Don\'t Panic. In the nearly 50 years since the discovery of Lyme disease, Borrelia burgdorferi has emerged as an unlikely workhorse of microbiology. Interest in studying host-pathogen interactions fueled significant progress in making the fastidious microbe approachable in laboratory settings, including the development of culture methods, animal models, and genetic tools. By developing these systems, insight has been gained into how the microbe is able to survive its enzootic cycle and cause human disease. Here, we discuss the discovery of B. burgdorferi and its development as a model organism before diving into the critical lessons we have learned about B. burgdorferi biology at pivotal stages of its lifecycle: gene expression changes during the tick blood meal, colonization of a new vertebrate host, and developing a long-lasting infection in that vertebrate until a new tick feeds. Our goal is to highlight the advancements that have facilitated B. burgdorferi research and identify gaps in our current understanding of the microbe.

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.

Lyme disease, the leading vector-borne disease in the United States and Europe, develops after infection with Borrelia burgdorferi sensu lato bacteria. Transmission of the spirochete from the tick vector to a vertebrate host requires global changes in gene expression that are controlled, in part, by the Rrp2/RpoN/RpoS alternative sigma factor cascade. Transcriptional studies defining the B. burgdorferi RpoS regulon have suggested that RpoS activates the transcription of paralogous family 52 (PFam52) genes. In strain B31, PFam52 genes (bbi42, bbk53, and bbq03) encode a set of conserved hypothetical proteins with >89% amino acid identity that are predicted to be surface-localized. Extensive homology among members of paralogous families complicates studies of protein contributions to pathogenicity as the potential for functional redundancy will obfuscate findings. Using a sequential mutagenesis approach, we generated clones expressing a single PFam52 paralog, as well as a strain deficient in all three. The single paralog expressing strains were used to confirm BBI42, BBK53, and BBQ03 surface localization and RpoS regulation. Surprisingly, the PFam52-deficient strain was able to infect mice and complete the enzootic cycle similar to the wild-type parental strain. Indeed, the presence of numerous pseudogenes that contain frameshifts or internal stop codons among the PFam52 genes suggests that they may be subjected to gene loss in B. burgdorferi\'s reduced genome. Alternatively, the lack of phenotype might reflect the limitations of the experimental mouse infection model.

This study aimed to detect, isolate and to characterize by molecular methods a relapsing fever group (RFG) Borrelia in white-eared opossums (Didelphis albiventris) from Brazil. During 2015-2018, when opossums (Didelphis spp.) were captured in six municipalities of the state of São Paulo, Brazil, molecular analyses revealed the presence of a novel RFG Borrelia sp. in the blood of seven opossums (Didelphis albiventris), out of 142 sampled opossums (4.9% infection rate). All seven infected opossums were from a single location (Ribeirão Preto municipality). In a subsequent field study in Ribeirão Preto during 2021, two new opossums (D. albiventris) were captured, of which one contained borrelial DNA in its blood. Macerated tissues from this infected opossum were inoculated into laboratory animals (rodents and rabbits) and two big-eared opossums (Didelphis aurita), which had blood samples examined daily via dark-field microscopy. No spirochetes were visualized in the blood of the laboratory animals. Contrastingly, spirochetes were visualized in the blood of the two D. aurita opossums between 12 and 25 days after inoculation. Blood samples from these opossums were used for a multi-locus sequencing typing (MLST) based on six borrelial loci. Phylogenies inferred from MLST genes positioned the sequenced Borrelia genotype into the RFG borreliae clade basally to borreliae of the Asian-African group, forming a monophyletic group with another Brazilian isolate, \"Candidatus B. caatinga\". Based on this concatenated phylogenetic analysis, which supports that the new borrelial isolate corresponds to a putative new species, we propose the name \"Candidatus Borrelia mimona\".

Emerging and re-emerging pathogens often stem from zoonotic origins, cycling between humans and animals, and are frequently vectored and maintained by hematophagous arthropod vectors. The efficiency by which these disease agents are successfully transmitted between vertebrate hosts is influenced by many factors, including the host on which a vector feeds. The Lyme disease bacterium Borrelia burgdorferi sensu lato has adapted to survive in complex host environments, vectored by Ixodes ticks, and maintained in multiple vertebrate hosts. The versatility of Lyme borreliae in disparate host milieus is a compelling platform to investigate mechanisms dictating pathogen transmission through complex networks of vertebrates and ticks. Squamata, one of the most diverse clade of extant reptiles, is comprised primarily of lizards, many of which are readily fed upon by Ixodes ticks. Yet, lizards are one of the least studied taxa at risk of contributing to the transmission and life cycle maintenance of Lyme borreliae. In this review, we summarize the current evidence, spanning from field surveillance to laboratory infection studies, supporting their contributions to Lyme borreliae circulation. We also summarize the current understanding of divergent lizard immune responses that may explain the underlying molecular mechanisms to confer Lyme spirochete survival in vertebrate hosts. This review offers a critical perspective on potential enzootic cycles existing between lizard-tick-Borrelia interactions and highlights the importance of an eco-immunology lens for zoonotic pathogen transmission studies.

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.

UNASSIGNED: Relapsing fever (RF) remains a neglected human disease that is caused by a number of diverse pathogenic Borrelia (B.) species. Characterized by high cell densities in human blood, relapsing fever spirochetes have developed plentiful strategies to avoid recognition by the host defense mechanisms. In this scenario, spirochetal lipoproteins exhibiting multifunctional binding properties in the interaction with host-derived molecules are known to play a key role in adhesion, fibrinolysis and complement activation.
UNASSIGNED: Binding of CihC/FbpC orthologs to different human proteins and conversion of protein-bound plasminogen to proteolytic active plasmin were examined by ELISA. To analyze the inhibitory capacity of CihC/FbpC orthologs on complement activation, a microtiter-based approach was performed. Finally, AlphaFold predictions were utilized to identified the complement-interacting residues.
UNASSIGNED: Here, we elucidate the binding properties of CihC/FbpC-orthologs from distinct RF spirochetes including B. parkeri, B. hermsii, B. turicatae, and B. recurrentis to human fibronectin, plasminogen, and complement component C1r. All CihC/FbpC-orthologs displayed similar binding properties to fibronectin, plasminogen, and C1r, respectively. Functional studies revealed a dose dependent binding of plasminogen to all borrelial proteins and conversion to active plasmin. The proteolytic activity of plasmin was almost completely abrogated by tranexamic acid, indicating that lysine residues are involved in the interaction with this serine protease. In addition, a strong inactivation capacity toward the classical pathway could be demonstrated for the wild-type CihC/FbpC-orthologs as well as for the C-terminal CihC fragment of B. recurrentis. Pre-incubation of human serum with borrelial molecules except CihC/FbpC variants lacking the C-terminal region protected serum-susceptible Borrelia cells from complement-mediated lysis. Utilizing AlphaFold2 predictions and existing crystal structures, we mapped the putative key residues involved in C1r binding on the CihC/FbpC orthologs attempting to explain the relatively small differences in C1r binding affinity despite the substitutions of key residues. Collectively, our data advance the understanding of the multiple binding properties of structural and functional highly similar molecules of relapsing fever spirochetes proposed to be involved in pathogenesis and virulence.

Borrelia theileri is a tick-borne spirochete causative agent of fever, apathy and reduced food consumption in cattle. Molecular diagnosis has expanded the understanding of Borrelia theileri with new hosts and geographical locations being described. The present study aimed to describe the first molecular detection of B. theileri in wild tapirs (Tapirus terrestris) from South America. Blood DNA samples obtained from 99 tapirs sampled in Pantanal (n = 61) and Cerrado (n = 38) biomes were screened using a qPCR assay based on the 16 S rRNA gene of Borrelia sp. Positive samples in the qPCR assay were subjected to PCR assays to allow characterization of fragments from 16 S rRNA and flaB genes. Two (2/99; 2.0%) animals from Pantanal biome were positive in the qPCR and one sample presented bands of expected size for the flaB protocol. Amplicons from this sample were successfully cloned and sequenced. In the phylogenetic analysis, Borrelia sp. from T. terrestris grouped together with B. theileri sequences previously detected in Rhipicephalus microplus ticks and cattle from Minas Gerais State in Brazil, Rhipicephalus geigyi from Mali, and R. microplus and Haemaphysalis sulcata from Pakistan. This finding contributes to our knowledge regarding susceptible hosts species for B. theileri. More studies are necessary to understand the potential effects of B. theileri on tapir\'s health.

UNASSIGNED: Borrelia miyamotoi is a spirochete species transmitted via hard ticks. Following its discovery in Japan, this pathogen has been detected around the world, and is increasingly confirmed as a human pathogen causing febrile disease, namely relapsing fever. Its presence has been confirmed in the Northeast China. However, there is little information regarding the presence of B. miyamotoi and other hard-tick-borne relapsing fever spirochetes in southern China including Yunnan province, where tick and animal species are abundant and many people both inhabit and visit for recreation.
UNASSIGNED: For the present study, we collected samples of ticks, wildlife, and domestic animal hosts from different counties in Yunnan province. Nucleic acids from samples were extracted, and the presence of B. miyamotoi and other relapsing fever spirochetes was confirmed using polymerase chain reaction (PCR) for the 16S rRNA specific target gene fragment. The positive samples were then amplified for partial genome of the flaB and glpQ genes. Statistical differences in its distribution were analyzed by SPSS 20 software. Sequence of partial 16S rRNA, flaB and glpQ genome were analyzed and phylogenetic trees were constructed.
UNASSIGNED: A total of 8260 samples including 2304 ticks, 4120 small mammals and 1836 blood of domestic animal hosts were collected for screening for infection of B. miyamotoi and other relapsing fever spirochetes. Cattle and sheep act as the main hosts and Rhipicephalus microplus, Haemaphysalis nepalensis, H. kolonini and Ixodes ovatus were identified as the important vector host with high prevalence or wide distribution. Only one Mus caroli (mouse) and one Sorex alpinus (shrew) were confirmed positive for relapsing fever spirochetes. Evidence of vertical transmission in ticks was also confirmed. Two known strains of B. miyamotoi and one novel relapsing fever spirochetes, B. theileri-like agent, were confirmed and described with their host adaptation, mutation, and potential risk of spreading and spillover for human beings.
UNASSIGNED: Our results provide new evidence of relapsing fever spirochetes in vector and animal hosts in Yunnan province based on large sample sizes, and offer guidance on further investigation, surveillance and monitoring of this pathogen.

Pathogenic spirochetes cause a range of serious human diseases such as Lyme disease (LD), syphilis, leptospirosis, relapsing fever (RF), and periodontal disease. Motility is a critical virulence factor for spirochetes. From the mechanical perspective of the infection, it has been widely believed that flagella are the sole key players governing the migration and dissemination of these pathogens in the host. Here, we highlight the important contribution of spirochetal surface-exposed adhesive molecules and their dynamic interactions with host molecules in the process of infection, specifically in spirochetal swimming and crawling migration. We believe that these recent findings overturn the prevailing view depicting the spirochetal body to be just an inert elastic bag, which does not affect spirochetal cell locomotion.