HadA is an atypical new multifunctional trimeric coiled-coil adhesin of Haemophilus influenzae biogroup aegyptius, which promotes entry into host cells

The Oca (Oligomeric coiled-coil adhesin) family is a subgroup of the bacterial trimeric autotransporter adhesins, which includes structurally related proteins, such as YadA of Yersinia enterocolitica and NadA of Neisseria meningitidis . In this study, we searched in silico for novel members of this family in bacterial genomes and identified HadA ( Haemophilus adhesin A), a trimeric autotransporter expressed only by Haemophilus influenzae biogroup aegyptius causing Brazilian purpuric fever (BPF), a fulminant septicemic disease of children. By comparative genomics and sequence analysis we predicted that the hadA gene is harboured on a mobile genetic element unique to BPF isolates. Biological analysis of HadA in the native background was limited because this organism is not amenable to genetic manipulation. Alternatively, we demonstrated that expression of HadA confers to a non-invasive Escherichia coli strain the ability to adhere to human cells and to extracellular matrix proteins and to induce in vitro bacterial aggregation and microcolony formation. Intriguingly, HadA is predicted to lack the typical N-terminal head domain of Oca proteins generally associated with cellular receptor binding. We propose here a structural model of the HadA coiled-coil stalk and show that the N-terminal region is still responsible of the binding activity and a KGD motif plays a role. Interestingly, HadA promotes bacterial entry into mammalian cells. Our results show a cytoskeleton re-arrangement and an involvement of clathrin in the HadA-mediated internalization. These data give new insights on the structure-function relationship of oligomeric coiled-coil adhesins and suggest a potential role of this protein in the pathogenesis of BPF.     

Neisseria meningitidis App,a new adhesin with autocatalytic serine protease activity

Neisseria meningitidis is a Gram-negative bacterium which colonizes the human upper respiratory tract. Occasionally, it translocates to the bloodstream causing sepsis and from there it can cross the blood-brain barrier and cause meningitis. Many of the molecules, which mediate the interaction of N. meningitidis to host cells, are still unknown. Recently, App (Adhesion and penetration protein) was described as a member of the autotransporter family and a homologue to the Hap (Haemophilus adhesion and penetration) protein of Haemophilus influenzae, a molecule that plays a role in the interaction with human epithelial cells. In this study we expressed app in Escherichia coli in order to analyse the functional properties of the protein. We show that the protein is exported to the E. coli surface, processed by an endogenous serine-protease activity and released in the culture supernatant. Escherichia coli expressing app adhere to Chang epithelial cells, showing that App is able to mediate bacterial adhesion to host cells. The serine protease activity is localized at the amino-terminal domain, whereas the binding domain is in the carboxy-terminal region. The role of App in adhesion was confirmed also in N. meningitidis.     

Meningococcal outer membrane protein NhhA triggers apoptosis in macrophages

Phagocytotic cells play a fundamental role in the defense against bacterial pathogens. One mechanism whereby bacteria evade phagocytosis is to produce factors that trigger apoptosis. Here we identify for the first time a meningococcal protein capable of inducing macrophage apoptosis. The conserved meningococcal outer membrane protein NhhA (Neisseria hia/hsf homologue A, also known as Hsf) mediates bacterial adhesion and interacts with extracellular matrix components heparan sulphate and laminin. Meningococci lacking NhhA fail to colonise nasal mucosa in a mouse model of meningococcal disease. We found that exposure of macrophages to NhhA resulted in a highly increased rate of apoptosis that proceeded through caspase activation. Exposure of macrophages to NhhA also led to iNOS induction and nitric oxide production. However, neither nitric oxide production nor TNF-α signaling was found to be a prerequisite for NhhA-induced apoptosis. Macrophages exposed to wildtype NhhA-expressing meningococci were also found to undergo apoptosis whereas NhhA-deficient meningococci had a markedly decreased capacity to induce macrophage apoptosis. These data provide new insights on the role of NhhA in meningococcal disease. NhhA-induced macrophage apoptosis could be a mechanism whereby meningococci evade immunoregulatory and phagocytotic actions of macrophages.     

A subfamily of Dr adhesins of Escherichia coli bind independently to decay-accelerating factor and the N-domain of carcinoembryonic antigen

Escherichia coli expressing the Dr family of adhesins adheres to epithelial cells by binding to decay-accelerating factor (DAF) and carcinoembryonic antigen (CEA)-related cell surface proteins. The attachment of bacteria expressing Dr adhesins to DAF induces clustering of DAF around bacterial cells and also recruitment of CEA-related cell adhesion molecules. CEA, CEACAM1, and CEACAM6 have been shown to serve as receptors for some Dr adhesins (AfaE-I, AfaE-III, DraE, and DaaE). We demonstrate that AfaE-I, AfaE-V, DraE, and DaaE adhesins bind to the N-domain of CEA. To identify the residues involved in the N-CEA/DraE interaction, we performed SPR binding analyses of naturally occurring variants and a number of randomly generated mutants in DraE and N-CEA. Additionally, we used chemical shift mapping by NMR to determine the surface of DraE involved in N-CEA binding. These results show a distinct CEA binding site located primarily in the A, B, E, and D strands of the Dr adhesin. Interestingly, this site is located opposite to the beta-sheet encompassing the previously determined binding site for DAF, which implies that the adhesin can bind simultaneously to both receptors on the epithelial cell surface. The recognition of CEACAMs from a highly diverse DrCEA subfamily of Dr adhesins indicates that interaction with these receptors plays an important role in niche adaptation of E. coli strains expressing Dr adhesins.     

The BtaF Trimeric Autotransporter of Brucella suis Is Involved in Attachment to Various Surfaces,Resistance to Serum and Virulence

The adhesion of bacterial pathogens to host cells is an event that determines infection, and ultimately invasion and intracellular multiplication. Several evidences have recently shown that this rule is also truth for the intracellular pathogen Brucella. Brucella suis displays the unipolar BmaC and BtaE adhesins, which belong to the monomeric and trimeric autotransporter (TA) families, respectively. It was previously shown that these adhesins are involved in bacterial adhesion to host cells and components of the extracellular matrix (ECM). In this work we describe the role of a new member of the TA family of B. suis (named BtaF) in the adhesive properties of the bacterial surface. BtaF conferred the bacteria that carried it a promiscuous adhesiveness to various ECM components and the ability to attach to an abiotic surface. Furthermore, BtaF was found to participate in bacterial adhesion to epithelial cells and was required for full virulence in mice. Similar to BmaC and BtaE, the BtaF adhesin was expressed in a small subpopulation of bacteria, and in all cases, it was detected at the new pole generated after cell division. Interestingly, BtaF was also implicated in the resistance of B. suis to porcine serum. Our findings emphasize the impact of TAs in the Brucella lifecycle.     

Architecture and adhesive activity of the Haemophilus influenzae Hsf adhesin

Haemophilus influenzae type b is an important cause of meningitis and other serious invasive diseases and initiates infection by colonizing the upper respiratory tract. Among the major adhesins in H. influenzae type b is a nonpilus protein called Hsf, a large protein that forms fiber-like structures on the bacterial surface and shares significant sequence similarity with the nontypeable H. influenzae Hia autotransporter. In the present study, we characterized the structure and adhesive activity of Hsf. Analysis of the predicted amino acid sequence of Hsf revealed three regions with high-level homology to the HiaBD1 and HiaBD2 binding domains in Hia. Based on examination of glutathione S-transferase fusion proteins corresponding to these regions, two of the three had adhesive activity and one was nonadhesive in assays with cultured epithelial cells. Structural modeling demonstrated that only the two regions with adhesive activity harbored an acidic binding pocket like the binding pocket identified in the crystal structure of HiaBD1. Consistent with these results, disruption of the acidic binding pockets in the adhesive regions eliminated adhesive activity. These studies advance our understanding of the architecture of Hsf and the family of trimeric autotransporters and provide insight into the structural determinants of H. influenzae type b adherence.     

Adhesin degradation accelerates delivery of heat-labile toxin by enterotoxigenic Escherichia coli

Many enteric pathogens, including enterotoxigenic Escherichia coli (ETEC), produce one or more serine proteases that are secreted via the autotransporter (or type V) bacterial secretion pathway. These molecules have collectively been referred to as SPATE proteins (serine protease autotransporter of the Enterobacteriaceae). EatA, an autotransporter previously identified in ETEC, possesses a functional serine protease motif within its secreted amino-terminal passenger domain. Although this protein is expressed by many ETEC strains and is highly immunogenic, its precise function is unknown. Here, we demonstrate that EatA degrades a recently characterized adhesin, EtpA, resulting in modulation of bacterial adhesion and accelerated delivery of the heat-labile toxin, a principal ETEC virulence determinant. Antibodies raised against the passenger domain of EatA impair ETEC delivery of labile toxin to epithelial cells suggesting that EatA may be an effective target for vaccine development.     

Evolutionary Conservation in Biogenesis of β-Barrel Proteins Allows Mitochondria to Assemble a Functional Bacterial Trimeric Autotransporter Protein

Yersinia adhesin A (YadA) belongs to a class of bacterial adhesins that form trimeric structures. Their mature form contains a passenger domain and a C-terminal β-domain that anchors the protein in the outer membrane (OM). Little is known about how precursors of such proteins cross the periplasm and assemble into the OM. In the present study we took advantage of the evolutionary conservation in the biogenesis of β-barrel proteins between bacteria and mitochondria. We previously observed that upon expression in yeast cells, bacterial β-barrel proteins including the transmembrane domain of YadA assemble into the mitochondrial OM. In the current study we found that when expressed in yeast cells both the monomeric and trimeric forms of full-length YadA were detected in mitochondria but only the trimeric species was fully integrated into the OM. The oligomeric form was exposed on the surface of the organelle in its native conformation and maintained its capacity to adhere to host cells. The co-expression of YadA with a mitochondria-targeted form of the bacterial periplasmic chaperone Skp, but not with SurA or SecB, resulted in enhanced levels of both forms of YadA. Taken together, these results indicate that the proper assembly of trimeric autotransporter can occur also in a system lacking the lipoproteins of the BAM machinery and is specifically enhanced by the chaperone Skp.     

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.     

A conserved glycine residue of trimeric autotransporter domains plays a key role in Yersinia adhesin A autotransport

The Yersinia adhesin A (YadA) is a trimeric autotransporter adhesin of enteric yersiniae. It consists of three major domains: a head mediating adherence to host cells, a stalk involved in serum resistance, and an anchor that forms a membrane pore and is responsible for the autotransport function. The anchor contains a glycine residue, nearly invariant throughout trimeric autotransporter adhesins, that faces the pore lumen. To address the role of this glycine, we replaced it with polar amino acids of increasing side chain size and expressed wild-type and mutant YadA in Escherichia coli. The mutations did not impair the YadA-mediated adhesion to collagen and to host cells or the host cell cytokine production, but they decreased the expression levels and stability of YadA trimers with increasing side chain size. Likewise, autoagglutination and resistance to serum were decreased in these mutants. We found that the periplasmic protease DegP is involved in the degradation of YadA and that in an E. coli degP deletion strain, mutant versions of YadA were expressed almost to wild-type levels. We conclude that the conserved glycine residue affects both the export and the stability of YadA and consequently some of its putative functions in pathogenesis.     

Characterization of BCAM0224, a Multifunctional Trimeric Autotransporter from the Human Pathogen Burkholderia cenocepacia

Members of the trimeric autotransporter adhesin (TAA) family play a crucial role in adhesion of Gram-negative pathogens to host cells. Moreover, these proteins are multifunctional virulence factors involved in several other biological traits, including invasion into host cells and evasion of the host immune system. In cystic fibrosis epidemic Burkholderia cenocepacia strain J2315, we identified a unique TAA (BCAM0224)-encoding gene, previously described as being implicated in virulence. Here, we characterized this multifunctional protein, trying to establish its role in B. cenocepacia pathogenicity. We show that BCAM0224 occurs on the bacterial surface and adopts a trimeric conformation. Furthermore, we demonstrated that BCAM0224 is needed for earlier stages of biofilm formation and is required for swarming motility. In addition, BCAM0224 plays an important role in evasion of the human innate immune system, providing resistance against the bactericidal activity of serum via the complement classical pathway. Finally, BCAM0224 mediates bacterial adhesion to and invasion of cultured human bronchial epithelial cells. Together, these data reveal the high versatility of the BCAM0224 protein as a virulence factor in the pathogenic bacterium B. cenocepacia.     

The head of Bartonella adhesin A is crucial for host cell interaction of Bartonella henselae

Human pathogenic Bartonella henselae cause cat scratch disease and vasculoproliferative disorders (e.g. bacillary angiomatosis). Expression of Bartonella adhesin A (BadA) is crucial for bacterial autoagglutination, adhesion to host cells, binding to extracellular matrix proteins and proangiogenic reprogramming via activation of hypoxia inducible factor (HIF)-1. Like the prototypic Yersinia adhesin A, BadA belongs to the class of trimeric autotransporter adhesins and is constructed modularly consisting of a head, a long and repetitive neck-stalk module and a membrane anchor. Until now, the exact biological role of these domains is not known. Here, we analysed the function of the BadA head by truncating the repetitive neck-stalk module of BadA (B. henselae badA(-)/pHN23). Like B. henselae Marseille wild type, B. henselae badA(-)/pHN23 showed autoagglutination, adhesion to collagen and endothelial cells and activation of HIF-1 in host cells. Remarkably, B. henselae badA(-)/pHN23 did not bind to fibronectin (Fn) suggesting a crucial role of the deleted stalk domain in Fn binding. Additionally, the recombinantly expressed BadA head adhered to human umbilical vein endothelial cells and to a lesser degree to epithelial (HeLa 229) cells. Our data suggest that the head represents the major functional domain of BadA responsible for host adhesion and angiogenic reprogramming.     

UpaG, a new member of the trimeric autotransporter family of adhesins in uropathogenic Escherichia coli

The ability of Escherichia coli to colonize both intestinal and extraintestinal sites is driven by the presence of specific virulence factors, among which are the autotransporter (AT) proteins. Members of the trimeric AT adhesin family are important virulence factors for several gram-negative pathogens and mediate adherence to eukaryotic cells and extracellular matrix (ECM) proteins. In this study, we characterized a new trimeric AT adhesin (UpaG) from uropathogenic E. coli (UPEC). Molecular analysis of UpaG revealed that it is translocated to the cell surface and adopts a multimeric conformation. We demonstrated that UpaG is able to promote cell aggregation and biofilm formation on abiotic surfaces in CFT073 and various UPEC strains. In addition, UpaG expression resulted in the adhesion of CFT073 to human bladder epithelial cells, with specific affinity to fibronectin and laminin. Prevalence analysis revealed that upaG is strongly associated with E. coli strains from the B2 and D phylogenetic groups, while deletion of upaG had no significant effect on the ability of CFT073 to colonize the mouse urinary tract. Thus, UpaG is a novel trimeric AT adhesin from E. coli that mediates aggregation, biofilm formation, and adhesion to various ECM proteins.     

Identification and characterization of App: an immunogenic autotransporter protein of Neisseria meningitidis

In a search for immunogenic virulence factors in Neisseria meningitidis, we have identified a gene encoding a predicted 160 kDa protein with homology to the autotransporter family of proteins. Members of this family are secreted or surface exposed and are often associated with virulence in Gram-negative bacterial pathogens. We named the gene adhesion and penetration protein (app), because of its extensive homology to the hap gene of Haemophilus influenzae. We reconstructed the gene with reference to genomic sequence data and cloned and expressed the protein in Escherichia coli. Rabbit antiserum raised against recombinant App reacted with proteins in all meningococcal isolates examined, which represented clonal groups responsible for the majority of meningococcal invasive disease. Antibodies to the protein were detected in the sera of patients convalescing from meningococcal infection. Purified App had strong stimulating activity for T cells isolated from a number of healthy donors and from one convalescent patient. We confirmed that App is surface localized, cleaved and secreted by N. meningitidis. Importantly, the rabbit anti-App serum killed the organism in the presence of complement. Thus, App is conserved among meningococci, immunogenic in humans and potentially involved in virulence. It therefore merits further investigation as a component of a future multivalent vaccine.     

Neisseria meningitidis producing the Opc adhesin binds epithelial cell proteoglycan receptors

Neisseria meningitidis possesses a repertoire of surface adhesins that promote bacterial adherence to and entry into mammalian cells. Here, we have identified heparan sulphate proteoglycans as epithelial cell receptors for the meningococcal Opc invasin. Binding studies with radiolabelled heparin and heparin affinity chromatography demonstrated that Opc is a heparin binding protein. Subsequent binding experiments with purified ³⁵SO₄-labelled epithelial cell proteoglycan receptors and infection assays with epithelial cells that had been treated with heparitinase to remove glycosaminoglycans confirmed that Opc-expressing meningococci exploit host cell-surface proteoglycans to gain access to the epithelial cell interior. Unexpectedly, Opa28-producing meningococci lacking Opc also bound proteoglycans. These bacteria also bound CEA receptors in contrast to the Opc-expressing phenotype, suggesting that Opa28 may possess domains with specificity for different receptors. Opa/Opc-negative meningococci did not bind either proteoglycan or CEA receptors. Using a set of genetically defined mutants with different lipopolysaccharide (LPS) and capsular phenotype, we were able to demonstrate that surface sialic acids interfere with the Opc–proteoglycan receptor interaction. This effect may provide the molecular basis for the reported modulatory effect of capsule and LPS on meningococcal adherence to and entry into various cell types.     

A BCAM0223 Mutant of Burkholderia cenocepacia Is Deficient in Hemagglutination, Serum Resistance, Adhesion to Epithelial Cells and Virulence

Burkholderia cepacia complex (Bcc) bacteria are a problematic group of microorganisms causing severe infections in patients with Cystic Fibrosis. In early stages of infection, Bcc bacteria must be able to adhere to and colonize the respiratory epithelium. Although this is not fully understood, this primary stage of infection is believed to be in part mediated by a specific type of adhesins, named trimeric autotransporter adhesins (TAAs). These homotrimeric proteins exist on the surface of many Gram negative pathogens and often mediate a number of critical functions, including biofilm formation, serum resistance and adherence to an invasion of host cells. We have previously identified in the genome of the epidemic clinical isolate B. cenocepacia J2315, a novel cluster of genes putatively encoding three TAAs (BCAM0219, BCAM0223 and BCAM0224). In this study, the genomic organization of the TAA cluster has been determined. To further address the direct role of the putative TAA BCAM0223 in B. cenocepacia pathogenicity, an isogenic mutant was constructed via insertional inactivation. The BCAM0223::Tp mutant is deficient in hemagglutination, affected in adherence to vitronectin and in biofilm formation and showed attenuated virulence in the Galleria mellonella model of infection. Moreover, the BCAM0223::Tp mutant also showed a significant reduction in its resistance to human serum as well as in adherence, but not in invasion of, cultured human bronchial epithelial cells. Altogether these results demonstrate that the BCAM0223 protein is a multifunctional virulence factor that may contribute to the pathogenicity of B. cenocepacia.     

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.     

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.     

Autodisplay: functional display of active beta-lactamase on the surface of Escherichia coli by the AIDA-I autotransporter

Members of the protein family of immunoglobulin A1 protease-like autotransporters comprise multidomain precursors consisting of a C-terminal autotransporter domain that promotes the translocation of N-terminally attached passenger domains across the cell envelopes of gram-negative bacteria. Several autotransporter domains have recently been shown to efficiently promote the export of heterologous passenger domains, opening up an effective tool for surface display of heterologous proteins. Here we report on the autotransporter domain of the Escherichia coli adhesin involved in diffuse adherence (AIDA-I), which was genetically fused to the C terminus of the periplasmic enzyme beta-lactamase, leading to efficient expression of the fusion protein in E. coli. The beta-lactamase moiety of the fusion protein was presented on the bacterial surface in a stable manner, and the surface-located beta-lactamase was shown to be enzymatically active. Enzymatic activity was completely removed by protease treatment, indicating that surface display of beta-lactamase was almost quantitative. The periplasmic domain of the outer membrane protein OmpA was not affected by externally added proteases, demonstrating that the outer membranes of E. coli cells expressing the beta-lactamase AIDA-I fusion protein remained physiologically intact.     

A structural overview of mycobacterial adhesins: Key biomarkers for diagnostics and therapeutics

Adherence, colonization, and survival of mycobacteria in host cells require surface adhesins, which are attractive pharmacotherapeutic targets. A large arsenal of pilus and non‐pilus adhesins have been identified in mycobacteria. These adhesins are capable of interacting with host cells, including macrophages and epithelial cells and are essential to microbial pathogenesis. In the last decade, several structures of mycobacterial adhesins responsible for adhesion to either macrophages or extra cellular matrix proteins have been elucidated. In addition, key structural and functional information have emerged for the process of mycobacterial adhesion to epithelial cells, mediated by the Heparin‐binding hemagglutinin (HBHA). In this review, we provide an overview of the structural and functional features of mycobacterial adhesins and discuss their role as important biomarkers for diagnostics and therapeutics. Based on the reported data, it appears clear that adhesins are endowed with a variety of different structures and functions. Most adhesins play important roles in the cell life of mycobacteria and are key virulence factors. However, they have adapted to an extracellular life to exert a role in host‐pathogen interaction. The type of interactions they form with the host and the adhesin regions involved in binding is partly known and is described in this review.