Lyme disease is the most common vector-borne disease in the United States. It is caused by the bacteria of the Borrelia species, Spirochaetaceae family. Bacteria are generally transmitted to humans through the bite of infected blacklegged ticks. Lyme disease may be easily overlooked, as symptoms like headache, fatigue, and muscle aches can be easily confused with other diagnoses while rushes only appear in approximately 70% of cases. Failing to identify the disease early results in the delayed treatment and therefore loss of medication effectiveness and the possibility of the development of long-term complications. Currently, there is no Lyme disease vaccine authorized in the United States. For these reasons, it is crucial to focus on the development of a Lyme disease vaccine that will prevent infection.
In this research, we evaluated the possibility of expressing two commonly conserved proteins of Borrelia burgdorferi on the surface of outer membrane vesicles (OMVs) using CytolysinA (ClyA). Outer membrane vesicles (OMVs) are naturally shed by gram-negative bacteria. These vesicles are abundant in immunostimulatory proteins while lacking the ability to replicate, which indicates their significantly higher safety profile compared to other types of vaccines. RevA is a highly conserved protein among Borrelia burgdorferi responsible for binding host fibronectin, a component of an extracellular matrix. Outer surface protein C (OspC) is a major surface lipoprotein in Borrelia burgdorferi essential for establishing infection by bacteria in mammals. These proteins are surface-exposed in most bacteria via an N-terminal signaling sequence associated with a localization of lipoproteins (LOL) pathway. ClyA - OspC and ClyA - RevA fusions with highly conserved N-terminus signaling sequence were successfully expressed in BL21 (DE3), but a cleavage of fusion proteins was observed. The identified cleavage was attributed to variations of the LOL signaling sequence located between the ClyA and the investigated antigens. Additionally, when we attempted to express the fusion proteins on the OMVs, the levels of the ClyA - OspC and ClyA - RevA were undetectable via Western blotting. Future studies will focus on recombinant proteins lacking the signaling sequence, which will prevent the cleavage between ClyA and the antigen of interest and enable proper protein expression.