A comprehensive genetic map of cytokine responses in Lyme borreliosis.

The incidence of Lyme borreliosis has risen, accompanied by persistent symptoms. The innate immune system and related cytokines are crucial in the host response and symptom development. We characterized cytokine production capacity before and after antibiotic treatment in 1,060 Lyme borreliosis patients. We observed a negative correlation between antibody production and IL-10 responses, as well as increased IL-1Ra responses in patients with disseminated disease. Genome-wide mapping the cytokine production allowed us to identify 34 cytokine quantitative trait loci (cQTLs), with 31 novel ones. We pinpointed the causal variant at the TLR1-6-10 locus and validated the regulation of IL-1Ra responses at transcritpome level using an independent cohort. We found that cQTLs contribute to Lyme borreliosis susceptibility and are relevant to other immune-mediated diseases. Our findings improve the understanding of cytokine responses in Lyme borreliosis and provide a genetic map of immune function as an expanded resource.

Development of a sequence-based in silico OspA typing method for Borrelia burgdorferi sensu lato.

Lyme disease (LD), caused by spirochete bacteria of the genus Borrelia burgdorferi sensu lato, remains the most common vector-borne disease in the northern hemisphere. Borrelia outer surface protein A (OspA) is an integral surface protein expressed during the tick cycle, and a validated vaccine target. There are at least 20 recognized Borrelia genospecies, that vary in OspA serotype. This study presents a new in silico sequence-based method for OspA typing using next-generation sequence data. Using a compiled database of over 400 Borrelia genomes encompassing the 4 most common disease-causing genospecies, we characterized OspA diversity in a manner that can accommodate existing and new OspA types and then defined boundaries for classification and assignment of OspA types based on the sequence similarity. To accommodate potential novel OspA types, we have developed a new nomenclature: OspA in silico type (IST). Beyond the ISTs that corresponded to existing OspA serotypes 1-8, we identified nine additional ISTs that cover new OspA variants in B. bavariensis (IST9-10), B. garinii (IST11-12), and other Borrelia genospecies (IST13-17). The IST typing scheme and associated OspA variants are available as part of the PubMLST Borrelia spp. database. Compared to traditional OspA serotyping methods, this new computational pipeline provides a more comprehensive and broadly applicable approach for characterization of OspA type and Borrelia genospecies to support vaccine development.

Members of the paralogous gene family 12 from the Lyme disease agent Borrelia burgdorferi are non-specific DNA-binding proteins.

Lyme disease is the most prevalent vector-borne infectious disease in Europe and the USA. Borrelia burgdorferi, as the causative agent of Lyme disease, is transmitted to the mammalian host during the tick blood meal. To adapt to the different encountered environments, Borrelia has adjusted the expression pattern of various, mostly outer surface proteins. The function of most B. burgdorferi outer surface proteins remains unknown. We determined the crystal structure of a previously uncharacterized B. burgdorferi outer surface protein BBK01, known to belong to the paralogous gene family 12 (PFam12) as one of its five members. PFam12 members are shown to be upregulated as the tick starts its blood meal. Structural analysis of BBK01 revealed similarity to the coiled coil domain of structural maintenance of chromosomes (SMC) protein family members, while functional studies indicated that all PFam12 members are non-specific DNA-binding proteins. The residues involved in DNA binding were identified and probed by site-directed mutagenesis. The combination of SMC-like proteins being attached to the outer membrane and exposed to the environment or located in the periplasm, as observed in the case of PFam12 members, and displaying the ability to bind DNA, represents a unique feature previously not observed in bacteria.

Characterization and genomic analysis of the Lyme disease spirochete bacteriophage ϕBB-1.

Lyme disease is a tick-borne infection caused by the spirochete Borrelia (Borreliella) burgdorferi. Borrelia species have highly fragmented genomes composed of a linear chromosome and a constellation of linear and circular plasmids some of which are required throughout the enzootic cycle. Included in this plasmid repertoire by almost all Lyme disease spirochetes are the 32-kb circular plasmid cp32 prophages that are capable of lytic replication to produce infectious virions called ϕBB-1. While the B. burgdorferi genome contains evidence of horizontal transfer, the mechanisms of gene transfer between strains remain unclear. While we know that ϕBB-1 transduces cp32 and shuttle vector DNA during in vitro cultivation, the extent of ϕBB-1 DNA transfer is not clear. Herein, we use proteomics and long-read sequencing to further characterize ϕBB-1 virions. Our studies identified the cp32 pac region and revealed that ϕBB-1 packages linear cp32s via a headful mechanism with preferential packaging of plasmids containing the cp32 pac region. Additionally, we find ϕBB-1 packages fragments of the linear chromosome and full-length plasmids including lp54, cp26, and others. Furthermore, sequencing of ϕBB-1 packaged DNA allowed us to resolve the covalently closed hairpin telomeres for the linear B. burgdorferi chromosome and most linear plasmids in strain CA-11.2A. Collectively, our results shed light on the biology of the ubiquitous ϕBB-1 phage and further implicates ϕBB-1 in the generalized transduction of diverse genes and the maintenance of genetic diversity in Lyme disease spirochetes.

Italian peninsula as a hybridization zone of Ixodes inopinatus and I. ricinus and the prevalence of tick-borne pathogens in I. inopinatus, I. ricinus, and their hybrids.

BACKGROUND: Ixodes inopinatus was described from Spain on the basis of morphology and partial sequencing of 16S ribosomal DNA. However, several studies suggested that morphological differences between I. inopinatus and Ixodes ricinus are minimal and that 16S rDNA lacks the power to distinguish the two species. Furthermore, nuclear and mitochondrial markers indicated evidence of hybridization between I. inopinatus and I. ricinus. In this study, we tested our hypothesis on tick dispersal from North Africa to Southern Europe and determined the prevalence of selected tick-borne pathogens (TBPs) in I. inopinatus, I. ricinus, and their hybrids. METHODS: Ticks were collected in Italy and Algeria by flagging, identified by sequencing of partial TROSPA and COI genes, and screened for Borrelia burgdorferi s.l., B. miyamotoi, Rickettsia spp., and Anaplasma phagocytophilum by polymerase chain reaction and sequencing of specific markers. RESULTS: Out of the 380 ticks, in Italy, 92 were I. ricinus, 3 were I. inopinatus, and 136 were hybrids of the two species. All 149 ticks from Algeria were I. inopinatus. Overall, 60% of ticks were positive for at least one TBP. Borrelia burgdorferi s.l. was detected in 19.5% of ticks, and it was significantly more prevalent in Ixodes ticks from Algeria than in ticks from Italy. Prevalence of Rickettsia spotted fever group (SFG) was 51.1%, with significantly greater prevalence in ticks from Algeria than in ticks from Italy. Borrelia miyamotoi and A. phagocytophilum were detected in low prevalence (0.9% and 5.2%, respectively) and only in ticks from Italy. CONCLUSIONS: This study indicates that I. inopinatus is a dominant species in Algeria, while I. ricinus and hybrids were common in Italy. The higher prevalence of B. burgdorferi s.l. and Rickettsia SFG in I. inopinatus compared with that in I. ricinus might be due to geographical and ecological differences between these two tick species. The role of I. inopinatus in the epidemiology of TBPs needs further investigation in the Mediterranean Basin.

Genome-wide analyses in Lyme borreliosis: identification of a genetic variant associated with disease susceptibility and its immunological implications.

BACKGROUND: Genetic variation underly inter-individual variation in host immune responses to infectious diseases, and may affect susceptibility or the course of signs and symptoms. METHODS: We performed genome-wide association studies in a prospective cohort of 1138 patients with physician-confirmed Lyme borreliosis (LB), the most common tick-borne disease in the Northern hemisphere caused by the bacterium Borrelia burgdorferi sensu lato. Genome-wide variants in LB patients-divided into a discovery and validation cohort-were compared to two healthy cohorts. Additionally, ex vivo monocyte-derived cytokine responses of peripheral blood mononuclear cells to several stimuli including Borrelia burgdorferi were performed in both LB patient and healthy control samples, as were stimulation experiments using mechanistic/mammalian target of rapamycin (mTOR) inhibitors. In addition, for LB patients, anti-Borrelia antibody responses were measured. Finally, in a subset of LB patients, gene expression was analysed using RNA-sequencing data from the ex vivo stimulation experiments. RESULTS: We identified a previously unknown genetic variant, rs1061632, that was associated with enhanced LB susceptibility. This polymorphism was an eQTL for KCTD20 and ETV7 genes, and its major risk allele was associated with upregulation of the mTOR pathway and cytokine responses, and lower anti-Borrelia antibody production. In addition, we replicated the recently reported SCGB1D2 locus that was suggested to have a protective effect on B. burgdorferi infection, and associated this locus with higher Borrelia burgdorferi antibody indexes and lower IL-10 responses. CONCLUSIONS: Susceptibility for LB was associated with higher anti-inflammatory responses and reduced anti-Borrelia antibody production, which in turn may negatively impact bacterial clearance. These findings provide important insights into the immunogenetic susceptibility for LB and may guide future studies on development of preventive or therapeutic measures. TRIAL REGISTRATION: The LymeProspect study was registered with the International Clinical Trials Registry Platform (NTR4998, registration date 2015-02-13).

SCGB1D2 inhibits growth of Borrelia burgdorferi and affects susceptibility to Lyme disease.

Lyme disease is a tick-borne disease caused by bacteria of the genus Borrelia. The host factors that modulate susceptibility for Lyme disease have remained mostly unknown. Using epidemiological and genetic data from FinnGen and Estonian Biobank, we identify two previously known variants and an unknown common missense variant at the gene encoding for Secretoglobin family 1D member 2 (SCGB1D2) protein that increases the susceptibility for Lyme disease. Using live Borrelia burgdorferi (Bb) we find that recombinant reference SCGB1D2 protein inhibits the growth of Bb in vitro more efficiently than the recombinant protein with SCGB1D2 P53L deleterious missense variant. Finally, using an in vivo murine infection model we show that recombinant SCGB1D2 prevents infection by Borrelia in vivo. Together, these data suggest that SCGB1D2 is a host defense factor present in the skin, sweat, and other secretions which protects against Bb infection and opens an exciting therapeutic avenue for Lyme disease.

Borrelia burgdorferi PlzA is a cyclic-di-GMP dependent DNA and RNA binding protein.

The PilZ domain-containing protein, PlzA, is the only known cyclic di-GMP binding protein encoded by all Lyme disease spirochetes. PlzA has been implicated in the regulation of many borrelial processes, but the effector mechanism of PlzA was not previously known. Here, we report that PlzA can bind DNA and RNA and that nucleic acid binding requires c-di-GMP, with the affinity of PlzA for nucleic acids increasing as concentrations of c-di-GMP were increased. A mutant PlzA that is incapable of binding c-di-GMP did not bind to any tested nucleic acids. We also determined that PlzA interacts predominantly with the major groove of DNA and that sequence length and G-C content play a role in DNA binding affinity. PlzA is a dual-domain protein with a PilZ-like N-terminal domain linked to a canonical C-terminal PilZ domain. Dissection of the domains demonstrated that the separated N-terminal domain bound nucleic acids independently of c-di-GMP. The C-terminal domain, which includes the c-di-GMP binding motifs, did not bind nucleic acids under any tested conditions. Our data are supported by computational docking, which predicts that c-di-GMP binding at the C-terminal domain stabilizes the overall protein structure and facilitates PlzA-DNA interactions via residues in the N-terminal domain. Based on our data, we propose that levels of c-di-GMP during the various stages of the enzootic life cycle direct PlzA binding to regulatory targets.

Genetic manipulation of an Ixodes scapularis cell line.

Although genetic manipulation is one of the hallmarks of model organisms, its applicability to non-model species has remained difficult due to our limited understanding of their fundamental biology. For instance, manipulation of a cell line originated from the black-legged tick Ixodes scapularis, an arthropod that serves as a vector for several human pathogens, has yet to be established. Here, we demonstrate the successful genetic modification of the commonly used tick ISE6 line through ectopic expression and clustered regularly interspaced palindromic repeats [(CRISPR)/CRISPR-associated protein 9 (Cas9)] genome editing. We performed ectopic expression using nucleofection and attained CRISPR-Cas9 editing via homology-dependent recombination. Targeting the E3 ubiquitin ligase x-linked inhibitor of apoptosis (xiap) and its substrate p47 led to an alteration in molecular signaling within the immune deficiency network and increased infection of the rickettsial agent Anaplasma phagocytophilum in I. scapularis ISE6 cells. Collectively, our findings complement techniques for the genetic engineering of I. scapularis ticks, which currently limit efficient and scalable molecular genetic screens in vivo.IMPORTANCEGenetic engineering in arachnids has lagged compared to insects, largely because of substantial differences in their biology. This study unveils the implementation of ectopic expression and CRISPR-Cas9 gene editing in a tick cell line. We introduced fluorescently tagged proteins in ISE6 cells and edited its genome via homology-dependent recombination. We ablated the expression of xiap and p47, two signaling molecules present in the immune deficiency (IMD) pathway of Ixodes scapularis. Impairment of the tick IMD pathway, an analogous network of the tumor necrosis factor receptor in mammals, led to enhanced infection of the rickettsial agent Anaplasma phagocytophilum. Altogether, our findings provide a critical technical resource to the scientific community to enable a deeper understanding of biological circuits in the black-legged tick I. scapularis.

A Fur family protein BosR is a novel RNA-binding protein that controls rpoS RNA stability in the Lyme disease pathogen.

The σ54-σS sigma factor cascade plays a central role in regulating differential gene expression during the enzootic cycle of Borreliella burgdorferi, the Lyme disease pathogen. In this pathway, the primary transcription of rpoS (which encodes σS) is under the control of σ54 which is activated by a bacterial enhancer-binding protein (EBP), Rrp2. The σ54-dependent activation in B. burgdorferi has long been thought to be unique, requiring an additional factor, BosR, a homologue of classical Fur/PerR repressor/activator. However, how BosR is involved in this σ54-dependent activation remains unclear and perplexing. In this study, we demonstrate that BosR does not function as a regulator for rpoS transcriptional activation. Instead, it functions as a novel RNA-binding protein that governs the turnover rate of rpoS mRNA. We further show that BosR directly binds to the 5' untranslated region (UTR) of rpoS mRNA, and the binding region overlaps with a region required for rpoS mRNA degradation. Mutations within this 5'UTR region result in BosR-independent RpoS production. Collectively, these results uncover a novel role of Fur/PerR family regulators as RNA-binding proteins and redefine the paradigm of the σ54-σS pathway in B. burgdorferi.

Vector competence of Ixodes ricinus instars for the transmission of Borrelia burgdorferi sensu lato in different small mammalian hosts.

BACKGROUND: Many pathogens and parasites can infect multiple host species, and the competence of different hosts as pathogen reservoirs is key to understanding their epidemiology. Small mammals are important hosts for the instar stages of Ixodes ricinus ticks, the principal vector of Lyme disease in Europe. Small mammals also act as reservoirs of Borrelia afzelii, the most common genospecies of the Borrelia burgdorferi sensu lato (s.l.) spirochetes causing Lyme disease in Europe. However, we lack quantitative estimates on whether different small mammal species are equally suitable hosts for feeding I. ricinus and whether they show differences in pathogen transmission from host to tick. METHODS: Here, we analysed the feeding success and prevalence of B. burgdorferi s.l. infections in 12,987 instar I. ricinus found on captured small mammals with known infection status in Norway (2018-2022). RESULTS: We found that larvae were more likely to acquire a blood meal from common shrews (Sorex araneus, 46%) compared to bank voles (Myodes glareolus, 36%) and wood mice (Apodemus sylvaticus, 31%). Nymphs tended to be more likely to acquire a blood meal from wood mice (66%) compared to bank voles (54%). Common shrews harboured few nymphs (n=19). Furthermore, we found that larvae feeding on infected bank voles (11%) were more likely to be infected with B. burgdorferi s.l. than larvae on infected common shrews (7%) or wood mice (4%). CONCLUSIONS: Our study provides quantitative evidence of differences in suitability for the instar stages of I. ricinus across taxa of small mammals and highlights how even known small mammal host species can differ in their ability to feed ticks and infect larval ticks with the pathogen causing Lyme disease.

BadR directly represses the expression of the glycerol utilization operon in the Lyme disease pathogen.

Glycerol utilization as a carbohydrate source by Borreliella burgdorferi, the Lyme disease spirochete, is critical for its successful colonization and persistence in the tick vector. The expression of the glpFKD (glp) operon, which encodes proteins for glycerol uptake/utilization, must be tightly regulated during the enzootic cycle of B. burgdorferi. Previous studies have established that the second messenger cyclic di-GMP (c-di-GMP) is required for the activation of glp expression, while an alternative sigma factor RpoS acts as a negative regulator for glp expression. In the present study, we report identification of a cis element within the 5´ untranslated region of glp that exerts negative regulation of glp expression. Further genetic screen of known and predicted DNA-binding proteins encoded in the genome of B. burgdorferi uncovered that overexpressing Borrelia host adaptation regulator (BadR), a known global regulator, dramatically reduced glp expression. Similarly, the badR mutant significantly increased glp expression. Subsequent electrophoretic mobility shift assay analyses demonstrated that BadR directly binds to this cis element, thereby repressing glp independent of RpoS-mediated repression. The efficiency of BadR binding was further assessed in the presence of c-di-GMP and various carbohydrates. This finding highlights multi-layered positive and negative regulatory mechanisms employed by B. burgdorferi to synchronize glp expression throughout its enzootic cycle.IMPORTANCEBorreliella burgdorferi, the Lyme disease pathogen, must modulate its gene expression differentially to adapt successfully to its two disparate hosts. Previous studies have demonstrated that the glycerol uptake and utilization operon, glpFKD, plays a crucial role in spirochetal survival within ticks. However, the glpFKD expression must be repressed when B. burgdorferi transitions to the mammalian host. In this study, we identified a specific cis element responsible for the repression of glpFKD. We further pinpointed Borrelia host adaptation regulator as the direct binding protein to this cis element, thereby repressing glpFKD expression. This discovery paves the way for a deeper exploration of how zoonotic pathogens sense distinct hosts and switch their carbon source utilization during transmission.

Use of Specific Borrelia Phages as a New Strategy for Improved Diagnostic Tests.

The high failure rate of tick-borne infection (TBI)-related testing underscores the need for novel approaches that do not rely on serology and two-tier testing. Delayed diagnosis of TBIs, especially Borrelia infections, results in high healthcare costs and great suffering. There is a significant need for a reliable blood test that can aid in the diagnosis of Lyme disease, particularly when the current FDA-approved serological test is not sensitive enough to detect early Lyme patients who have not yet produced antibodies against Borrelia. Bacteriophages are viruses that specifically associate with their bacterial hosts, particularly prophages, bacteriophages residing in bacteria, and have proven to be tightly correlated with their bacterial hosts. They are poised to have wider applications as markers to detect bacteria, particularly in infectious disease. The gene of choice depends on the prevalence of phages within a particular group of bacteria. Phage genes that have been used as molecular markers to examine phage diversity include structural genes encoding the major capsid protein, the portal protein, the DNA polymerase, and the terminase. Borrelia species carry specific phage sequences that can be used as a proxy to identify the bacteria. Using phages as a proxy for bacteria is beneficial, as phages can be detected more easily than bacteria and can be used to bypass the cryptic and tissue-bound feature that typifies human Borrelia infections.We explored a completely new way of detecting Borrelia using Borrelia-specific bacteriophages as a diagnostic tool. Our detection method, patented by Phelix R&D and Leicester University (WO2018083491A1), could potentially transform infectious disease diagnostics through the innovative use of real-time PCR to target circulating bacteriophage DNA in blood from patients with Lyme disease. Firstly, this bacteriophage-based approach offers increased sensitivity since bacteriophages are typically present in five- to tenfold excess over bacterial cells, making it more accurate and sensitive than conventional bacteria-targeting PCR tests. One of the reasons bacteria-based PCR tests are frequently negative is due to the low bacterial concentration in the blood. Bacteriophage-based PCR surpasses this barrier and offers a direct test, as phages are part of bacteria's own genetic material, in contrast to all existing indirect tests (ELISA, Western BLOT, LTT/ELISPOT test). Secondly, a phage-based test can differentiate between different Lyme disease-causing and relapsing fever-causing Borrelia subtypes (B. burgdorferi s. l., B. miyamotoi, etc.), given that bacteriophages are indicators of bacterial identity. Finally, this test can detect Lyme disease in both early and late stages.

Establishing a Zebrafish Model for Borrelia burgdorferi Infection Using Immersion and Microinjection Methods.

Borrelia burgdorferi is the spirochetal bacterium that causes Lyme disease. Even though antimicrobial sensitivity of B. burgdorferi has been widely studied, there is still a need to develop an affordable, practical, high-throughput in vivo model which can be used to find effective antibiotic therapies, especially for the recently discovered persister and biofilm forms. Here, we describe the immersion and microinjection methods to introduce B. burgdorferi spirochetes into zebrafish larvae. The B. burgdorferi-zebrafish model can be produced by immersing 5-day post-fertilization (dpf) zebrafish in a B. burgdorferi culture, or by injecting B. burgdorferi into the hindbrain of zebrafish at 28 h post-fertilization (hpf). To demonstrate that B. burgdorferi indeed infect the fish, nested polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), live fluorescence imaging, histological staining, and wholemount immunohistochemical (IHC) methods can be used on B. burgdorferi-infected zebrafish.

A systemic approach to identify non-abundant immunogenic proteins in Lyme disease pathogens.

Borrelia burgdorferi, the pathogen of Lyme disease, differentially produces many outer surface proteins (Osp), some of which represent the most abundant membrane proteins, such as OspA, OspB, and OspC. In cultured bacteria, these proteins can account for a substantial fraction of the total cellular or membrane proteins, posing challenges to the identification and analysis of non-abundant proteins, which could serve as novel pathogen detection markers or as vaccine candidates. Herein, we introduced serial mutations to remove these abundant Osps and generated a B. burgdorferi mutant deficient in OspA, OspB, and OspC in an infectious 297-isolate background, designated as OspABC- mutant. Compared to parental isolate, the mutant did not reflect growth defects in the cultured medium but showed differential mRNA expression of representative tested genes, in addition to gross changes in cellular and membrane protein profiles. The analysis of differentially detectable protein contents of the OspABC- mutant, as compared to the wild type, by two-dimensional gel electrophoresis followed by liquid chromatography-mass spectrometry, identified several spirochete proteins that are dominated by proteins of unknown functions, as well as membrane transporters, chaperons, and metabolic enzymes. We produced recombinant forms of two of these represented proteins, BBA34 and BB0238, and showed that these proteins are detectable during spirochete infection in the tick-borne murine model of Lyme borreliosis and thus serve as potential antigenic markers of the infection.IMPORTANCEThe present manuscript employed a systemic approach to identify non-abundant proteins in cultured Borrelia burgdorferi that are otherwise masked or hidden due to the overwhelming presence of abundant Osps like OspA, OspB, and OspC. As these Osps are either absent or transiently expressed in mammals, we performed a proof-of-concept study in which their removal allowed the analysis of otherwise less abundant antigens in OspABC-deficient mutants and identified several immunogenic proteins, including BBA34 and BB0238. These antigens could serve as novel vaccine candidates and/or genetic markers of Lyme borreliosis, promoting new research in the clinical diagnosis and prevention of Lyme disease.

Genome-wide DNA Methylation Profiling in Lyme Neuroborreliosis Reveals Altered Methylation Patterns of HLA Genes.

Lyme neuroborreliosis (LNB) is a complex neuroinflammatory disorder caused by Borrelia burgdorferi, which is transmitted through tick bites. Epigenetic alterations, specifically DNA methylation (DNAm), could play a role in the host immune response during infection. In this study, we present the first genome-wide analysis of DNAm in peripheral blood mononuclear cells from patients with LNB and those without LNB. Using a network-based approach, we highlighted HLA genes at the core of these DNAm changes, which were found to be enriched in immune-related pathways. These findings shed light on the role of epigenetic modifications in the LNB pathogenesis that should be confirmed and further expanded upon in future studies.

Targeting Borrelia burgdorferi HtpG with a berserker molecule, a strategy for anti-microbial development.

Conventional antimicrobial discovery relies on targeting essential enzymes in pathogenic organisms, contributing to a paucity of new antibiotics to address resistant strains. Here, by targeting a non-essential enzyme, Borrelia burgdorferi HtpG, to deliver lethal payloads, we expand what can be considered druggable within any pathogen. We synthesized HS-291, an HtpG inhibitor tethered to the photoactive toxin verteporfin. Reactive oxygen species, generated by light, enables HS-291 to sterilize Borrelia cultures by causing oxidation of HtpG, and a discrete subset of proteins in proximity to the chaperone. This caused irreversible nucleoid collapse and membrane blebbing. Tethering verteporfin to the HtpG inhibitor was essential, since free verteporfin was not retained by Borrelia in contrast to HS-291. For this reason, we liken HS-291 to a berserker, wreaking havoc upon the pathogen's biology once selectively absorbed and activated. This strategy expands the druggable pathogenic genome and offsets antibiotic resistance by targeting non-essential proteins.

Structure of the Borrelia burgdorferi ATP-dependent metalloprotease FtsH in its functionally relevant hexameric form.

ATP-dependent proteases FtsH are conserved in bacteria, mitochondria, and chloroplasts, where they play an essential role in degradation of misfolded/unneeded membrane and cytosolic proteins. It has also been demonstrated that the FtsH homologous protein BB0789 is crucial for mouse and tick infectivity and in vitro growth of the Lyme disease-causing agent Borrelia burgdorferi. This is not surprising, considering B. burgdorferi complex life cycle, residing in both in mammals and ticks, which requires a wide range of membrane proteins and short-lived cytosolic regulatory proteins to invade and persist in the host organism. In the current study, we have solved the crystal structure of the cytosolic BB0789166-614, lacking both N-terminal transmembrane α-helices and the small periplasmic domain. The structure revealed the arrangement of the AAA+ ATPase and the zinc-dependent metalloprotease domains in a hexamer ring, which is essential for ATPase and proteolytic activity. The AAA+ domain was found in an ADP-bound state, while the protease domain showed coordination of a zinc ion by two histidine residues and one aspartic acid residue. The loop region that forms the central pore in the oligomer was poorly defined in the crystal structure and therefore predicted by AlphaFold to complement the missing structural details, providing a complete picture of the functionally relevant hexameric form of BB0789. We confirmed that BB0789 is functionally active, possessing both protease and ATPase activities, thus providing novel structural-functional insights into the protein, which is known to be absolutely necessary for B. burgdorferi to survive and cause Lyme disease.

Diversity and host specificity of Borrelia burgdorferi's outer surface protein C (ospC) alleles in synanthropic mammals, with a notable ospC allele U absence from mixed infections.

Interactions among pathogen genotypes that vary in host specificity may affect overall transmission dynamics in multi-host systems. Borrelia burgdorferi, a bacterium that causes Lyme disease, is typically transmitted among wildlife by Ixodes ticks. Despite the existence of many alleles of B. burgdorferi's sensu stricto outer surface protein C (ospC) gene, most human infections are caused by a small number of ospC alleles ["human infectious alleles" (HIAs)], suggesting variation in host specificity associated with ospC. To characterize the wildlife host association of B. burgdorferi's ospC alleles, we used metagenomics to sequence ospC alleles from 68 infected individuals belonging to eight mammalian species trapped at three sites in suburban New Brunswick, New Jersey (USA). We found that multiple allele ("mixed") infections were common. HIAs were most common in mice (Peromyscus spp.) and only one HIA was detected at a site where mice were rarely captured. ospC allele U was exclusively found in chipmunks (Tamias striatus), and although a significant number of different alleles were observed in chipmunks, including HIAs, allele U never co-occurred with other alleles in mixed infections. Our results suggest that allele U may be excluding other alleles, thereby reducing the capacity of chipmunks to act as reservoirs for HIAs.

Detection of Borrelia burgdorferi sensu lato species in host-seeking Ixodes species ticks in the United States.

Lyme disease is the most commonly reported vector-borne disease in the United States and is transmitted by Ixodes scapularis in the eastern US and I. pacificus in the west. The causative agents, Borrelia burgdorferi sensu stricto (Bbss) and B. mayonii belong to the B. burgdorferi sensu lato (Bbsl) species complex. An additional eight species of Bbsl have been identified in Ixodes species ticks in the US, but their geographic distribution, vector associations, human encounter rates and pathogenicity in humans are poorly defined. To better understand the geographic distribution and vector associations of Bbsl spirochetes in frequent and infrequent human-biting Ixodes species ticks in the US, we previously screened 29,517 host-seeking I. scapularis or I. pacificus ticks and 692 ticks belonging to eight other Ixodes species for Borrelia spirochetes using a previously described tick testing algorithm that utilizes a combination of real-time PCR and Sanger sequencing for Borrelia species identification. The assay was designed to detect known human pathogens spread by Ixodes species ticks, but it was not optimized to detect Bbsl co-infections. To determine if such co-infections were overlooked particularly in ticks infected with Bbss, we retested and analyzed a subsample of 845 Borrelia infected ticks using a next generation sequencing multiplex PCR amplicon sequencing (MPAS) assay that can identify Borrelia species and Bbsl co-infections. The assay also includes targets that can molecularly confirm identifications of Ixodes species ticks to better inform pathogen-vector associations. We show that Bbss is the most prevalent species in I. scapularis and I. pacificus; other Bbsl species were rarely detected in I. scapularis and the only Bbsl co-infections identified in I. scapularis were with Bbss and B. mayonii. We detected B. andersonii in I. dentatus in the Mid-Atlantic and Upper Midwest regions, B. kurtenbachii in I. scapularis in the Upper Midwest, B. bissettiae in I. pacificus and I. spinipalpis in the Northwest, and B. carolinensis in I. affinis in the Mid-Atlantic and Southeast, and B. lanei in I. spinipalpis in the Northwest. Twelve of 62 (19.4%) Borrelia-infected I. affinis from the Mid-Atlantic region were co-infected with Bbss and B. carolinensis. Our data support the notion that Bbsl species are maintained in largely independent enzootic cycles, with occasional spill-over resulting in multiple Bbsl species detected in Ixodes species ticks.