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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.
Many snake venoms are known for their antithrombotic activity. They contain components that specifically target different platelet-activating receptors such as the collagen-binding integrin α2β1 and the von Willebrand factor receptor GPIb. In a search for an α2β1 integrin-blocking component from the venom of the habu snake (Trimeresurus flavoviridis), we employed two independent purification protocols. First, we used the integrin α2A domain, a major collagen-binding domain, as bait for affinity purification of an α2β1 integrin-binding toxin from the crude venom. Second, in parallel, we used classical protein separation protocols and tested for α2β1 integrin-inhibiting capabilities by ELISA. Using both approaches, we identified flavocetin-A as an inhibitor of α2β1 integrin. Hitherto, flavocetin-A has been reported as a GPIb inhibitor. However, flavocetin-A inhibited collagen-induced platelet aggregation even after GPIb was blocked with other inhibitors. Moreover, flavocetin-A antagonized α2β1 integrin-mediated adhesion and migration of HT1080 human fibrosarcoma cells, which lack any GPIb, on collagen. Protein chemical analyses proved that flavocetin-A binds to α2β1 integrin and its α2A domain with high affinity and in a cooperative manner, which most likely is due to its quaternary structure. Kinetic measurements confirmed the formation of a strong complex between integrin and flavocetin-A, which dissociates very slowly. This study proves that flavocetin-A, which has long been known as a GPIb inhibitor, efficiently targets α2β1 integrin and thus blocks collagen-induced platelet activation. Moreover, our findings suggest that the separation of GPIb- and α2β1 integrin-blocking members within the C-type lectin-related protein family is less strict than previously assumed.