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Abstract

Introduction: Bartonella henselae is a facultative intracellular zoonotic pathogen and is the causative agent of cat scratch disease and other clinical entities such as endocarditis and bacillary angiomatosis. Bacterial adhesion is crucial in the early stages of pathogenic infections. Accordingly, B. henselae principally targets endothelial cells in which the Bartonella adhesin A (BadA) is essential for adherence to host cells and extracellular matrix proteins such as fibronectin (Fn). Furthermore, BadA is a trimeric autotransporter adhesin (TAA), is modularly structured including a head domain, a long and repetitive neck/stalk region, and a conserved C-terminal anchor domain, and is responsible for angiogenic reprograming of host infected cells. The identification of BadA-Fn binding regions would support the development of new "anti-adhesive" components that inhibit the initial adherence.
Methods: The badA genomic island of eight B. henselae strains was analysed using long-read PacBio SMRT sequencing. Various truncated BadA (fusion) proteins were constructed in a B. henselae Marseille BadA-transposon mutant (∆BadA-T) via Gibson Assembly® technology. Surface expression of BadA proteins was assessed via confocal laser scanning microscopy using rabbit anti- BadA IgG antibodies and transmission electron microscopy. Identification of potential Fn binding site(s) was performed via ELISA and fluorescence microscopy. Immunogenicity of few truncated BadA proteins was assessed via Western blotting using human sera of 13 patients diagnosed with for example cat scratch disease.
Results: The badA gene (8.7 to 13.2 kb long) and flanking badA pseudogenes show a high intraspecies variability, occasionally including a peculiar 18 nt-long repeat sequence motif. B. henselae Marseille contains a badA gene of 11,922 bp and consist of 30 neck/stalk domains displaying a repetitive superimposed domain sequence pattern. Furthermore, six additional truncated BadA mutants were each expressed on the outer membrane of B. henselae Marseille ∆BadA-T. Truncated BadA constructs retain their immunodominant characteristic. The BadA neck/stalk domain composition influences Fn binding capacity with a focus on residue motifs present in domains 19 and 27.
Discussion: The variable and repetitive genomic badA island indicates frequent reshuffling of homologous domain regions, facilitated via active recombination mechanisms, potentially contributing to differences in virulence and host immune evasion. Presumably, BadA adheres to Fn in a cumulative effort with quick saturation, primarily via species-conserved unpaired β-sheet hairpins of particular domains repeatedly present throughout the neck/stalk region. The localisation of the main BadA-Fn binding regions provides a basis towards new "anti-adhesive" components in preventing initial adherence of B. henselae and other TAA-expressing pathogens in the course of infection.