Conserved lysine residues in decorin binding proteins of Borrelia garinii are critical in adhesion to human brain microvascular endothelial cells.

Lyme borreliosis is a tick-borne disease caused by Borrelia burgdorferi sensu lato spirochetes (Lyme borreliae). When the disease affects the central nervous system, it is referred to as neuroborreliosis. In Europe, neuroborreliosis is most often caused by Borrelia garinii. Although it is known that in the host Lyme borreliae spread from the tick bite site to distant tissues via the blood vasculature, the adherence of Lyme borreliae to human brain microvascular endothelial cells has not been studied before. Decorin binding proteins are adhesins expressed on Lyme borreliae. They mediate the adhesion of Lyme borreliae to decorin and biglycan, and the lysine residues located in the binding site of decorin binding proteins are important to the binding activity. In this study, we show that lysine residues located in the canonical binding site can also be found in decorin binding proteins of Borrelia garinii, and that these lysines contribute to biglycan and decorin binding. Most importantly, we show that the lysine residues are crucial for the binding of Lyme borreliae to decorin and biglycan expressing human brain microvascular endothelial cells, which in turn suggests that they are involved in the pathogenesis of neuroborreliosis.

Borrelia burgdorferi Infection in Biglycan Knockout Mice.

BACKGROUND: Borrelia burgdorferi sensu lato spirochetes (Borrelia) causing Lyme borreliosis are able to disseminate from the initial entry site to distant organs in the host. Outer-surface adhesins are crucial in the bacterial dissemination and adhesion to various tissues. Two well-characterized Borrelia adhesins, decorin-binding proteins A and B, have been shown to bind to 2 host receptors, decorin and biglycan. However, the role of biglycan in Borrelia infection has not been characterized in vivo. METHODS: We infected biglycan knockout (KO) and wild-type (WT) C3H mice with strains representing 3 Borrelia genospecies, Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii. The infection was monitored by measuring joint swelling, Borrelia culture, polymerase chain reaction analysis, and serologic analysis. The host immune responses were analyzed by histological scoring of the inflammation in tissues and by cytokine profiling. RESULTS: B. burgdorferi sensu stricto and B. garinii established long-term infection in mice of both genotypes, while B. afzelii failed to disseminate in KO mice. Further, the B. burgdorferi sensu stricto-infected KO mice had persistent inflammation in the joints. CONCLUSIONS: The dissemination and tissue colonization of Borrelia and the inflammatory response of the host differ in a mouse biglycan expression- and Borrelia genospecies-dependent manner.

Borrelia afzelii alters reproductive success in a rodent host.

The impact of a pathogen on the fitness and behaviour of its natural host depends upon the host-parasite relationship in a given set of environmental conditions. Here, we experimentally investigated the effects of Borrelia afzelii, one of the aetiological agents of Lyme disease in humans, on the fitness of its natural rodent host, the bank vole (Myodes glareolus), in semi-natural conditions with two contrasting host population densities. Our results show that B. afzelii can modify the reproductive success and spacing behaviour of its rodent host, whereas host survival was not affected. Infection impaired the breeding probability of large bank voles. Reproduction was hastened in infected females without alteration of the offspring size at birth. At low density, infected males produced fewer offspring, fertilized fewer females and had lower mobility than uninfected individuals. Meanwhile, the infection did not affect the proportion of offspring produced or the proportion of mating partner in female bank voles. Our study is the first to show that B. afzelii infection alters the reproductive success of the natural host. The effects observed could reflect the sickness behaviour due to the infection or they could be a consequence of a manipulation of the host behaviour by the bacteria.

Flow-Tolerant Adhesion of a Bacterial Pathogen to Human Endothelial Cells Through Interaction With Biglycan.

BACKGROUND: Bacterial pathogens causing systemic infections disseminate from the initial infection focus to the target organs usually through the blood vasculature. To be able to colonize various organs, bacteria need to adhere to the endothelial cells of the vascular wall, and the adhesion must be strong enough to resist the shear force of the blood flow.Borrelia burgdorferi sensu lato spirochetes, the causative agents of the tick-borne disease Lyme borreliosis, disseminate hematogenously from the tick bite site to the joints, the heart, and the central nervous system of the patient. METHODS: We used both wild-type and genetically modified B. burgdorferi s. l. bacteria, recombinant borrelia adhesins, and an array of adhesion assays carried out both under stationary and flow conditions to investigate the molecular mechanisms of borrelial adhesion to human endothelial cells. RESULTS: Borrelia garinii, a member of the B. burgdorferi s. l. complex, adhered to biglycan expressed by human endothelial cells in a flow-tolerant manner. The adhesion was mediated by the decorin-binding protein A (DbpA) and DbpB surface molecules of B. garinii. CONCLUSIONS: The proteoglycan biglycan is a receptor molecule for flow-resistant adhesion of the bacterial pathogen B. garinii on human endothelial cells.

CXCL13 and neopterin concentrations in cerebrospinal fluid of patients with Lyme neuroborreliosis and other diseases that cause neuroinflammation.

BACKGROUND: Laboratory diagnosis of Lyme neuroborreliosis (LNB) is partly based on the detection of intrathecal Borrelia burgdorferi-specific antibody production (increased antibody index (AI)). However, AI can be negative in patients with early LNB and, conversely, can remain elevated for months after antibiotic treatment. Recent studies suggested that the chemokine CXCL13 in the cerebrospinal fluid (CSF) is a biomarker for active LNB. Also, CSF neopterin-level determination has been used to assess the degree of neuroinflammation in a wide variety of diseases. METHODS: CXCL13 concentrations were analyzed in CSF samples of 366 retrospectively identified individuals. The samples represented pretreatment LNB (38 patients), non-LNB comparison patients, tick-borne encephalitis, central nervous system (CNS) varicella zoster virus infection, CNS herpes simplex virus infection, CNS HHV6 infection, CNS enterovirus infection, and untreated neurosyphilis. The panel included also samples from patients with multiple sclerosis and other neuroinflammatory conditions. Of the LNB patients, 24 posttreatment CSF samples were available for CXCL13 analysis. Neopterin concentrations were determined in a subset of these samples. RESULTS: The CXCL13 concentrations in CSF samples of untreated LNB patients were significantly higher (median, 6,480 pg/ml) than the concentrations in the non-LNB group (median, <7.8 pg/ml), viral CNS infection samples (median, <7.8 pg/ml), or samples from patients with noninfectious neuroinflammatory conditions (median, <7.8 pg/ml). The use of cut-off 415 pg/ml led to a sensitivity of 100% and specificity of 99.7% for the diagnosis of LNB in these samples. CSF CXCL13 median concentrations declined significantly from 16,770 pg/ml before to 109 pg/ml after the treatment.CSF neopterin concentration was significantly higher among the untreated LNB patients than in the non-LNB group. The use of neopterin concentration 10.6 nM as the cut-off led to a sensitivity of 88.6% and a specificity of 65.0% for the diagnosis of LNB. The CSF neopterin concentrations decreased statistically significantly with the treatment. CONCLUSIONS: These results clearly indicate that highly elevated CSF CXCL13 levels are strongly associated with untreated LNB. CXCL13 outperformed neopterin and appears to be an excellent biomarker in differentiating LNB from viral CNS infections and from other neuroinflammatory conditions.

Decorin binding by DbpA and B of Borrelia garinii, Borrelia afzelii, and Borrelia burgdorferi sensu Stricto.

BACKGROUND: Decorin adherence is crucial in the pathogenesis of Lyme borreliosis. Decorin-binding proteins (Dbp) A and B are the adhesins that mediate this interaction. DbpA and B of Borrelia garinii, Borrelia afzelii, and Borrelia burgdorferi sensu stricto (ss) differ in their amino acid sequence, but little attention has been paid to the potential difference in their decorin binding. METHODS: We expressed recombinant DbpA and DbpB of B. garinii, B. afzelii, and B. burgdorferi ss and studied their binding to decorin. We also generated recombinant Borrelia strains to study the role of DbpA and DbpB in the adhesion of live spirochetes to decorin and decorin-expressing cells. RESULTS. Recombinant DbpA of B. garinii and DbpB of B. garinii and B. burgdorferi ss showed strong binding to decorin, whereas DbpA of B. burgdorferi ss and both DbpA and DbpB of B. afzelii exhibited no or only minor binding activity. DbpA and DbpB of B. garinii and B. burgdorferi ss also supported the adhesion of whole spirochetes to decorin and decorin-expressing cells, whereas DbpA and DbpB of B. afzelii did not exhibit this activity. CONCLUSIONS: Dbp A and B of B. garinii and B. burgdorferi ss mediate the interaction between the spirochete and decorin, whereas the same adhesins of B. afzelii show only negligible activity.

Nanomechanical mechanisms of Lyme disease spirochete motility enhancement in extracellular matrix.

As opposed to pathogens passively circulating in the body fluids of their host, pathogenic species within the Spirochetes phylum are able to actively coordinate their movement in the host to cause systemic infections. Based on the unique morphology and high motility of spirochetes, we hypothesized that their surface adhesive molecules might be suitably adapted to aid in their dissemination strategies. Designing a system that mimics natural environmental signals, which many spirochetes face during their infectious cycle, we observed that a subset of their surface proteins, particularly Decorin binding protein (Dbp) A/B, can strongly enhance the motility of spirochetes in the extracellular matrix of the host. Using single-molecule force spectroscopy, we disentangled the mechanistic details of DbpA/B and decorin/laminin interactions. Our results show that spirochetes are able to leverage a wide variety of adhesion strategies through force-tuning transient molecular binding to extracellular matrix components, which concertedly enhance spirochetal dissemination through the host.

Preperitoneal Fat Grafting Inhibits the Formation of Intra-abdominal Adhesions in Mice.

BACKGROUND: Adhesion formation contributes to postoperative complications in abdominal and gynaecological surgery. Thus far, the prevention and treatment strategies have focused on mechanical barriers in solid and liquid form, but these methods are not in routine use. As autologous fat grafting has become popular in treatment of hypertrophic scars because of its immunomodulatory effects, we postulated that fat grafting could also prevent peritoneal adhesion through similar mechanisms. METHODS: This was a control versus intervention study to evaluate the effect of fat grafting in the prevention on peritoneal adhesion formation. An experimental mouse model for moderate and extensive peritoneal adhesions was used (n = 4-6 mice/group). Adhesions were induced mechanically, and a free epididymal fat graft from wild type or CAG-DsRed mice was injected preperitoneally immediately after adhesion induction. PET/CT imaging and scaling of the adhesions were performed, and samples were taken for further analysis at 7 and 30 days postoperation. Macrophage phenotyping was further performed from peritoneal lavage samples, and the expression of inflammatory cytokines and mesothelial layer recovery were analysed from peritoneal tissue samples. RESULTS: Fat grafting significantly inhibited the formation of adhesions. PET/CT results did not show prolonged inflammation in any of the groups. While the expression of anti-inflammatory and anti-fibrotic IL-10 was significantly increased in the peritoneum of the fat graft-treated group at 7 days, tissue-resident and repairing M2 macrophages could no longer be detected in the fat graft at this time point. The percentage of the continuous, healed peritoneum as shown by Keratin 8 staining was greater in the fat graft-treated group after 7 days. CONCLUSIONS: Fat grafting can inhibit the formation of peritoneal adhesions in mice. Our results suggest that fat grafting promotes the peritoneal healing process in a paracrine manner thereby enabling rapid regeneration of the peritoneal mesothelial cell layer.

Decorin binding proteins of Borrelia burgdorferi promote arthritis development and joint specific post-treatment DNA persistence in mice.

Decorin binding proteins A and B (DbpA and B) of Borrelia burgdorferi are of critical importance for the virulence of the spirochete. The objective of the present study was to further clarify the contribution of DbpA and B to development of arthritis and persistence of B. burgdorferi after antibiotic treatment in a murine model of Lyme borreliosis. With that goal, mice were infected with B. burgdorferi strains expressing either DbpA or DbpB, or both DbpA and B, or with a strain lacking the adhesins. Arthritis development was monitored up to 15 weeks after infection, and bacterial persistence was studied after ceftriaxone and immunosuppressive treatments. Mice infected with the B. burgdorferi strain expressing both DbpA and B developed an early and prominent joint swelling. In contrast, while strains that expressed DbpA or B alone, or the strain that was DbpA and B deficient, were able to colonize mouse joints, they caused only negligible joint manifestations. Ceftriaxone treatment at two or six weeks of infection totally abolished joint swelling, and all ceftriaxone treated mice were B. burgdorferi culture negative. Antibiotic treated mice, which were immunosuppressed by anti-TNF-alpha, remained culture negative. Importantly, among ceftriaxone treated mice, B. burgdorferi DNA was detected by PCR uniformly in joint samples of mice infected with DbpA and B expressing bacteria, while this was not observed in mice infected with the DbpA and B deficient strain. In conclusion, these results show that both DbpA and B adhesins are crucial for early and prominent arthritis development in mice. Also, post-treatment borrelial DNA persistence appears to be dependent on the expression of DbpA and B on B. burgdorferi surface. Results of the immunosuppression studies suggest that the persisting material in the joints of antibiotic treated mice is DNA or DNA containing remnants rather than live bacteria.