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).

Borrelia burgdorferi Is a Poor Inducer of Gamma Interferon: Amplification Induced by Interleukin-12.

Laboratory diagnosis of Lyme borreliosis (LB) is mainly based on serology, which has limitations, particularly in the early stages of the disease. In recent years there have been conflicting reports concerning a new diagnostic tool using the cytokine interferon-gamma (IFN-γ). Previous studies have generally found low concentrations of IFN-γ in early LB infection. The goal of this study is to investigate IFN-γ regulation during early LB and provide insights into the host response to B. burgdorferi. We performed in vitro experiments with whole blood assays and peripheral blood mononuclear cells (PBMCs) of LB patients and healthy volunteers exposed to B. burgdorferi and evaluated the IFN-γ response using ELISA and related interindividual variation in IFN-γ production to the presence of single nucleotide polymorphisms. IFN-γ production of B. burgdorferi-exposed PBMCs and whole blood was amplified by the addition of interleukin-12 (IL-12) to the stimulation system. This effect was observed after 24 h of B. burgdorferi stimulation in both healthy individuals and LB patients. The effect was highly variable between individuals, but was significantly higher in LB patients 6 weeks since the start of antibiotic treatment compared to healthy individuals. IL-12 p40 and IL-18 mRNA were upregulated upon exposure to B. burgdorferi, whereas IL-12 p35 and IFN-γ mRNA expression remained relatively unchanged. SNP Rs280520 in the downstream IL-12 pathway, Tyrosine Kinase 2, was associated with increased IFN-γ production. This study shows that IL-12 evokes an IFN-γ response in B. burgdorferi exposed cells, and that LB patients and healthy controls respond differently to this stimulation.

Identifying platelet-derived factors as amplifiers of B. burgdorferi-induced cytokine production.

Previous studies have shown that monocytes can be 'trained' or tolerized by certain stimuli to respond stronger or weaker to a secondary stimulation. Rewiring of glucose metabolism was found to be important in inducing this phenotype. As we previously found that Borrelia burgdorferi (B. burgdorferi), the causative agent of Lyme borreliosis (LB), alters glucose metabolism in monocytes, we hypothesized that this may also induce long-term changes in innate immune responses. We found that exposure to B. burgdorferi decreased cytokine production in response to the TLR4-ligand lipopolysaccharide (LPS). In addition, B. burgdorferi exposure decreased baseline levels of glycolysis, as assessed by lactate production. Using GWAS analysis, we identified a gene, microfibril-associated protein 3-like (MFAP3L) as a factor influencing lactate production after B. burgdorferi exposure. Validation experiments proved that MFAP3L affects lactate- and cytokine production following B. burgdorferi stimulation. This is mediated by functions of MFAP3L, which includes activating ERK2 and through activation of platelet degranulation. Moreover, we showed that platelets and platelet-derived factors play important roles in B. burgdorferi-induced cytokine production. Certain platelet-derived factors, such chemokine C-X-C motif ligand 7 (CXCL7) and (C-C motif) ligand 5 (CCL5), were elevated in the circulation of LB patients in comparison to healthy individuals.

A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis.

Lyme disease, the most common vector-borne illness in North America, is caused by the spirochete Borrelia burgdorferi. Infection begins in the skin following a tick bite and can spread to the hearts, joints, nervous system, and other organs. Diverse host responses influence the level of B. burgdorferi infection in mice and humans. Using a systems biology approach, we examined potential molecular interactions between human extracellular and secreted proteins and B. burgdorferi. A yeast display library expressing 1031 human extracellular proteins was probed against 36 isolates of B. burgdorferi sensu lato. We found that human Peptidoglycan Recognition Protein 1 (PGLYRP1) interacted with the vast majority of B. burgdorferi isolates. In subsequent experiments, we demonstrated that recombinant PGLYRP1 interacts with purified B. burgdorferi peptidoglycan and exhibits borreliacidal activity, suggesting that vertebrate hosts may use PGLYRP1 to identify B. burgdorferi. We examined B. burgdorferi infection in mice lacking PGLYRP1 and observed an increased spirochete burden in the heart and joints, along with splenomegaly. Mice lacking PGLYRP1 also showed signs of immune dysregulation, including lower serum IgG levels and higher levels of IFNγ, CXCL9, and CXCL10.Taken together, our findings suggest that PGLYRP1 plays a role in the host's response to B. burgdorferi and further demonstrate the utility of expansive yeast display screening in capturing biologically relevant interactions between spirochetes and their hosts.

Borrelia miyamotoi Activates Human Dendritic Cells and Elicits T Cell Responses.

The spirochete Borrelia miyamotoi has recently been shown to cause relapsing fever. Like the Lyme disease agent, Borrelia burgdorferi, B. miyamotoi is transmitted through the bite of infected ticks; however, little is known about the response of the immune system upon infection. Dendritic cells (DCs) play a central role in the early immune response against B. burgdorferi We investigated the response of DCs to two different strains of B. miyamotoi using in vitro and ex vivo models and compared this to the response elicited by B. burgdorferi. Our findings show that B. miyamotoi is phagocytosed by monocyte-derived DCs, causing upregulation of activation markers and production of proinflammatory cytokines in a similar manner to B. burgdorferi. Recognition of B. miyamotoi was demonstrated to be partially mediated by TLR2. DCs migrated out of human skin explants upon inoculation of the skin with B. miyamotoi. Finally, we showed that B. miyamotoi-stimulated DCs induced proliferation of naive CD4+ and CD8+ T cells to a larger extent than B. burgdorferi. In conclusion, we show in this study that DCs respond to and mount an immune response against B. miyamotoi that is similar to the response to B. burgdorferi and is able to induce T cell proliferation.

An intervention in general practice to improve the management of Lyme borreliosis in Denmark.

BACKGROUND: Our objectives were to improve the following outcomes in patients with Lyme borreliosis (LB) through an educational intervention in general practice: (i) increase the number of hospital referrals on suspicion of LB, (ii) increase the number of cerebrospinal fluid (CSF) tests examined for Borrelia burgdorferi antibody index, (iii) decrease the number of serum-B. burgdorferi antibody tests ordered, (iv) shorten delay from symptom onset to hospital in Lyme neuroborreliosis (LNB) patients, (v) increase LB knowledge among general practitioners. METHODS: A prospective non-blinded non-randomized intervention trial on the island of Funen, Denmark. The intervention included oral and written education about LB and was carried out in areas with an LNB incidence ≥4.7/100.000 between 22 January 2019 and 7 May 2019. Results were compared between the intervention group (49 general practices) and the remaining general practices in Funen (71 practices) 2 years before and after the intervention. RESULTS: In the study period, 196 patients were referred on suspicion of LB, a 28.9% increase in the intervention group post-intervention, 59.5% increase in the control group (P = 0.47). The number of CSF-Borrelia-antibody index tests increased 20.8% in the intervention group, 18.0% in the control group (P = 0.68), while ordered serum-B. burgdorferi antibody tests declined 43.1% in the intervention group, 34.5% in the control group (P = 0.30). 25.1% had the presence of serum-B. burgdorferi antibodies. We found no difference in LNB pre-hospital delay before and after intervention or between groups (P = 0.21). The intervention group performed significantly better on a follow-up questionnaire (P = 0.02). CONCLUSION: We found an overall improvement in LB awareness and referrals among general practitioners but could not show any effect of the intervention on clinical outcomes of LNB.

Pathogenesis of Relapsing Fever.

Relapsing fever (RF) is caused by several species of Borrelia; all, except two species, are transmitted to humans by soft (argasid) ticks. The species B. recurrentis is transmitted from one human to another by the body louse, while B. miyamotoi is vectored by hard-bodied ixodid tick species. RF Borrelia have several pathogenic features that facilitate invasion and dissemination in the infected host. In this article we discuss the dynamics of vector acquisition and subsequent transmission of RF Borrelia to their vertebrate hosts. We also review taxonomic challenges for RF Borrelia as new species have been isolated throughout the globe. Moreover, aspects of pathogenesis including symptomology, neurotropism, erythrocyte and platelet adhesion are discussed. We expound on RF Borrelia evasion strategies for innate and adaptive immunity, focusing on the most fundamental pathogenetic attributes, multiphasic antigenic variation. Lastly, we review new and emerging species of RF Borrelia and discuss future directions for this global disease.

Diagnostic Performance of the Novel BioPlex Lyme Serological Assays in European Patients with Lyme Disease.

Serodiagnosis of Lyme borreliosis (LB) comes with several drawbacks, among which is limited sensitivity in early disease. This study assesses the sensitivity and specificity of the novel BioPlex 2200 Lyme IgG and Lyme IgM assays. It also assesses potential improvements to the assays through receiver-operating characteristic (ROC) analysis. The BioPlex assays were performed on sera of 158 Dutch patients with physician-confirmed LB (both early localized and disseminated), 800 healthy blood donors from the Netherlands, and 90 cross-reactive controls. The BioPlex (Biopl) assays were compared with two commercial enzyme immunoassays (Euroimmun [Eur]/C6-ELISA) and one immunoblot (recomLine). The highest sensitivity in early LB was achieved with the BioPlex assays, which outperformed the Euroimmun and C6-ELISA (Biopl: 81/88, 92.1%; Eur: 64/88, 72.7%; C6: 72/88, 81.8%). Sensitivity of all assays was comparable in patients with disseminated LB. The BioPlex assays were outperformed in terms of specificity (all healthy blood donors, Biopl: 571/800, 71.4%; Eur: 711/800, 88.9%; C6: 727/800, 90.9%), but further analyses showed promising avenues following cutoff optimization. ROC analysis showed that 2/6 antigens of the combined BioPlex IgG and IgM assays had significantly higher areas under the curve (AUCs) than those of the other analyses. Potential modified versions of the assays based on these antigens largely outperformed the Euroimmun and C6-ELISA in EM patients (Biopl: 81/80, 92.1%) while maintaining a comparable or even higher specificity (Biopl: 714/800, 89.3%). The BioPlex 2200 Lyme IgG and Lyme IgM assays are promising tools for the serodiagnosis of early LB, with the potential to be used as a standalone test. Further research is necessary to validate the findings of this discovery cohort.

Borrelia burgdorferi sensu lato seroconversion after intravenous immunoglobulin treatment: A cohort study.

OBJECTIVE: Intravenous immunoglobulin (IVIg) consists of pooled donor immunoglobulins (IgG), possibly including anti-Borrelia burgdorferi (Bbsl) antibodies. Apparent IVIg-related Bbsl seroconversion could lead to incorrect diagnosis of Lyme borreliosis. This cohort study was designed to determine how often IVIg treatment leads to apparent Bbsl seroconversion and whether antibodies disappear post-treatment. METHODS: Sera from chronic inflammatory demyelinating polyneuropathy (CIDP) and myositis patients were analyzed, drawn pre-treatment and 6-12 weeks after the start of IVIg. In patients with apparent seroconversion, follow-up samples after treatment withdrawal were analyzed, if available. Patients treated with corticosteroids were included as controls. A two-tier protocol was used for serological testing consisting of the C6 Lyme ELISA (Oxford Immunotec) and confirmation by immunoglobulin M (IgM) and immunoglobulin G (IgG) immunoblot (Mikrogen® ). RESULTS: We included 61 patients: 51 patients were treated with IVIg and 10 with dexamethasone. Of the patients treated with IVIg, 42 had CIDP (82%) and were treated with Nanogam® (Sanquin Plasma Products). Nine patients had myositis (18%) and were treated with Privigen® (CSL Behring). Anti-Bbsl IgG seroprevalence pre-treatment was 3% (2/61). Apparent seroconversion during IVIg treatment occurred in 39% (20/51) of patients, all treated with Nanogam. Post-treatment seroreversion occurred in 92% (12/13) of patients with available follow-up samples; in 78% (7/9) seroreversion was observed within 3 months. CONCLUSIONS: Transient presence of anti-Bbsl IgG antibodies after IVIg is regularly observed. This effect appears to be dependent on the IVIg brand, probably reflecting variation in Bbsl exposure of plasma donors. Lyme borreliosis serological testing during, and weeks to months after, IVIg is therefore of limited utility.

Whole genome sequencing of Borrelia miyamotoi isolate Izh-4: reference for a complex bacterial genome.

BACKGROUND: The genus Borrelia comprises spirochaetal bacteria maintained in natural transmission cycles by tick vectors and vertebrate reservoir hosts. The main groups are represented by a species complex including the causative agents of Lyme borreliosis and relapsing fever group Borrelia. Borrelia miyamotoi belongs to the relapsing fever group of spirochetes and forms distinct populations in North America, Asia, and Europe. As all Borrelia species B. miyamotoi possess an unusual and complex genome consisting of a linear chromosome and a number of linear and circular plasmids. The species is considered an emerging human pathogen and an increasing number of human cases are being described in the Northern hemisphere. The aim of this study was to produce a high quality reference genome that will facilitate future studies into genetic differences between different populations and the genome plasticity of B. miyamotoi. RESULTS: We used multiple available sequencing methods, including Pacific Bioscience single-molecule real-time technology (SMRT) and Oxford Nanopore technology (ONT) supplemented with highly accurate Illumina sequences, to explore the suitability for whole genome assembly of the Russian B. miyamotoi isolate, Izh-4. Plasmids were typed according to their potential plasmid partitioning genes (PF32, 49, 50, 57/62). Comparing and combining results of both long-read (SMRT and ONT) and short-read methods (Illumina), we determined that the genome of the isolate Izh-4 consisted of one linear chromosome, 12 linear and two circular plasmids. Whilst the majority of plasmids had corresponding contigs in the Asian B. miyamotoi isolate FR64b, there were only four that matched plasmids of the North American isolate CT13-2396, indicating differences between B. miyamotoi populations. Several plasmids, e.g. lp41, lp29, lp23, and lp24, were found to carry variable major proteins. Amongst those were variable large proteins (Vlp) subtype Vlp-α, Vlp-γ, Vlp-δ and also Vlp-ß. Phylogenetic analysis of common plasmids types showed the uniqueness in Russian/Asian isolates of B. miyamotoi compared to other isolates. CONCLUSIONS: We here describe the genome of a Russian B. miyamotoi clinical isolate, providing a solid basis for future comparative genomics of B. miyamotoi isolates. This will be a great impetus for further basic, molecular and epidemiological research on this emerging tick-borne pathogen.

The predictive value of CXCL13 in suspected Lyme neuroborreliosis: a retrospective cross-sectional study.

The role of CXCL13 as a marker of Lyme neuroborreliosis (LNB) is under investigation, and CXCL13 is not part of routine diagnostics in suspicion of LNB. Our aim was to find the optimal cut-off value of CXCL13 for LNB in a Danish population and to investigate the role of CXCL13 both in early LNB and as a discriminatory marker between LNB and other neuroinflammatory disorders. We conducted a retrospective cross-sectional study including all patients with a cerebrospinal CXCL13 test performed at the Department of Clinical Immunology, Odense University Hospital, Denmark, between 1 January 2015 and 31 December 2018. We included 619 patients, of which 51 had definite LNB, 14 patients had possible LNB with neurological symptoms suggestive of LNB and pleocytosis but no intrathecal Borrelia antibodies, eight patients had prior LNB and 546 had no LNB. With an optimal CXCL13 cut-off of 49 ng/L we found a sensitivity of 100% and specificity of 94% (AUC 0.988, 95% CI 0.980-0.996) when patients treated with antibiotics prior to lumbar puncture were excluded (n = 130). All patients with possible LNB had a CXCL13 value above the cut-off value; 18/546 patients (3.3%) without LNB had a CXCL13 value ≥ 50 ng/L. While CXCL13 cannot be used as a stand-alone test, it can be used as a reliable additional marker in treatment-naive patients suspected of LNB. CXCL13 can be used to monitor treatment response in LNB patients.

Two Cases of Borrelia miyamotoi Meningitis, Sweden, 2018.

We report 2 human cases of Borrelia miyamotoi disease diagnosed in Sweden, including 1 case of meningitis in an apparently immunocompetent patient. The diagnoses were confirmed by 3 different independent PCR assays and DNA sequencing from cerebrospinal fluid, supplemented by serologic analyses.

Prevalence and determinants of persistent symptoms after treatment for Lyme borreliosis: study protocol for an observational, prospective cohort study (LymeProspect).

BACKGROUND: After antibiotic treatment of Lyme borreliosis, a subset of patients report persistent symptoms, also referred to as post-treatment Lyme disease syndrome. The reported prevalence of persistent symptoms varies considerably, and its pathophysiology is under debate. The LymeProspect study has been designed to investigate the prevalence, severity, and a wide range of hypotheses on the etiology of persistent symptoms among patients treated for Lyme borreliosis in the Netherlands. METHODS: LymeProspect is a prospective, observational cohort study among adults with proven or probable Lyme borreliosis, either erythema migrans or disseminated manifestations, included at the start of antibiotic treatment. During one year of follow-up, participants are subjected to questionnaires every three months and blood is collected repeatedly during the first three months. The primary outcome is the prevalence of persistent symptoms after treatment, assessed by questionnaires online focusing on fatigue (CIS, subscale fatigue severity), pain (SF-36, subscale pain) and neurocognitive dysfunction (CFQ). Potential microbiological, immunological, genetic, epidemiological and cognitive-behavioral determinants for persistent symptoms are secondary outcome measures. Control cohorts include patients with long-lasting symptoms and unconfirmed Lyme disease, population controls, and subjects having reported a tick bite not followed by Lyme borreliosis. DISCUSSION: This article describes the background and design of the LymeProspect study protocol. This study is characterized by a prospective, explorative and multifaceted design. The results of this study will provide insights into the prevalence and determinants of persistent symptoms after treatment for Lyme borreliosis, and may provide a rationale for preventive and treatment recommendations. TRIAL REGISTRATION: NTR4998 (Netherlands Trial Register). Date of registration: 13 February 2015.

Borrelia miyamotoi Disease in an Immunocompetent Patient, Western Europe.

Borrelia miyamotoi disease is a hard tick-borne relapsing fever illness that occurs across the temperate climate zone. Human B. miyamotoi disease in immunocompetent patients has been described in Russia, North America, and Japan. We describe a case of B. miyamotoi disease in an immunocompetent patient in western Europe.

In Vitro Antimicrobial Susceptibility of Clinical Isolates of Borrelia miyamotoi.

Borrelia miyamotoi is an emerging relapsing fever (RF) Borrelia species that is reported to cause human disease in regions in which Lyme borreliosis is endemic. We recently showed that B. miyamotoi tick isolates are resistant to amoxicillin in vitro; however, clinical isolates have not been studied. Therefore, our aim was to show the antimicrobial susceptibility of recently obtained clinical isolates of B. miyamotoi A dilution series of various antibiotics was made in modified Kelly-Pettenkofer medium with 10% fetal calf serum. The susceptibilities of different B. miyamotoi clinical, B. miyamotoi tick, RF Borrelia, and Borrelia burgdorferisensu lato isolates were tested by measuring MICs through colorimetric changes and by counting motile spirochetes by dark-field microscopy after 72 h of incubation. The ceftriaxone and azithromycin MIC ranges of the six B. miyamotoi clinical isolates tested were 0.03 to 0.06 mg/liter and 0.0016 to 0.0032 mg/liter, respectively. These values are similar to MICs for RF Borrelia strains and B. miyamotoi tick isolates. All tested RF Borrelia strains were susceptible to doxycycline (microscopic MIC range, 0.0625 to 0.25 mg/liter). In contrast to the MICs of the tested B. burgdorferi sensu lato strains and in line with our previous findings, the amoxicillin MICs (range, 8 to 32 mg/liter) of all RF Borrelia strains, including B. miyamotoi clinical isolates, were above the clinical breakpoint for resistance (≤4 mg/liter). Clinical isolates of B. miyamotoi are highly susceptible to doxycycline, azithromycin, and ceftriaxone in vitro Interestingly, as described previously for tick isolates, amoxicillin shows poor in vitro activity against B. miyamotoi clinical isolates.

Variable Major Proteins as Targets for Specific Antibodies against Borrelia miyamotoi.

Borrelia miyamotoi is a relapsing fever spirochete in Ixodes ticks that has been recently identified as a human pathogen causing hard tick-borne relapsing fever (HTBRF) across the Northern Hemisphere. No validated serologic test exists, and current serologic assays have low sensitivity in early HTBRF. To examine the humoral immune response against B. miyamotoi, we infected C3H/HeN mice with B. miyamotoi strain LB-2001 expressing variable small protein 1 (Vsp1) and demonstrated that spirochetemia was cleared after 3 d, coinciding with anti-Vsp1 IgM production. Clearance was also observed after passive transfer of immune sera to infected SCID mice. Next, we showed that anti-Vsp1 IgG eliminates Vsp1-expressing B. miyamotoi, selecting for spirochetes expressing a variable large protein (VlpC2) resistant to anti-Vsp1. The viability of Asian isolate B. miyamotoi HT31, expressing Vlp15/16 and Vlp18, was also unaffected by anti-Vsp1. Finally, in nine HTBRF patients, we demonstrated IgM reactivity to Vsp1 in two and against Vlp15/16 in four ∼1 wk after these patients tested positive for B. miyamotoi by PCR. Our data show that B. miyamotoi is able to express various variable major proteins (VMPs) to evade humoral immunity and that VMPs are antigenic in humans. We propose that serologic tests based on VMPs are of additional value in diagnosing HTBRF.

In Vitro Susceptibility of the Relapsing-Fever Spirochete Borrelia miyamotoi to Antimicrobial Agents.

Hard-tick-borne relapsing fever (HTBRF) is an emerging infectious disease throughout the temperate zone caused by the relapsing-fever spirochete Borrelia miyamotoi Antibiotic treatment of HTBRF is empirically based on the treatment of Lyme borreliosis; however, the antibiotic susceptibility of B. miyamotoi has not been studied to date. Thus, we set out to determine the in vitro antimicrobial susceptibility of B.miyamotoi A microdilution method with 96-well microtiter plates was used to determine the antibiotic susceptibilities of two B.miyamotoi strains isolated on two different continents (Asia and North America), two Borrelia burgdorferisensu lato strains, and one Borrelia hermsii isolate for purposes of comparison. The MIC and minimal bactericidal concentration (MBC) were determined by both microscopy and colorimetric assays. We were able to show that relative to the B. burgdorferi sensu lato isolates, both B.miyamotoi strains and B. hermsii demonstrated greater susceptibility to doxycycline and azithromycin, equal susceptibility to ceftriaxone, and resistance to amoxicillin in vitro The MIC and MBC of amoxicillin for B. miyamotoi evaluated by microscopy were 16 to 32 mg/liter and 32 to 128 mg/liter, respectively. Since B. miyamotoi is susceptible to doxycycline, azithromycin, and ceftriaxone in vitro, our data suggest that these antibiotics can be used for the treatment of HTBRF. Oral amoxicillin is currently used as an alternative for the treatment of HTBRF; however, since we found that the B. miyamotoi strains tested were resistant to amoxicillin in vitro, this issue warrants further study.

A tick gut protein with fibronectin III domains aids Borrelia burgdorferi congregation to the gut during transmission.

Borrelia burgdorferi transmission to the vertebrate host commences with growth of the spirochete in the tick gut and migration from the gut to the salivary glands. This complex process, involving intimate interactions of the spirochete with the gut epithelium, is pivotal to transmission. We utilized a yeast surface display library of tick gut proteins to perform a global screen for tick gut proteins that might interact with Borrelia membrane proteins. A putative fibronectin type III domain-containing tick gut protein (Ixofin3D) was most frequently identified from this screen and prioritized for further analysis. Immunization against Ixofin3D and RNA interference-mediated reduction in expression of Ixofin3D resulted in decreased spirochete burden in tick salivary glands and in the murine host. Microscopic examination showed decreased aggregation of spirochetes on the gut epithelium concomitant with reduced expression of Ixofin3D. Our observations suggest that the interaction between Borrelia and Ixofin3D facilitates spirochete congregation to the gut during transmission, and provides a "molecular exit" direction for spirochete egress from the gut.

Factor Xa activation of factor V is of paramount importance in initiating the coagulation system: lessons from a tick salivary protein.

BACKGROUND: Generation of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme activated factor X (FXa) to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation, and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. METHODS AND RESULTS: Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied with the use of plasma, whole blood, and purified systems. Here, we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. Accordingly, tick feeding is impaired on TIX-5 immune rabbits, displaying the in vivo importance of TIX-5. CONCLUSIONS: Our data elucidate a unique molecular mechanism by which ticks inhibit the host's coagulation system. From our data, we propose a revised blood coagulation scheme in which direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.