Trimeric autotransporter adhesins: variable structure, common function

Trimeric autotransporter adhesins (TAAs) are important virulence factors in gram-negative pathogens. Despite the variety of hosts ranging from plants to mammals and the specialized regulation of TAAs, their molecular organization follows surprisingly simple rules: they form trimeric surface structures with a head-stalk-anchor architecture. The head and stalk are composed of a small set of domains, building blocks that are frequently arranged repetitively. We propose that this repetitive arrangement facilitates recombination of domains to modulate the specificity of the common function: adhesion to the host.     

A Neisserial autotransporter NalP modulating the processing of other autotransporters

Autotransporters constitute a relatively simple secretion system in Gram-negative bacteria, depending for their translocation across the outer membrane only on a C-terminal translocator domain. We have studied a novel autotransporter serine protease, designated NalP, from Neisseria meningitidis strain H44/76, featuring a lipoprotein motif at the signal sequence cleavage site. Indeed, lipidation of NalP could be demonstrated, but the secreted 70 kDa domain of NalP lacked the lipid-moiety as a result of additional N-terminal processing. A nalP mutant showed a drastically altered profile of secreted proteins. Mass-spectrometric analysis of tryptic fragments identified the autotransporters IgA protease and App, a homologue of the adhesin Hap of Haemophilus influenzae, as the major secreted proteins. Two forms of both of these proteins were found in the culture supernatant of the wild-type strain, whereas only the lower molecular-weight forms predominated in the culture supernatant of the nalP mutant. The serine-protease active site of NalP was required for the modulation of the processing of these autotransporters. We propose that, apart from the autoproteolytic processing, NalP can process App and IgA protease and hypothesize that this function of NalP could contribute to the virulence of the organism.     

Trimeric autotransporters require trimerization of the passenger domain for stability and adhesive activity

In recent years, structural studies have identified a number of bacterial, viral, and eukaryotic adhesive proteins that have a trimeric architecture. The prototype examples in bacteria are the Haemophilus influenzae Hia adhesin and the Yersinia enterocolitica YadA adhesin. Both Hia and YadA are members of the trimeric-autotransporter subfamily and are characterized by an internal passenger domain that harbors adhesive activity and a short C-terminal translocator domain that inserts into the outer membrane and facilitates delivery of the passenger domain to the bacterial surface. In this study, we examined the relationship between trimerization of the Hia and YadA passenger domains and the capacity for adhesive activity. We found that subunit-subunit interactions and stable trimerization are essential for native folding and stability and ultimately for full-level adhesive activity. These results raise the possibility that disruption of the trimeric architecture of trimeric autotransporters, and possibly other trimeric adhesins, may be an effective strategy to eliminate adhesive activity.     

Surface display of proteins by Gram-negative bacterial autotransporters

Expressing proteins of interest as fusions to proteins of the bacterial envelope is a powerful technique with many biotechnological and medical applications. Autotransporters have recently emerged as a good tool for bacterial surface display. These proteins are composed of an N-terminal signal peptide, followed by a passenger domain and a translocator domain that mediates the outer membrane translocation of the passenger. The natural passenger domain of autotransporters can be replaced by heterologous proteins that become displayed at the bacterial surface by the translocator domain. The simplicity and versatility of this system has made it very attractive and it has been used to display functional enzymes, vaccine antigens as well as polypeptides libraries. The recent advances in the study of the translocation mechanism of autotransporters have raised several controversial issues with implications for their use as display systems. These issues include the requirement for the displayed polypeptides to remain in a translocation-competent state in the periplasm, the requirement for specific signal sequences and "autochaperone" domains, and the influence of the genetic background of the expression host strain. It is therefore important to better understand the mechanism of translocation of autotransporters in order to employ them to their full potential. This review will focus on the recent advances in the study of the translocation mechanism of autotransporters and describe practical considerations regarding their use for bacterial surface display.     

Extracellular production of recombinant proteins using bacterial autotransporters

Escherichia coli is still a very popular host for the production of recombinant proteins at an analytical or industrial scale. Secretion of the proteins into the culture medium or display at the cell surface would be preferred in many applications but is hampered by the complex two-layered cell envelope. The autotransporter pathway is used by E. coli to secrete virulence factors via a relatively simple but efficient and specific mechanism. Here we discuss recent progress in the structural and mechanistic analysis of this pathway and the implications for future development of a versatile platform for secretion and display of heterologous proteins.     

Trimeric G proteins and vesicle formation

Among the proteins regulating vesicular traffic, the small, Ras-like GTPases have received particular attention. Several recent reports indicate that another class of GTP-binding (G) protein, the heterotrimeric G proteins, also participates in the regulation of vesicular traffic. Thus, studies using transfected cells and cell-free systems show that a pertussis toxin-sensitive trimeric G protein, G(i3), is involved in the formation of secretory vesicles from the Golgi complex. These results raise the intriguing possibility that signal transduction processes across intracellular membranes play a role in vesicle formation, and provide important clues about the molecular machinery involved in this process.     

TOX defines a conserved subfamily of HMG-box proteins

Background  

HMG-box proteins are a large and diverse superfamily of architectural factors that share one or more copies of a sequence- and structurally-related DNA binding domain. These proteins can modify chromatin structure by bending and unwinding DNA. HMG-box proteins can be divided into two subfamilies based on whether they recognize DNA in a sequence-dependent or sequence-independent manner. We recently identified an HMG-box protein involved in T cell development, designated TOX, which is highly conserved in humans and mice.     

Identification and characterization of autotransporter proteins of Yersinia pestis KIM

Yersinia pestis is a Gram-negative bacterium that causes plague. Currently, plague is considered a re-emerging infectious disease and Y. pestis a potential bioterrorism agent. Autotransporters (ATs) are virulence proteins translocated by a variety of pathogenic Gram-negative bacteria across the cell envelope to the cell surface or extracellular environment. In this study, we screened the genome of Yersinia pestis KIM for AT genes whose expression might be relevant for the pathogenicity of this plague-causing organism. By in silico analyses, we identified ten putative AT genes in the genomic sequence of Y. pestis KIM; two of these genes are located within known pathogenicity islands. The expression of all ten putative AT genes in Y. pestis KIM was confirmed by RT-PCR. Five genes, designated yapA, yapC, yapG, yapK and yapN, were subsequently cloned and expressed in Escherichia coli K12 for protein secretion studies. Two forms of the YapA protein (130 kDa and 115 kDa) were found secreted into the culture medium. Protease cleavage at the C terminus of YapA released the protein from the cell surface. Outer membrane localization of YapC (65 kDa), YapG (100 kDa), YapK (130 kDa), and YapN (60 kDa) was established by cell fractionation, and cell surface localization of YapC and YapN was demonstrated by protease accessibility experiments. In functional studies, YapN and YapK showed hemagglutination activity and YapC exhibited autoagglutination activity. Data reported here represent the first study on Y. pestis ATs.     

Identification and characterization of autotransporter proteins of Yersinia pestis KIM

Yersinia pestis is a Gram-negative bacterium that causes plague. Currently, plague is considered a re-emerging infectious disease and Y. pestis a potential bioterrorism agent. Autotransporters (ATs) are virulence proteins translocated by a variety of pathogenic Gram-negative bacteria across the cell envelope to the cell surface or extracellular environment. In this study, we screened the genome of Yersinia pestis KIM for AT genes whose expression might be relevant for the pathogenicity of this plague-causing organism. By in silico analyses, we identified ten putative AT genes in the genomic sequence of Y. pestis KIM; two of these genes are located within known pathogenicity islands. The expression of all ten putative AT genes in Y. pestis KIM was confirmed by RT-PCR. Five genes, designated yapA, yapC, yapG, yapK and yapN, were subsequently cloned and expressed in Escherichia coli K12 for protein secretion studies. Two forms of the YapA protein (130 kDa and 115 kDa) were found secreted into the culture medium. Protease cleavage at the C terminus of YapA released the protein from the cell surface. Outer membrane localization of YapC (65 kDa), YapG (100 kDa), YapK (130 kDa), and YapN (60 kDa) was established by cell fractionation, and cell surface localization of YapC and YapN was demonstrated by protease accessibility experiments. In functional studies, YapN and YapK showed hemagglutination activity and YapC exhibited autoagglutination activity. Data reported here represent the first study on Y. pestis ATs.     

Characterization of a subfamily of beetle odorant-binding proteins found in hemolymph

In insects, hydrophobic odorants are transported through the sensillar lymph to receptors on sensory neurons by odorant-binding proteins (OBPs). The beetle Tenebrio molitor, which is a pest of stored grain products, produces a set of 12-14-kDa OBP-like proteins in its hemolymph. The structure of one of these proteins and that of a moth pheromone-binding protein have been solved. Both proteins have at least six alpha-helices with an internal, hydrophobic, ligand-binding pocket, but the beetle OBP lacks one of the disulfide bonds immediately adjacent to this pocket. To explore this difference and to sample isoform diversity, T. molitor hemolymph OBPs were fractionated by size-exclusion chromatography and reversed-phase high performance liquid chromatography. Selected fractions were reduced and alkylated, and tryptic peptides were sequenced by tandem mass spectrometry. Partial sequences of 7 different isoforms were obtained and used to clone 9 new cDNAs encoding OBPs with identities from 32 to 99%. The more divergent isoforms have numerous substitutions of hydrophobic residues that presumably alter the shape and specificity of the ligand-binding pocket. These isoforms all lack the same third disulfide bridge and are more similar to one another than to any of the 38 OBPs in Drosophila melanogaster. They have presumably arisen via gene duplication following separation of the major insect orders.     

Polar localization of the autotransporter family of large bacterial virulence proteins

Autotransporters are an extensive family of large secreted virulence-associated proteins of gram-negative bacteria. Secretion of such large proteins poses unique challenges to bacteria. We demonstrate that autotransporters from a wide variety of rod-shaped pathogens, including IcsA and SepA of Shigella flexneri, AIDA-I of diffusely adherent Escherichia coli, and BrkA of Bordetella pertussis, are localized to the bacterial pole. The restriction of autotransporters to the pole is dependent on the presence of a complete lipopolysaccharide (LPS), consistent with known effects of LPS composition on membrane fluidity. Newly synthesized and secreted BrkA is polar even in the presence of truncated LPS, and all autotransporters examined are polar in the cytoplasm prior to secretion. Together, these findings are consistent with autotransporter secretion occurring at the poles of rod-shaped gram-negative organisms. Moreover, NalP, an autotransporter of spherically shaped Neisseria meningitidis contains the molecular information to localize to the pole of Escherichia coli. In N. meningitidis, NalP is secreted at distinct sites around the cell. These data are consistent with a model in which the secretion of large autotransporters occurs via specific conserved pathways located at the poles of rod-shaped bacteria, with profound implications for the underlying physiology of the bacterial cell and the nature of bacterial pathogen-host interactions.     

Misfolding of a bacterial autotransporter

The adhesin involved in diffuse adherence (AIDA) is an autotransporter protein that confers the diffuse adherence phenotype to certain diarrheagenic Escherichia coli strains. It consists of a 49 amino acid signal peptide, a 797 amino acid passenger domain, and a 440 amino acid beta-domain integrated into the outer membrane. The beta-domain consists of two parts: the beta(1)-domain, which is predicted to form two beta-strands on the bacterial cell surface, and the beta(2)-domain, which constitutes the transmembrane domain. We have previously shown that the beta-domain can be folded from the urea-denatured state when bound to a nickel column during purification. It has not been possible to achieve proper refolding of the beta-domain in solution; instead, a misfolded state C is formed. Here, we characterize this misfolded state in greater detail, showing that despite being misfolded, C can be analyzed as a conventional conformational state, with cooperative unfolding in urea and SDS as well as showing simple exponential kinetics during its formation in the presence of lipid vesicles and detergent micelles. The kinetics of formation of C is sensitive to the lipid composition in vesicles. We have also attempted to identify biological factors that might aid folding of the beta-domain to the properly folded state. However, no purified periplasmic or cytosolic chaperone was found to increase folding yields, and no factor in a periplasmic extract was identified that could bind to C. We conclude that it is the exposure to the unique spatial arrangement of the bacterial cell that leads to proper refolding of the beta-domain.     

Requirement for YaeT in the outer membrane assembly of autotransporter proteins

Autotransporters constitute the largest group of secreted proteins in gram-negative bacteria. Autotransporter secretion involves the insertion of a carboxy-terminal beta barrel into and the translocation of an amino-terminal domain across the outer membrane. Here, we demonstrate that secretion of autotransporters from several organisms requires the outer membrane assembly factor YaeT.     

Distorted octahedral coordination of tungstate in a subfamily of specific binding proteins

Bacteria and archaea import molybdenum and tungsten from the environment in the form of the oxyanions molybdate (MoO₄ ²⁻) and tungstate (WO₄ ²⁻). These substrates are captured by an external, high-affinity binding protein, and delivered to ATP binding cassette transporters, which move them across the cell membrane. We have recently reported a crystal structure of the molybdate/tungstate binding protein ModA/WtpA from Archaeoglobus fulgidus, which revealed an octahedrally coordinated central metal atom. By contrast, the previously determined structures of three bacterial homologs showed tetracoordinate molybdenum and tungsten atoms in their binding pockets. Until then, coordination numbers above four had only been found for molybdenum/tungsten in metalloenzymes where these metal atoms are part of the catalytic cofactors and coordinated by mostly non-oxygen ligands. We now report a high-resolution structure of A. fulgidus ModA/WtpA, as well as crystal structures of four additional homologs, all bound to tungstate. These crystal structures match X-ray absorption spectroscopy measurements from soluble, tungstate-bound protein, and reveal the details of the distorted octahedral coordination. Our results demonstrate that the distorted octahedral geometry is not an exclusive feature of the A. fulgidus protein, and suggest distinct binding modes of the binding proteins from archaea and bacteria. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. K. Hollenstein and M. Comellas-Bigler contributed equally to this work.     

The crystal structure of Streptococcus pyogenes uridine phosphorylase reveals a distinct subfamily of nucleoside phosphorylases

Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2'-deoxyuridine to uracil and ribose 1-phosphate or 2'-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 ? resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an α/β monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild-type SpUP showed that its substrate specificity is similar to that of EcUP, while EcUP is ～7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.     

Structure and dynamics of the human pleckstrin DEP domain: distinct molecular features of a novel DEP domain subfamily

Pleckstrin1 is a major substrate for protein kinase C in platelets and leukocytes, and comprises a central DEP (disheveled, Egl-10, pleckstrin) domain, which is flanked by two PH (pleckstrin homology) domains. DEP domains display a unique alpha/beta fold and have been implicated in membrane binding utilizing different mechanisms. Using multiple sequence alignments and phylogenetic tree reconstructions, we find that 6 subfamilies of the DEP domain exist, of which pleckstrin represents a novel and distinct subfamily. To clarify structural determinants of the DEP fold and to gain further insight into the role of the DEP domain, we determined the three-dimensional structure of the pleckstrin DEP domain using heteronuclear NMR spectroscopy. Pleckstrin DEP shares main structural features with the DEP domains of disheveled and Epac, which belong to different DEP subfamilies. However, the pleckstrin DEP fold is distinct from these structures and contains an additional, short helix alpha4 inserted in the beta4-beta5 loop that exhibits increased backbone mobility as judged by NMR relaxation measurements. Based on sequence conservation, the helix alpha4 may also be present in the DEP domains of regulator of G-protein signaling (RGS) proteins, which are members of the same DEP subfamily. In pleckstrin, the DEP domain is surrounded by two PH domains. Structural analysis and charge complementarity suggest that the DEP domain may interact with the N-terminal PH domain in pleckstrin. Phosphorylation of the PH-DEP linker, which is required for pleckstrin function, could regulate such an intramolecular interaction. This suggests a role of the pleckstrin DEP domain in intramolecular domain interactions, which is distinct from the functions of other DEP domain subfamilies found so far.     

Directed evaluation of enterotoxigenic Escherichia coli autotransporter proteins as putative vaccine candidates

Background

Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in developing countries, where it accounts for millions of infections and hundreds of thousands of deaths annually. While vaccine development to prevent diarrheal illness due to ETEC is feasible, extensive effort is needed to identify conserved antigenic targets. Pathogenic Escherichia coli, including ETEC, use the autotransporter (AT) secretion mechanism to export virulence factors. AT proteins are comprised of a highly conserved carboxy terminal outer membrane beta barrel and a surface-exposed amino terminal passenger domain. Recent immunoproteomic studies suggesting that multiple autotransporter passenger domains are recognized during ETEC infection prompted the present studies.

Methodology

Available ETEC genomes were examined to identify AT coding sequences present in pathogenic isolates, but not in the commensal E. coli HS strain. Passenger domains of the corresponding autotransporters were cloned and expressed as recombinant antigens, and the immune response to these proteins was then examined using convalescent sera from patients and experimentally infected mice.

Principal Findings

Potential AT genes shared by ETEC strains, but absent in the E. coli commensal HS strain were identified. Recombinant passenger domains derived from autotransporters, including Ag43 and an AT designated pAT, were recognized by antibodies from mice following intestinal challenge with {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407, and both Ag43 and pAT were identified on the surface of ETEC by flow cytometry. Likewise, convalescent sera from patients with ETEC diarrhea recognized Ag43 and pAT, suggesting that these proteins are expressed during both experimental and naturally occurring ETEC infections and that they are immunogenic. Vaccination of mice with recombinant passenger domains from either pAT or Ag43 afforded protection against intestinal colonization with ETEC.

Conclusions

Passenger domains of conserved autotransporter proteins could contribute to protective immune responses that develop following infection with ETEC, and these antigens consequently represent potential targets to explore in vaccine development.     

The passenger‐associated transport repeat promotes virulence factor secretion efficiency and delineates a distinct autotransporter subtype

Autotransporters are a superfamily of virulence factors secreted by Gram negative bacteria. They are comprised of an N‐terminal passenger domain that is translocated across the outer membrane and a C‐terminal domain that inserts into the outer membrane forming a β‐barrel anchor. It is still poorly understood how the passenger is efficiently translocated in the absence of external energy inputs. Several mechanisms have been proposed in solution of this problem, yet due to the vast diversity of size, sequence and function of the passenger, it is not clear how widely these mechanisms are employed. In this study we functionally characterize a conserved repeat found in many passengers that we designate the Passenger‐associated Transport Repeat (PATR). Using the autotransporter IcsA from the enteropathogen Shigella flexneri, we identified conserved PATR residues that are required for efficient export of the passenger during growth and infection. Furthermore, PATR‐containing autotransporters are significantly larger than non‐PATR autotransporters, with PATR copy number correlating with passenger size. We also show that PATR‐containing autotransporters delineate a subgroup that associates with specific virulence traits and architectures. These results advance our understanding of autotransporter composition and indicate that an additional transport mechanism is important for thousands of these proteins.