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Interaction of variable bacterial outer membrane lipoproteins with brain endothelium.

Gandhi G, Londoño D, Whetstine CR, Sethi N, Kim KS, Zückert WR, Cadavid D - PLoS ONE (2010)

Bottom Line: The results showed that heterologous expression of LVsp1 or LVsp2 in B. burgdorferi increased its association with HBMEC to a similar degree.The association of LVsp2 with HBMEC and its movement from blood to brain parenchyma significantly increased in the presence of LVsp1.Variable 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Neuroscience and Center for the Study of Emerging Pathogens, UMDNJ-New Jersey Medical School, Newark, New Jersey, USA.

ABSTRACT

Background: Previously 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 findings: We 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/significance: Variable 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.

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Related in: MedlinePlus

Lipidated Vsp1 or Vsp2 inhibit the association of LVsp1 with brain endothelium non-specifically.1×105 HBMEC in suspension in Eppendorf tubes were incubated with 0.2 µg of Eu-LVsp1 or Eu-LVsp2 for 2 h at 37°C in the presence or absence of 10-fold excess unlabeled homologous or heterologous Vsp protein. Results are expressed as the mean ± SD ratio of counts per second (cps) of Eu associated to HBMEC relative to unbound Eu and represent data from at least two separate experiments using 8 wells per protein. Notice that the association of Eu-LVsp1 with HBMEC was blocked by 10-fold excess unlabeled LVsp1 (panel A) or LVsp2 (panel C) but not by 10-fold excess unlabeled rVsp1 or albumin (panel A). This did not occur for Eu-LVsp2 (Panel B). Also notice that excess unlabeled LVsp1 increased the association of Eu-LVsp2 with HBMEC (panel C).
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pone-0013257-g007: Lipidated Vsp1 or Vsp2 inhibit the association of LVsp1 with brain endothelium non-specifically.1×105 HBMEC in suspension in Eppendorf tubes were incubated with 0.2 µg of Eu-LVsp1 or Eu-LVsp2 for 2 h at 37°C in the presence or absence of 10-fold excess unlabeled homologous or heterologous Vsp protein. Results are expressed as the mean ± SD ratio of counts per second (cps) of Eu associated to HBMEC relative to unbound Eu and represent data from at least two separate experiments using 8 wells per protein. Notice that the association of Eu-LVsp1 with HBMEC was blocked by 10-fold excess unlabeled LVsp1 (panel A) or LVsp2 (panel C) but not by 10-fold excess unlabeled rVsp1 or albumin (panel A). This did not occur for Eu-LVsp2 (Panel B). Also notice that excess unlabeled LVsp1 increased the association of Eu-LVsp2 with HBMEC (panel C).

Mentions: Next we compared the association of Vsp1 and Vsp2 with HBMEC. For this we used the same Eppendorf cell suspension assay as before. The results showed that both proteins showed similar association with HBMEC: the mean ± SD ratios of bound to unbound cps were 2.16±(0.93) for Eu-LVsp1 and 2.7±(0.4) for Eu-LVsp2 (p = 0.30, Figure 7A&B). We also examined the mechanism of association of LVsp1 and LVsp2 with HBMEC using protein competition experiments. First we incubated Eu-LVsp1 with HBMEC in the presence of 10-fold higher concentrations of unlabeled LVsp1, rVsp1, or albumin. The results showed that only excess unlabeled LVsp1 interfered with the association (Figure 7A). Next we did the same with Eu-LVsp2 by incubating it with 10 times excess unlabeled LVsp2, rVsp2, and albumin. Unexpectedly, the results showed that none of them interfered with the association (Figure 7B). Therefore, we proceeded to study the degree to which excess unlabeled heterologous Vsp proteins interfered with the association each of the two Vsp proteins with HBMEC. The results showed that 10-fold excess unlabeled LVsp2 interfered with the association of Eu-LVsp1 with HBMEC but that 10-fold excess unlabeled LVsp1 had the opposite effect: it increased the association of LVsp2 with HBMEC (Figure 7C).


Interaction of variable bacterial outer membrane lipoproteins with brain endothelium.

Gandhi G, Londoño D, Whetstine CR, Sethi N, Kim KS, Zückert WR, Cadavid D - PLoS ONE (2010)

Lipidated Vsp1 or Vsp2 inhibit the association of LVsp1 with brain endothelium non-specifically.1×105 HBMEC in suspension in Eppendorf tubes were incubated with 0.2 µg of Eu-LVsp1 or Eu-LVsp2 for 2 h at 37°C in the presence or absence of 10-fold excess unlabeled homologous or heterologous Vsp protein. Results are expressed as the mean ± SD ratio of counts per second (cps) of Eu associated to HBMEC relative to unbound Eu and represent data from at least two separate experiments using 8 wells per protein. Notice that the association of Eu-LVsp1 with HBMEC was blocked by 10-fold excess unlabeled LVsp1 (panel A) or LVsp2 (panel C) but not by 10-fold excess unlabeled rVsp1 or albumin (panel A). This did not occur for Eu-LVsp2 (Panel B). Also notice that excess unlabeled LVsp1 increased the association of Eu-LVsp2 with HBMEC (panel C).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2962627&req=5

pone-0013257-g007: Lipidated Vsp1 or Vsp2 inhibit the association of LVsp1 with brain endothelium non-specifically.1×105 HBMEC in suspension in Eppendorf tubes were incubated with 0.2 µg of Eu-LVsp1 or Eu-LVsp2 for 2 h at 37°C in the presence or absence of 10-fold excess unlabeled homologous or heterologous Vsp protein. Results are expressed as the mean ± SD ratio of counts per second (cps) of Eu associated to HBMEC relative to unbound Eu and represent data from at least two separate experiments using 8 wells per protein. Notice that the association of Eu-LVsp1 with HBMEC was blocked by 10-fold excess unlabeled LVsp1 (panel A) or LVsp2 (panel C) but not by 10-fold excess unlabeled rVsp1 or albumin (panel A). This did not occur for Eu-LVsp2 (Panel B). Also notice that excess unlabeled LVsp1 increased the association of Eu-LVsp2 with HBMEC (panel C).
Mentions: Next we compared the association of Vsp1 and Vsp2 with HBMEC. For this we used the same Eppendorf cell suspension assay as before. The results showed that both proteins showed similar association with HBMEC: the mean ± SD ratios of bound to unbound cps were 2.16±(0.93) for Eu-LVsp1 and 2.7±(0.4) for Eu-LVsp2 (p = 0.30, Figure 7A&B). We also examined the mechanism of association of LVsp1 and LVsp2 with HBMEC using protein competition experiments. First we incubated Eu-LVsp1 with HBMEC in the presence of 10-fold higher concentrations of unlabeled LVsp1, rVsp1, or albumin. The results showed that only excess unlabeled LVsp1 interfered with the association (Figure 7A). Next we did the same with Eu-LVsp2 by incubating it with 10 times excess unlabeled LVsp2, rVsp2, and albumin. Unexpectedly, the results showed that none of them interfered with the association (Figure 7B). Therefore, we proceeded to study the degree to which excess unlabeled heterologous Vsp proteins interfered with the association each of the two Vsp proteins with HBMEC. The results showed that 10-fold excess unlabeled LVsp2 interfered with the association of Eu-LVsp1 with HBMEC but that 10-fold excess unlabeled LVsp1 had the opposite effect: it increased the association of LVsp2 with HBMEC (Figure 7C).

Bottom Line: The results showed that heterologous expression of LVsp1 or LVsp2 in B. burgdorferi increased its association with HBMEC to a similar degree.The association of LVsp2 with HBMEC and its movement from blood to brain parenchyma significantly increased in the presence of LVsp1.Variable 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Neuroscience and Center for the Study of Emerging Pathogens, UMDNJ-New Jersey Medical School, Newark, New Jersey, USA.

ABSTRACT

Background: Previously 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 findings: We 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/significance: Variable 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.

Show MeSH
Related in: MedlinePlus