Strong interactions between Salp15 homologues from the tick I. ricinus and distinct types of the outer surface OspC protein from Borrelia.

Ticks belonging to the genus Ixodes are parasites feeding on vertebrate blood and vectors for many pathogenic microbes, including Borrelia burgdorferi sensu lato spirochetes, the causative agent of Lyme borreliosis. The tick saliva contains a mixture of bioactive molecules showing a wide range of properties for efficient engorgement. One of the most extensively studied components of tick saliva is a 15-kDa salivary gland protein (Salp15) from Ixodes scapularis. This multifunctional protein suppresses the immune response of hosts through pleiotropic action on a few crucial defense pathways. Salp15 and its homologue from I. ricinus Iric1 have been also shown to bind to Borrelia burgdorferi sensu stricto outer surface protein C (OspC) permitting the spirochetes to evade antibody-mediated killing in the human host. Further studies revealed that Salp15 and Iric1 protected B. burgdorferi s. s. and B. garinii expressing OspC against the complement system. OspC is the most variable protein on the outer surface of Borrelia, which in addition to Salp15 can also bind other ligands, such as plasminogen, fibrinogen, fibronectin or complement factor 4. So far several OspC variants produced by B. burgdorferi s. l. spirochetes were shown to be capable of binding Salp15 or its homologue, but the protection against borreliacidal antibodies has only been proven in the case of B. burgdorferi s. s. The question of Salp15 contribution to Borrelia survival during the infection has been comprehensively studied during the last decades. In contrast, the organization of the OspC-Salp15 complex has been poorly explored. This report describes the binding between three Salp15 homologues from the tick Ixodes ricinus (Iric1, Iric2 and Iric3) and OspC from four B. burgdorferi sensu lato strains in terms of the binding parameters, analyzed with two independent biophysical methods - Microscale thermophoresis (MST) and Biolayer interferometry (BLI). The results of both experiments show a binding constant at the nanomolar level, which indicates very strong interactions. While the Iric1-OspC binding has been reported before, we show in this study that also Iric2 and Iric3 are capable of OspC binding with high affinity. This observation suggests that these two Salp15 homologues might be used by B. burgdorferi s. l. in a way analogous to Iric1. A comparison of the results from the two methods let us propose that N-terminal immobilization of OspC significantly increases the affinity between the two proteins. Finally, our results indicate that the Iric binding site is located in close proximity of the OspC epitopes recognized by human antibodies, which may have important biological and medical implications.

Borrelia outer surface protein C is capable of human fibrinogen binding.

Outer surface protein C (OspC) is one of the most abundant surface lipoproteins produced during early infection by the Borrelia spirochete, the causative agent of Lyme disease. The high sequence variability of the ospC gene results in the production of several and strongly divergent OspC types. One of the known roles of OspC is the recruitment of blood components, including complement regulators, to facilitate the bloodstream survival of Borrelia at an essential stage of host infection. Here, we identify and describe a new interaction between OspC and human fibrinogen. To test the ability of OspC to bind fibrinogen, we developed a microscale thermophoresis assay using four fluorescently labeled types of OspC. We show that OspC binds fibrinogen tightly, with nanomolar Kd , and that the binding depends on the OspC type. The binding assays combined with SAXS studies allowed us to map the OspC-binding site on the fibrinogen molecule. Spectrometric measurements of fibrinogen clotting in the presence of OspC indicate that OspC negatively influences the clot formation process. Taken together, our findings are consistent with the hypothesis that OspC interacts with blood protein partners to facilitate Borrelia spreading by the hematogenous route.

BMV Propagation, Extraction and Purification Using Chromatographic Methods.

Brome mosaic virus (BMV) is a well-known plant virus representing single-stranded RNA (ssRNA) positive-sense viruses. It has been widely used as a model in multiple studies concerning plant virus biology, epidemiology and the application of viral capsids in nanotechnology. Herein, we describe a method for BMV purification based on ion-exchange and size-exclusion chromatography. The presented method is of similar efficiency to previously described protocols relying on differential centrifugation and can easily be scaled up. The resulting BMV capsids are stable and monodisperse and can be used for further applications.

Biophysical analysis of BMV virions purified using a novel method.

Brome mosaic virus (BMV) has been successfully loaded with different types of nanoparticles. However, studies concerning its application as a nanoparticle carrier demand high-purity virions in large amounts. Existing BMV purification protocols rely on multiple differential ultracentrifugation runs of the initially purified viral preparation. Herein, we describe an alternative method for BMV purification based on ion-exchange chromatography and size-exclusion chromatography (SEC) yielding 0.2mg of virus from 1g of plant tissue. Our method is of similar efficiency to previously described protocols and can easily be scaled up. The method results in high-quality BMV preparations as confirmed by biophysical analyses, including cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), static light scattering (SLS), and circular dichroism (CD) measurements and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy. Our results revealed that purified BMV capsids are stable and monodisperse and can be used for further downstream applications. In this work, we also characterize secondary structure and size fluctuations of the BMV virion at different pH values.