Interaction of Variable Bacterial Outer Membrane Lipoproteins with Brain Endothelium

Abstract

BackgroundPreviously we reported that the variable outer membrane lipoprotein Vsp1 from the relapsing fever spirochete Borrelia turicatae disseminates from blood to brain better than the closely related Vsp2 [1]. Here we studied the interaction between Vsp1 and Vsp2 with brain endothelium in more detail.Methodology/Principal FindingsWe compared Vsp1 to Vsp2 using human brain microvascular endothelial cell (HBMEC) association assays with aminoacid radiolabeled Vsp-expressing clones of recombinant Borrelia burgdorferi and lanthanide-labeled purified lipidated Vsp1 (LVsp1) and Vsp2 (LVsp2) and inoculations of the lanthanide-labeled proteins into mice. The results showed that heterologous expression of LVsp1 or LVsp2 in B. burgdorferi increased its association with HBMEC to a similar degree. Purified lanthanide-labeled lipidated Vsp1 (LVsp1) and LVsp2 by themselves were capable of associating with HBMEC. The association of LVsp1 with brain endothelium was time-dependent, saturable, and required the lipidation. The association of Vsp1 with HBMEC was inhibited by incubation at lower temperature or with excess unlabeled LVsp1 or LVsp2 but not with excess rVsp1 or mouse albumin or an anti Vsp1 monoclonal antibody. The association of LVsp2 with HBMEC and its movement from blood to brain parenchyma significantly increased in the presence of LVsp1.Conclusions/SignificanceVariable bacterial outer membrane lipoproteins interact with brain endothelium differently; the lipidation and variable features at the protein dome region are key modulators of this interaction.

IL-10 Prevents Apoptosis of Brain Endothelium during Bacteremia

“…Another limitation of the current study is that most experiments were done with a single bacterial species, the Oz1 strain of B. turicatae. Further studies are necessary to investigate the susceptibility of brain endothelial cells to apoptosis with other bacterial species and with isogenic serotypes within the same species (25,32). The finding that incubation with purified outer membrane lipoprotein was sufficient to cause apoptosis of HBMEC indicates that lipoprotein recognition receptors are likely involved in signaling apoptosis in brain endothelial cells.…”

“…For studies of brain endothelial cells we used SV-40-transformed HBMEC; HBMEC were chosen because they have been found to express features of blood-brain barrier endothelial cells and their interaction with bacterial and viral pathogens have been studied extensively (12,(21)(22)(23)(24)(25). HBMEC passages 3-5 were cultured in RPMI 1640 medium (Sigma-Aldrich), supplemented with 10% NuSerum (BD Biosciences, Mountain View, CA), 10% FBS (Sigma-Aldrich), MEM vitamins (BioWhittaker), MEM nonessential amino acids (Sigma-Aldrich), 1 mM sodium pyruvate (BioWhittaker), 2 mM L-glutamine (Sigma-Aldrich), 30 mg/ml endothelial growth supplement (Sigma-Aldrich), and 5 U/ml heparin (Sigma-Aldrich) at 37˚C in 5% CO 2 as described previously (12).…”

“…Variable surface lipoprotein 2 (LVsp2), the major outer membrane lipoproteins of Bt2, was prepared by detergent extraction, followed by purification as described previously (9,14,25,28). Briefly, suspensions containing ∼5 3 10 9 spirochetes were centrifuged at 8000 3 g for 20 min, washed in 30 ml Tris-EDTA buffer (50 mM Tris [pH 7.6] and 5 mM EDTA), and centrifuged a second time, and the pellet was resuspended in 20 ml lysis and solubilization solution consisting of 2% octylglucopyranoside (O8001; Sigma-Aldrich) and 50 mM DTT in Tris-NaCl-EDTA buffer (50 mM Tris [pH 7.6], 150 mM NaCl, and 5 mM EDTA) (28).…”

Surface Localization Determinants of Borrelia OspC/Vsp Family Lipoproteins

“…They contribute to chronic infection of mammalian hosts by participating in an elaborate scheme of multiphasic antigenic variation designed to repeatedly evade the host's immune response (5). Vsps have also been shown to be the determinants of B. turicatae tissue tropism (14,15,22,48,49) and may enhance invasion of tissues by binding to glycosaminoglycans (36).Our previous investigations into the secretion of the major Borrelia surface lipoproteins led to some intriguing discoveries. We first noticed that any known OM lipoprotein secretion modules, i.e., LolB, type II or type V systems, were missing from Borrelia genomes.…”

“…They contribute to chronic infection of mammalian hosts by participating in an elaborate scheme of multiphasic antigenic variation designed to repeatedly evade the host's immune response (5). Vsps have also been shown to be the determinants of B. turicatae tissue tropism (14,15,22,48,49) and may enhance invasion of tissues by binding to glycosaminoglycans (36).…”