Strain Variation of Lyme Disease Spirochetes Isolated from Ixodes ricinus Ticks and Rodents Collected in Two Endemic Areas in Switzerland

The relationship among Lyme borreliosis, Borrelia genospecies, rodent reservoirs, and Ixodes ricinus L. ticks was studied in two endemic areas in Switzerland. Ear punch biopsies and sampling of internal organs were used to isolate Borrelia hurgdorferi (Johnson, Schmid, Hyde, Steigerwalt and Brenner) from small mammals, Apodemus sylvaticus L., A. flavicollis Melchior, Clethrionomys glareolus Schreber. Spirochetes were isolated from ear tissue and spleen of the rodents. Isolates were homogeneous and belonged to typing group II identified as B. afzelii (Canica, Nato, du Merle, Mazie, Baranton and Postic). Our data show that a specific association exists between B. afzelii and rodent reservoirs in European foci. Borreliae were also isolated from field-collected I. ricinus ticks from the same study areas. Proteinic and antigenic analysis indicated that more than one genospecies were present in the tick population. This suggests that other vertebrate hosts may serve as reservoirs of other Borrelia genospecies implicated with Lyme diseas

Contribution of the Infection-Associated Complement Regulator-Acquiring Surface Protein 4 (ErpC) to Complement Resistance of \u3cem\u3eBorrelia Burgdorferi\u3c/em\u3e

Borrelia burgdorferi evades complement-mediated killing by interacting with complement regulators through distinct complement regulator-acquiring surface proteins (CRASPs). Here, we extend our analyses to the contribution of CRASP-4 in mediating complement resistance of B. burgdorferi and its interaction with human complement regulators. CRASP-4 (also known as ErpC) was immobilized onto magnetic beads and used to capture proteins from human serum. Following Western blotting, factor H (CFH), CFH-related protein 1 (CFHR1), CFHR2, and CFHR5 were identified as ligands of CRASP-4. To analyze the impact of native CRASP-4 on mediating survival of serum-sensitive cells in human serum, a B. garinii strain was generated that ectopically expresses CRASP-4. CRASP-4-producing bacteria bound CFHR1, CFHR2, and CFHR5 but not CFH. In addition, transformed spirochetes deposited significant amounts of lethal complement components on their surface and were susceptible to human serum, thus indicating that CRASP-4 plays a subordinate role in complement resistance of B. burgdorferi

Contribution of the Infection-Associated Complement Regulator-Acquiring Surface Protein 4 (ErpC) to Complement Resistance of Borrelia burgdorferi

Borrelia burgdorferi evades complement-mediated killing by interacting with complement regulators through distinct complement regulator-acquiring surface proteins (CRASPs). Here, we extend our analyses to the contribution of CRASP-4 in mediating complement resistance of B. burgdorferi and its interaction with human complement regulators. CRASP-4 (also known as ErpC) was immobilized onto magnetic beads and used to capture proteins from human serum. Following Western blotting, factor H (CFH), CFH-related protein 1 (CFHR1), CFHR2, and CFHR5 were identified as ligands of CRASP-4. To analyze the impact of native CRASP-4 on mediating survival of serum-sensitive cells in human serum, a B. garinii strain was generated that ectopically expresses CRASP-4. CRASP-4-producing bacteria bound CFHR1, CFHR2, and CFHR5 but not CFH. In addition, transformed spirochetes deposited significant amounts of lethal complement components on their surface and were susceptible to human serum, thus indicating that CRASP-4 plays a subordinate role in complement resistance of B. burgdorferi

Apoptotic pathways are selectively activated by granzyme A and/or granzyme B in CTL-mediated target cell lysis

Purified cytolytic T lymphocyte (CTL) proteases granzyme (gzm)A and gzmB with sublytic dose of perforin (perf) initiate distinct proapoptotic pathways. Their physiological relevance in CTL-mediated target cell apoptosis is elusive. Using ex vivo virus-immune CD8+ T cells from mice deficient in perf, gzmA and/or gzmB, and the Fas-resistant EL4.F15 tumor target cell, we show that (a) CTL from gzmA−/− or gzmB−/− mice similarly induced early proapoptotic features, such as phosphatidyl serine (PS) exposure on plasma membrane, ΔΨm loss, and reactive oxygen radical generation, though with distinct kinetics; (b) CTL from gzmA−/− but not from gzmB−/− mice activate caspase 3 and 9; (c) PS exposure induced by CTL from gzmA−/− or gzmB−/− mice is prevented, respectively, by caspase inhibitors or by reactive oxygen scavengers without interfering with target cell death; and (d) all gzm-induced apoptotic features analyzed depend critically on perf. Thus, perf is the principal regulator in CTL-mediated and gzm-facilitated intracellular processes. The ability of gzmA and gzmB to induce multiple independent cell death pathways may be the hosts response to circumvent evasion strategies of pathogens and tumors

Human Complement Regulators C4b-Binding Protein and C1 Esterase Inhibitor Interact with a Novel Outer Surface Protein of Borrelia recurrentis

The spirochete Borrelia recurrentis is the causal agent of louse-borne relapsing fever and is transmitted to humans by the infected body louse Pediculus humanus. We have recently demonstrated that the B. recurrentis surface receptor, HcpA, specifically binds factor H, the regulator of the alternative pathway of complement activation, thereby inhibiting complement mediated bacteriolysis. Here, we show that B. recurrentis spirochetes express another potential outer membrane lipoprotein, termed CihC, and acquire C4b-binding protein (C4bp) and human C1 esterase inhibitor (C1-Inh), the major inhibitors of the classical and lectin pathway of complement activation. A highly homologous receptor for C4bp was also found in the African tick-borne relapsing fever spirochete B. duttonii. Upon its binding to B. recurrentis or recombinant CihC, C4bp retains its functional potential, i.e. facilitating the factor I-mediated degradation of C4b. The additional finding that ectopic expression of CihC in serum sensitive B. burgdorferi significantly increased spirochetal resistance against human complement suggests this receptor to substantially contribute, together with other known strategies, to immune evasion of B. recurrentis

First insights in the variability of Borrelia recurrentis genomes

Background: Borrelia recurrentis is the causative agent of louse-borne relapsing fever, endemic to the Horn of Africa. New attention was raised in Europe, with the highest number of cases (n = 45) reported among migrants in 2015 in Germany and sporadically from other European countries. So far only one genome was sequenced, hindering the development of specific molecular diagnostic and typing tools. Here we report on modified culture conditions for B. recurrentis and the intraspecies genome variability of six isolates isolated and cultured in different years in order to explore the possibility to identify new targets for typing and examine the molecular epidemiology of the pathogen. Methodology/Principal findings: Two historical isolates from Ethiopia and four isolates from migrants from Somalia (n = 3) and Ethiopia (n = 1) obtained in 2015 were cultured in MPK-medium supplemented with 50% foetal calf serum. Whole DNA was sequenced using Illumina MiSeq technology and analysed using the CLC Genomics Workbench and SPAdes de novo assembler. Compared to the reference B. recurrentis A1 29-38 SNPs were identified in the genome distributed on the chromosome and plasmids. In addition to that, plasmids of differing length, compared to the available reference genome were identified. Conclusions/Significance: The observed low genetic variability of B. recurrentis isolates is possibly due to the adaptation to a very conserved vector-host (louse-human) cycle, or influenced by the fastidious nature of the pathogen and their resistance to in vitro growth. Nevertheless, isolates obtained in 2015 were bearing the same chromosomal SNPs and could be distinguished from the historical isolates by means of whole genome sequencing, but not hitherto used typing methods. This is the first study examining the molecular epidemiology of B. recurrentis and provides the necessary background for the development of better diagnostic tools

Direct Toll-like receptor 2 mediated co-stimulation of T cells in the mouse system as a basis for chronic inflammatory joint disease

The pathogenesis of chronic inflammatory joint diseases such as adult and juvenile rheumatoid arthritis and Lyme arthritis is still poorly understood. Central to the various hypotheses in this respect is the notable involvement of T and B cells. Here we develop the premise that the nominal antigen-independent, polyclonal activation of preactivated T cells via Toll-like receptor (TLR)-2 has a pivotal role in the initiation and perpetuation of pathogen-induced chronic inflammatory joint disease. We support this with the following evidence. Both naive and effector T cells express TLR-2. A prototypic lipoprotein, Lip-OspA, from the etiological agent of Lyme disease, namely Borrelia burgdorferi, but not its delipidated form or lipopolysaccharide, was able to provide direct antigen-nonspecific co-stimulatory signals to both antigen-sensitized naive T cells and cytotoxic T lymphocyte (CTL) lines via TLR-2. Lip-OspA induced the proliferation and interferon (IFN)-γ secretion of purified, anti-CD3-sensitized, naive T cells from C57BL/6 mice but not from TLR-2-deficient mice. Induction of proliferation and IFN-γ secretion of CTL lines by Lip-OspA was independent of T cell receptor (TCR) engagement but was considerably enhanced after suboptimal TCR activation and was inhibitable by monoclonal antibodies against TLR-2