Characterization of the family-level Borreliaceae pan-genome and development of an episomal typing protocol

Abstract

The Borreliaceae family includes many obligate parasitic bacterial species etiologically associated with a myriad of zoonotic borrelioses, including Lyme disease and vector-borne relapsing fevers. Borreliaceae infections are difficult to detect by both direct and indirect methods, often leading to delayed and missed diagnoses. Efforts to improve diagnostics center around the development of molecular diagnostics (MDx), but due to deep tissue sequestration and the lack of persistent bacteremias, even MDx assays suffer from a lack of sensitivity. Additionally, the extensive genomic heterogeneity among isolates, even within the same species, contributes to the lack of assay sensitivity, as single target assays, whether nucleic acid-based or serologically based, cannot provide universal coverage. This within-species heterogeneity is partly due to differences in replicon repertoires and genomic structures that have likely arisen to support the complex Borreliaceae life cycle necessary for these parasites to survive in multiple hosts, each with unique immune responses. We constructed a Borreliaceae family-level pan-genome and characterized the phylogenetic relationships among the constituent taxa, which supports the recent, although contested, taxonomy of splitting the family into at least two genera. Gene content profiles were created for the majority of the Borreliaceae replicons, providing for the first time their unambiguous molecular typing. Our characterization of the Borreliaceae pan-genome supports the splitting of the former Borrelia genus into two genera and provides for the phylogenetic placement of several non-species designated isolates. Mining this family-level pan-genome will enable the development of precision diagnostics corresponding to gene content-driven clinical outcomes while also providing targets for interventions.

Importance: Using whole genome sequencing, we demonstrated that the bacteria that are transmitted by ticks and other arthropod vectors that cause Lyme disease and relapsing fevers, while related, do not belong within the same genus classification. In addition, through characterization of their highly atypical genomic structure, we were able to develop a genetic typing system that will help with future studies of how they cause disease while also providing targets for medical interventions.

Keywords: Borrelia; Borreliaceae; Borreliella; comparative genomics; distributed genome hypothesis; pan-genome; phylogenetics; spirochete; supragenome; taxonomy.

Fig 1

In silico comparison of the genomes of two B. hermsii HS1 strains shows homology throughout several replicons and indicates regions of heterogeneity. (A) Alignments of a pair of B. hermsii HS1 strains show nearly identical genomes. Similar pairwise locally collinear blocks are indicated in the same colors (pink to pink, blue to blue, etc.). Contig breaks are indicated by red lines. (B) D-Genies dot plot of whole-genome complete reference B. hermsii strain HS1 (query, x-axis) to our sequenced B. hermsii strain HS1d (target, y-axis). Overall percent similarity was indicated from red (low = 0) to green (high = 100%). All contigs of the two whole genome sequences are portrayed in the figure. Each contig pair is separated by a dotted line. The position of the matches between strains is indicated by colored dots and lines (yellow, orange, and green). Green lines perpendicular to the diagonal are the result of the linear episomal hairpin ends.

Fig 2

Self-alignment of B. hermsii strain HS1d contigs reveals inverted repeats at linear contig ends. A faceted pafr dot plot of each contig within the B. hermsii strain HS1d whole-genome sequence. The overall percent similarity was indicated from light blue (low = 0) to dark blue (high = 100%). All contigs were represented within each individual facet of the plot. Within each plot, x- and y-axes are the overall lengths of the compared contigs.

Fig 3

Blast search of B. hermsii strain HS1 linear contigs reveals ResT sites located at the ends of replicons. A graphic depiction of Pacific Biosciences long-read sequencing platform and modified prepared Borreliaceae template (replicon image modified from Pacific Biosciences 2022 using Procreate). Alongside the sequencing schematic is one of the faceted plots of B. hermsii strain HS1d (contig 3). This plot shows the percent similarity of the replicon along its entire length with regions of similarity removed. Perpendicular lines are inverted repeats. The overall percent similarity was indicated from light blue (low = 0) to dark blue (high = 100%). All contigs were represented within each individual facet of the plot.

Fig 4

Average nucleotide identity (ANI) of all Borreliaceae species and strains examined demonstrated genera-specific clustering. The range of ANI is from least (yellow) to greatest (blue). Each species is represented on both the x- and y-axes. The diagonal line represents identity. The dendrograms were made with complete-linkage hierarchical clustering. The key color is noted on the right-hand side. The annotations indicate the generic groupings.

Fig 5

Average nucleotide identity (ANI) analyses of Borreliella genomes show a high degree of similarity amongst the major species of the genus. The range of ANI is from least (yellow) to greatest (blue). Each of the Borreliella spirochetes is represented on both the x- and y-axes. The diagonal line represents identities. The dendrograms were made with complete-linkage hierarchical clustering. The color bar key is noted on the right-hand side. The annotations indicate the species group.

Fig 6

Average nucleotide identity (ANI) of Borrelia genomes shows separation of the major species. The range of ANI is from least (yellow) to greatest (blue). Each Borrelia spirochete was represented on both the x- and y-axes. The diagonal line represents identities. The dendrograms were made with complete-linkage hierarchical clustering. The color bar key is noted on the right-hand side. The annotations indicate the species group.

Fig 7

Average nucleotide identity (ANI) of Borreliaceae genomes from monotreme/reptile-associated isolates (which included isolates from ticks recovered from reptiles) suggests their placement within the family tree. The range of ANI is from least (yellow) to greatest (blue). Each isolate is represented on both the x- and y-axes. The diagonal line represents identities. The dendrograms were made with complete-linkage hierarchical clustering. The color bar key is noted on the right-hand side. The annotations indicate the species group

Fig 8

Midpoint-rooted single-copy core gene tree of the Borreliaceae shows the division between the Borreliella and Borrelia genera. The origin of the spirochete is indicated by the color of the nodes: blue (Virginia Commonwealth University); red (NCBI). The annotation bar indicates the species.

Fig 9

The gene cluster frequency of the Borreliaceae family pan-genome reveals a large pan-genome. Each colored circle signifies different components of the pan-genome [core (blue), soft-core (orange), shell (green), and cloud (red)]. The x-axis signifies the BlastP percent homology threshold. The y-axis signifies the number of genes within the pan-genome.

Fig 10

Borreliaceae gene presence/absence heatmap. Genes are colored blue if present and white if absent. Homologs were defined as having Roary BlastP of ≥75%. The y-axis represents gene clusters within the Borreliaceae pan-genome. The x-axis depicts all Borreliaceae isolates included in the study. The red annotations indicate the gene groups. The Borreliaceae pan-genome has a relatively small core, with each of the two genera-defined core genomes being more than twice as large as the family core genome. Only the most prevalent of the distributed genes (not present in either of the three core genomes) are included in the heatmap.

Fig 11

Large-scale average nucleotide identity (ANI) of Borreliaceae replicons reveals definitive groupings across the family. The range of ANI is from least (yellow) to greatest (blue). Each of the Borreliaceae replicons is represented on the x- and y-axes based on complete-linkage hierarchical clustering. The annotations indicate the replicon group.