Proteomic analysis of Lyme disease: global protein comparison of three strains of Borrelia burgdorferi

The Borrelia burgdorferi spirochete is the causative agent of Lyme disease, the most common tick-borne disease in the United States. It has been studied extensively to help understand its pathogenicity of infection and how it can persist in different mammalian hosts. We report the proteomic analysis of the archetype B. burgdorferi B31 strain and two other strains (ND40, and JD-1) having different Borrelia pathotypes using strong cation exchange fractionation of proteolytic peptides followed by high-resolution, reversed phase capillary liquid chromatography coupled with ion trap tandem mass spectrometric analysis. Protein identification was facilitated by the availability of the complete B31 genome sequence. A total of 665 Borrelia proteins were identified representing approximately 38% coverage of the theoretical B31 proteome. A significant overlap was observed between the identified proteins in direct comparisons between any two strains (>72%), but distinct differences were observed among identified hypothetical and outer membrane proteins of the three strains. Such a concurrent proteomic overview of three Borrelia strains based upon only the B31 genome sequence is shown to provide significant insights into the presence or absence of specific proteins and a broad overall comparison among strains.     

Protein profile determination of <Emphasis Type="Italic">Borrelia afzelii</Emphasis> and <Emphasis Type="Italic">Borrelia garinii</Emphasis> isolated from skin and cerebrospinal fluid

In Europe the initial skin manifestation of Lyme borreliosis (erythema migrans) is predominantly caused by Borrelia afzelii while nervous system involvement is usually associated with Borrelia garinii. The aim of our study was to compare protein profiles of B. afzelii and B. garinii isolated from skin of patients with erythema migrans and from cerebrospinal fluid (CSF) of patients with Lyme neuroborreliosis. We analyzed 187 Borrelia strains, 74 B. afzelii and 113 B. garinii, for the presence of flagellin, and outer surface proteins A, B and C. Their protein profiles were obtained by SDS–PAGE and analyzed by computer program Gel Doc. Differences in the presence of proteins were found comparing isolates according to species (B. afzelii and B. garinii) and to their origin (skin and CSF isolates); the heterogeneity regarding the presence or absence of borrelial proteins was established within particular species. Protein profiles of the analyzed strains showed differences in the number, amount and molecular mass of analyzed proteins. Distinctions in the synthesis of outer surface proteins may play a role in the dispersal of borreliae within and between animal reservoir and vector ticks, as well as in pathogenesis of Lyme borreliosis in humans.     

Preparation and purification of recombinant outer surface protein A (rOspA) of Borrelia burgdorferi sensu stricto and Borrelia afzelii

The recombinant Outer surface protein A (rOspA) from Borrelia burgdorferi is a possible immunogen for protection of infected humans and animals against development of Lyme borreliosis (Lyme disease), a chronic tick-borne disease characterised by diverse dermatologic, neurologic, rheumatic, and cardiac manifestations. For several years, research and development have been directed towards a vaccine for the prevention of this debilitating disease. Numerous animal studies demonstrate that pre-existing antibodies against the outer surface proteins of B. burgdorferi can prevent infection and disease caused by this organism. In this communication, using recombinant DNA technology, genes from B. burgdorferi sensu stricto and B. afzelii were inserted into E. coli-expression vectors and the rOspA were produced. Our aim was to obtain rOspA protein in a purity and quantity desirable for immunization of experimental animals. rOspA is currently the most developed, molecularly-defined vaccine candidate for the prevention of Lyme borreliosis.     

The complement regulator factor H binds to the surface protein OspE of Borrelia burgdorferi

Spirochete bacteria of the Borrelia burgdorferi sensu lato complex cause Lyme borreliosis. The three pathogenic subspecies Borrelia garinii, Borrelia afzelii, and Borrelia burgdorferi sensu stricto differ in their disease profiles and susceptibility to complement lysis. We investigated whether complement resistance of Borreliae could be due to acquisition of the main soluble inhibitors of the alternative complement pathway, factor H and the factor H-like protein 1. When exposed to nonimmune EDTA-plasma, the serum-resistant B. afzelii and B. burgdorferi sensu stricto strains bound factor H/factor H-like protein 1 to their surfaces. Assays with radiolabeled proteins showed that factor H bound strongly to the B. burgdorferi sensu stricto strain. To identify factor H ligands on the borrelial surface, we analyzed a panel of outer surface proteins of B. burgdorferi sensu stricto with the surface plasmon resonance technique. The outer surface lipoprotein OspE was identified as a specific ligand for factor H. Using recombinant constructs of factor H, the binding site for OspE was localized to the C-terminal short consensus repeat domains 15-20. Specific binding of factor H to B. burgdorferi sensu stricto OspE may help the pathogen to evade complement attack and phagocytosis.     

Prevalence of IgG reactivity in Lyme borreliosis patients versus Borrelia garinii and Borrelia afzelii in a restricted area of Northern Italy

Abstract This survey evaluates the antibody band patterns of sera taken from clinically defined cases of Lyme borreliosis, towards three locally isolated strains of Borrelia burgdorferi , belonging to the three species: Borrelia sensu stricto, Borrelia garinii and Borrelia afzelii , by means of Western blot. The sera were taken from patients resident in a limited area of Friuli Venezia Giulia (FVG) region. The data indicated that, besides a different feature of the band reactivity which correlated to the different stages of Lyme borreliosis, there was a preferential reactivity to the species Borrelia afzelii and Borrelia garinii . An immunodominant band at 51 kDa, corresponding to a protein visible in the electrophoretic profile of strain BL3 ( B. afzelii ), behaved like a marker of an early infection, because it was present exclusively in the sera of patient with ECM. The overall findings would indicate that B. afzelii and B. garinii are the prevalent genospecies in the FVG area, even if strains belonging to B. sensu stricto have been also isolated in this area. Consequently strains representative of these two species must be used as antigens in Western blot.     

Coinfection with Borrelia burgdorferi sensu stricto and Borrelia garinii alters the course of murine Lyme borreliosis

Ixodes ricinus ticks and mice can be infected with both Borrelia burgdorferi sensu stricto and Borrelia garinii. The effect of coinfection with these two Borrelia species on the development of murine Lyme borreliosis is unknown. Therefore, we investigated whether coinfection with the nonarthritogenic B. garinii strain PBi and the arthritogenic B. burgdorferi sensu stricto strain B31 alters murine Lyme borreliosis. Mice simultaneously infected with PBi and B31 showed significantly more paw swelling and arthritis, long-standing spirochetemia, and higher numbers of B31 spirochetes than did mice infected with B31 alone. However, the number of PBi spirochetes was significantly lower in coinfected mice than in mice infected with PBi alone. In conclusion, simultaneous infection with B. garinii and B. burgdorferi sensu stricto results in more severe Lyme borreliosis. Moreover, we suggest that competition of the two Borrelia species within the reservoir host could have led to preferential maintenance, and a rising prevalence, of B. burgdorferi sensu stricto in European I. ricinus populations.     

Outer surface protein polymorphisms linked to host‐spirochete association in Lyme borreliae

Lyme borreliosis is caused by multiple species of the spirochete bacteria Borrelia burgdorferi sensu lato. The spirochetes are transmitted by ticks to vertebrate hosts, including small‐ and medium‐sized mammals, birds, reptiles, and humans. Strain‐to‐strain variation in host‐specific infectivity has been documented, but the molecular basis that drives this differentiation is still unclear. Spirochetes possess the ability to evade host immune responses and colonize host tissues to establish infection in vertebrate hosts. In turn, hosts have developed distinct levels of immune responses when invaded by different species/strains of Lyme borreliae. Similarly, the ability of Lyme borreliae to colonize host tissues varies among different spirochete species/strains. One potential mechanism that drives this strain‐to‐strain variation of immune evasion and colonization is the polymorphic outer surface proteins produced by Lyme borreliae. In this review, we summarize research on strain‐to‐strain variation in host competence and discuss the evidence that supports the role of spirochete‐produced protein polymorphisms in driving this variation in host specialization. Such information will provide greater insights into the adaptive mechanisms driving host and Lyme borreliae association, which will lead to the development of interventions to block pathogen spread and eventually reduce Lyme borreliosis health burden.     

Comparative proteome analysis of subcellular fractions from Borrelia burgdorferi by NEPHGE and IPG

Borrelia burgdorferi, the cause of Lyme disease, produces excessive amounts of membrane lipoproteins such as outer surface protein A (OspA) when grown in vitro, and consequently many low or moderately abundant proteins are underrepresented when cell lysates are examined by 2-DE. We analyzed the B. burgdorferi B31 proteome computationally and by IPG or modified NEPHGE after subcellular fractionation into membrane-associated and soluble proteins. The B. burgdorferi B31 theoretical proteome is comprised of 1623 proteins and has a mean pI of 8.36 and a median pI of 9.03 with 68% of the proteome possessing a pI >/=7.5. Separation of soluble proteins by IPG resulted in 205 individual spots and identification of 78 protein spots by MALDI-TOF MS. Separation by modified NEPHGE routinely resulted in approximately 185 soluble and 160 membrane protein spots with the identification of 88 individual protein spots combined by MALDI-TOF MS. Homologues to GroEL and aminopeptidase I were present in greater amounts in the membrane faction, with enolase at nearly equivalent amounts in the soluble and membrane fractions. Identification of proteins isolated and separated by such methods will enable future determination of proteome changes in membrane and soluble protein fractions as spirochetes adapt to their changing environments.     

Comparative cryo-electron tomography of pathogenic Lyme disease spirochetes

Spirochetes of the Borrelia burgdorferi sensu lato group, the causative agents of Lyme borreliosis, exhibit a complex biology evolved in its zoonotic cycle. Cryo-electron tomography was used to investigate structural features of three species, B. burgdorferi , B. garinii and B. afzelii , known to cause different clinical manifestations in humans. All three organisms revealed an overall similar architecture and showed different numbers of periplasmic flagellar filaments, polar periplasmic void regions, vesicles budding from the outer membrane sheath, which was covered by an amorphous slime layer. The latter was shown to be distinct in its density when comparing the three human-pathogenic Lyme disease spirochetes and Borrelia hermsii , a species causing relapsing fever. Tomograms of dividing bacteria revealed vesicles near the site of division and new basal bodies that were attached at each end of newly establishing cytoplasmic cylinder poles, while periplasmic flagellar filaments still passed the impending site of division. Two different kinds of cytoplasmic filaments showed similarities to MreB or FtsZ filaments of other bacteria. The similar and distinct structural features of Borrelia and the previously investigated pathogenic and non-pathogenic Treponema species emphasize the importance of further studying phylogenetically distant spirochetes.     

Proteomic analysis of three Borrelia burgdorferi sensu lato native species and disseminating clones: Relevance for Lyme vaccine design

Lyme borreliosis is the most important vector‐borne disease in the Northern hemisphere. It is caused by Borrelia burgdorferi sensu lato bacteria transmitted to humans by the bite of hard ticks, Ixodes spp. Although antibiotic treatments are efficient in the early stage of the infection, a significant number of patients develop disseminated manifestations (articular, neurological, and cutaneous) due to unnoticed or absence of erythema migrans, or to inappropriate treatment. Vaccine could be an efficient approach to decrease Lyme disease incidence. We have developed a proteomic approach based on a one dimensional gel electrophoresis followed by LC‐MS/MS strategy to identify new vaccine candidates. We analyzed a disseminating clone and the associated wild‐type strain for each major pathogenic Borrelia species: B. burgdorferi sensu stricto, B. garinii, and B. afzelii. We identified specific proteins and common proteins to the disseminating clones of the three main species. In parallel, we used a spectral counting strategy to identify upregulated proteins common to the clones. Finally, 40 proteins were found that could potentially be involved in bacterial virulence and of interest in the development of a new vaccine. We selected the three proteins specifically detected in the disseminating clones of the three Borrelia species and checked by RT‐PCR whether they are expressed in mouse skin upon B. burgdorferi ss inoculation. Interestingly, BB0566 appears as a potential vaccine candidate. All MS data have been deposited in the ProteomeXchange with identifier PXD000876 ( http://proteomecentral.proteomexchange.org/dataset/PXD000876 ).     

Molecular identification and analysis of Borrelia burgdorferi sensu lato in lizards in the southeastern United States

Lyme borreliosis (LB) group spirochetes, collectively known as Borrelia burgdorferi sensu lato, are distributed worldwide. Wild rodents are acknowledged as the most important reservoir hosts. Ixodes scapularis is the primary vector of B. burgdorferi sensu lato in the eastern United States, and in the southeastern United States, the larvae and nymphs mostly parasitize certain species of lizards. The primary aim of the present study was to determine whether wild lizards in the southeastern United States are naturally infected with Lyme borreliae. Blood samples obtained from lizards in Florida and South Carolina were tested for the presence of LB spirochetes primarily by using B. burgdorferi sensu lato-specific PCR assays that amplify portions of the flagellin (flaB), outer surface protein A (ospA), and 66-kDa protein (p66) genes. Attempts to isolate spirochetes from a small number of PCR-positive lizards failed. However, PCR amplification and sequence analysis of partial flaB, ospA, and p66 gene fragments confirmed numerous strains of B. burgdorferi sensu lato, including Borrelia andersonii, Borrelia bissettii, and B. burgdorferi sensu stricto, in blood from lizards from both states. B. burgdorferi sensu lato DNA was identified in 86 of 160 (54%) lizards representing nine species and six genera. The high infection prevalence and broad distribution of infection among different lizard species at different sites and at different times of the year suggest that LB spirochetes are established in lizards in the southeastern United States.     

Surface localization determinants of Borrelia OspC/Vsp family lipoproteins

The dimeric OspC/Vsp family surface lipoproteins of Borrelia spirochetes are crucial to the transmission and persistence of Lyme borreliosis and tick-borne relapsing fever. However, the requirements for their proper surface display remained undefined. In previous studies, we showed that localization of Borrelia burgdorferi monomeric surface lipoprotein OspA was dependent on residues in the N-terminal "tether" peptide. Here, site-directed mutagenesis of the B. burgdorferi OspC tether revealed two distinct regions affecting either release from the inner membrane or translocation through the outer membrane. Determinants of both of these steps appear consolidated within a single region of the Borrelia turicatae Vsp1 tether. Periplasmic OspC mutants still were able to form dimers. Their localization defect could be rescued by the addition of an apparently structure-destabilizing C-terminal epitope tag but not by coexpression with wild-type OspC. Furthermore, disruption of intermolecular Vsp1 salt bridges blocked dimerization but not surface localization of the resulting Vsp1 monomers. Together, these results suggest that Borrelia OspC/Vsp1 surface lipoproteins traverse the periplasm and the outer membrane as unfolded monomeric intermediates and assemble into their functional multimeric folds only upon reaching the spirochetal surface.     

Molecular analysis and expression of a Borrelia burgdorferi gene encoding a 22kDa protein (pC) in Escherichia coli

We describe the cloning and expression of the pc gene which encodes a major immunodominant protein of Borrelia burgdorferi, the causative agent of Lyme borreliosis. The pC protein was purified from lysates of B. burgdorferi strain PKo. After tryptic digestion of the pC protein the resulting oligopeptides were applied to a gas-phase sequenator. Thus partial amino acid sequences were obtained. The deduced oligonucleotides were used as hybridization probes. After Southern blotting a reactive band in the 3 kb range of PstI-digested genomic DNA was detected. The insertion of these fragments into pUC vectors finally resulted in pc-positive Escherichia coli clones. The gene (encoding a protein with 212 amino acids) was expressed in E. coli with varying deletions at the 5' end. A sequence comparison with other outer membrane proteins of B. burgdorferi indicates a processing of pC that is similar to that of lipoproteins.     

Controlled activation of the Cpx system is essential for growth of Yersinia enterocolitica

Until recently, three spirochete genospecies were considered to be the causative agents of Lyme borreliosis (LB) in Europe: Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii . However, the DNA of Borrelia valaisiana, Borrelia lusitaniae, Borrelia spielmanii and Borrelia bissettii has already been detected in samples of human origin, or the spirochetes were isolated from the patients with symptoms of LB. Molecular analysis of 12 selected serum samples collected in the regional hospital confirmed the presence of B. bissettii DNA in cases of single and multiple infection in patients with symptomatic borreliosis or chronic borrelial infection. The presence of B. bissettii as a single strain in patients provides strong support of the fact that B. bissettii might be a causative agent of the disease. After the first isolation of B. bissettii from the samples of human origin in Slovenia, following the detection of this species in cardiac valve tissue of the patient with endocarditis and aortic valve stenosis in the Czech Republic, here we present additional molecular data supporting the involvement of B. bissettii in LB in Europe.     

Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis.

We studied 48 Borrelia isolates that were associated with Lyme borreliosis or were isolated from ticks and identified three DNA relatedness groups by using the S1 nuclease method. The three DNA groups (genospecies) were associated with specific rRNA gene restriction patterns, protein electrophoresis patterns, and patterns of reactivity with murine monoclonal antibodies. Genospecies I corresponded to Borrelia burgdorferi sensu stricto since it contained the type strain of this species (strain ATCC 35210); this genospecies included 28 isolates from Europe and the United States. Genospecies II was named Borrelia garinii sp. nov. and included 13 isolates from Europe and Japan. Genospecies III (group VS461) included seven isolates from Europe and Japan.     

DipA, a pore-forming protein in the outer membrane of Lyme disease spirochetes exhibits specificity for the permeation of dicarboxylates

Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a single-channel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl) were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species.     

Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi.

The outer membrane-spanning (Oms) proteins of Borrelia burgdorferi have been visualized by freeze-fracture analysis but, until recently, not further characterized. We developed a method for the isolation of B. burgdorferi outer membrane vesicles and described porin activities with single-channel conductances of 0.6 and 12.6 nS in 1 M KCI. By using both nondenaturing isoelectric focusing gel electrophoresis and fast-performance liquid chromatography separation after detergent solubilization, we found that the 0.6-nS porin activity resided in a 28-kDa protein, designated Oms28. The oms28 gene was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence of Oms28 predicted a 257-amino-acid precursor protein with a putative 24-amino-acid leader peptidase I signal sequence. Processed Oms28 yielded a mature protein with a predicted molecular mass of 25,363 Da. When overproduced in Escherichia coli, the Oms28 porin fractionated in part to the outer membrane. Sodium dodecyl sulfate-polyacrylamide gel-purified recombinant Oms28 from E. coli retained functional activity as demonstrated by an average single-channel conductance of 1.1 nS in the planar lipid bilayer assay. These findings confirmed that Oms28 is a B. burgdorferi porin, the first to be described. As such, it is potential relevance to the pathogenesis of Lyme borreliosis and to the physiology of the spirochete.     

The role of Borrelia burgdorferi outer surface proteins

Human pathogenic spirochetes causing Lyme disease belong to the Borrelia burgdorferi sensu lato complex. Borrelia burgdorferi organisms are extracellular pathogens transmitted to humans through the bite of Ixodes spp. ticks. These spirochetes are unique in that they can cause chronic infection and persist in the infected human, even though a robust humoral and cellular immune response is produced by the infected host. How this extracellular pathogen is able to evade the host immune response for such long periods of time is currently unclear. To gain a better understanding of how this organism persists in the infected human, many laboratories have focused on identifying and characterizing outer surface proteins of B.?burgdorferi. As the interface between B.?burgdorferi and its human host is its outer surface, proteins localized to the outer membrane must play an important role in dissemination, virulence, tissue tropism, and immune evasion. Over the last two decades, numerous outer surface proteins from B.?burgdorferi have been identified, and more recent studies have begun to elucidate the functional role(s) of many borrelial outer surface proteins. This review summarizes the outer surface proteins identified in B.?burgdorferi to date and provides detailed insight into the functions of many of these proteins as they relate to the unique parasitic strategy of this spirochetal pathogen.     

Molecular diagnosis of Borrelia burgdorferi infection (Lyme disease).

In spite of significant advances in immunologically based testing, accurate diagnosis of Lyme borreliosis remains problematic. To address this issue, a DNA amplification-based diagnostic test was developed utilizing the polymerase chain reaction (PCR) and oligonucleotide primers specific for the OspA and OspB genes of Borrelia burgdorferi. In this approach, a relatively large DNA fragment is amplified with an outer set of primers, and a "nested" internal sequence of the PCR product subsequently reamplified with an inner set of primers. This nested approach coupled with simple differential centrifugation allowed specific detection of as few as four B. burgdorferi organisms mixed in 2 ml of blood. This methodology was utilized on patients' samples, and it allowed detection of B. burgdorferi in the peripheral blood and urine of several individuals with clinical evidence of Lyme borreliosis. PCR became negative and symptoms improved following antibiotic therapy of treated individuals. These studies suggest that direct detection of Borrelia in infected individuals can aid in diagnosis and evaluation of therapy for Lyme borreliosis.     

环介导恒温扩增对莱姆病病原伯氏疏螺旋体的分型鉴定

使用环介导恒温扩增技术,基于莱姆病病原伯氏疏螺旋体的外膜蛋白A(OspA)基因,针对伯氏疏螺旋体不同的基因型设计特异性引物,对国内主要的莱姆病病原伯氏疏螺旋体的3个基因型进行分型鉴定。研究结果表明,设计的引物具有良好的特异性,可以对狭义伯氏疏螺旋体(Borrelia burgdorferi sensu strict)、嘎氏疏螺旋体(B.afzelii)和伽氏疏螺旋体(B.garinii)进行分型鉴定。伯氏疏螺旋体的分型鉴定可以对不同临床症状莱姆病患者的治疗和莱姆病的控制提供一定的依据。