Bacterial Viruses as Drivers of Genetic and Antigenic Variation in Lyme Disease
Margie Kinnersley | The University of Montana
Lyme disease, a bacterial infection transmitted to humans by ticks of the genus Ixodes, accounts for nearly 80% of tickborne illness and is a significant source of morbidity across the US and Europe. The bacterium that causes the majority of Lyme disease cases in the US, Borrelia burgdorferi, expresses a variety of outer surface proteins (Osp) that assist in immune-system evasion. In order for B. burgdorferi to infect a host that has already developed an adaptive immune response against related strains, B. burgdorferi mutates Osp through a process called antigenic variation. One of the major drivers of Osp heterogeneity is horizontal gene transfer (HGT) between B. burgdorferi.
Several Osp are encoded by a bacteriophage (virus) called φBB-1 that chronically infects all Lyme-disease Borrelia. The φBB-1 genome is maintained in the cytoplasm of B. burgdorferi as a multicopy 32-kb circular plasmid called cp32. Cp32s encode conserved phage structural genes, but also encode Osp involved in immune system evasion such as Bdr, OspE/F and Mlp. These genes are up-regulated under conditions that simulate tick feeding and the passage of B. burgdorferi into a mammalian host and show evidence of sequence recombination as might be expected following HGT. φBB-1 can transfer DNA between Borrelia strains both in laboratory culture and in nature.
TEM imaging of B. burgdorferi supernatants often reveals more than one phage particle type, raising the possibility that phages other than φBB-1 can contribute to horizontal gene transfer in Borrelia. Although φBB-1 is currently the only characterized phage, other B. burgdorferi plasmids (e.g. circular plasmids cp9/cp18 and linear plasmids lp54, lp56 and lp28-2) harbor phage-like sequences.
The primary goal of this proposal is to study the population genomics of phage and phage-like particles from B. burgdorferi in order to build a framework in which to study their ecology and role as mediators of gene exchange in Lyme disease spirochetes. The feasibility of this project is high: φBB-1 can facilitate transfer of selectable markers and small foreign plasmids in laboratory culture when phage production is chemically induced with ethanol. I routinely grow B. burgdorferi in the lab and have adapted a tangential flow filtration (TFF) method to concentrate/purify φBB-1 for ongoing studies using phage as novel antigens for vaccine development.
I hypothesize that φBB-1 facilitates horizontal gene transfer and drives antigenic variation in Borrelia. To test this hypothesis, I propose the following aims:
- Characterize phage virion diversity in B. burgdorferi. I will test the hypothesis that B. burgdorferi are infected by multiple phage types beyond φBB-1 that are derived from distinct genetic elements. Phages will be induced, purified and analyzed using a combination of TFF, SDS-PAGE and mass spectrometry. Completion of this aim will answer outstanding questions regarding the potential for phages other than φBB-1 to participate in HGT and the Lyme disease lifecycle.
- Define the role of B. burgdorferi phages in HGT and antigenic variation. I will test the hypothesis that phages facilitate HGT between and promote antigenic variation in different B. burgdorferi strains. To address this hypothesis, I will sequence phage and plasmids from B. burgdorferi cultures grown in isolation and from B. burgdorferi cultures that have been exposed to concentrated, purified phage from a different strain (lateral transfer). From sequence data I will be able to identify horizontally acquired plasmids, recombined regions of DNA and non-cp32 DNA that is packaged into phage capsids. I predict that Osp genes will show high sequence variation while the genes that encode phage capsid proteins will remain unchanged.
If successful, this work will provide new insight into the role that phages play in HGT and antigenic variation in Lyme-disease Borrelia, and could lead to new strategies to limit transmission and decrease disease severity in people. This work also has the potential to produce new genetic tools or enhance exiting tools to manipulate B. burgdorferi. Because other spirochetes like relapsing fever Borrelia (e.g. B. hermsii) have φBB-1 homologs, these studies will also shed light on the role of phages in the pathogenesis of other tick-borne diseases.