proteins. The platform has potential with regard to the development of recombinant live vaccines and may be useful for other biotechnological applications that require high-level secretion or display of recombinant proteins by bacteria.

Serine protease autotransporters fromShigella flexneriand pathogenicEscherichia colitarget a broad range of leukocyte glycoproteins

The serine protease autotransporters of Enterobacteriaceae (SPATEs) are secreted by pathogenic Gram-negative bacteria through the autotransporter pathway. We previously classified SPATE proteins into two classes: cytotoxic (class 1) and noncytotoxic (class 2). Here, we show that Pic, a class 2 SPATE protein produced by Shigella flexneri 2a, uropathogenic and enteroaggregative Escherichia coli strains, targets a broad range of human leukocyte adhesion proteins. Substrate specificity was restricted to glycoproteins rich in O-linked glycans, including CD43, CD44, CD45, CD93, CD162 (PSGL-1; P-selectin glycoprotein ligand 1), and the surface-attached chemokine fractalkine, all implicated in leukocyte trafficking, migration, and inflammation. N-terminal sequencing of proteolytic products revealed Pic (protease involved in colonization) cleavage sites to occur before Thr or Ser residues. The purified carbohydrate sLewis-X implied in inflammation and malignancy inhibited cleavage of PSGL-1 by Pic. Exposure of human leukocytes to purified Pic resulted in polymorphonuclear cell activation, but impaired chemotaxis and transmigration; Pic-treated T cells underwent programmed cell death. We also show that the Pic-related protease Tsh/Hbp, implicated in extraintestinal infections, exhibited a spectrum of substrates similar to those cleaved by Pic. In the guinea pig keratoconjunctivitis model, a Shigella pic mutant induced greater inflammation than its parent strain. We suggest that the class-2 SPATEs represent unique immune-modulating bacterial virulence factors.

A conserved stable core structure in the passenger domain β‐helix of autotransporter virulence proteins

In Gram-negative bacteria, a wide variety of virulence factors are secreted via the autotransporter (AT) pathway. Intriguingly, there is no significant concentration of ATP in the periplasm, nor a proton gradient across the OM, so the energetic origin of efficient secretion of AT proteins is unknown. More than 97% of AT proteins are predicted to contain right-handed parallel beta-helical structure, and the three crystal structures available for AT passenger domains each contain a long right-handed parallel beta-helix. Previous studies have shown that pertactin, an AT from Bordetella pertussis, exhibits three-state folding and has a C-terminal stable core structure. Here, we show that Pet, an unrelated AT from Escherichia coli, also exhibits three-state unfolding and also has a stable core structure. Deletion mutants, mass spectrometry, and N-terminal sequencing demonstrate that the Pet stable core is also located near the C-terminus of the passenger domain. Moreover, sequence analysis suggests that three-state folding and a C-terminal stable core structure could be important general features of the biogenesis of AT proteins in vivo.

The Autodisplay Story, from Discovery to Biotechnical and Biomedical Applications

Among the pathways used by gram-negative bacteria for protein secretion, the autotransporter pathway represents a solution of impressive simplicity. Proteins are transported, independent of their nature as recombinant or native passengers, as long as the coding nucleotide sequence is inserted in frame between those of an N-terminal signal peptide and a C-terminal domain, referred to as the beta-barrel of the outer membrane translocation unit. The immunoglobulin A1 (IgA1) protease from Neisseria gonorrhoeae was the first identified member of the autotransporter family of secreted proteins. The IgA1 protease was employed in initial experiments investigating autotransporter-mediated surface display of recombinant proteins and to investigate structural and functional requirements. Various other autotransporter proteins have since been described, and the autodisplay system was developed on the basis of the natural Escherichia coli autotransporter protein AIDA-I (adhesin involved in diffuse adherence). Autodisplay has been used for the surface display of random peptide libraries to successfully screen for novel enzyme inhibitors. The autodisplay system was also used for the surface display of functional enzymes, including esterases, oxidoreductases, and electron transfer proteins. Whole E. coli cells displaying enzymes have been utilized to efficiently synthesize industrially important rare organic compounds with specific chirality. Autodisplay of epitopes on the surface of attenuated Salmonella carriers has also provided a novel way to induce immune protection after oral vaccination. This review summarizes the structural and functional features of the autodisplay system, illustrating its discovery and most recent applications. Autodisplay facilitates the export of more than 100,000 recombinant molecules per single cell and permits the oligomerization of subunits on the cell surface as well as the incorporation of inorganic prosthetic groups after transport of apoproteins onto the bacterial surface without disturbing bacterial integrity or viability. We discuss future biotechnical and biomedical applications in the light of these achievements.

Crystal structure of the Helicobacter pylori vacuolating toxin p55 domain

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

Protein Secretion in Gram-Negative Bacteria via the Autotransporter Pathway

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

Autotransporter Proteins.

This review focuses on the function of the Escherichia coli and Salmonella autotransporters for which a considerable amount of literature is available. Members of the serine protease autotransporters of the Enterobacteriaceae (SPATEs) family are proteins from E. coli and Shigella spp., which, like the Neisseria and Haemophilus influenzae IgA1 proteases and Hap, possess a consensus serine protease motif. The largest subfamily of autotransporters is defined by the AidA conserved domain COG3468 and consists of members from a diverse range of animal and plant pathogens including E. coli, S. enterica, Yersinia pestis. This subfamily, which is composed of more than 55 proteins, possesses some of the best-characterized autotransporter proteins including the S. flexneri mediator of motility IcsA, the major phase-variable E. coli outer membrane protein antigen 43 (Ag43) and the diffuse adhering E. coli (DAEC) adhesin AIDA-I, from which this subfamily derives its name. Another member of the AIDA-I family, and one of the most studied autotransporter proteins, is IcsA. The autotransporter pathway is emerging as the most common mechanism of protein translocation across the gram-negative outer membrane.

Structure of the translocator domain of a bacterial autotransporter.

Autotransporters are virulence-related proteins of Gram-negative bacteria that are secreted via an outer-membrane-based C-terminal extension, the translocator domain. This domain supposedly is sufficient for the transport of the N-terminal passenger domain across the outer membrane. We present here the crystal structure of the in vitro-folded translocator domain of the autotransporter NalP from Neisseria meningitidis, which reveals a 12-stranded beta-barrel with a hydrophilic pore of 10 x 12.5 A that is filled by an N-terminal alpha-helix. The domain has pore activity in vivo and in vitro. Our data are consistent with the model of passenger-domain transport through the hydrophilic channel within the beta-barrel, and inconsistent with a model for transport through a central channel formed by an oligomer of translocator domains. However, the dimensions of the pore imply translocation of the secreted domain in an unfolded form. An alternative model, possibly covering the transport of folded domains, is that passenger-domain transport involves the Omp85 complex, the machinery required for membrane insertion of outer-membrane proteins, on which autotransporters are dependent.

PicU, a second serine protease autotransporter of uropathogenic Escherichia coli

Escherichia coli is the major ætiological agent of urinary tract infections (UTI). Like diarrhoeagenic strains of E. coli, uropathogenic isolates possess virulence determinants that distinguish them from commensal strains and allow them to produce the clinical manifestations associated with UTI. Several autotransporter proteins have been associated with the ability of E. coli, and other Gram-negative bacteria, to cause disease. Recently, we described the existence within uropathogenic E. coli (UPEC) strains of Sat, a toxin of the serine protease autotransporter of Enterobacteriaceae (SPATE) subfamily. Using features common to proteins secreted via the autotransporter pathway we have identified nine additional autotransporter proteins from the genomic sequence data of UPEC CFT073. Surprisingly, two additional members of the SPATE subfamily were identified. One protein, designated PicU, was homologous to the Pic protein identified in Shigella flexneri and enteroaggregative E. coli. The PicU protein was expressed and investigated for functional activity.