The Bartonella autotransporter BafA activates the host VEGF pathway to drive angiogenesis.

Pathogenic bacteria of the genus Bartonella can induce vasoproliferative lesions during infection. The underlying mechanisms are unclear, but involve secretion of an unidentified mitogenic factor. Here, we use functional transposon-mutant screening in Bartonella henselae to identify such factor as a pro-angiogenic autotransporter, called BafA. The passenger domain of BafA induces cell proliferation, tube formation and sprouting of microvessels, and drives angiogenesis in mice. BafA interacts with vascular endothelial growth factor (VEGF) receptor-2 and activates the downstream signaling pathway, suggesting that BafA functions as a VEGF analog. A BafA homolog from a related pathogen, Bartonella quintana, is also functional. Our work unveils the mechanistic basis of vasoproliferative lesions observed in bartonellosis, and we propose BafA as a key pathogenic factor contributing to bacterial spread and host adaptation.

The rhizobial autotransporter determines the symbiotic nitrogen fixation activity of Lotus japonicus in a host-specific manner.

Leguminous plants establish endosymbiotic associations with rhizobia and form root nodules in which the rhizobia fix atmospheric nitrogen. The host plant and intracellular rhizobia strictly control this symbiotic nitrogen fixation. We recently reported a Lotus japonicus Fix- mutant, apn1 (aspartic peptidase nodule-induced 1), that impairs symbiotic nitrogen fixation. APN1 encodes a nodule-specific aspartic peptidase involved in the Fix- phenotype in a rhizobial strain-specific manner. This host-strain specificity implies that some molecular interactions between host plant APN1 and rhizobial factors are required, although the biological function of APN1 in nodules and the mechanisms governing the interactions are unknown. To clarify how rhizobial factors are involved in strain-specific nitrogen fixation, we explored transposon mutants of Mesorhizobium loti strain TONO, which normally form Fix- nodules on apn1 roots, and identified TONO mutants that formed Fix+ nodules on apn1 The identified causal gene encodes an autotransporter, part of a protein secretion system of Gram-negative bacteria. Expression of the autotransporter gene in M. loti strain MAFF3030399, which normally forms Fix+ nodules on apn1 roots, resulted in Fix- nodules. The autotransporter of TONO functions to secrete a part of its own protein (a passenger domain) into extracellular spaces, and the recombinant APN1 protein cleaved the passenger protein in vitro. The M. loti autotransporter showed the activity to induce the genes involved in nodule senescence in a dose-dependent manner. Therefore, we conclude that the nodule-specific aspartic peptidase, APN1, suppresses negative effects of the rhizobial autotransporter in order to maintain effective symbiotic nitrogen fixation in root nodules.

A Chimeric EccB-MycP Fusion Protein is Functional and a Stable Component of the ESX-5 Type VII Secretion System Membrane Complex.

The mycosin protease (MycP) is widely conserved in type VII secretion (T7S) systems throughout Actinobacteria. Within the T7S systems of mycobacteria, also known as the ESX systems, MycP is essential for secretion, which is probably linked to its stabilizing effect on the ESX membrane complex. However, it is unknown how this is mediated, as MycP is not a stable component of this complex. In this study, we set out to create a chimeric fusion protein of EccB5 and MycP5, based on a chimeric gene of eccB and mycP in the T7S locus of Bifidobacterium dentium. We show that this fusion protein is functional and capable of complementing ESX-5 secretion in both an eccB5 and a mycP5 knockout in Mycobacterium marinum. To study the ESX complex containing this fusion protein in more detail, we replaced the original eccB5 and mycP5 of the Mycobacterium xenopi esx-5 locus, reconstituted in Mycobacterium smegmatis, with the chimeric gene. The EccB5-MycP5 fusion construct also restored ESX-5 secretion under these double knockout conditions. Subsequent protein pulldowns on the central complex component EccC5 showed that under these conditions, the EccB5-MycP5 fusion was specifically copurified and a stable component of the ESX-5 complex. Based on our results, we can conclude that MycP5 carries out its essential function in secretion in close proximity to EccB5, indicating that EccB5 is the direct interaction partner of MycP5.

Recent advances in the understanding of trimeric autotransporter adhesins.

Adhesion is the initial step in the infection process of gram-negative bacteria. It is usually followed by the formation of biofilms that serve as a hub for further spread of the infection. Type V secretion systems engage in this process by binding to components of the extracellular matrix, which is the first step in the infection process. At the same time they provide protection from the immune system by either binding components of the innate immune system or by establishing a physical layer against aggressors. Trimeric autotransporter adhesins (TAAs) are of particular interest in this family of proteins as they possess a unique structural composition which arises from constraints during translocation. The sequence of individual domains can vary dramatically while the overall structure can be very similar to one another. This patchwork approach allows researchers to draw conclusions of the underlying function of a specific domain in a structure-based approach which underscores the importance of solving structures of yet uncharacterized TAAs and their individual domains to estimate the full extent of functions of the protein a priori. Here, we describe recent advances in understanding the translocation process of TAAs and give an overview of structural motifs that are unique to this class of proteins. The role of BpaC in the infection process of Burkholderia pseudomallei is highlighted as an exceptional example of a TAA being at the centre of infection initiation.

Insights into the autotransport process of a trimeric autotransporter, Yersinia Adhesin A (YadA).

Trimeric autotransporter adhesins (TAAs) are a subset of a larger protein family called the type V secretion systems. They are localized on the cell surface of Gram-negative bacteria, function as mediators of attachment to inorganic surfaces and host cells, and thus include important virulence factors. Yersinia adhesin A (YadA) from Yersinia enterocolitica is a prototypical TAA that is used extensively to study the structure and function of the type Vc secretion system. A solid-state NMR study of the membrane anchor domain of YadA previously revealed a flexible stretch of small residues, termed the ASSA region, that links the membrane anchor to the stalk domain. In this study, we present evidence that single amino acid proline substitutions produce two different conformers of the membrane anchor domain of YadA; one with the N-termini facing the extracellular surface, and a second with the N-termini located in the periplasm. We propose that TAAs adopt a hairpin intermediate during secretion, as has been shown before for other subtypes of the type V secretion system. As the YadA transition state intermediate can be isolated from the outer membrane, future structural studies should be possible to further unravel details of the autotransport process.

Identification of a novel post-insertion step in the assembly of a bacterial outer membrane protein.

Although the barrel assembly machinery (Bam) complex has been shown to facilitate the insertion of ß barrel proteins into the bacterial outer membrane (OM), the stage at which ß barrels fold is unknown. Here, we describe insights into ß barrel assembly that emerged from an analysis of a member of the autotransporter family of OM proteins (EspP) in Escherichia coli. EspP contains an extracellular 'passenger' domain that is translocated across the OM and then released from the covalently linked ß barrel domain in an intra-barrel cleavage reaction. We found that the mutation of an unusual lipid-exposed lysine residue impairs a previously unidentified late folding step that follows both the membrane insertion of the ß barrel domain and the secretion of the passenger domain but that precedes proteolytic maturation. Our results demonstrate that ß barrel assembly can be completed at a post-insertion stage and raise the possibility that interactions with membrane lipids can promote folding in vivo. Furthermore, by showing that the passenger domain is secreted before the ß barrel domain is fully assembled, our results also provide evidence against the long-standing hypothesis that autotransporters are autonomous protein secretion systems.

Hop-family Helicobacter outer membrane adhesins form a novel class of Type 5-like secretion proteins with an interrupted ß-barrel domain.

The human stomach pathogen Helicobacter pyloriattaches to healthy and inflamed gastric tissue through members of a paralogous family of 'Helicobacter outer membrane proteins' (Hops), including adhesins BabA, SabA, HopQ, LabA and HopZ. Hops share a conserved 25 kDa C-terminal region that is thought to form an autotransporter-like transmembrane domain. Instead, our results show that Hops contain a non-continuous transmembrane domain, composed of seven predicted ß-strands at the C-terminus and one at the N-terminus. Folding and outer membrane localization of the C-terminal ß-domain critically depends on a predicted transmembrane ß-strand within the first 16 N-terminal residues. The N-terminus is shown to reside in the periplasm, and our crystal and small angle X-ray scattering structures for the SabA extracellular domain reveal a conserved coiled-coil stem domain that connects to transmembrane ß-strand 1 and 2. Taken together, our data show that Hop adhesins represent a novel outer membrane protein topology encompassing an OmpA-like 8-stranded ß-barrel that is interrupted by a 15-108 kDa domain inserted inside the first extracellular loop. The insertion of large, folded domains in an extracellular loop is unprecedented in bacterial outer membrane proteins and is expected to have important consequences on how these proteins reach the cell surface.

Molecular basis for the folding of ß-helical autotransporter passenger domains.

Bacterial autotransporters comprise a C-terminal ß-barrel domain, which must be correctly folded and inserted into the outer membrane to facilitate translocation of the N-terminal passenger domain to the cell exterior. Once at the surface, the passenger domains of most autotransporters are folded into an elongated ß-helix. In a cellular context, key molecules catalyze the assembly of the autotransporter ß-barrel domain. However, how the passenger domain folds into its functional form is poorly understood. Here we use mutational analysis on the autotransporter Pet to show that the ß-hairpin structure of the fifth extracellular loop of the ß-barrel domain has a crucial role for passenger domain folding into a ß-helix. Bioinformatics and structural analyses, and mutagenesis of a homologous autotransporter, suggest that this function is conserved among autotransporter proteins with ß-helical passenger domains. We propose that the autotransporter ß-barrel domain is a folding vector that nucleates folding of the passenger domain.

Utilization of Variant and Fusion Proteins To Functionally Map the Aggregatibacter actinomycetemcomitans Trimeric Autotransporter Protein ApiA.

The Gram-negative bacterium Aggregatibacter actinomycetemcomitans is a causative agent of localized aggressive periodontitis. Critical to its infection process is the first and essential step of attachment, which is related to the coordinated functions of surface components comprised of proteins and extracellular polysaccharides. One such protein is the outer membrane trimeric autotransporter protein ApiA, a versatile virulence factor with numerous functions, including cell binding, invasion, serum resistance, autoaggregation, and induction of cytokine release. Here we report on the use of Escherichia coli strains expressing protein variants to define the separate functions ascribed to the N terminus and those related to the C terminus. Importantly, a hybrid protein that comprised the N terminus of trimeric ApiA and the ß-barrel domain of monomeric autotransporter Aae was constructed, which allowed the expression of a monomer surface-exposed domain of ApiA. Functional and phenotypic analyses demonstrated that the C terminus of ApiA forms an independent domain that is crucial for general stability and trimer formation, which appears to be associated with autoaggregation, biofilm formation, and surface expression. Importantly, the results show that the monomeric form of the N-terminal passenger domain of ApiA, while surface exposed, is sufficient for binding to buccal epithelial cells; however, it is not sufficient to allow aggregation and biofilm formation, strengthening the importance of the role of trimerization in these phenotypes.

A chemical and biological toolbox for Type Vd secretion: Characterization of the phospholipase A1 autotransporter FplA from Fusobacterium nucleatum.

Fusobacterium nucleatum is an oral pathogen that is linked to multiple human infections and colorectal cancer. Strikingly, F. nucleatum achieves virulence in the absence of large, multiprotein secretion systems (Types I, II, III, IV, and VI), which are widely used by Gram-negative bacteria for pathogenesis. By contrast, F. nucleatum strains contain genomic expansions of Type V secreted effectors (autotransporters) that are critical for host cell adherence, invasion, and biofilm formation. Here, we present the first characterization of an F. nucleatum Type Vd phospholipase class A1 autotransporter (strain ATCC 25586, gene FN1704) that we hereby rename Fusobacterium phospholipase autotransporter (FplA). Biochemical analysis of multiple Fusobacterium strains revealed that FplA is expressed as a full-length 85-kDa outer membrane-embedded protein or as a truncated phospholipase domain that remains associated with the outer membrane. Whereas the role of Type Vd secretion in bacteria remains unidentified, we show that FplA binds with high affinity to host phosphoinositide-signaling lipids, revealing a potential role for this enzyme in establishing an F. nucleatum intracellular niche. To further analyze the role of FplA, we developed an fplA gene knock-out strain, which will guide future in vivo studies to determine its potential role in F. nucleatum pathogenesis. In summary, using recombinant FplA constructs, we have identified a biochemical toolbox that includes lipid substrates for enzymatic assays, potent inhibitors, and chemical probes to detect, track, and characterize the role of Type Vd secreted phospholipases in Gram-negative bacteria.

Structural basis of the PE-PPE protein interaction in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, has developed multiple strategies to adapt to the human host. The five type VII secretion systems, ESX-1-5, direct the export of many virulence-promoting protein effectors across the complex mycobacterial cell wall. One class of ESX substrates is the PE-PPE family of proteins, which is unique to mycobacteria and essential for infection, antigenic variation, and host-pathogen interactions. The genome of Mtb encodes 168 PE-PPE proteins. Many of them are thought to be secreted through ESX-5 secretion system and to function in pairs. However, understanding of the specific pairing of PE-PPE proteins and their structure-function relationship is limited by the challenging purification of many PE-PPE proteins, and our knowledge of the PE-PPE interactions therefore has been restricted to the PE25-PPE41 pair and its complex with the ESX-5 secretion system chaperone EspG5. Here, we report the crystal structure of a new PE-PPE pair, PE8-PPE15, in complex with EspG5. Our structure revealed that the EspG5-binding sites on PPE15 are relatively conserved among Mtb PPE proteins, suggesting that EspG5-PPE15 represents a more typical model for EspG5-PPE interactions than EspG5-PPE41. A structural comparison with the PE25-PPE41 complex disclosed conformational changes in the four-helix bundle structure and a unique binding mode in the PE8-PPE15 pair. Moreover, homology-modeling and mutagenesis studies further delineated the molecular determinants of the specific PE-PPE interactions. These findings help develop an atomic algorithm of ESX-5 substrate recognition and PE-PPE pairing.

Identification of a surface epitope specific of virulent strains of Haemophilus parasuis.

Haemophilus parasuis is a bacterium from the Pasteurellaceae family that comprises strains of different degree of virulence. Non-virulent strains are considered components of the upper respiratory tract microbiota, while virulent strains can invade systemic organs and cause fibrinous polyserositis (Glässer's disease). Genomic comparison of virulent and non-virulent strains led to the identification of a family of genes differentially associated to virulence, the virulence-associated trimeric autotransporters (vtaA). Monoclonal antibody 69C6 reacted with the surface of virulent strains and has allowed now the identification of an epitope in the C terminus of the passenger domain of the VtaAs from virulent strains. Protein modelling indicated that the epitope is probably exposed, although sera from pigs vaccinated with the passenger domain of VtaA9 and from convalescent animals did not react with the 69C6 epitope. Induction of antibodies against the 69C6 epitope by vaccination would allow a response targeting specifically virulent strains of H. parasuis.

Structure of the Bacterial Sex F Pilus Reveals an Assembly of a Stoichiometric Protein-Phospholipid Complex.

Conjugative pili are widespread bacterial appendages that play important roles in horizontal gene transfer, in spread of antibiotic resistance genes, and as sites of phage attachment. Among conjugative pili, the F "sex" pilus encoded by the F plasmid is the best functionally characterized, and it is also historically the most important, as the discovery of F-plasmid-mediated conjugation ushered in the era of molecular biology and genetics. Yet, its structure is unknown. Here, we present atomic models of two F family pili, the F and pED208 pili, generated from cryoelectron microscopy reconstructions at 5.0 and 3.6 Å resolution, respectively. These structures reveal that conjugative pili are assemblies of stoichiometric protein-phospholipid units. We further demonstrate that each pilus type binds preferentially to particular phospholipids. These structures provide the molecular basis for F pilus assembly and also shed light on the remarkable properties of conjugative pili in bacterial secretion and phage infection.

Type V Secretion Systems in Bacteria.

Type V secretion denotes a variety of secretion systems that cross the outer membrane in Gram-negative bacteria but that depend on the Sec machinery for transport through the inner membrane. They are possibly the simplest bacterial secretion systems, because they consist only of a single polypeptide chain (or two chains in the case of two-partner secretion). Their seemingly autonomous transport through the outer membrane has led to the term "autotransporters" for various subclasses of type V secretion. In this chapter, we review the structure and function of these transporters and review recent findings on additional factors involved in the secretion process, which have put the term "autotransporter" to debate.

Surface display of Aggregatibacter actinomycetemcomitans autotransporter Aae and dispersin B hybrid act as antibiofilm agents.

Among the various proteins expressed by the periodontopathogen Aggregatibacter actinomycetemcomitans, two proteins play important roles for survival in the oral cavity. The autotransporter Aae facilitates the attachment of the pathogen to oral epithelial cells, which act as a reservoir, while the biofilm-degrading glycoside hydrolase dispersin B facilitates the movement of daughter cells from the mature biofilm to a new site. The objective of this study was to use the potential of these two proteins to control biofilms. To this end, we generated a hybrid construct between the Aae C-terminal translocating domain and dispersin B, and mobilized it into Escherichia coli Rosetta (DE3) pLysS cells. Immunofluorescence analysis of the modified E. coli cells confirmed the presence of dispersin B on the surface. Further, the membrane localization of the displayed dispersin B was confirmed with Western blot analysis. The integrity of the E. coli cells displaying the dispersin B was confirmed through FACS analysis. The hydrolytic activity of the surface-displayed dispersin B was confirmed by using 4-methylumbelliferyl-ß-d-glucopyranoside as the substrate. The detachment ability of the dispersin B surface-displaying E. coli cells was shown using Staphylococcus epidermidis and Actinobacillus pleuropneumoniae biofilms in a microtiter assay. We concluded that the Aae ß-domain is sufficient to translocate foreign enzymes in the native folded form and that the method of Aae-mediated translocation of surface displayed enzymes might be useful for control of biofilms.

Structural Basis for Toughness and Flexibility in the C-terminal Passenger Domain of an Acinetobacter Trimeric Autotransporter Adhesin.

Trimeric autotransporter adhesins (TAAs) on the cell surface of Gram-negative pathogens mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific high adhesiveness to abiotic material surfaces as well as to biotic surfaces. It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain (TM), and the passenger domain contains an N-terminal head, N-terminal stalk, C-terminal head (Chead), and C-terminal stalk (Cstalk). The Chead-Cstalk-TM fragment, which is conserved in many Acinetobacter TAAs, has by itself the head-stalk-anchor architecture of a complete TAA. Here, we show the crystal structure of the Chead-Cstalk fragment, AtaA_C-terminal passenger domain (CPSD), providing the first view of several conserved TAA domains. The YadA-like head (Ylhead) of the fragment is capped by a unique structure (headCap), composed of three ß-hairpins and a connector motif; it also contains a head insert motif (HIM1) before its last inner ß-strand. The headCap, Ylhead, and HIM1 integrally form a stable Chead structure. Some of the major domains of the CPSD fragment are inherently flexible and provide bending sites for the fiber between segments whose toughness is ensured by topological chain exchange and hydrophobic core formation inside the trimer. Thus, although adherence assays using in-frame deletion mutants revealed that the characteristic adhesive sites of AtaA reside in its N-terminal part, the flexibility and toughness of the CPSD part provide the resilience that enables the adhesive properties of the full-length fiber across a wide range of conditions.

Analyzing the Role of Periplasmic Folding Factors in the Biogenesis of OMPs and Members of the Type V Secretion System.

The outer membrane (OM) of gram-negative bacteria is highly packed with OM proteins (OMPs) and the trafficking and assembly of OMPs in gram-negative bacteria is a subject of intense research. Structurally, OMPs vary in the number of ß-strands and in the size and complexity of extra-membrane domains, with extreme examples being the members of the type V protein secretion system (T5SS), such as the autotransporter (AT) and intimin/invasin families of secreted proteins, in which a large extracellular "passenger" domain is linked to a ß-barrel that inserts in the OM. Despite their structural and functional diversity, OMPs interact in the periplasm with a relatively small set of protein chaperones that facilitate their transport from the inner membrane (IM) to the ß-barrel assembly machinery (BAM complex), preventing aggregation and assisting their folding in various aspects including disulfide bond formation. This chapter is focused on the periplasmic folding factors involved in the biogenesis of integral OMPs and members of T5SS in E. coli, which are used as a model system in this field. Background information on these periplasmic folding factors is provided along with genetic methods to generate conditional mutants that deplete these factors from E. coli and biochemical methods to analyze the folding, surface display, disulfide formation and oligomerization state of OMPs/T5SS in these mutants.

New findings on the function and potential applications of the trimeric autotransporter adhesin.

Trimeric autotransporter adhesins (TAAs) are located on the surface of many pathogenic Gram-negative bacteria. TAAs belong to the autotransporter protein family and consist of three identical monomers. These obligate homotrimeric proteins are secreted through the bacterial type Vc secretion system and share a common molecular organization that each monomer consists of a N-terminal "passenger" domain and a C-terminal translocation domain. TAAs are important virulence factors that are involved in bacterial life cycle and participate in mediating infection, invasion, dissemination and evasion of host immune responses. TAAs have also proved to be useful for many applications, such as vaccines and disease biomarkers. We here mainly focused on new findings on bio-function and application of TAAs in addition to their common structure and secretion mechanisms.

The Autotransporter BpaB Contributes to the Virulence of Burkholderia mallei in an Aerosol Model of Infection.

Burkholderia mallei is a highly pathogenic bacterium that causes the zoonosis glanders. Previous studies indicated that the genome of the organism contains eight genes specifying autotransporter proteins, which are important virulence factors of Gram-negative bacteria. In the present study, we report the characterization of one of these autotransporters, BpaB. Database searches identified the bpaB gene in ten B. mallei isolates and the predicted proteins were 99-100% identical. Comparative sequence analyses indicate that the gene product is a trimeric autotransporter of 1,090 amino acids with a predicted molecular weight of 105-kDa. Consistent with this finding, we discovered that recombinant bacteria expressing bpaB produce a protein of ≥ 300-kDa on their surface that is reactive with a BpaB-specific monoclonal antibody. Analysis of sera from mice infected with B. mallei indicated that animals produce antibodies against BpaB during the course of disease, thus establishing production of the autotransporter in vivo. To gain insight on its role in virulence, we inactivated the bpaB gene of B. mallei strain ATCC 23344 and determined the median lethal dose of the mutant in a mouse model of aerosol infection. These experiments revealed that the bpaB mutation attenuates virulence 8-14 fold. Using a crystal violet-based assay, we also discovered that constitutive production of BpaB on the surface of B. mallei promotes biofilm formation. To our knowledge, this is the first report of a biofilm factor for this organism.

Structural and functional studies of BapC protein of Bordetella pertussis.

Bordetella pertussis, the causative agent of whooping cough, attaches to mucosal surface in upper respiratory tract, where it produces a variety of surface associated and secreted autotransporter molecules among others. In this study we have cloned newly identified member of autotransporter family BapC (B. pertussis autotransporter protein C); expressed it in Escherichia coli and characterized it for its different properties. We have also raised antisera to BapC protein; the antisera were used in immunofluorescence assay to determine the surface association of the protein. Results suggest that BapC in B. pertussis Taberman parent is surface exposed when compared with the respective BapC mutant. The neutralizing effect of anti-BapC serum was also evaluated in the presence of active complement proteins and results suggest that antiserum can potentiate the killing of B. pertussis cells in the presence of added source of complement. Structure of the protein was also studied, both α and ß domains of the protein were modeled, ß domain exhibits typical transmembrane ß-barrel porin topology whereas α domain behaves as a characteristic bacterial autotransporter passenger domain.