Interaction between Borrelia miyamotoi variable major proteins Vlp15/16 and Vlp18 with plasminogen and complement

Borrelia miyamotoi, a relapsing fever spirochete transmitted by Ixodid ticks causes B. miyamotoi disease (BMD). To evade the human host´s immune response, relapsing fever borreliae, including B. miyamotoi, produce distinct variable major proteins. Here, we investigated Vsp1, Vlp15/16, and Vlp18 all of which are currently being evaluated as antigens for the serodiagnosis of BMD. Comparative analyses identified Vlp15/16 but not Vsp1 and Vlp18 as a plasminogen-interacting protein of B. miyamotoi. Furthermore, Vlp15/16 bound plasminogen in a dose-dependent fashion with high affinity. Binding of plasminogen to Vlp15/16 was significantly inhibited by the lysine analog tranexamic acid suggesting that the protein–protein interaction is mediated by lysine residues. By contrast, ionic strength did not have an effect on binding of plasminogen to Vlp15/16. Of relevance, plasminogen bound to the borrelial protein cleaved the chromogenic substrate S-2251 upon conversion by urokinase-type plasminogen activator (uPa), demonstrating it retained its physiological activity. Interestingly, further analyses revealed a complement inhibitory activity of Vlp15/16 and Vlp18 on the alternative pathway by a Factor H-independent mechanism. More importantly, both borrelial proteins protect serum sensitive Borrelia garinii cells from complement-mediated lysis suggesting multiple roles of these two variable major proteins in immune evasion of B. miyamotoi.

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-β. 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.

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-β. 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.

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 a relatively new 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 methods (SMRT and ONT), we determined that the genome of the Izh-4 consisted of one linear chromosome, 12 linear and two circular plasmids. Whilst the majority of plasmids had corresponding assembly fragments 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-β. 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.

BACTERICIDAL EFFECT OF HUMAN SERUM ON BORRELIA MIYAMOTOI, CAUSATIVE AGENT OF IXODES TICK-BORNE BORRELIOSIS (ITBB-BM)

Aim. Our aim was to study the bactericidal effect of human serum on Borrelia miyamotoi in vitro. Materials and methods. B. miyamotoi spirochetes (strains HT31 and LB-2001) were incubated in non-immune serum of healthy donors (SHD) and in heat inactivated complement-depleted SHD, as well as in serum samples of the patients recovered from ITBB-BM. The viability, that is motility, of borrelia after incubation was investigated by dark-field microscopy. The levels ofserum antibody to B.miyamofoi-specificproteins (GlpQ enzyme and four variable major proteins Vlpl5/16, Vlpl8, Vspl, and Vlp5) were measured by specially designed plane protein microarray. Results. Borrelia fully retain their viability in non-immune SHD, but their motility is partially or completely suppressed by the addition of serum from ITBB-BM convalescents or rabbit antibodies to Д. miyamotoi. The immobilizing effect of the immune serum is substantially inhibited by its heat-inactivation, which indicates that immobilizing effect is mediated by the complement system. Conclusion. Antibody-dependent complement-mediated bactericidal action ofhuman blood serum is probably not the only and 100% effective mechanism for human defense against B. miyamotoi infection, but requires support from cellular immunity.

Clinical presentation of Ixodes tick-borne borreliosis caused by Borrelia miyamotoi in the context of an immune response to the pathogen

Ixodes tick-borne borreliosis caused by Borrelia miyamotoi (ITBB-BM) is a previously unknown infectious disease discovered in Russia. Aim. The present study continues the investigation of the clinical features of ITBB-BM in the context of an immune system-pathogen interaction. Subjects and methods. The study enrolled 117 patients with ITBB-BM and a comparison group of 71 patients with Lyme disease (LD) that is ITBB with erythema migrans. All the patients were treated at the New Hospital, Yekateringburg. More than 100 clinical, epidemiological and laboratory parameters were obtained from each patient’s medical history and included in the general database. A subset of patients hospitalized in 2015 and 2016 underwent additional laboratory examinations. Namely, the levels of B. miyamotoi-specific IgM and IgG antibodies were measured by the protein microarray containing GlpQ protein and four variable major proteins (VMPs): Vlp15/16, Vlp18, Vsp1, and Vlp5. The blood concentration of Borrelia was estimated by quantitative real-time PCR. Results. In contrast to LD, first of all (p

Alp, an Arthropod-Associated Outer Membrane Protein of Borrelia Species That Cause Relapsing Fever

ABSTRACTBorrelia hermsiiand other relapsing fever (RF) species are noted for their highly polymorphic surface antigens, the variable major proteins (VMP). Less is known about other surface proteins of these pathogens in either their vertebrate reservoirs or arthropod vectors. To further characterize these proteins, we elicited antibodies against VMP-less cells, noted antibody reactions against whole cells and cell components, and then subjected selected antigens to mass spectroscopy for amino acid sequencing for comparison against aB. hermsiigenome database. One of the derived monoclonal antibodies, H0120, agglutinated spirochetes, and in Western blot analyses, it bound to a 14-kDa protein of whole cells and their membrane fractions but not after protease treatment. A search of open reading frames of theB. hermsiigenome with extracted peptides identified the 14-kDa protein withbha128, a 453-nucleotide gene of the 175-kb linear plasmid. Thebha128gene was synthesized and expressed inEscherichia coli. The protein product was bound by antibody H0120. Genes homologous tobha128occur in the RF speciesBorrelia turicatae,B. duttonii, andB. recurrentisbut not in Lyme diseaseBorreliaspecies or other organisms. The following findings indicated an association of BHA128, renamed Alp, with the tick environment: (i) Alp was produced at higher levels at 23°C than at 34°C; (ii) almost all spirochetes in tick salivary glands were bound by the H0120 antibody, but only ∼1% of spirochetes in the blood of infected mice were bound; and (iii) infected mice produced antibodies to severalB. hermsiiantigens but not detectably to native or recombinant Alp.

Variable Tick Protein in Two Genomic Groups of the Relapsing Fever Spirochete Borrelia hermsii in Western North America

ABSTRACT Borrelia hermsii is the primary cause of tick-borne relapsing fever in North America. When its tick vector, Ornithodoros hermsi, acquires these spirochetes from the blood of an infected mammal, the bacteria switch their outer surface from one of many bloodstream variable major proteins (Vmps) to a unique protein, Vtp (Vsp33). Vtp may be critical for successful tick transmission of B. hermsii; however, the gene encoding this protein has been described previously in only one isolate. Here we identified and sequenced the vtp gene in 31 isolates of B. hermsii collected over 40 years from localities throughout much of its known geographic distribution. Seven major Vtp types were found. Little or no sequence variation existed within types, but between them significant variation was observed, similar to the pattern of diversity described for the outer surface protein C (OspC) gene in Lyme disease spirochetes. The pattern of sequence relatedness among the Vtp types was incongruent in two branches compared to two genomic groups identified among the isolates by multilocus sequence typing of the 16S rRNA, flaB, gyrB, and glpQ genes. Therefore, both horizontal transfer and recombination within and between the two genomic groups were responsible for some of the variation observed in the vtp gene. O. hermsi ticks were capable of transmitting spirochetes in the newly identified genomic group. Therefore, given the longevity of the tick vector and persistent infection of spirochetes in ticks, these arthropods rather than mammals may be the likely host where the exchange of spirochetal DNA occurs.