Trimeric Autotransporter Research Articles

Molecular Analysis of Aggregatibacter actinomycetemcomitans ApiA, a Multi-Functional Protein.

Aggregatibacter actinomycetemcomitans ApiA is a trimeric autotransporter outer membrane protein (Omp) that participates in multiple functions, enabling A. actinomycetemcomitans to adapt to a variety of environments. The goal of this study is to identify regions in the apiA gene responsible for three of these functions: auto-aggregation, buccal epithelial cell binding, and complement resistance. Initially, apiA was expressed in Escherichia coli. Finally, wild-type A. actinomycetemcomitans and an apiA-deleted version were tested for their expression in the presence and absence of serum and genes related to stress adaptation, such as oxygen regulation, catalase activity, and Omp proteins. Sequential deletions in specific regions in the apiA gene as expressed in E. coli were examined for membrane proteins, which were confirmed by microscopy. The functional activity of epithelial cell binding, auto-aggregation, and complement resistance were then assessed, and regions in the apiA gene responsible for these functions were identified. A region spanning amino acids 186-217, when deleted, abrogated complement resistance and Factor H (FH) binding, while a region spanning amino acids 28-33 was related to epithelial cell binding. A 13-amino-acid peptide responsible for FH binding was shown to promote serum resistance. An apiA deletion in a clinical isolate (IDH781) was created and tested in the presence and/or absence of active and inactive serum and genes deemed responsible for prominent functional activity related to A. actinomycetemcomitans survival using qRT-PCR. These experiments suggested that apiA expression in IDH781 is involved in global regulatory mechanisms that are serum-dependent and show complement resistance. This is the first study to identify specific apiA regions in A. actinomycetemcomitans responsible for FH binding, complement resistance, and other stress-related functions. Moreover, the role of apiA in overall gene regulation was observed.

Identification of Veillonella parvula and Streptococcus gordonii adhesins mediating co-aggregation and its impact on physiology and mixed biofilm structure.

Our research explores the mechanisms of bacterial adhesion within the dental plaque, focusing on Veillonella parvula, a key player in the oral microbiome. Dependent on lactate from streptococci, V. parvula plays a crucial bridging role in the formation of dental biofilms by co-aggregating with other bacteria. Despite its importance, the understanding of the underlying mechanisms of co-aggregation remains limited. Our study shows that V. parvula uses different trimeric autotransporters to adhere to oral Streptococci and Actinomyces. We additionally identify a novel adhesin from S. gordonii, VisA (SGO_2004) facilitating this interaction. We found that although co-aggregation does not affect cell-cell communication, it is critical for biofilm structure and species distribution. This research opens up new avenues for exploring microbial interactions in dental health and diseases.

Virulence-linked adhesin drives mutualist colonization of the bee gut via biofilm formation.

Bacterial biofilms are stable multicellular structures that can enable long term host association. Yet, the role of biofilms in supporting gut mutualism is still not fully understood. Here, we investigate Snodgrassella alvi, a beneficial bacterial symbiont of honey bees, and find that biofilm formation is required for its colonization of the bee gut. We constructed fifteen S. alvi mutants containing knockouts of genes known to promote colonization with putative roles in biofilm formation. Genes required for colonization included staA and staB, encoding trimeric autotransporter adhesins (TAAs) and mltA, encoding a lytic transglycosylase. Intriguingly, TAAs are considered virulence factors in pathogens but support mutualism by the symbiont S. alvi. In vitro, biofilm formation was reduced in ΔstaB cells and abolished in the other two mutants. Loss of staA also reduced auto-aggregation and cell-cell connections. Based on structural predictions, StaA/B are massive (>300 nm) TAAs with many repeats in their stalk regions. Further, we find that StaA/B are conserved across Snodgrassella species, suggesting that StaA/B-dependent colonization is characteristic of this symbiont lineage. Finally, staA deletion increases sensitivity to bactericidal antimicrobials, suggesting that the biofilm indirectly buffers against antibiotic stress. In all, the inability of two biofilm-deficient strains (ΔstaA and ΔmltA) to effectively mono-colonize bees indicates that S. alvi biofilm formation is required for colonization of the bee gut. We envision the bee gut system as a genetically tractable model for studying the physical basis of biofilm-mutualist-gut interactions.

Transcriptome analysis reveals a new virulence-associated trimeric autotransporter responsible for Glaesserella parasuis autoagglutination.

Capsular polysaccharide is an important virulence factor of Glaesserella parasuis. An acapsular mutant displays multiple phenotype variations, while the underlying mechanism for these variations is unknown. In this study, we created an acapsular mutant by deleting the wza gene in the capsule locus. We then used transcriptome analysis to compare the gene expression profiles of the wza deletion mutant with those of the parental strain to understand the possible reasons for the phenotypic differences. The mutant Δwza, which has a deleted wza gene, secreted less polysaccharide and lost its capsule structure. The Δwza exhibited increased autoagglutination, biofilm formation and adherence to eukaryotic cells, while the complementary strain C-Δwza partially restored the phenotype. Transcriptome analysis revealed several differentially expressed genes (DEGs) in Δwza, including up-regulated outer membrane proteins and proteins involved in peptidoglycan biosynthesis, suggesting that wza deletion affects the cell wall homeostasis of G. parasuis. Transcriptome analysis revealed the contribution of non-coding RNAs in the regulation of DEGs. Moreover, a new virulence-associated trimeric autotransporter, VtaA31 is upregulated in Δwza. It is responsible for enhanced autoagglutination but not for enhanced biofilm formation and adherence to eukaryotic cells in Δwza. In conclusion, these data indicate that wza affects the expression of multiple genes, especially those related to cell wall synthesis. Furthermore, they provide evidence that vtaA31 is involved in the autoagglutination of G. parasuis.

The Trimeric Autotransporter Adhesin SadA from Salmonella spp. as a Novel Bacterial Surface Display System.

Bacterial surface display platforms have been developed for applications such as vaccine delivery and peptide library screening. The type V secretion system is an attractive anchoring motif for the surface expression of foreign proteins in gram-negative bacteria. SadA belongs to subtype C of the type V secretion system derived from Salmonella spp. and promotes biofilm formation and host cell adherence. The inner membrane lipoprotein SadB is important for SadA translocation. In this study, SadA was used as an anchoring motif to expose heterologous proteins in Salmonella typhimurium using SadB. The ability of SadA to display heterologous proteins on the S. typhimurium surface in the presence of SadB was approximately three-fold higher than that in its absence of SadB. Compared to full-length SadA, truncated SadAs (SadA877 and SadA269) showed similar display capacities when exposing the B-cell epitopes of urease B from Helicobacter pylori (UreB158-172aa and UreB349-363aa). We grafted different protein domains, including mScarlet (red fluorescent protein), the urease B fragment (UreBm) from H. pylori SS1, and/or protective antigen domain 4 from Bacillus anthracis A16R (PAD4), onto SadA877 or SadA1292. Whole-cell dot blotting, immunofluorescence, and flow cytometric analyses confirmed the localization of Flag×3-mScarlet (~30 kDa) and Flag×3-UreBm-mScarlet (~58 kDa) to the S. typhimurium surface using truncated SadA877 or SadA1292 as an anchoring motif. However, Flag×3-UreBm-PAD4-mScarlet (~75 kDa) was displayed on S. typhimurium using SadA1292. The oral administrated pSadBA1292-FUM/StmΔygeAΔmurI and pSadBA877-FUM/StmΔygeAΔmurI could elicit a significant mucosal and humoral immunity response. SadA could thus be used as an anchoring motif for the surface expression of large heterologous proteins as a potential strategy for attenuated bacterial vaccine development.

Adhesion preference of the sticky bacterium Acinetobacter sp. Tol 5.

Gram-negative bacterium Acinetobacter sp. Tol 5 exhibits high adhesiveness to various surfaces of general materials, from hydrophobic plastics to hydrophilic glass and metals, via AtaA, an Acinetobacter trimeric autotransporter adhesin Although the adhesion of Tol 5 is nonspecific, Tol 5 cells may have prefer materials for adhesion. Here, we examined the adhesion of Tol 5 and other bacteria expressing different TAAs to various materials, including antiadhesive surfaces. The results highlighted the stickiness of Tol 5 through the action of AtaA, which enabled Tol 5 cells to adhere even to antiadhesive materials, including polytetrafluoroethylene with a low surface free energy, a hydrophilic polymer brush with steric hindrance, and mica with an ultrasmooth surface. Single-cell force spectroscopy as an atomic force microscopy technique revealed the strong cell adhesion force of Tol 5 to these antiadhesive materials. Nevertheless, Tol 5 cells showed a weak adhesion force toward a zwitterionic 2-methacryloyloxyethyl-phosphorylcholine (MPC) polymer-coated surface. Dynamic flow chamber experiments revealed that Tol 5 cells, once attached to the MPC polymer-coated surface, were exfoliated by weak shear stress. The underlying adhesive mechanism was presumed to involve exchangeable, weakly bound water molecules. Our results will contribute to the understanding and control of cell adhesion of Tol 5 for immobilized bioprocess applications and other TAA-expressing pathogenic bacteria of medical importance.

Dual function of the O-antigen WaaL ligase of Aggregatibacter actinomycetemcomitans.

Protein glycosylation is critical to the quaternary structure and collagen-binding activity of the extracellular matrix protein adhesin A (EmaA) associated with Aggregatibacter actinomycetemcomitans. The glycosylation of this large, trimeric autotransporter adhesin is postulated to be mediated by WaaL, an enzyme with the canonical function to ligate the O-polysaccharide (O-PS) antigen with a terminal sugar of the lipid A-core oligosaccharide of lipopolysaccharide (LPS). In this study, we have determined that the Escherichia coli waaL ortholog (rflA) does not restore collagen binding of a waaL mutant strain of A. actinomycetemcomitans but does restore O-PS ligase activity following transformation of a plasmid expressing waaL. Therefore, a heterologous E. coli expression system was developed constituted of two independently replicating plasmids expressing either waaL or emaA of A. actinomycetemcomitans to directly demonstrate the necessity of ligase activity for EmaA collagen binding. Proper expression of the protein encoded by each plasmid was characterized, and the individually transformed strains did not promote collagen binding. However, coexpression of the two plasmids resulted in a strain with a significant increase in collagen binding activity and a change in the biochemical properties of the protein. These results provide additional data supporting the novel hypothesis that the WaaL ligase of A. actinomycetemcomitans shares a dual role as a ligase in LPS biosynthesis and is required for collagen binding activity of EmaA.

Immune responses following neonatal vaccination with conserved F4 fragment of VtaA proteins from virulent Glaesserella parasuis adjuvanted with CAF®01 or CDA

Glaesserella parasuis is a Gram-negative bacterium that colonizes the upper airways of swine, capable of causing a systemic infection called Glässer’s disease. This disease is more frequent in young post-weaning piglets. Current treatments against G. parasuis infection are based on the use of antimicrobials or inactivated vaccines, which promote limited cross-protection against different serovars. For this reason, there is an interest in developing novel subunit vaccines with the capacity to confer effective protection against different virulent strains. Herein, we characterize the immunogenicity and the potential benefits of neonatal immunization with two different vaccine formulations based on the F4 polypeptide, a conserved immunogenic protein fragment from the virulence-associated trimeric autotransporters of virulent G. parasuis strains. With this purpose, we immunized two groups of piglets with F4 combined with cationic adjuvant CAF®01 or cyclic dinucleotide CDA. Piglets immunized with a commercial bacterin and non-immunized animals served as control groups. The vaccinated piglets received two doses of vaccine, at 14 days old and 21 days later. The immune response induced against the F4 polypeptide varied depending on the adjuvant used. Piglets vaccinated with the F4+CDA vaccine developed specific anti-F4 IgGs, biased towards the induction of IgG1 responses, whereas no anti-F4 IgGs were de novo induced after immunization with the CAF®01 vaccine. Piglets immunized with both formulations displayed balanced memory T-cell responses, evidenced upon in vitro re-stimulation of peripheral blood mononuclear cells with F4. Interestingly, pigs immunized with F4+CAF®01 controlled more efficiently a natural nasal colonization by a virulent serovar 4 G. parasuis that spontaneously occurred during the experimental procedure. According to the results, the immunogenicity and the protection afforded by F4 depend on the adjuvant used. F4 may represent a candidate to consider for a Glässer‘s disease vaccine and could contribute to a better understanding of the mechanisms involved in protection against virulent G. parasuis colonization.

Adh Promotes Actinobacillus pleuropneumoniae Survival in Porcine Alveolar Macrophages by Inhibiting CHAC2-Mediated Respiratory Burst and Inflammatory Cytokine Expression.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) causes porcine pleuropneumonia that seriously endangers pig's health. Adh, located in the head region of trimeric autotransporter adhesion of A. pleuropneumoniae, affects bacterial adhesion and pathogenicity. However, how Adh mediates A. pleuropneumoniae immune invasion is still unclear. Here, we established the A. pleuropneumoniae strain L20 or L20 ΔAdh-infected porcine alveolar macrophages (PAM) model, and applied protein overexpression, RNA interference, qRT-PCR, Western blot and immunoflourescence techniques to dissect the effects of Adh on PAM during A. pleuropneumoniae infection. We found that Adh could increase the A. pleuropneumoniae adhesion and intracellular survival in PAM. Gene chip analysis of piglet lungs further showed that Adh significantly induced cation transport regulatory-like protein 2 (CHAC2) expression, whose overexpression suppressed the phagocytic capacity of PAM. Furthermore, CHAC2 overexpression dramatically increased glutathione (GSH) expression, decreased reactive oxygen species (ROS), and promoted A. pleuropneumoniae survival in PAM, while the knockdown of CHAC2 reversed these phenomena. Meanwhile, CHAC2 silence activated the NOD1/NF-κB pathway, resulting in an increase in IL-1β, IL-6, and TNF-α expression, whereas this effect was weakened by CHAC2 overexpression and addition of NOD1/NF-κB inhibitor ML130. Moreover, Adh enhanced the secretion of LPS of A. pleuropneumoniae, which regulated the expression of CHAC2 via TLR4. In conclusion, through a LPS-TLR4-CHAC2 pathway, Adh inhibits respiratory burst and inflammatory cytokines expression to promote A. pleuropneumoniae survival in PAM. This finding may provide a novel target for the prevention and treatment of A. pleuropneumoniae.

Molecular Characterization and Phylogenetic Analysis of Outer membrane protein P2 (OmpP2) of Glaesserella (Haemophilus) parasuis Isolates in Central State of Peninsular Malaysia

Glaesserella (Haemophilus) parasuis, the etiological agent of Glässer's disease, is an economically significant pathogen commonly associated with serofibrinous polyserositis, arthritis, fibrinous bronchopneumonia and/or meningitis. This study is the first attempt to molecularly characterize and provide a detailed overview of the genetic variants of G. parasuis present in Malaysia, in reference to its serotype, virulence-associated trimeric autotransporters (vtaA) gene and outer membrane protein P2 (OmpP2) gene. The G. parasuis isolates (n = 11) from clinically sick field samples collected from two major pig producing states (Selangor and Perak) were selected for analysis. Upon multiplex PCR, the majority of the isolates (eight out of 11) were identified to be serotype 5 or 12, and interestingly, serotypes 3, 8 and 15 were also detected, which had never been reported in Malaysia prior to this. Generally, virulent vtaA was detected for all isolates, except for one, which displayed a nonvirulent vtaA. A phylogenetic analysis of the OmpP2 gene revealed that the majority of Malaysian isolates were clustered into genotype 1, which could be further divided into Ia and Ib, while only one isolate was clustered into genotype 2.

Comparison of Biological Activities of BafA Family Autotransporters within Bartonella Species Derived from Cats and Rodents.

Bartonella species are hemotropic, facultative intracellular bacteria, some of which cause zoonoses, that are widely disseminated among many mammals, including humans. During infection in humans, vascular endothelial cells play a crucial role as a replicative niche for Bartonella, and some are capable of promoting vascular proliferation. Along with well-studied pathogenic factors such as a trimeric autotransporter adhesin BadA or VirB/D4 type IV secretion system, bacteria-secreted protein BafA is also involved in Bartonella-induced vasoproliferation. Genes encoding BafA orthologs have been found in the genomes of most Bartonella species, but their functionality remains unclear. In this study, we focused on three cat-derived zoonotic species (B. henselae, B. koehlerae, and B. clarridgeiae) and two rodent-derived species (B. grahamii and B. doshiae) and compared the activity of BafA derived from each species. Recombinant BafA proteins of B. henselae, B. koehlerae, B. clarridgeiae, and B. grahamii, species that also cause human disease, induced cell proliferation and tube formation in cultured endothelial cells, while BafA derived from B. doshiae, a species that is rarely found in humans, showed neither activity. Additionally, treatment of cells with these BafA proteins increased phosphorylation of both vascular endothelial growth factor receptor 2 and extracellular signal-regulated kinase 1/2, with the exception of B. doshiae BafA. Differential bafA mRNA expression and BafA secretion among the species likely contributed to the differences in the cell proliferation phenotype of the bacteria-infected cells. These findings suggest that the biological activity of BafA may be involved in the infectivity or pathogenicity of Bartonella species in humans.

Identification of the adhesive domain of AtaA from Acinetobacter sp. Tol 5 and its application in immobilizing Escherichia coli.

Cell immobilization is an important technique for efficiently utilizing whole-cell biocatalysts. We previously invented a method for bacterial cell immobilization using AtaA, a trimeric autotransporter adhesin from the highly sticky bacterium Acinetobacter sp. Tol 5. However, except for Acinetobacter species, only one bacterium has been successfully immobilized using AtaA. This is probably because the heterologous expression of large AtaA (1MDa), that is a homotrimer of polypeptide chains composed of 3,630 amino acids, is difficult. In this study, we identified the adhesive domain of AtaA and constructed a miniaturized AtaA (mini-AtaA) to improve the heterologous expression of ataA. In-frame deletion mutants were used to perform functional mapping, revealing that the N-terminal head domain is essential for the adhesive feature of AtaA. The mini-AtaA, which contains a homotrimer of polypeptide chains from 775 amino acids and lacks the unnecessary part for its adhesion, was properly expressed in E. coli, and a larger amount of molecules was displayed on the cell surface than that of full-length AtaA (FL-AtaA). The immobilization ratio of E. coli cells expressing mini-AtaA on a polyurethane foam support was significantly higher compared to the cells with or without FL-AtaA expression, respectively. The expression of mini-AtaA in E. coli had little effect on the cell growth and the activity of another enzyme reflecting the production level, and the immobilized E. coli cells could be used for repetitive enzymatic reactions as a whole-cell catalyst.

Serotype-Specific Sugars Impact Structure but Not Functions of the Trimeric Autotransporter Adhesin EmaA of Aggregatibacter actinomycetemcomitans.

The human oral pathobiont Aggregatibacter actinomycetemcomitans expresses multiple virulence factors, including the trimeric, extracellular matrix protein adhesin A (EmaA). The posttranslational modification of EmaA is proposed to be dependent on the sugars and enzymes associated with O-polysaccharide (O-PS) synthesis of the lipopolysaccharide (LPS). This modification is important for the structure and function of this adhesin. To determine if the composition of the sugars alters structure and/or function, the prototypic 202-kDa protein was expressed in a non-serotype b, emaA mutant strain. The transformed strain displayed EmaA adhesins similar in appearance to the prototypic adhesin as observed by two-dimensional (2D) electron microscopy of whole-mount negatively stained bacterial preparations. Biochemical analysis indicated that the protein monomers were posttranslationally modified. 3D electron tomographic reconstruction and structure analyses of the functional domain revealed three well-defined subdomains (SI, SII, and SIII) with a linker region between SII and SIII. Structural changes were observed in all three subdomains and the linker region of the adhesins synthesized compared with the known structure. These changes, however, did not affect the ability of the strain to bind collagen or form biofilms. The data suggest that changes in the composition of the glycan moiety alter the 3D structure of the molecule without negatively affecting the function(s) associated with this adhesin. IMPORTANCE The human oral pathogen A. actinomycetemcomitans is a causative agent of periodontal and several systemic diseases. EmaA is a trimeric autotransporter protein adhesin important for colonization by this pathobiont in vivo. This adhesin is modified with sugars associated with the O-polysaccharide (O-PS), and the modification is mediated using the enzymes involved in lipopolysaccharide (LPS) biosynthesis. The interaction with collagen is not mediated by the specific binding between the glycans and collagen but is attributed to changes in the final quaternary structure necessary to maintain an active adhesin. In this study, we have determined that the composition of the sugars utilized in the posttranslational modification of this adhesin is exchangeable without compromising functional activities.

Pathogenic determinants of Kingella kingae disease.

Kingella kingae is an emerging pediatric pathogen and is increasingly recognized as a leading etiology of septic arthritis, osteomyelitis, and bacteremia and an occasional cause of endocarditis in young children. The pathogenesis of K. kingae disease begins with colonization of the upper respiratory tract followed by breach of the respiratory epithelial barrier and hematogenous spread to distant sites of infection, primarily the joints, bones, and endocardium. As recognition of K. kingae as a pathogen has increased, interest in defining the molecular determinants of K. kingae pathogenicity has grown. This effort has identified numerous bacterial surface factors that likely play key roles in the pathogenic process of K. kingae disease, including type IV pili and the Knh trimeric autotransporter (adherence to the host), a potent RTX-family toxin (epithelial barrier breach), and multiple surface polysaccharides (complement and neutrophil resistance). Herein, we review the current state of knowledge of each of these factors, providing insights into potential approaches to the prevention and/or treatment of K. kingae disease.

Adhesion of Bartonella henselae to Fibronectin Is Mediated via Repetitive Motifs Present in the Stalk of Bartonella Adhesin A.

ABSTRACTAdhesion to host cells is the first and most crucial step in infections with pathogenic Gram-negative bacteria and is often mediated by trimeric autotransporter adhesins (TAAs). Bartonella henselae targets the extracellular matrix glycoprotein fibronectin (Fn) via the Bartonella adhesin A (BadA) attaching the bacteria to the host cell. The TAA BadA is characterized by a highly repetitive passenger domain consisting of 30 neck/stalk domains with various degrees of similarity. To elucidate the motif sequences mediating Fn binding, we generated 10 modified BadA constructs and verified their expression via Western blotting, confocal laser scanning, and electron microscopy. We analyzed their ability to bind human plasma Fn using quantitative whole-cell enzyme-linked immunosorbent assays (ELISAs) and fluorescence microscopy. Polyclonal antibodies targeting a 15-mer amino acid motif sequence proved to reduce Fn binding. We suggest that BadA adheres to Fn in a cumulative effort with quick saturation primarily via unpaired β-strands appearing in motifs repeatedly present throughout the neck/stalk region. In addition, we demonstrated that the length of truncated BadA constructs correlates with the immunoreactivity of human patient sera. The identification of BadA-Fn binding regions will support the development of new “antiadhesive” compounds inhibiting the initial adherence of B. henselae and other TAA-expressing pathogens to host cells.IMPORTANCE Trimeric autotransporter adhesins (TAAs) are important virulence factors and are widely present in various pathogenic Gram-negative bacteria. TAA-expressing bacteria cause a wide spectrum of human diseases, such as cat scratch disease (Bartonella henselae), enterocolitis (Yersinia enterocolitica), meningitis (Neisseria meningitis), and bloodstream infections (multidrug-resistant Acinetobacter baumannii). TAA-targeted antiadhesive strategies (against, e.g., Bartonella adhesin A [BadA], Yersinia adhesin A [YadA], Neisseria adhesin A [NadA], and Acinetobacter trimeric autotransporter [Ata]) might represent a universal strategy to counteract such bacterial infections. BadA is one of the best characterized TAAs, and because of its high number of (sub)domains, it serves as an attractive adhesin to study the domain-function relationship of TAAs in the infection process. The identification of common binding motifs between TAAs (here, BadA) and their major binding partner (here, fibronectin) provides a basis toward the design of novel “antiadhesive” compounds preventing the initial adherence of Gram-negative bacteria in infections.

The XadA Trimeric Autotransporter Adhesins in Xylella fastidiosa Differentially Contribute to Cell Aggregation, Biofilm Formation, Insect Transmission and Virulence to Plants.

Surface adhesion strategies are widely employed by bacterial pathogens during establishment and systemic spread in their host. A variety of cell-surface appendages such as pili, fimbriae, and afimbrial adhesins are involved in these processes. The phytopathogen Xylella fastidiosa employs several of these structures for efficient colonization of its insect and plant hosts. Among the adhesins encoded in the X. fastidiosa genome, three afimbrial adhesins, XadA1, Hsf/XadA2, and XadA3, are predicted to be trimeric autotransporters with a C-terminal YadA-anchor membrane domain. We analyzed the individual contributions of XadA1, XadA2, and XadA3 to various cellular behaviors both in vitro and in vivo. Using isogenic X. fastidiosa mutants, we found that cell-cell aggregation and biofilm formation were severely impaired in the absence of XadA3. No significant reduction of cell-surface attachment was found with any mutant under flow conditions. Acquisition by insect vectors and transmission to grapevines were reduced in the XadA3 deletion mutant. While the XadA3 mutant was hypervirulent in grapevines, XadA1 or XadA2 deletion mutants conferred lower disease severity than the wild-type strain. This insight of the importance of these adhesive proteins and their individual contributions to different aspects of X. fastidiosa biology should guide new approaches to reduce pathogen transmission and disease development. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The C-terminal head domain of Burkholderia pseudomallei BpaC has a striking hydrophilic core with an extensive solvent network.

Gram‐negative pathogens like Burkholderia pseudomallei use trimeric autotransporter adhesins such as BpaC as key molecules in their pathogenicity. Our 1.4 Å crystal structure of the membrane‐proximal part of the BpaC head domain shows that the domain is exclusively made of left‐handed parallel β‐roll repeats. This, the largest such structure solved, has two unique features. First, the core, rather than being composed of the canonical hydrophobic Ile and Val, is made up primarily of the hydrophilic Thr and Asn, with two different solvent channels. Second, comparing BpaC to all other left‐handed parallel β‐roll structures showed that the position of the head domain in the protein correlates with the number and type of charged residues. In BpaC, only negatively charged residues face the solvent—in stark contrast to the primarily positive surface charge of the left‐handed parallel β‐roll “type” protein, YadA. We propose extending the definitions of these head domains to include the BpaC‐like head domain as a separate subtype, based on its unusual sequence, position, and charge. We speculate that the function of left‐handed parallel β‐roll structures may differ depending on their position in the structure.

Identification of Glaesserella parasuis and Differentiation of Its 15 Serovars Using High-Resolution Melting Assays.

Glaesserella parasuis is the etiological agent of Glässer’s disease, which is associated with polyserositis and arthritis and has a significant impact on the economy of the pig production industry. For the optimal surveillance of this pathogen, as well as for the investigation of G. parasuis-associated diseases, it is crucial to identify G. parasuis at the serovar level. In this work, we designed and developed new high-resolution melting (HRM) approaches, namely, the species-specific GPS-HRM1 and two serovar-specific HRM assays (GPS-HRM2 and GPS-HRM3), and evaluated the sensitivity and specificity of the assays. The HRM assays demonstrated good sensitivity, with 12.5 fg–1.25 pg of input DNA for GPS-HRM1 and 125 fg–12.5 pg for GPS-HRM2 and GPS-HRM3, as well as a specificity of 100% for the identification of all recognized 15 G. parasuis serovars. Eighteen clinical isolates obtained between 2014 and 2022 in Switzerland were tested by applying the developed HRM assays, which revealed a heterogeneous distribution of serovars 2, 7, 4, 13, 1, and 14. The combination with virulence marker vtaA (virulence-associated trimeric autotransporters) allows for the prediction of potentially virulent strains. The assays are simple to execute and enable a reliable low-cost approach, thereby refining currently available diagnostic tools.

Long-Read Sequencing Reveals Genetic Adaptation of Bartonella Adhesin A Among Different Bartonella henselae Isolates.

Bartonella henselae is the causative agent of cat scratch disease and other clinical entities such as endocarditis and bacillary angiomatosis. The life cycle of this pathogen, with alternating host conditions, drives evolutionary and host-specific adaptations. Human, feline, and laboratory adapted B. henselae isolates often display genomic and phenotypic differences that are related to the expression of outer membrane proteins, for example the Bartonella adhesin A (BadA). This modularly-structured trimeric autotransporter adhesin is a major virulence factor of B. henselae and is crucial for the initial binding to the host via the extracellular matrix proteins fibronectin and collagen. By using next-generation long-read sequencing we demonstrate a conserved genome among eight B. henselae isolates and identify a variable genomic badA island with a diversified and highly repetitive badA gene flanked by badA pseudogenes. Two of the eight tested B. henselae strains lack BadA expression because of frameshift mutations. We suggest that active recombination mechanisms, possibly via phase variation (i.e., slipped-strand mispairing and site-specific recombination) within the repetitive badA island facilitate reshuffling of homologous domain arrays. The resulting variations among the different BadA proteins might contribute to host immune evasion and enhance long-term and efficient colonisation in the differing host environments. Considering the role of BadA as a key virulence factor, it remains important to check consistently and regularly for BadA surface expression during experimental infection procedures.

The Trimeric Autotransporter Adhesin YadA of Yersinia enterocolitica Serotype O:9 Binds Glycan Moieties.

Yersinia adhesin A (YadA) is a key virulence factor of Yersinia enterocolitica and Yersinia pseudotuberculosis. YadA is a trimeric autotransporter adhesin, a class of adhesins that have been shown to enable many Gram-negative pathogens to adhere to/interact with the host extracellular matrix proteins such as collagen, vitronectin, and fibronectin. Here, we show for the first time that YadA of Yersinia enterocolitica serotype O:9 not only interacts with proteinaceous surface molecules but can also attach directly to glycan moieties. We show that YadA from Y. enterocolitica serotype O:9 does not interact with the vitronectin protein itself but exclusively with its N-linked glycans. We also show that YadA can target other glycan moieties as found in heparin, for example. So far, little is known about specific interactions between bacterial autotransporter adhesins and glycans. This could potentially lead to new antimicrobial treatment strategies, as well as diagnostic applications.