Analysis of the Borreliaceae Pangenome Reveals a Distinct Genomic Architecture Conserved Across Phylogenetic Scales

Abstract

The family Borreliaceae contains arthropod-borne spirochetes that cause two widespread human diseases, Lyme disease and relapsing fever. Lyme disease is a subacute, progressive illness with variable stage and tissue manifestations. Relapsing fever is an acute febrile illness with prominent bacteremia that may recur and disseminate, particularly to the nervous system. Clinical heterogeneity is a hallmark of both diseases. While human clinical manifestations are influenced by a wide variety of factors, including immune status and host genetic susceptibility, there is evidence that Borreliaceae microbial factors influence the clinical manifestations of human disease caused by this family of spirochetes. Despite these associations, the spirochete genes that influence the severity and manifestations of human disease are, for the most part, unknown. Recent work has identified lineage-specific expansions of lipoproteome-rich accessory genome elements in virulent clones of Borrelia burgdorferi. Using publicly available genome assemblies, it is shown that all Borreliaceae lineages for which sufficient sequence data are available harbor a similar pattern of strongly structured, lineage-specific expansions in their accessory genomes, particularly among lipoproteins, and that this pattern holds across phylogenetic scales including genera, species, and genotypes. The relationships among pangenome elements suggest that infrequent episodes of marked genomic change followed by clonal expansion in geographically and enzootically structured populations may account for the unique lineage structure of Borreliaceae. This analysis informs future genotype-phenotype studies among Borreliaceae and lays a foundation for studies of individual gene function guided by phylogenetic patterns of conservation, diversification, gain, and/or loss.

Keywords: Borreliaceae; Genomics; Lyme disease; Relapsing fever; Spirochetes.

Figure 1.

Core genome phylogenetic relationships among Borreliaceae. Radial tree of Borreliaceae with tips annotated by species. Nodes with bootstrap support >90% are colored with a dot, demonstrating robust phylogenetic support for the genus- and species-specific divisions on the tree. The clade of Lyme disease (LD)–causing spirochetes, Borreliella/Borrelia burgdorferi sensu lato, has been highlighted in red; relapsing fever (RF)–causing spirochetes and the reptile-associated group are shaded in blue and orange, respectively. Old World and New World subgroupings of RF spirochetes are shown with a dotted line. Within the LD spirochetes, the clades associated with distinct manifestations are shown. The scale bar denotes nucleotide substitutions per site. Abbreviations: ACA, acrodermatitis chronicum atrophicans; LA, Lyme arthritis; LD, Lyme disease; LNB, Lyme neuroborreliosis; REP, reptile-associated group; RF, relapsing fever.

Figure 2.

Accessory genome content among Borreliaceae is strongly structured by lineage. Patterns of open reading frame presence are shown according to a phylogenetic tree produced from core genome sequences. A matrix showing the presence or absence of pangenome homology groups in each strain is shown at right. Pangenome homology groups that are present are shaded in solid color, and groups that are absent are uncolored. Each row corresponds to an individual strain, and each column corresponds to individual homology groups. The rows are ordered according to a phylogenetic tree, with tips annotated by the Borreliaceae genus and species. Relevant taxa are also labeled. As in Figure 1, the Borreliaceae that cause Lyme disease (Borreliella/Borrelia burgdorferi sensu lato) are shaded and labeled in red; the relapsing fever and reptile-associated groups are shaded and labeled in blue and orange, respectively. The columns are clustered with hierarchical clustering to reflect similarity in the patterns of gene presence among lineages. The tree scale bar denotes nucleotide substitutions per site. Abbreviations: Bbsl, Borrelia burgdorferi sensu lato; LD, Lyme disease; ORF, open reading frame; REP, reptile-associated group; RF, relapsing fever.

Figure 3.

The Borreliaceae lipoproteome is strongly structured by lineage. Patterns of open reading frames annotated as putative lipoproteins, as classified by SpLip [49], are shown according to a phylogenetic tree produced from core genome sequences. A matrix showing the presence or absence of probable lipoproteins in each strain is shown. Each row corresponds to an individual strain and each column corresponds to a homology group classified as a probable lipoprotein. The rows are ordered according to a phylogenetic tree, with tips annotated by the Borreliaceae genus and species. Relevant taxa are also labeled. As in Figure 1, the Borreliaceae that cause Lyme disease (Borreliella/Borrelia burgdorferi sensu lato) are shaded and labeled in red; the relapsing fever and reptile-associated groups are shaded and labeled in blue and orange, respectively. The columns are clustered with hierarchical clustering to reflect similarity in the patterns of gene presence among lineages. The tree scale bar denotes nucleotide substitutions per site. Abbreviations: Bbsl, Borrelia burgdorferi sensu lato; LD, Lyme disease; ORF, open reading frame; REP, reptile-associated group; RF, relapsing fever.

Figure 4.

Borreliaceae lipoproteins are preferentially encoded on plasmids. The genomic location of pangenome elements is shown. Counts are stratified by whether they are classified by SpLip [49] as probable lipoproteins, possible lipoproteins, or not lipoproteins. Test of association by Fisher exact test, P < 2.2 × 10⁻¹⁶.

Figure 5.

Borrelia pangenomes contain an expanded number of open reading frames (ORFs) and putative lipoproteins. A, The number of ORF homology groups for each genome assessed, grouped by genus. B, The number of ORF homology groups for each genome assessed, grouped by species. C, The number of ORF homology groups annotated as probable lipoproteins for each genome assessed, grouped by genus. D, The number of ORF groups annotated as probable lipoproteins for each genome assessed, grouped by species. P values report the results of a Wilcoxon rank-sum test with a 2-sided alternative. Abbreviations: **P < .01; ****P < .0001.