Fig 1.
Progression of Lyme disease in humans following B. burgdorferi infection.
Spirochetes are inoculated in the skin through the bite of an infected hard-shelled Ixodes tick. CNS, central nervous system; PNS, peripheral nervous system. Reprinted from Trends in Microbiology, Vol. 21, No. 8, Coburn, J., Leong, J. and Chaconas, G., Illuminating the roles of the Borrelia burgdorferi adhesins, Pages 372–379, Copyright 2013, with permission from Elsevier. [7]
Fig 2.
Intravital imaging of transmigrated B. burgdorferi in the knee joint-proximal tissue of a living mouse.
Infectious GFP-expressing (green) B. burgdorferi (left and right panels GCB726, central panel GCB966, 4x108 spirochetes per mouse) were injected into the tail vein of Cd1d-/- mice. At 20 hours post-inoculation, vascular transmigration was scored in the knee joint-proximal tissue of living mice by intravital microscopy using a spinning disk laser confocal microscope. Blood vessels were stained with Alexa Fluor 555-conjugated PECAM-1 antibody (red). Video footage of transmigrated spirochetes is shown in S1 and S2 Videos.
Fig 3.
Kinetics of transmigration by fluorescent infectious B. burgdorferi in Cd1d-/- mice.
Infectious GFP-expressing B. burgdorferi (GCB847) were injected into the tail vein of Cd1d-/- mice, 4x108 spirochetes per mouse (n = 2/group for all time points except 20h where n = 6). At the indicated times vascular transmigration was scored in the knee joint-proximal tissue of living mice by intravital microscopy using a spinning disk laser confocal microscope. Blood vessels were stained with PE-conjugated PECAM-1 antibody. Green fluorescent spirochetes outside of the vasculature were counted in at least five fields of view (FOV) per mouse.
Fig 4.
The effect of bbK32 deletion in an infectious strain background on vascular transmigration and clearance in Cd1d-/- mice.
A) GFP-expressing B. burgdorferi strains, infectious (GCB966), a bbk32 deletion strain (GCB971) and a high-passage non-infectious strain (GCB706) were injected into the tail vein of Cd1d-/- mice (n = 6/group, 4x108 spirochetes were injected per mouse). After 24 hours, vascular transmigration was scored in the knee joint-proximal tissue in the living mice by intravital microscopy using a spinning disk laser confocal microscope. Blood vessels were stained with PE-conjugated or Alexa Fluor 555-conjugated PECAM-1 antibody and green fluorescent spirochetes outside of the vasculature were counted in at least five fields of view (FOV) per mouse. Statistical significance was analyzed using the non-parametric Mann-Whitney test; ns denotes not significant (P-values >0.05). B) Concentrations of B. burgdorferi in mouse plasma after iv inoculation. Cd1d-/- mice were injected with B. burgdorferi through the tail vein and blood was withdrawn at 5 and 60 minutes post-inoculation (n = 3/group). Blood cells were allowed to settle overnight as described in Materials and Methods and spirochetes in the plasma were directly counted by dark-field microscopy. The change in spirochete concentration between 5 and 60 minutes was determined for each mouse as the percentage of spirochetes present at 60 minutes relative to the initial 5 minute time point. Statistical significance was analyzed using the non-parametric Mann-Whitney test; ns denotes not significant (P-values >0.05).
Fig 5.
The effect of p66 deletion in an infectious background on vascular transmigration and clearance in CD1d-/- mice.
A) GFP-expressing B. burgdorferi strains, infectious wild type (GCB847), a p66 deletion strain (GCB849) and a strain where the wild-type p66 gene was reintroduced into the p66 mutant (GCB851), were injected into the tail vein of Cd1d-/- mice (n = 6-16/group, 4x108 spirochetes were injected per mouse). After 24 hours, vascular transmigration was scored in the knee joint-proximal tissue in the living mice by intravital microscopy using a spinning disk laser confocal microscope. Blood vessels were stained with PE-conjugated PECAM-1 antibody and green fluorescent spirochetes outside of the vasculature were counted in at least five fields of view (FOV) per mouse. Statistical significance was analyzed using non-parametric Kruskal-Wallis ANOVA followed by Dunn's multiple comparisons test. P-values for select pairwise comparisons are shown; ns denotes not significant (P-values >0.05). B). Concentrations of B. burgdorferi in mouse plasma after iv inoculation. Cd1d-/- mice were injected with 4x108 B. burgdorferi through the tail vein and blood was withdrawn at 5 and 60 minutes post-inoculation (n = 5-11/group). Blood cells were allowed to settle overnight as described in Materials and Methods and spirochetes in the plasma were directly counted by dark-field microscopy. The change in spirochete concentration between 5 and 60 minutes was determined for each mouse as the percentage of spirochetes present at 60 minutes relative to the initial 5 minute time point. Statistical significance was analyzed using non-parametric Kruskal-Wallis ANOVA followed by Dunn's multiple comparisons test. P-values for select pairwise comparisons are shown; ns denotes not significant (P-values >0.05).
Fig 6.
The effect of p66 site-directed integrin binding mutants on vascular transmigration and clearance in Cd1d-/- mice.
A) GFP-expressing B. burgdorferi strains, infectious wild type (GCB847), p66D205A,D207A (GCB3003) and p66Δ202–208 (GCB3004) were injected into the tail vein of Cd1d-/- mice (n = 3/group, 4x108 spirochetes were injected per mouse). After 24 hours, vascular transmigration was scored in the knee joint-proximal tissue in the living mice by intravital microscopy using a spinning disk laser confocal microscope. Blood vessels were stained with PE-conjugated PECAM-1 antibody and green fluorescent spirochetes outside of the vasculature were counted in at least five fields of view (FOV) per mouse. Statistical significance was analyzed using non-parametric Kruskal-Wallis ANOVA followed by Dunn's multiple comparisons test. P-values for select pairwise comparisons are shown; ns denotes not significant (P-values >0.05). B). Concentrations of B. burgdorferi in mouse plasma after iv inoculation. Cd1d-/- mice were injected with B. burgdorferi through the tail vein and blood was withdrawn at 5 and 60 minutes post-inoculation (n = 3/group). Blood cells were allowed to settle overnight as described in Materials and Methods and spirochetes in the plasma were directly counted by dark-field microscopy. The change in spirochete concentration between 5 and 60 minutes was determined for each mouse as the percentage of spirochetes present at 60 minutes relative to the initial 5 minute time point. Statistical significance was analyzed using non-parametric Kruskal-Wallis ANOVA followed by Dunn's multiple comparisons test. P-values for select pairwise comparisons are shown; ns denotes not significant (P-values >0.05).
Fig 7.
Visualization of β3 integrin in post-capillary venules in knee joint-proximal tissue by multi-channel spinning disk intravital microscopy.
GFP-expressing infectious B. burgdorferi (GCB847) was injected into the jugular vein of BALB/c mice as noted for other intravital experiments and data were acquired between 5–60 minutes postinfection. Blood vessels were stained with PE-conjugated PECAM-1 antibody and Alexa Fluor 647-conjugated β3 integrin antibody. The upper panel shows tethering and stationary interactions of B. burgdorferi (green), with PECAM-1 expressing endothelium visualized inside the blood vessels (red). The lower panel shows tethering and stationary interactions of the B. burgdorferi (green), with β3 integrin visualized inside the blood vessels (blue). The β3 integrin staining is also visible in smooth muscles (blue) surrounding the blood vessels.
Fig 8.
The effect of p66 deletion on vascular adhesion and clearance in a high- passage B. burgdorferi strain in BALB/c mice.
Non-infectious GFP-expressing B. burgdorferi wild type (GCB3212), Δp66 (GCB3214), and a strain where the wild-type p66 gene was reintroduced into the p66 deletion mutant (Δp66/comp, GCB3218) were injected into the jugular vein of BALB/c, 4x108 spirochetes per mouse (n = 7/group). Over a period of up to 45 minutes, microvascular interaction rates A) (tethering + dragging), and stationary adhesions B) were enumerated in the knee joint-proximal tissue by intravital microscopy using spinning disk laser confocal microscopy as described in Materials and Methods. Blood vessels were stained with PE-conjugated PECAM-1 antibody. Statistical significance was analyzed using the non-parametric Kruskal-Wallis test; ns denotes not significant (P-values >0.05). C) Concentrations of B. burgdorferi in mouse plasma after iv inoculation. BALB/c mice were inoculated with B. burgdorferi through the tail vein as above and blood was withdrawn at 3 and 18 minutes post-inoculation (n = 7/group). Blood cells were allowed to settle overnight as described in Materials and Methods and spirochetes in the plasma were directly counted by dark-field microscopy. The change in spirochete concentration between 3 and 18 minutes was determined for each mouse as the percentage of spirochetes present at 18 minutes relative to the initial 3 minute time point. Statistical significance was analyzed using the non-parametric Kruskal-Wallis test; ns denotes not significant (P-values >0.05).