Borreliella burgdorferi factor H-binding proteins are not required for serum resistance and infection in mammals.

The causative agent of Lyme disease (LD), Borreliella burgdorferi, binds factor H (FH) and other complement regulatory proteins to its surface. B. burgdorferi B31 (type strain) encodes five FH-binding proteins (FHBPs): CspZ, CspA, and the OspE paralogs OspEBBN38, OspEBBL39, and OspEBBP38. This study assessed potential correlations between the production of individual FHBPs, FH-binding ability, and serum resistance using a panel of infectious B. burgdorferi clonal populations recovered from dogs. FHBP production was assessed in cultivated spirochetes and by antibody responses in naturally infected humans, dogs, and eastern coyotes (wild canids). FH binding specificity and sensitivity to dog and human serum were also assessed and compared. No correlation was observed between the production of individual FHBPs and FH binding with serum resistance, and CspA was determined to not be produced in animals. Notably, one or more clones isolated from dogs lacked CspZ or the OspE proteins (a finding confirmed by genome sequence determination) and did not bind FH derived from canines. The data presented do not support a correlation between FH binding and the production of individual FHBPs with serum resistance and infectivity. In addition, the limited number and polymorphic nature of cp32s in B. burgdorferi clone DRI85A that were identified through genome sequencing suggest no strict requirement for a defined set of these replicons for infectivity. This study reveals that the immune evasion mechanisms employed by B. burgdorferi are diverse, complex, and yet to be fully defined.

Simulations of cross-amyloid aggregation of amyloid-ß and islet amyloid polypeptide fragments.

Amyloid-ß (Aß) and islet amyloid polypeptide (IAPP) are small peptides, classified as amyloids, that have the potential to self-assemble and form cytotoxic species, such as small soluble oligomers and large insoluble fibrils. The formation of Aß aggregates facilitates the progression of Alzheimer's disease (AD), while IAPP aggregates induce pancreatic ß-cell apoptosis, leading to exacerbation of type 2 diabetes (T2D). Cross-amyloid interactions between Aß and IAPP have been described both in vivo and in vitro, implying the role of Aß or IAPP as modulators of cytotoxic self-aggregation of each species, and suggesting that Aß-IAPP interactions are a potential molecular link between AD and T2D. Using molecular dynamics (MD) simulations, "hotspot" regions of the two peptides were studied to understand the formation of hexamers in a heterogeneous and homogeneous peptide-containing environment. Systems of only Aß(16-22) peptides formed antiparallel, ß-barrel-like structures, while systems of only IAPP(20-29) peptides formed stacked, parallel ß-sheets and had relatively unstable aggregation structures after 2 µs of simulation time. Systems containing both Aß and IAPP (1:1 ratio) hexamers showed antiparallel, ß-barrel-like structures, with an interdigitated arrangement of Aß(16-22) and IAPP(20-29). These ß-barrel structures have features of cytotoxic amyloid species identified in previous literature. Ultimately, this work seeks to provide atomistic insight into both the mechanism behind cross-amyloid interactions and structural morphologies of these toxic amyloid species.

FtlA and FtlB Are Candidates for Inclusion in a Next-Generation Multiantigen Subunit Vaccine for Lyme Disease.

Lyme disease (LD) is a tick-transmitted bacterial infection caused by Borreliella burgdorferi and other closely related species collectively referred to as the LD spirochetes. The LD spirochetes encode an uncharacterized family of proteins originally designated protein family twelve (PF12). In B. burgdorferi strain B31, PF12 consists of four plasmid-carried genes, encoding BBK01, BBG01, BBH37, and BBJ08. Henceforth, we designate the PF12 proteins family twelve lipoprotein (Ftl) A (FtlA) (BBK01), FtlB (BBG01), FtlC (BBH37), and FtlD (BBJ08). The goal of this study was to assess the potential utility of the Ftl proteins in subunit vaccine development. Immunoblot analyses of LD spirochete cell lysates demonstrated that one or more of the Ftl proteins are produced by most LD isolates during cultivation. The Ftl proteins were verified to be membrane associated, and nondenaturing PAGE revealed that FtlA, FtlB, and FtlD formed dimers, while FtlC formed hexamers. Analysis of serum samples from B. burgdorferi antibody (Ab)-positive client-owned dogs (n = 50) and horses (n = 90) revealed that a majority were anti-Ftl Ab positive. Abs to the Ftl proteins were detected in serum samples from laboratory-infected dogs out to 497 days postinfection. Anti-FtlA and FtlB antisera displayed potent complement-dependent Ab-mediated killing activity, and epitope localization revealed that the bactericidal epitopes reside within the N-terminal domain of the Ftl proteins. This study suggests that FtlA and FtlB are potential candidates for inclusion in a multivalent vaccine for LD.

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

Background: The Borreliaceae family includes many obligate parasitic bacterial species which are etiologically associated with a myriad of zoonotic borrelioses including Lyme disease and vector-borne relapsing fevers. Infections by the Borreliaceae are difficult to detect by both direct and indirect methods, often leading to delayed and missed diagnoses. Efforts to improve diagnoses center around the development of molecular diagnostics (MDx), but due to deep tissue sequestration of the causative spirochaetes and the lack of persistent bacteremias, even MDx assays suffer from a lack of sensitivity. Additionally, the highly extensive genomic heterogeneity among isolates, even within the same species, contributes to the lack of assay sensitivity as single target assays 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 lifecycle in which these parasites have to survive in multiple hosts each with unique immune responses. Results: We constructed a Borreliaceae family-level pangenome and characterized the phylogenetic relationships among the constituent taxa which supports the recent taxonomy of splitting the family into at least two genera. Gene content pro les were created for the majority of the Borreliaceae replicons, providing for the first time their unambiguous molecular typing. Conclusion: 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 pangenome will enable precision diagnostics corresponding to gene content-driven clinical outcomes while also providing targets for interventions.

Serological survey for maternal antibodies to Borreliella burgdorferi and Anaplasma phagocytophilum in dogs from endemic and non-endemic regions of the United States.

In North America, the tick-borne pathogens Borreliella burgdorferi (Lyme disease; LD) and Anaplasma phagocytophilum (anaplasmosis) are a significant health threat to dogs. Little is known regarding the seroprevalence of maternal antibodies (Abs) to these pathogens in young dogs. The analysis of maternal antibody (Ab) profiles is important as it could bear on the interpretation of currently available diagnostic assays and the potential for vaccine interference in pups. In this pilot study, sera from 32 client-owned dogs (6-24 weeks of age; 3 serum samples per dog) from four veterinary hospitals in the United States were screened for IgG against B. burgdorferi and A. phagocytophilum using whole cell lysate immunoblots and recombinant protein-based ELISAs. As a control, the sera were also screened for Abs to canine parvovirus and canine distemper virus using a commercially available colorimetric assay. Maternally derived Abs against B. burgdorferi including the diagnostic antigen VlsE were detected in 2 of the 32 dogs, accounting for 12.5 % of dogs from LD endemic regions, and as expected, the Ab levels declined over time. Differentiating between maternal Ab and infection-induced Ab is of importance in interpreting serological tests for tick-borne diseases in young dogs and in making decisions regarding treatment and timing of vaccination.

Large-Scale Sequencing of Borreliaceae for the Construction of Pan-Genomic-Based Diagnostics.

The acceleration of climate change has been associated with an alarming increase in the prevalence and geographic range of tick-borne diseases (TBD), many of which have severe and long-lasting effects-particularly when treatment is delayed principally due to inadequate diagnostics and lack of physician suspicion. Moreover, there is a paucity of treatment options for many TBDs that are complicated by diagnostic limitations for correctly identifying the offending pathogens. This review will focus on the biology, disease pathology, and detection methodologies used for the Borreliaceae family which includes the Lyme disease agent Borreliella burgdorferi. Previous work revealed that Borreliaceae genomes differ from most bacteria in that they are composed of large numbers of replicons, both linear and circular, with the main chromosome being the linear with telomeric-like termini. While these findings are novel, additional gene-specific analyses of each class of these multiple replicons are needed to better understand their respective roles in metabolism and pathogenesis of these enigmatic spirochetes. Historically, such studies were challenging due to a dearth of both analytic tools and a sufficient number of high-fidelity genomes among the various taxa within this family as a whole to provide for discriminative and functional genomic studies. Recent advances in long-read whole-genome sequencing, comparative genomics, and machine-learning have provided the tools to better understand the fundamental biology and phylogeny of these genomically-complex pathogens while also providing the data for the development of improved diagnostics and therapeutics.