Fig 2: Effect of cytokines on microvascular interactions of the doubly deficient BBK32, VlsE strain of B. burgdorferi in BALB/c mice.Mice were injected with the wild-type strain (GCB726, n = 5) cultured with 1% blood (see Materials and Methods) for a positive control. For the analysis, they were injected with PBS (n = 5) or the specific cytokine 3 hours before infection with the GFP-expressing VlsE, BBK32 doubly deficient strain (GCB4036 or GCB4038) cultured with blood. Vascular interactions were detected using high acquisition rate spinning disk confocal intravital microscopy 5-50 minutes post-infection. The number of interactions per minute for transient adhesions was analyzed using intravital microscopy. The graph shows the number of microvascular interactions in mouse venules. Statistical significance was determined using the Kruskal-Wallis test with Dunn’s multiple comparison test. p < 0.05 was considered significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. The cytokines used were: TNF-α (45 ng/25 g mouse, n = 4), MCP-1 (5 μg/25 g mouse, n = 6), IL-10 (2.5 μg/25 g mouse, n = 5), IL-1β (250 ng/25 g mouse, n = 5), GM-CSF (15 ng/25 g mouse, n = 4) injected intravenously, and IL-17A (0.5 μg/25 g mouse, n = 3) injected intraperitoneally.

Fig 3: Effect of MCP-1 on microvascular interactions in BALB/c mice.Mice were injected with PBS or MCP-1 (5 μg/25g mouse) 3 hours before infection with strains carrying the indicated surface-exposed proteins. Vascular adhesion interactions were detected using high acquisition rate spinning disk confocal intravital microscopy 5-50 minutes post-infection. The number of interactions per minute for transient adhesions was analyzed. The graph shows the comparison of the number of microvascular interactions with or without MCP-1 in mouse venules for each protein. Statistical significance was determined using the Mann-Whitney test with for each strain comparison (with/without MCP-1). In all cases P < 0.05 was considered significant, *p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant.

Fig 4: Effect of blood on the BBK32, VlsE minus strain of B. burgdorferi.A) Effect of blood exposure on microvascular interactions of the Borrelia burgdorferi doubly deficient strain in MCP-1–treated BALB/c mice. The GFP-expressing wild-type strain (GCB726), cultured with blood, was used as a positive control. GCB4038, which is deficient in both VlsE and BBK32, was grown with or without mouse blood for 48 hours prior to infection. To evaluate the impact of blood exposure on vascular adhesion, BALB/c mice (n = 3) were pretreated with 5 μg/25 g body weight of MCP-1 via intravenous injection 3 hours before infection. Vascular interactions were visualized by intravital microscopy as described in Fig. 1. Error bars represent standard deviation (SD). Statistical significance was assessed using the Kruskal–Wallis test followed by Dunn’s multiple comparisons; p < 0.05 was considered significant (****p < 0.0001, ns = not significant). B) Effect of proteinase K on microvascular transient interactions of the BBK32, VlsE minus strain of B. burgdorferi grown in blood in MCP-1 treated BALB/c mice. GFP-expressing doubly deficient strain GCB4036 was grown with mouse blood 48 hours before infection. Mice (n=3-4) received 5 μg/25 g mouse MCP-1 by i.v. injection 3 hr prior to infection with GCB4036. GCB 4036 was incubated with or without proteinase K 150 μg/ml one hour before infection. After infection, vascular transient adhesion interactions were detected by intravital microscopy as in Fig. 1. Error bars represent SD, and statistical significance was analyzed using the Mann-Whitney test; p < 0.05 was considered significant (****p < 0.0001). C) Differential gene expression in the doubly deficient B. burgdorferi GCB4036 following exposure to whole blood. Cultures were incubated with whole blood for 48 hours prior to RNA sequencing. A total of 39 genes were differentially expressed, including 30 upregulated genes (violet and grey dots) and 9 downregulated genes (red dots). Among the upregulated genes, 16 (shown in violet and listed on the right with the fold change values in parentheses) are predicted to encode surface-exposed proteins, based on the surface proteome (Dowdell et al., 2017).