Expression of Endothelial Cell Adhesion Molecules in Joints and Heart during Borrelia burgdorferi Infection of Mice

The expression of adhesion molecules on endothelia was examined during chronic arthritis and carditis in SCID and immunocompetent susceptible AKR/N mice infected with Borrelia burgdorferi (B. burgdorferi). All stages of disease were associated with the upregulation or new expression of ICAM-1 and P-selectin and of VCAM-1 and E-selectin, respectively, on blood vessels of affected joint tissues of SCID and AKR/N mice as well as on heart tissue of SCID mice but not in other tissues. Moreover, ICAM-1 was also found on infiltrating mononuclear cells. The overall staining intensity for each of the four adhesion molecules on individual tissue sections of joint and heart increased with time of infection and was associated with the presence of spirochetes in the tissue. In addition it is shown that in both mouse strains inflammation of joints but not heart is accompanied by vascular proliferation. Synovial but not heart tissues of infected SCID mice were found to express both, peripheral-(PNAd) and mucosal (MAdCAM-1) lymph node high endothelia venule associated vascular addressins as detected by mAb Meca-79 and Meca-367, respectively, but only at later stages of the disease and only on newly generated small venules. However, neither of the two addressins were evident in synovial lesions of AKR/N mice. Together the data suggest that the concomittant induction of ICAM-1, VCAM-1, E-selectin and P-selectin in lesions of infected mice provide a means for enhanced cellular infiltration into affected organs and that the regulation of these structures is conserved in the absence of a functional immune system. Furthermore, the differential induction of vascular proliferation in joint and heart tissues as well as the restricted expression patterns of vascular addressins indicate that the pathogenetic processes induced by B. burgdorferi are distinct for joint and heart.     

First molecular detection of Borrelia afzelii in clinical samples in Korea

Borrelia afzelii nucleic acids were detected in the sera of febrile disease patients by a nested PCR that targeted the rrf (5S)-rrl (23S) spacer of B. burgdorferi sensu lato. The B. afzelii-specific DNA fragment was detected in 8 out of 283 sera which were proven to have immunoglobulin G or M antibodies against B. burgdorferi antigens through IFA. The results were further confirmed through restriction fragment length polymorphism and sequencing analysis of the DNA fragments. The results indicated for the first time that Lyme borreliosis is prevalent in Korea.     

Borrelia burgdorferi potently activates bone marrow-derived conventional dendritic cells for production of IL-23 required for IL-17 release by T cells

Lyme borreliosis is characterized by cellular inflammatory responses at multiple body sites. Recently, an association of interleukin-17 (IL-17) and Lyme arthritis was suggested. In this context, it is of special interest that the heterodimeric cytokine IL-23 can act on T cells and initiate the up-regulation of effector cytokines such as IL-17. To determine the role of this specific cytokine cascade for the induction of subsequently induced proinflammatory events we developed an in vitro system to investigate the IL-23-inducing capacity of Borrelia burgdorferi and the potential of the spirochete for inducing the IL-23/IL-17 axis. We used cells derived from mice deficient for IL-23 or IL-12 only or deficient for both IL-12 and IL-23 to define precisely the function of these cytokines. Experiments with bone marrow-derived dendritic cells (BMDC) identified these cells as sources for IL-23 but not for IL-12 after B. burgdorferi exposure. Subsequent investigations with T cell-depleted splenocyte fractions revealed a tight IL-23/IL-17 axis in response to the spirochetes. Monoclonal antibodies that block IL-23 showed further that BMDC-derived IL-23 was required for production of IL-17 in this experimental model. These in vitro data describing a spirochete-induced release of IL-23 may help to define IL-17-dependent inflammatory responses in the disease.     

Borrelia burgdorferi protein interactions critical for microbial persistence in mammals

Borrelia burgdorferi is the causative agent of Lyme disease that persists in a complex enzootic life cycle, involving Ixodes ticks and vertebrate hosts. The microbe invades ticks and vertebrate hosts in spite of active immune surveillance and potent microbicidal responses, and establishes long‐term infection utilising mechanisms that are yet to be unravelled. The pathogen can cause multi‐system disorders when transmitted to susceptible mammalian hosts, including in humans. In the past decades, several studies identified a limited number of B. burgdorferi gene‐products critical for pathogen persistence, transmission between the vectors and the host, and host–pathogen interactions. This review will focus on the interactions between B. burgdorferi proteins, as well as between microbial proteins and host components, protein and non‐protein components, highlighting their roles in pathogen persistence in the mammalian host. A better understanding of the contributions of protein interactions in the microbial virulence and persistence of B. burgdorferi would support development of novel therapeutics against the infection.     

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.     

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 uridine kinase: an enzyme of the pyrimidine salvage pathway for endogenous use of nucleotides

The 621 bp udk gene encoding Borrelia burgdorferi potential uridine kinase, involved in the pyrimidine salvage pathway, was cloned and sequenced. The B. burgdorferi protein has a molecular mass of 24 kDa in sodium dodecyl sulfate-polyacrylamide gel. The N-terminal sequence of the protein, Ala-Lys-Ile-Ile, is identical to that predicted but lacks N-terminal methionine. udk is located at around 15 kb from the left telomere and forms an operon with an upstream ORF. A likely hypothesis for the role of the pyrimidine salvage pathway is the sole use of endogenous nucleotides for Borrelia.     

The Lon-2 protease of Borrelia burgdorferi is critical for infection in the mammalian host

Borrelia burgdorferi encodes a functional homolog of canonical Lon protease termed Lon-2. To date, the contribution of Lon-2 to B. burgdorferi fitness and infection remains unexplored. Herein, we showed that expression of lon-2 was highly induced during animal infection, suggesting that Lon-2 is important for B. burgdorferi infection. We further generated a lon-2 deletion mutant. Compared with that of wild-type (WT) strain, the infectivity of the mutant was severely attenuated in a murine infection model. Although no growth defect was observed for the mutant in normal BSK-II medium, resistance of the lon-2 mutant to osmotic stress was markedly reduced. In addition, when exposed to tert-Butyl hydroperoxide, survival of the lon-2 mutant was impaired. In addition, we found that the protein levels of RpoS and RpoS-dependent OspC were decreased in the mutant. All these phenotypes were restored to WT or near-WT levels when lon-2 mutation was complemented in cis. Taken together, these results demonstrate that Lon-2 is critical for B. burgdorferi to establish infection and to cope with environmental stresses. This study provides a foundation for further uncovering the direct link between the dual roles of Lon-2 in protein quality control and bacterial pathogenesis.     

Diversity of Lyme borreliosis spirochetes isolated from ticks in Serbia

Spirochetes from the Borrelia burgdorferi sensu lato (s.l.). (Spirochaetales: Spirochaetaceae) species complex, including the causative agents of Lyme borreliosis, have been isolated from ticks, vertebrate reservoirs and humans. Previous analyses based on direct molecular detection in ticks indicated a considerable diversity of B. burgdorferi s.l. complex in Serbia. The present study aimed (a) to isolate borrelia strains from Serbia; (b) to determine their genotypic characteristics; and (c) to establish a collection of viable B. burgdorferi s.l. strains for further biological, ecological and genetic studies. For the present study, 231 adult Ixodes ricinus (Ixodida: Ixodidae) ticks from 16 ecologically different localities in Serbia were individually processed to cultivate B. burgdorferi s.l. This led to the isolation of 36 strains. A hbb gene quantitative real‐time polymerase chain reaction (PCR) based on melting temperature determination and ospA gene sequencing were used to genotype the isolated spirochetes. The species identified based on the hbb gene real‐time PCR were: Borrelia lusitaniae (44.4%), Borrelia afzelii (36.1%), Borrelia garinii (13.9%) and Borrelia valaisiana (5.6%), whereas the ospA sequence analysis revealed the occurrence of Borrelia bavariensis. This is the first report of the isolation of B. lusitaniae, B. garinii, B. bavariensis and B. valaisiana strains in Serbia.     

Preliminary Observations on Ultrastructure of Borreliae in Tissues of Ixodes persulcatus

We observed Lyme borrelia by electron microscopy in the tissues of the ticks, Ixodes persulcatus, which were indicated positive for borreliae by BSK cultures of their internal organs. Borreliae (0.25 μm in diameter) were found only in the lumen of the midgut. They were closely associated with the microvilli on the midgut epithelium but never penetrated into the epithelial cells. Ultrastructural features common to Lyme borreliae., i.e., the three-layered membranes surrounding the cytoplasm and orientation of the flagella insertions, were obviously confirmed. The present results are useful to understand tick tissue-borrelia interface.