Molecular Mechanisms Involved in Vascular Interactions of the Lyme Disease Pathogen in a Living Host

Hematogenous dissemination is important for infection by many bacterial pathogens, but is poorly understood because of the inability to directly observe this process in living hosts at the single cell level. All disseminating pathogens must tether to the host endothelium despite significant shear forces caused by blood flow. However, the molecules that mediate tethering interactions have not been identified for any bacterial pathogen except E. coli, which tethers to host cells via a specialized pillus structure that is not found in many pathogens. Furthermore, the mechanisms underlying tethering have never been examined in living hosts. We recently engineered a fluorescent strain of Borrelia burgdorferi, the Lyme disease pathogen, and visualized its dissemination from the microvasculature of living mice using intravital microscopy. We found that dissemination was a multistage process that included tethering, dragging, stationary adhesion and extravasation. In the study described here, we used quantitative real-time intravital microscopy to investigate the mechanistic features of the vascular interaction stage of B. burgdorferi dissemination. We found that tethering and dragging interactions were mechanistically distinct from stationary adhesion, and constituted the rate-limiting initiation step of microvascular interactions. Surprisingly, initiation was mediated by host Fn and GAGs, and the Fn- and GAG-interacting B. burgdorferi protein BBK32. Initiation was also strongly inhibited by the low molecular weight clinical heparin dalteparin. These findings indicate that the initiation of spirochete microvascular interactions is dependent on host ligands known to interact in vitro with numerous other bacterial pathogens. This conclusion raises the intriguing possibility that fibronectin and GAG interactions might be a general feature of hematogenous dissemination by other pathogens

Kinetic profiles of signaling pathway activation in response to TLR2 ligands in BMDMs.

<p>Primary bone marrow derived macrophages (BMDMs), isolated from either wildtype or TLR2-/- mice, treated with LPS (100 ng/mL), LTA (1 μg/mL), R-FSL-1 (100 ng/mL, R), S-FSL-1 (100 ng/mL, S), Pam2CSK4 (100 ng/mL, P2), Pam3CSK4 (100 ng/mL, P3) for 0, 5, 15, 30,45, or 60 minutes were used to perform Western blot analysis of A) phospho-SAPK/JNK activation signal and total-SAPK/JNK protein amounts, B) Phospho-p38 activation signal and total-p38 protein amounts, and C) total IκBα protein amounts. In D) RT-PCR was performed on mRNA isolated from wildtype or TLR2-/- bone marrow derived macrophage treated with each ligand for 4 hours and the TLR2-dependence of cell activation leading to TNFα mRNA transcription is shown. All results are representative of n = 3 independent experiments.</p

Evaluation of the in vivo pro-inflammatory capacity of distinct TLR2 ligands.

<p>Wildtype, male, C57/B6 mice were given intrascrotal injections of either saline alone, or saline containing TNFα (20 ng/g), LPS (10 ng/g), Pam3CSK4 (5 ng/g), Pam2CSK4 (5 ng/g), S-FSL1 (5 ng/g, S), R-FSL1 (5 ng/g, R), LTA (5 or 25 ng/g). After 4 hours the cremaster muscle was exteriorized and the degree of leukocyte A) adhesion and B) emigration was measured. In C) total circulating leukocytes were counted in the blood. In D) the relative percentages of lymphocytes (Ly), monocytes (M), and neutrophils (N) within the post-capillary venules were determined from haematoxylin and eosin stained cremaster tissue sections. All results are representative of n≥3 independent experiments. (*** p<0.001).</p