The Haemophilus influenzae Hia autotransporter contains an unusually short trimeric translocator domain

Gram-negative bacterial autotransporter proteins are a growing group of virulence factors that are characterized by their ability to cross the outer membrane without the help of accessory proteins. A conserved C-terminal beta-domain is critical for targeting of autotransporters to the outer membrane and for translocation of the N-terminal "passenger" domain to the bacterial surface. We have demonstrated previously that the Haemophilus influenzae Hia adhesin belongs to the autotransporter family, with translocator activity residing in the C-terminal 319 residues. To gain further insight into the mechanism of autotransporter protein translocation, we performed a structure-function analysis on Hia. In initial experiments, we generated a series of in-frame deletions and a set of chimeric proteins containing varying regions of the Hia C terminus fused to a heterologous passenger domain and discovered that the final 76 residues of Hia are both necessary and sufficient for translocation. Analysis by flow cytometry revealed that the region N-terminal to this shortened translocator domain is surface localized, further suggesting that this region is not involved in beta-barrel formation or in translocation of the passenger domain. Western analysis demonstrated that the translocation-competent regions of the C terminus migrated at masses consistent with trimers, suggesting that the Hia C terminus oligomerizes. Furthermore, fusion proteins containing a heterologous passenger domain demonstrated that similarly small C-terminal regions of Yersinia sp. YadA and Neisseria meningitidis NhhA are translocation-competent. These data provide experimental support for a unique subclass of autotransporters characterized by a short trimeric translocator domain.     

Repetitive architecture of the Haemophilus influenzae Hia trimeric autotransporter

The Hia autotransporter of Haemophilus influenzae belongs to the trimeric autotransporter subfamily and mediates bacterial adherence to the respiratory epithelium. In this report, we show that the structure of Hia is characterized by a modular architecture containing repeats of structurally distinct domains. Comparison of the structures of HiaBD1 and HiaBD2 adhesive repeats and a nonadhesive repeat (a novel fold) shed light on the structural determinants of Hia adhesive function. Examination of the structure of an extended version of the Hia translocator domain revealed the structural transition between the C-terminal translocator domain and the N-terminal passenger domain, highlighting a highly intertwined domain that is ubiquitous among trimeric autotransporters. Overall, this study provides important insights into the mechanism of Hia adhesive activity and the overall structure of trimeric autotransporters.     

Structural basis for host recognition by the Haemophilus influenzae Hia autotransporter

Haemophilus influenzae is an important human pathogen that initiates infection by colonizing the upper respiratory tract. The H. influenzae Hia autotransporter is an adhesive protein that promotes adherence to respiratory epithelial cells. Hia adhesive activity resides in two homologous binding domains, called HiaBD1 and HiaBD2. These domains interact with the same host cell receptor, but bind with different affinities. In this report, we describe the crystal structure of the high-affinity HiaBD1 binding domain, which has a novel trimeric architecture with three-fold symmetry and a mushroom shape. The subunit constituents of the trimer are extensively intertwined. The receptor-binding pocket is formed by an acidic patch that is present on all three faces of the trimer, providing potential for a multivalent interaction with the host cell surface, analogous to observations with the trimeric tumor necrosis factor superfamily of proteins. Hia is a novel example of a bacterial trimeric adhesin and may be the prototype member of a large family of bacterial virulence proteins with a similar architecture.     

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.     

The Haemophilus influenzae Hia autotransporter harbours two adhesive pockets that reside in the passenger domain and recognize the same host cell receptor

Haemophilus influenzae is a human-specific pathogen and a major source of morbidity worldwide. Infection with this organism begins with colonization of the nasopharynx, a process that probably depends on adherence to respiratory epithelium. The Hia autotransporter protein is the major adhesin ex-pressed by a subset of non-typeable H. influenzae strains and promotes high-level adherence to a variety of human epithelial cell lines. In the current study, we discovered that the Hia passenger domain contains two distinct binding pockets, including one at the C-terminal end and a second at the N-terminal end. Competition assays revealed that the two binding pockets interact with the same host cell receptor structure, although with differing affinities. Additional experiments demonstrated that both binding domains are required for full-level bacterial adherence. These observations are reminiscent of eukaryotic cell adhesion molecules and highlight the first example of a bacterial adhesin with two domains that participate in a bivalent interaction with identical host cell receptors. Such an interaction increases avidity, thus stabilizing bacterial adherence to the epithelial surface, despite physical forces such as coughing, sneezing and mucociliary clearance.     

The Haemophilus influenzae Hia adhesin is an autotransporter protein that remains uncleaved at the C terminus and fully cell associated

Nontypeable Haemophilus influenzae is a gram-negative commensal organism that is commonly associated with localized respiratory tract disease. The pathogenesis of disease begins with colonization of the nasopharynx, a process that likely depends on bacterial adherence to respiratory epithelial cells. Hia is the major adhesin expressed by a subset of nontypeable H. influenzae strains and promotes efficient adherence to a variety of human epithelial cell lines. Based on previous work, Hia is transported to the surface of Escherichia coli transformants and is capable of mediating E. coli adherence without the assistance of other H. influenzae proteins. In the present study, we examined the mechanism of Hia secretion. PhoA fusions, deletional mutagenesis, and N-terminal amino acid sequencing established that the signal for Hia export from the cytoplasm resides in the first 49 amino acids, including a 24-amino-acid stretch with striking similarity to the N terminus of a number of proteins belonging to the autotransporter family. Immunoelectron microscopy demonstrated that the Hia internal region defined by amino acids 221 to 779 is exposed on the bacterial surface. Secondary-structure analysis predicted that the C terminus of Hia forms a beta-barrel with a central hydrophilic channel, and site-specific mutagenesis and fusion protein analysis demonstrated that the C terminus targets Hia to the outer membrane and functions as an outer membrane translocator, analogous to observations with autotransporter proteins. In contrast to typical autotransporter proteins, Hia undergoes no cleavage between the internal and C-terminal domains and remains fully cell associated. Together, these results suggest that Hia is the prototype of an important subfamily of autotransporter proteins.     

Characteristics of major outer membrane proteins of Haemophilus influenzae.

Several properties of Haemophilus influenzae outer membrane proteins were analyzed to define related proteins in various isolates. H. influenzae type b 760705 had six major outer membrane proteins with the following characteristics. Protein a (Mr, 47,000) demonstrated heat modifiability in sodium dodecyl sulfate; its apparent molecular weight was 34,000 at temperatures below 60 degrees C. This protein was extracted from cell envelopes by using Triton X-100-10 mM MgCl2; in cell envelope preparations, the protein was degraded by trypsin. Proteins b (Mr, 41,000) and c (Mr, 40,000) were insensitive to trypsin degradation, were not heat modifiable in sodium dodecyl sulfate, and were peptidoglycan associated in 0.5% Triton X-100-0.2% sodium dodecyl sulfate. The amount of protein b was reduced in ultrasonically obtained cell envelopes. Protein d (Mr, 37,000) was heat modifiable in sodium dodecyl sulfate with an Mr of 28,000 at temperatures below 100 degrees C and was degraded by trypsin, leaving a membrane-bound fragment of Mr, 27,000. Both the intact and degraded proteins were immunologically cross-reactive with the heat-modifiable OmpA protein of Escherichia coli K-12. Protein d was absent in LiCl-EDTA extracts of cells. Protein e (Mr, 30,000), invariably present in all H. influenzae strains tested, was insensitive to trypsin and absent in LiCl-EDTA extracts of cells. Protein k (Mr, 58,000) was extracted from cell envelopes with 2% Triton X-100-10 mM MgCl2 and, in cell envelopes, appeared to be sensitive to trypsin degradation. Proteins with similar properties to those of proteins a to k were found in 10 other H. influenzae b strains, reference strains with serotype a, c, d, e, and f capsules, and 18 of 20 nonencapsulated strains. Their relative molecular weights, however, varied.     

Identification of a novel trimeric autotransporter adhesin in the cryptic genospecies of Haemophilus

Haemophilus biotype IV strains belonging to the recently recognized Haemophilus cryptic genospecies are an important cause of maternal genital tract and neonatal systemic infections and initiate infection by colonizing the genital or respiratory epithelium. To gain insight into the mechanism of Haemophilus cryptic genospecies colonization, we began by examining prototype strain 1595 and three other strains for adherence to genital and respiratory epithelial cell lines. Strain 1595 and two of the three other strains demonstrated efficient adherence to all of the cell lines tested. With a stably adherent variant of strain 1595, we generated a Mariner transposon library and identified 16 nonadherent mutants. All of these mutants lacked surface fibers and contained an insertion in the same open reading frame, which encodes a 157-kDa protein designated Cha for cryptic haemophilus adhesin. Analysis of the predicted amino acid sequence of Cha revealed the presence of an N-terminal signal peptide and a C-terminal domain bearing homology to YadA-like and Hia-like trimeric autotransporters. Examination of the C-terminal 120 amino acids of Cha demonstrated mobility as a trimer on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the capacity to present the passenger domain of the Hia trimeric autotransporter on the bacterial surface. Southern analysis revealed that the gene that encodes Cha is conserved among clinical isolates of the Haemophilus cryptic genospecies and is absent from the closely related species Haemophilus influenzae. We speculate that Cha is important in the pathogenesis of disease due to the Haemophilus cryptic genospecies and is in part responsible for the apparent tissue tropism of this organism.     

Stability and membrane interactions of an autotransport protein: MD simulations of the Hia translocator domain in a complex membrane environment

Hia is a trimeric autotransporter found in the outer membrane of Haemphilus influenzae. The X-ray structure of Hia translocator domain revealed each monomer to consist of an α-helix connected via a loop to a 4-stranded β-sheet, thus the topology of the trimeric translocator domain is a 12-stranded β-barrel containing 3 α-helices that protrude from the mouth of the β-barrel into the extracellular medium. Molecular dynamics simulations of the Hia monomer and trimer have been employed to explore the interactions between the helices, β-barrel and connecting loops that may contribute to the stability of the trimer. In simulations of the Hia monomer we show that the central α-helix may stabilise the fold of the 4-stranded β-sheet. In simulations of the Hia trimer, a H-bond network involving residues in the β-barrel, α-helices and loops has been identified as providing stability for the trimeric arrangement of the monomers. Glutamine residues located in the loops connecting the α-helices to the β-barrel are orientated in a triangular arrangement such that each forms 2 hydrogen bonds to each of the corresponding glutamines in the other loops. In the absence of the loops, the β‐barrel becomes distorted. Simulations show that while the trimeric translocator domain β-barrel is inherently flexible, it is unlikely to accommodate the passenger domain in a folded conformation. Simulations of Hia in an asymmetric model of the outer membrane have revealed membrane–protein interactions that anchor the protein within its native membrane environment.     

Structure of the Haemophilus influenzae HMW1B translocator protein: evidence for a twin pore

Secretion of the Haemophilus influenzae HMW1 adhesin occurs via the two-partner secretion pathway and requires the HMW1B outer membrane translocator. HMW1B has been subjected to extensive biochemical studies to date. However, direct examination of the structure of HMW1B has been lacking, leaving fundamental questions about the oligomeric state, the membrane-embedded beta-barrel domain, the approximate size of the beta-barrel pore, and the mechanism of translocator activity. In the current study, examination of purified HMW1B by size exclusion chromatography and negative staining electron microscopy revealed that the predominant species was a dimer. In the presence of lipid, purified HMW1B formed two-dimensional crystalline sheets. Examination of these crystals by cryo-electron microscopy allowed determination of a projection structure of HMW1B to 10 A resolution. The native HMW1B structure is a dimer of beta-barrels, with each beta-barrel measuring 40 A by 50 A in the two orthogonal directions and appearing largely occluded, leaving only a narrow pore. These observations suggest that HMW1B undergoes a large conformational change during translocation of the 125-kDa HMW1 adhesin.     

Immunogenicity of a Haemophilus influenzae polysaccharide-Neisseria meningitidis outer membrane protein complex conjugate vaccine

Polysaccharide-protein conjugate vaccines made with different carriers vary in their ability to elicit antipolysaccharide IgG antibody responses in young infants and an adult mouse model, suggesting that the carrier proteins used in the conjugate vaccines differ in their ability to act as carriers, or that additional mechanisms of immunogenicity play a role. A conjugate vaccine of Haemophilus influenzae PRP coupled to the outer membrane protein complex (OMPC) of Neisseria meningitidis serogroup B is immunogenic in children as young as 2 mo of age and is immunogenic in infant rhesus monkeys, an animal model for infant humans. In the present study, PRP-OMPC was found to induce efficient IgM to IgG switching of anti-PRP serum antibody in adult mice, whereas PRP conjugated to two other protein carriers did not. Thus the PRP-OMPC conjugate was examined in order to determine why PRP coupled to OMPC was so immunogenic, even more immunogenic than conjugates made with other carrier proteins. The OMPC carrier differs from the other protein carriers in that the proteins are present in a liposomal form containing lipids (including LPS) derived from the outer membrane of N. meningitidis. We studied the OMPC to see whether the different components or the nature of the OMPC carrier could contribute to its enhanced immunogenicity. Specifically we evaluated the OMPC for both classic Th cell carrier activity and adjuvanticity, and the LPS component of OMPC for systemic polyclonal B cell activation. Carrier recognition of the OMPC moiety of PRP-OMPC was demonstrated. In addition the PRP-OMPC conjugate vaccine was observed to have adjuvant properties for both T cell-dependent and T cell-independent Ag in the absence of LPS-induced systemic polyclonal B cell activation. These observations suggest that in addition to functioning as a classic protein carrier whereby the proteins in OMPC provide Th cell epitopes, the OMPC also has adjuvant activity that distinguishes it from other protein carriers and may contribute to the increased immunogenicity of PRP-OMPC conjugates in animal models.     

Channel properties of the translocator domain of the autotransporter Hbp of Escherichia coli

Autotransporters produced by Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain (TD), and a passenger domain in between. The TD facilitates the secretion of the passenger across the outer membrane. It generally consists of a channel-forming β-barrel that can be plugged by an α-helix that is formed by a polypeptide fragment immediately N-terminal to the barrel domain in the sequence. In this work, we characterized the TD of the hemoglobin protease Hbp of Escherichia coli by comparing its properties with the TDs of NalP of Neisseria meningitidis and IgA protease of Neisseria gonorrhoeae. All TDs were produced in inclusion bodies and folded in vitro. In the case of the TD of Hbp, this procedure resulted in autocatalytic intramolecular processing, which mimicked the in vivo processing. Liposome-swelling assays and planar lipid bilayer experiments revealed that the pore of the Hbp TD was largely obstructed. In contrast, an Hbp TD variant that lacked only one amino-acid residue from the N terminus showed the opening and closing of a channel comparable to what was reported for the TD of NalP. Additionally, the naturally processed helix contributed to the stability of the TD, as shown by chemical denaturation monitored by tryptophan fluorescence. Overall these results show that Hbp is processed by an autocatalytic intramolecular mechanism resulting in the stable docking of the α-helix in the barrel. In addition, we could show that the α-helix contributes to the stability of TDs.     

Channel properties of the translocator domain of the autotransporter Hbp of Escherichia coli

Abstract

Autotransporters produced by Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain (TD), and a passenger domain in between. The TD facilitates the secretion of the passenger across the outer membrane. It generally consists of a channel-forming β-barrel that can be plugged by an α-helix that is formed by a polypeptide fragment immediately N-terminal to the barrel domain in the sequence. In this work, we characterized the TD of the hemoglobin protease Hbp of Escherichia coli by comparing its properties with the TDs of NalP of Neisseria meningitidis and IgA protease of Neisseria gonorrhoeae. All TDs were produced in inclusion bodies and folded in vitro. In the case of the TD of Hbp, this procedure resulted in autocatalytic intramolecular processing, which mimicked the in vivo processing. Liposome-swelling assays and planar lipid bilayer experiments revealed that the pore of the Hbp TD was largely obstructed. In contrast, an Hbp TD variant that lacked only one amino-acid residue from the N terminus showed the opening and closing of a channel comparable to what was reported for the TD of NalP. Additionally, the naturally processed helix contributed to the stability of the TD, as shown by chemical denaturation monitored by tryptophan fluorescence. Overall these results show that Hbp is processed by an autocatalytic intramolecular mechanism resulting in the stable docking of the α-helix in the barrel. In addition, we could show that the α-helix contributes to the stability of TDs.     

Lactoperoxidase and Iodo-Gen-catalyzed iodination labels inner and outer membrane proteins of Haemophilus influenzae

Both inner and outer membrane proteins of Haemophilus influenzae type b were labeled by iodination procedures believed to be specific for exposed surface proteins only. It is suggested that this is due to specific properties of the outer membrane of H. influenzae and that use of these procedures with other gram-negative bacteria be evaluated carefully.     

Influence of the passenger domain of a model autotransporter on the properties of its translocator domain

Autotransporters are a superfamily of proteins secreted by Gram-negative bacteria including many virulence factors. They are modular proteins composed of an N-terminal signal peptide, a surface-exposed ‘passenger’ domain carrying the activity of the protein, and a C-terminal ‘translocator’ domain composed of an α-helical linker region and a transmembrane β-barrel. The translocator domain plays an essential role for the secretion of the passenger domain across the outer membrane; however, the mechanism of autotransport remains poorly understood. The whooping cough agent Bordetella pertussis produces an autotransporter serine-protease, SphB1, which is involved in the maturation of an adhesin at the bacterial surface. SphB1 also mediates the proteolytic maturation of its own precursor. We used SphB1 as a model autotransporter and performed the first comparisons of the biochemical and biophysical properties of an isolated translocator domain with those of the same domain preceded by the C-terminal moiety of its natural passenger. By using cross-linking and dynamic light scattering, we provide evidence that the passenger domain promotes the auto-association of SphB1, although these interactions appear rather labile. Electrophysiological studies revealed that the passenger domain of the autotransporter appears to maintain the translocator channel in a low-conductance conformation, most likely by stabilizing the α-helix inside the pore. That the passenger may significantly influence AT physicochemical properties is likely to be relevant for the in vivo maturation and stability of AT proteins.     

Influence of the passenger domain of a model autotransporter on the properties of its translocator domain

Autotransporters are a superfamily of proteins secreted by Gram-negative bacteria including many virulence factors. They are modular proteins composed of an N-terminal signal peptide, a surface-exposed 'passenger' domain carrying the activity of the protein, and a C-terminal 'translocator' domain composed of an alpha-helical linker region and a transmembrane beta-barrel. The translocator domain plays an essential role for the secretion of the passenger domain across the outer membrane; however, the mechanism of autotransport remains poorly understood. The whooping cough agent Bordetella pertussis produces an autotransporter serine-protease, SphB1, which is involved in the maturation of an adhesin at the bacterial surface. SphB1 also mediates the proteolytic maturation of its own precursor. We used SphB1 as a model autotransporter and performed the first comparisons of the biochemical and biophysical properties of an isolated translocator domain with those of the same domain preceded by the C-terminal moiety of its natural passenger. By using cross-linking and dynamic light scattering, we provide evidence that the passenger domain promotes the auto-association of SphB1, although these interactions appear rather labile. Electrophysiological studies revealed that the passenger domain of the autotransporter appears to maintain the translocator channel in a low-conductance conformation, most likely by stabilizing the alpha-helix inside the pore. That the passenger may significantly influence AT physicochemical properties is likely to be relevant for the in vivo maturation and stability of AT proteins.     

Human antibody response to outer membrane proteins and fimbriae of Haemophilus influenzae type b

We investigated the prevalence of antibodies in childrens' sera directed against outer membrane proteins (OMP) and fimbriae of Haemophilus influenzae type b. Invasive isolates of H. influenzae type b were enriched for fimbriae production; OMP and fimbriae were resolved by SDS-PAGE. After blotting to nitrocellulose, the proteins were incubated with homologous patient sera or with sera from healthy children. IgG antibodies bound to OMP were detected by immunoperoxidase staining. Immunoblotting was also performed using purified, nondenatured fimbriae as antigen. Nine of the 10 patients studied had antibodies in the acute serum directed against one or more of the OMP. Neither the acute nor the convalescent serum of the remaining patient contained antibodies against OMP. Antibodies against a greater number of OMP were present in the convalescent serum, in comparison to the acute serum, in 4 of the 10 patients. Five of 10 patients had antibodies against the purified fimbriae of an unrelated invasive isolate in either the acute or the convalescent serum. Acute sera from patients more frequently contained antibodies directed against OMP 60K (p less than or equal to 0.01) and OMP 51K (p less than or equal to 0.003) compared with the sera of healthy controls. In contrast, the sera of healthy children more frequently contained antibodies directed against OMP 40K (p less than or equal to 0.04). Sera from both patients and controls contained antibodies against commensal Haemophilus. We conclude that although antibodies against OMP are commonly present in healthy children, antibodies against certain OMP may be markers for susceptibility or protection.     

Effect of Haemophilus influenzae polysaccharide outer membrane protein complex conjugate vaccine on macrophages.

Haemophilus influenzae type b polysaccharide-conjugate vaccines elicit protective antibody responses in young infants. One of these conjugates, polysaccharide linked to outer membrane protein complex (PRP-OMPC), is produced by linking the capsular polysaccharide to an outer membrane protein complex derived from group B Neisseria meningitidis. The outer membrane protein complex contains T cell carrier epitopes that elicit T cell-dependent antibody responses. OMPC also has been shown to increase the antibody response to other proteins administered concurrently that are not covalently linked (i.e., acts as an adjuvant). In this study PRP-OMPC immunized mice demonstrated significant increases in spleen size as well as in splenocyte number as compared to saline controls (p < 0.01, p < 0.001, respectively). No such increase was noted after immunization with another H. influenzae type b-conjugate vaccine, oligosaccharide linked to a variant of diphtheria toxin. By analytic flow cytometry, the mice immunized with PRP-OMPC demonstrated an increase in large splenocytes expressing the Ag Mac-1 (CD11b, CR3). Furthermore, the spleens on histologic examination were characterized by an increase in the red pulp area consisting predominantly of cells of macrophage morphology. By immunohistochemical staining, the cells were identified as macrophages due to expression of Mac-1 and p150,95 (CD11C) Ag. After PRP-OMPC immunization, severe combined immunodeficient mice also demonstrated significant splenomegaly with an increase in macrophages identified by expression of Mac-1 and MHC class II Ag. Thus PRP-OMPC vaccine resulted in T cell-independent splenomegaly with an increase number of macrophages. We propose that this unique property may confer increased immunogenicity to PRP-OMPC through macrophage activation and cytokine release. Furthermore, the effect on macrophages may explain the "adjuvant" capacity of OMPC.     

The periplasmic folding of a cysteineless autotransporter passenger domain interferes with its outer membrane translocation

Autotransporters are single polypeptides consisting of an outer membrane translocation domain mediating the translocation of a passenger domain. The periplasmic folding state of the passenger domain is controversial. By comparisons of passenger domains differing in their folding properties, our results suggest that periplasmic folding of passenger domains interferes with translocation.