Introduction: Bacterial infections pose a significant global health threat. The initial step involves bacterial adhesion to host cell extracellular matrix (ECM) proteins such as fibronectin (Fn). Trimeric autotransporter adhesins (TAAs) are important pathogenicity factors of Gram-negative bacteria. Bartonella adhesin A (BadA), a prototypic TAA from B. henselae, is exceptionally long (monomer: 3,974 aa) and extensively characterized, making it a prime example of a TAA. B. henselae, the causative agent of cat scratch disease and bacillary angiomatosis, adheres to endothelial cells and ECM proteins via BadA interaction. This study aimed to develop novel anti-virulence components to inhibit TAA and Fn interaction.

Material & Methods: BadA domains with reported Fn affinity were expressed and purified. Single domain antibodies (sybodies) binding to the purified BadA domains, were selected using a combination of ribosome and phage display technologies. Affinity and specificity of the candidate sybodies were evaluated using ELISA assays and qPCR. Binding inhibition with sybodies was evaluated by ELISA, qPCR and immunofluorescence microscopy.

Results: Specific BadA domains with reported Fn affinity were purified. Sybodies targeting a major Fn binding domain were selected and purified. Functional evaluation of the sybodies demonstrated their ability to bind the specific BadA domain and hinder BadA and Fn interaction, providing evidence of their anti-ligand potential.

Conclusion: Targeting TAA-function is a promising strategy for fighting Gram-negative infections. Sybodies seem to represent promising "antiligand" tools awaiting future assessments in various in vitro and ex vivo models. The herein described Bartonella spp. experiments serve as proof-of-concept, paving the way for new therapeutic concepts ("antiligands") also targeting highly drug-resistant TAA-expressing A. baumannii and other TAA-expressing bacteria in the future.