Utilizing Two Borrelia bavariensis Isolates Naturally Lacking the PFam54 Gene Array To Elucidate the Roles of PFam54-Encoded Proteins.

Lyme borreliosis is the most common vector-borne disease in the Northern Hemisphere, caused by spirochetes belonging to the Borrelia burgdorferi sensu lato species complex, which are transmitted by ixodid ticks. B. burgdorferi sensu lato species produce a family of proteins on the linear plasmid 54 (PFam54), some of which confer the functions of cell adhesion and inactivation of complement, the first line of host defense. However, the impact of PFam54 in promoting B. burgdorferi sensu lato pathogenesis remains unclear because of the hurdles to simultaneously knock out all PFam54 proteins in a spirochete. Here, we describe two Borrelia bavariensis strains, PBN and PNi, isolated from patients naturally lacking PFam54 but maintaining the rest of the genome with greater than 95% identity to the reference B. bavariensis strain, PBi. We found that PBN and PNi less efficiently survive in human serum than PBi. Such defects were restored by introducing two B. bavariensis PFam54 recombinant proteins, BGA66 and BGA71, confirming the role of these proteins in providing complement evasion of B. bavariensis. Further, we found that all three strains remain detectable in various murine tissues 21 days post-subcutaneous infection, supporting the nonessential role of B. bavariensis PFam54 in promoting spirochete persistence. This study identified and utilized isolates deficient in PFam54 to associate the defects with the absence of these proteins, building the foundation to further study the role of each PFam54 protein in contributing to B. burgdorferi sensu lato pathogenesis. IMPORTANCE To establish infections, Lyme borreliae utilize various means to overcome the host's immune system. Proteins encoded by the PFam54 gene array play a role in spirochete survival in vitro and in vivo. Moreover, this gene array has been described in all currently available Lyme borreliae genomes. By investigating the first two Borrelia bavariensis isolates naturally lacking the entire PFam54 gene array, we showed that both patient isolates display an increased susceptibility to human serum, which can be rescued in the presence of two PFam54 recombinant proteins. However, both isolates remain infectious to mice after intradermal inoculation, suggesting the nonessential role of PFam54 during the long-term, but may differ slightly in the colonization of specific tissues. Furthermore, these isolates show high genomic similarity to type strain PBi (>95%) and could be used in future studies investigating the role of each PFam54 protein in Lyme borreliosis pathogenesis.

Unprecedented genetic variability of PFam54 paralogs among Eurasian Lyme borreliosis-causing spirochetes.

Lyme borreliosis (LB) is the most common vector-borne disease in the Northern Hemisphere caused by spirochetes belonging to the Borrelia burgdorferi sensu lato (Bbsl) complex. Borrelia spirochetes circulate in obligatory transmission cycles between tick vectors and different vertebrate hosts. To successfully complete this complex transmission cycle, Bbsl encodes for an arsenal of proteins including the PFam54 protein family with known, or proposed, influences to reservoir host and/or vector adaptation. Even so, only fragmentary information is available regarding the naturally occurring level of variation in the PFam54 gene array especially in relation to Eurasian-distributed species. Utilizing whole genome data from isolates (n = 141) originated from three major LB-causing Borrelia species across Eurasia (B. afzelii, B. bavariensis, and B. garinii), we aimed to characterize the diversity of the PFam54 gene array in these isolates to facilitate understanding the evolution of PFam54 paralogs on an intra- and interspecies level. We found an extraordinarily high level of variation in the PFam54 gene array with 39 PFam54 paralogs belonging to 23 orthologous groups including five novel paralogs. Even so, the gene array appears to have remained fairly stable over the evolutionary history of the studied Borrelia species. Interestingly, genes outside Clade IV, which contains genes encoding for proteins associated with Borrelia pathogenesis, more frequently displayed signatures of diversifying selection between clades that differ in hypothesized vector or host species. This could suggest that non-Clade IV paralogs play a more important role in host and/or vector adaptation than previously expected, which would require future lab-based studies to validate.