Question

Relapsing fever (RF) remains a neglected human disease caused by different Borrelia species. Characterized by high cell densities in human blood, relapsing fever Borrelia have developed strategies to avoid recognition by the host defense mechanisms. CihC orthologs, in particular, are known to interact with host-derived proteins involved in adhesion, fibrinolysis and complement activation.

Here, we elucidate the binding properties of CihC orthologs from distinct RF spirochetes including B. parkeri, B. hermsii, B. turicatae, and B. recurrentis to fibronectin, plasminogen, and complement C1r.

Methods

Binding of CihC orthologs to human proteins was measured by ELISA. A microtiter-based approach was also performed to analyze the inhibitory capacity of borrelial proteins on complement. AlphaFold predictions were used to identified the complement-interacting residues in four CihC orthologs. Functional analyses was conducted to explore conversion of protein-bound plasminogen to proteolytic active plasmin.

Results

All CihC orthologs displayed similar binding properties to fibronectin, plasminogen, and C1r, respectively. Regarding complement inactivation, CihC orthologs and the C-terminal CihC fragment exhibited a strong inactivation capacity toward the classical complement pathway. Moreover, pre-incubation of human serum with these molecules protected serum-susceptible Borrelia cells from complement-mediated lysis. Utilizing Alphafold predictions, we mapped the putative key residues involved in C1r binding on the CihC orthologs attempting to explain the relatively small differences in C1r binding affinity. In addition, functional studies revealed a dose dependent binding of plasminogen to all borrelial proteins selected and conversion to active plasmin. The proteolytic activity of plasmin was almost completely abrogated by the lysine analog tranexamic acid, indicating that lysine residues are involved in the interaction.

Conclusions

Collectively, our data advance the understanding of the multiple binding properties of structural and functional highly similar molecules of diverse RF spirochetes proposed to be involved in pathogenesis and virulence.