Carbohydrate utilization by the Lyme borreliosis spirochete, Borrelia burgdorferi

Growth kinetic analyses of Borrelia burgdorferi indicated that this bacterium can utilize a limited number of carbon sources for energy: the monosaccharides glucose, mannose, and N-acetylglucosamine, the disaccharides maltose and chitobiose, and glycerol. All of these carbohydrates are likely to be available to B. burgdorferi during infection of either vertebrate and arthropod hosts, enabling development of a model describing energy sources potentially used by the Lyme borreliosis spirochete during its natural infectious cycle.     

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.     

Relationship between infectivity and OspC expression in Lyme disease Borrelia

Abstract Borrelia burgdorferi sensu stricto strain 297 and B. garinii strains HP1 and 12–92 were serially subcultured for 36–50 passages in vitro for 1 year. All low-passage strains showed abundant expression of outer surface protein C (OspC) in the 22–23-kDa range, but the high-passage strains lost or showed reduced expression of OspC in comparison with the low-passage strains. The low-passage strains efficiently infected outbred ddY mice when inoculated into the hind footpad or peritoneal cavity. In contrast, the incidence of infection with the high-passage strains was low. Isolates from the bladders of mice inoculated with the high-passage strains expressed large amounts of OspC in comparison with those originally inoculated. These results indicate that OspC expression is related to the infectivity of Lyme disease borreliae.     

Problems of isolating Borrelia burgdorferi from ticks collected in United Kingdom foci of Lyme disease

Abstract. Many isolates of Borrelia burgdorferi have been obtained from ticks and vertebrate tissues collected in North America and continental Europe but only one established culture of United Kingdom Borrelia burgdorferi has been recorded. In this paper we report the isolation of B.burgdorferi from one of 108 tick pools representing 733 ticks and eighty-four tissue samples from twenty-six rodents collected in the U.K., and the subsequent failure to establish the isolate (from ticks collected in Fordingbridge) in culture. In contrast, using identical techniques and culture medium, B.burgdorferi was isolated from one of seven tick pools collected in Switzerland, and from a single pool of ticks collected in Slovakia, and both isolates were successfully passaged. Analysis of questing I.ricinus collected from Fordingbridge by direct immunofluorescence showed 6/32 (19%) of adults and 8/108 (7%) of nymphs were positive for B. burgdorferi , although only one nymph contained ≥ 1000 spirochaetes. To examine further the problem of isolating U.K. B.burgdorferi , twelve Ixodes ricinus tick samples from Fordingbridge, a recognized focus of Lyme disease, were subjected to isolation and culturing techniques, and the procedures monitored by use of the polymerase chain reaction (PCR). Whereas 11/12 samples were PCR positive after 2 weeks in culture, only one was PCR positive after 4 weeks. Motile spirochaetes were not visible by dark-field microscopy in any of the cultures. The results indicate that the standard BSK II medium routinely used to isolate and culture B. burgdorferi does not readily support the replication of the Borrelia species endemic to the U.K.     

Simplified method for the interpretation of immunoblots for Lyme borreliosis

Abstract Interpretation of immunoblot results for Lyme borreliosis remains an area of controversy. This study was undertaken to devise a simple method which incorporated distinctions between specific and non-specific bands. A numerical value was assigned to bands reflecting their relative importance. This scheme was evaluated with sera from 61 forestry workers, which were previously tested by immunoblotting and interpreted subjectively. These results were reported as five separate categories representing levels of reactivity. A total of 72% (44) of sera fell into the same categories as they were originally reported. The method was also assessed with 40 sera routinely submitted for Lyme borreliosis serology. These were interpreted by two members of staff without previous experience of immunoblotting. Their results gave 80% and 72.5% agreement with those of an experienced person used to reading immunoblots for Lyme borreliosis.     

A murine IgG1 monoclonal antibody thatbinds specifically to outer surface protein A of Lyme disease spirochete Borrelia burgdorferi

Abstract A murine monoclonal antibody, designated MA-2G9, directed against outer surface protein A (OspA) of the Lyme disease spirochete, Borrelia burgdorferi , has been produced. Antibody MA-2G9, IgG1 subclass, was purified by affinity chromatography on protein G Sepharose column and used for purification of OspA antigen from Borrelia burgdorferi cell lysate. Epitope specificity was studied by Western immunoblotting, using several strains of B. burgdorferi and non-Lyme disease bacteria such as Treponema pallidum and B. hermsii . The MA-2G9 monoclonal antibody reacted specifically with recombinant OspA aas well as with native OspA in sonicated B. burgdorferi strains. No reaction was observed with T. pallidum, Escherichia coli, Staphylococcus aureus and B. hermsii lysates. The MA-2G9 antibody also recognized the denatured form of OspA indicating that it is directed against sequential epitope and not conformational epitope.     

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.     

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.     

Exploring a possibility of using Raman spectroscopy for detection of Lyme disease

Lyme disease (LD), one of the most prevalent tick-borne diseases in the United States (US), is caused by Borreliella burgdorferi sensu stricto (Bb). To date, in the US, LD diagnostics is primarily based on validated two-tiered serological testing, which overall exhibits low sensitivity among other drawbacks. In the present study, a potential of Raman spectroscopy (RS) to detect Bb infection in mice has been explored. For that, C3H mice were infected with wild-type Bb strains, 297, B31, or B31-derived mutant, ∆vlsE. Blood samples taken prior to and post Bb infection were subjected to RS. The data demonstrated that RS did not directly detect Bb spirochetes in blood, but rather sensed biochemical changes associated with Bb infection. Despite Bb infection-associated blood changes detectable by RS were very limited, the partial least square discriminant analysis showed that the average true positive rates were 86% for 297 and 89% for B31 and ∆vlsE.     

Lyme disease in transgenic mice expressing the Borrelia burgdorferi flagellin epitope implicated in human neuroborreliosis

Because of an association of human neuroborreliosis with the development of an antibody response against an antigen in neural tissue that cross-reacts with an epitope on the flagellin protein of Borrelia burgdorferi, C3H transgenic mice were created that expressed the flagellin epitope (amino acids 213–224) as a fusion protein with myelin basic protein. The transgenic mice expressed the flagellin epitope selectively in myelinated regions of the nervous system. Both transgenic and non-transgenic mice developed an antibody response to the flagellin epitope during B. burgdorferi infection and both developed arthritis and carditis. However, no lesions were found in the central nervous system of either type of mouse for up to 8 weeks after infection. The data indicate that expression of the flagellin 213–24 epitope in mice does not result in neurologic disease, suggesting that B. burgdorferi flagellin antibodies may not be directly implicated in neuroborreliosis.     

Borrelia burgdorferi infection in Ixodes ricinus from habitats in Denmark

A total of 2647 ticks of the genus Ixodes was sampled by flagging the vegetation in thirty-one sites in eastern Jutland, Denmark. All ticks were identified as Ixodes ricinus Linnaeus. A total of 317 ticks (202 nymphs and 115 adults) from three different sites were examined for the spirochaete Borrelia burgdorferi Johnson et al. by indirect fluorescent antibody staining; the frequency of infection varied from 7% to 22%. It is concluded that I. ricinus, known to be the most common tick in Denmark, is the vector largely responsible for the transmission of B. burgdorferi in this country.     

Ticks and Lyme borreliosis in an alpine area in northeast Italy

A 2‐year study was conducted in a mountainous area of northeast Italy to evaluate the occurrence and distribution of ticks, as well as to assess the prevalence of the spirochaete Borrelia burgdorferi sensu lato. All ticks collected were Ixodes ricinus L. (Parasitiformes: Ixodidae). In general, most nymphs and adult ticks were collected from April to July. Tick density was highly variable among sites; however, two areas with different infestation levels were recognized. Prevalences of B. burgdorferi s.l. in nymphal stages were rather variable between sites; overall the prevalence of infected nymphs in the whole area was slightly higher than 20%. The prevalence of B. burgdorferi s.l. in nymphs does not seem to be correlated with nymph density. The correlation between the incidence of Lyme borreliosis (reported human cases/1000 inhabitants/year) and Borrelia prevalence in nymphs was not significant, although a significant correlation was found between borreliosis incidence and nymph density.     

Molecular analysis of a 66-kDa protein associated with the outer membrane of Lyme disease Borrelia

Abstract A 66-kDa protein (p66) associated with the outer membrane of Lyme disease Borrelia was analysed at the molecular level. The chromosomal genes encoding p66 in B. burgdorferi B31, B. afzelii ACAI, and B. garinii Ip90 were sequenced. Database searches revealed that the p66 gene sequences were homologous to a previously reported gene fragment of unknown function. The deduced amino acid sequences of p66 in different Lyme disease borreliae were 92–94% identical and had no homologs in the databases. Proteolytic cleavage patterns of p66 and a computer-predicted single trans-membrane helix suggested the presence of surface-exposed epitopes on the C-terminus.     

Evidence for frequent OspC gene transfer between Borrelia valaisiana sp. nov. and other Lyme disease spirochetes

IMP-1 metallo-beta-lactamase is a transferable carbapenem-hydrolyzing enzyme found in some clinical isolates of Pseudomonas aeruginosa, Serratia marcescens and Klebsiella pneumoniae. Bacteria that express IMP-1 show significantly reduced sensitivity to carbapenems and other beta-lactam antibiotics. A series of thioester derivatives has been shown to competitively inhibit purified IMP-1. As substrates for IMP-1, the thioesters yielded thiol hydrolysis products which themselves were reversible competitive inhibitors. The thioesters also increased sensitivity to the carbapenem L-742,728 in an IMP-1-producing laboratory stain of Escherichia coli, but will need further modification to improve their activity in less permeable organisms such as Pseudomonas and Serratia. Nonetheless, the thioester IMP-1 inhibitors offer an encouraging start to overcoming metallo-beta-lactamase-mediated resistance in bacteria.     

A mutagenic PCR identifies isolates of Borrelia garinii responsible for Lyme borreliosis

Borrelia garinii is one of the three major Borreliae responsible for Lyme borreliosis in Europe. We have characterized a protein of B. garinii (VS102) and a genomic fragment from the gene encoding this protein was cloned. The DNA sequence of the fragment showed high homology with a known gene of B. burgdorferi sensu stricto. The protein encoded by this gene in B. burgdorferi sensu stricto is a phosphocarrier protein (histidine-containing protein). A mutation T to G polymorphism at codon 57 was found to be specific to B. garinii. A PCR-based approach that allows the rapid detection of this mutation made it possible to specifically discriminate B. garinii from other B. burgdorferi genospecies with high sensitivity and specificity.     

Borrelia burgdorferi possesses a collagenolytic activity

Abstract Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi , an invasive spirochete. B. burgdorferi has a predilection for collagenous tissue and one major clinical manifestation of the disease is arthritis. We have identified a collagenolytic activity in B. burgdorferi detergent lysates using iodinated gelatin as well as iodinated pepsinized human collagen types II and IV as protein substrates. In addition, we describe several proteolytic activities in B. burgdorferi with molecular masses greater than 200 kDa on sodium dodecyl sulfate polyacrylamide gels containing copolymerized gelatin. We propose that the collagenolytic activity of B. burgdorferi has a role in invasion, in the pathogenesis of Lyme arthritis, and perhaps also in other manifestations of Lyme borreliosis.     

Lyme disease transmission by severely impaired ticks

It has been demonstrated that impairing protein synthesis using drugs targeted against tRNA amino acid synthetases presents a promising strategy for the treatment of a wide variety of parasitic diseases, including malaria and toxoplasmosis. This is the first study evaluating tRNA synthetases as potential drug targets in ticks. RNAi knock-down of all tested tRNA synthetases had a strong deleterious phenotype on Ixodes ricinus feeding. Our data indicate that tRNA synthetases represent attractive, anti-tick targets warranting the design of selective inhibitors. Further, we tested whether these severely impaired ticks were capable of transmitting Borrelia afzelii spirochaetes. Interestingly, biologically handicapped I. ricinus nymphs transmitted B. afzelii in a manner quantitatively sufficient to develop a systemic infection in mice. These data suggest that initial blood-feeding, despite the incapability of ticks to fully feed and salivate, is sufficient for activating B. afzelii from a dormant to an infectious mode, enabling transmission and dissemination in host tissues.