Whole genome sequencing of human Borrelia burgdorferi isolates reveals linked blocks of accessory genome elements located on plasmids and associated with human dissemination

Abstract

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 B. burgdorferi (Bb) isolates derived from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bb isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bb isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ~900 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination in humans and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, have increased rates of dissemination in humans. OspC type A strains possess a unique set of strongly linked genetic elements including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. These features of OspC type A strains reflect a broader paradigm across Bb isolates, in which near-clonal genotypes are defined by strain-specific clusters of linked genetic elements, particularly those encoding surface-exposed lipoproteins. These clusters of genes are maintained by strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection. Author summary Different genotypes of B. burgdorferi have been associated with different rates of dissemination, but the genetic basis of these differences is not known. We report the genomes of 299 B. burgdorferi isolates from patients with Lyme disease. We find that whole genome sequence (WGS) type A isolates are a genetically divergent group of isolates characterized by an enlarged pan-genome, an expanded surface lipoproteome encoded on a unique set of plasmids, including lp28-1 and lp56, and increased rates of dissemination. Using genome-wide association methods applied to the B. burgdorferi pan-genome, we identify loci associated with dissemination. The near-clonal nature of B. burgdorferi populations means that relationships of individual loci to dissemination are relatively weak after adjusting for the lineage structure among the isolates, implying that experimental studies and larger cohorts are needed to identify the causal alleles within a lineage mediating these effects. Across the isolates studied, an increasing number of surface-expressed lipoproteins was associated with an increased probability of dissemination in humans. The results underscore how strain-specific genetic variation—particularly among surface lipoproteins located on plasmids—is linked to the phenotype of human dissemination. More broadly, this approach provides a foundation for future studies linking spirochete genotype to the diverse clinical phenotypes of Lyme disease in humans.