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

Adhesion 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 ß-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.

Interaction of Bartonella henselae with Fibronectin Represents the Molecular Basis for Adhesion to Host Cells.

Bacterial adhesion to the host is the most decisive step in infections. Trimeric autotransporter adhesins (TAA) are important pathogenicity factors of Gram-negative bacteria. The prototypic TAA Bartonella adhesin A (BadA) from human-pathogenic Bartonella henselae mediates bacterial adherence to endothelial cells (ECs) and extracellular matrix proteins. Here, we determined the interaction between BadA and fibronectin (Fn) to be essential for bacterial host cell adhesion. BadA interactions occur within the heparin-binding domains of Fn. The exact binding sites were revealed by mass spectrometry analysis of chemically cross-linked whole-cell bacteria and Fn. Specific BadA interactions with defined Fn regions represent the molecular basis for bacterial adhesion to ECs and these data were confirmed by BadA-deficient bacteria and CRISPR-Cas knockout Fn host cells. Interactions between TAAs and the extracellular matrix might represent the key step for adherence of human-pathogenic Gram-negative bacteria to the host. IMPORTANCE Deciphering the mechanisms of bacterial host cell adhesion is a clue for preventing infections. We describe the underestimated role that the extracellular matrix protein fibronectin plays in the adhesion of human-pathogenic Bartonella henselae to host cells. Fibronectin-binding is mediated by a trimeric autotransporter adhesin (TAA) also present in many other human-pathogenic Gram-negative bacteria. We demonstrate that both TAA and host-fibronectin contribute significantly to bacterial adhesion, and we present the exact sequence of interacting amino acids from both proteins. Our work shows the domain-specific pattern of interaction between the TAA and fibronectin to adhere to host cells and opens the perspective to fight bacterial infections by inhibiting bacterial adhesion which represents generally the first step in infections.

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.

Host-Pathogen Adhesion as the Basis of Innovative Diagnostics for Emerging Pathogens.

Infectious diseases are an existential health threat, potentiated by emerging and re-emerging viruses and increasing bacterial antibiotic resistance. Targeted treatment of infectious diseases requires precision diagnostics, especially in cases where broad-range therapeutics such as antibiotics fail. There is thus an increasing need for new approaches to develop sensitive and specific in vitro diagnostic (IVD) tests. Basic science and translational research are needed to identify key microbial molecules as diagnostic targets, to identify relevant host counterparts, and to use this knowledge in developing or improving IVD. In this regard, an overlooked feature is the capacity of pathogens to adhere specifically to host cells and tissues. The molecular entities relevant for pathogen-surface interaction are the so-called adhesins. Adhesins vary from protein compounds to (poly-)saccharides or lipid structures that interact with eukaryotic host cell matrix molecules and receptors. Such interactions co-define the specificity and sensitivity of a diagnostic test. Currently, adhesin-receptor binding is typically used in the pre-analytical phase of IVD tests, focusing on pathogen enrichment. Further exploration of adhesin-ligand interaction, supported by present high-throughput "omics" technologies, might stimulate a new generation of broadly applicable pathogen detection and characterization tools. This review describes recent results of novel structure-defining technologies allowing for detailed molecular analysis of adhesins, their receptors and complexes. Since the host ligands evolve slowly, the corresponding adhesin interaction is under selective pressure to maintain a constant receptor binding domain. IVD should exploit such conserved binding sites and, in particular, use the human ligand to enrich the pathogen. We provide an inventory of methods based on adhesion factors and pathogen attachment mechanisms, which can also be of relevance to currently emerging pathogens, including SARS-CoV-2, the causative agent of COVID-19.

Complete Genome Sequence of Bartonella alsatica Strain IBS 382 (CIP 105477).

Bartonella alsatica causes bacteremia in rabbits and, rarely, human infections. Here, we announce the complete and closed genome of B. alsatica IBS 382 (CIP 105477), generated by long-read Pacific Biosciences single-molecule real-time (SMRT) sequencing. The availability of this genome sequence allows future work on understanding the zoonotic potential of this pathogen.

Interaction with the host: the role of fibronectin and extracellular matrix proteins in the adhesion of Gram-negative bacteria.

The capacity of pathogenic microorganisms to adhere to host cells and avoid clearance by the host immune system is the initial and most decisive step leading to infections. Bacteria have developed different strategies to attach to diverse host surface structures. One important strategy is the adhesion to extracellular matrix (ECM) proteins (e.g., collagen, fibronectin, laminin) that are highly abundant in connective tissue and basement membranes. Gram-negative bacteria express variable outer membrane proteins (adhesins) to attach to the host and to initiate the process of infection. Understanding the underlying molecular mechanisms of bacterial adhesion is a prerequisite for targeting this interaction by "anti-ligands" to prevent colonization or infection of the host. Future development of such "anti-ligands" (specifically interfering with bacteria-host matrix interactions) might result in the development of a new class of anti-infective drugs for the therapy of infections caused by multidrug-resistant Gram-negative bacteria. This review summarizes our current knowledge about the manifold interactions of adhesins expressed by Gram-negative bacteria with ECM proteins and the use of this information for the generation of novel therapeutic antivirulence strategies.

Immunogenicity of trimeric autotransporter adhesins and their potential as vaccine targets.

The current problem of increasing antibiotic resistance and the resurgence of numerous infections indicate the need for novel vaccination strategies more than ever. In vaccine development, the search for and the selection of adequate vaccine antigens is the first important step. In recent years, bacterial outer membrane proteins have become of major interest, as they are the main proteins interacting with the extracellular environment. Trimeric autotransporter adhesins (TAAs) are important virulence factors in many Gram-negative bacteria, are localised on the bacterial surface, and mediate the first adherence to host cells in the course of infection. One example is the Neisseria adhesin A (NadA), which is currently used as a subunit in a licensed vaccine against Neisseria meningitidis. Other TAAs that seem promising vaccine candidates are the Acinetobacter trimeric autotransporter (Ata), the Haemophilus influenzae adhesin (Hia), and TAAs of the genus Bartonella. Here, we review the suitability of various TAAs as vaccine candidates.