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Integrin binding by Borrelia burgdorferi P66 facilitates dissemination but is not required for infectivity.

Ristow LC, Bonde M, Lin YP, Sato H, Curtis M, Wesley E, Hahn BL, Fang J, Wilcox DA, Leong JM, Bergström S, Coburn J - Cell. Microbiol. (2015)

Bottom Line: Neither change affected surface localization or channel-forming activity of P66, but both significantly reduced binding to αv β3 .The delay in tissue colonization correlated with reduced migration of the Del202-208 strains across microvascular endothelial cells, similar to Δp66 bacteria.These results indicate that integrin binding by P66 is important to efficient dissemination of B. burgdorferi, which is critical to its ability to cause disease manifestations in incidental hosts and to its maintenance in the enzootic cycle.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Microbiology, Immunology, and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI, USA.

No MeSH data available.


Related in: MedlinePlus

Mutation of P66 affects the ability of Borrelia burgdorferi to cross an endothelial monolayer of cells. HMEC-1 human microvascular endothelial cells were plated in 3 μm pore size Transwell inserts and grown to confluence in antibiotic free medium. B31-A3 (wild-type), Δp66, p66cc, BD205A,D207A or BDel202–208 bacteria were added to the insert at an MOI of 20 and the ability of bacteria to cross the monolayer of cells was assessed over 48 h by taking an aliquot from the insert and from the well below. Bacteria were counted manually using a Petroff-Hausser counting chamber under dark-field microscopy, and the percentage of bacteria that crossed the cell monolayer was calculated. Inserts without a cell monolayer were assessed as controls for bacterial crossing of the membrane alone. A two-way repeated measures ANOVA with Bonferroni’s post-test was performed. Asterisks indicate comparison to wild-type bacteria, plus signs indicate comparison to p66cc bacteria. **/++P < 0.01, ***P < 0.001, n = 5.
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Figure 5: Mutation of P66 affects the ability of Borrelia burgdorferi to cross an endothelial monolayer of cells. HMEC-1 human microvascular endothelial cells were plated in 3 μm pore size Transwell inserts and grown to confluence in antibiotic free medium. B31-A3 (wild-type), Δp66, p66cc, BD205A,D207A or BDel202–208 bacteria were added to the insert at an MOI of 20 and the ability of bacteria to cross the monolayer of cells was assessed over 48 h by taking an aliquot from the insert and from the well below. Bacteria were counted manually using a Petroff-Hausser counting chamber under dark-field microscopy, and the percentage of bacteria that crossed the cell monolayer was calculated. Inserts without a cell monolayer were assessed as controls for bacterial crossing of the membrane alone. A two-way repeated measures ANOVA with Bonferroni’s post-test was performed. Asterisks indicate comparison to wild-type bacteria, plus signs indicate comparison to p66cc bacteria. **/++P < 0.01, ***P < 0.001, n = 5.

Mentions: The effect of the Del202–208 mutation on B. burgdorferi burdens in some tissues suggests a potential defect in at least some steps in the complex dissemination process. The spread of B. burgdorferi to tissues distant from the site of inoculation may occur by migration through tissues without entry into the vasculature, but is likely to be facilitated by entry into and dissemination through the vasculature. Colonization of distal sites would then involve crossing endothelial barriers while the bacteria are entering and exiting the vascular system. The potential role of the integrin-binding function of P66 in the ability to cross endothelial cell monolayers was therefore investigated in vitro based on the Boyden chamber assay (Boyden, 1962). A microvascular endothelial cell line (HMEC-1) was used to represent the cells present in small vessels or capillary beds in vivo, where B. burgdorferi can successfully exit the vasculature (Moriarty et al., 2008; 2012). Bacterial transmigration was assessed using confluent layers of HMEC-1 cells in Transwell inserts. Transwells without cells were used as controls for bacteria crossing the membrane alone. The role of P66 in this activity was quantified by comparing the ability of B31-A3 (WT), Δp66, p66cc, BD205A,D207A and BDel202–208 bacteria to cross the cell monolayer at a multiplicity of infection (MOI) of 20. Migration of all B. burgdorferi strains in wells without cells was significantly higher than that of any strain crossing wells with a HMEC-1 cell monolayer at time points ≥24 h (data not shown). By 48 h, there was a significant defect in the ability of Δp66 bacteria to cross the cell monolayer compared with WT and p66cc bacteria (Fig. 5). A similar defect was observed in BDel202–208 bacterial transmigration of the cell monolayer compared with WT and p66cc bacteria. The ability of the BD205A,D207A bacteria to cross the cell layers was not significantly different from that of WT bacteria, consistent with the decreased severity of phenotypic effects in vivo in this mutant as compared with the BDel202–208 bacteria and with the in vitro integrin-binding results. Transmigration was analysed up to 72 h post-inoculation, but the health of all B. burgdorferi strains diminished after 48 h in the cell growth medium, which is relatively nutrient poor compared with the bacterial culture medium, and results were not reproducible after 48 h.


Integrin binding by Borrelia burgdorferi P66 facilitates dissemination but is not required for infectivity.

Ristow LC, Bonde M, Lin YP, Sato H, Curtis M, Wesley E, Hahn BL, Fang J, Wilcox DA, Leong JM, Bergström S, Coburn J - Cell. Microbiol. (2015)

Mutation of P66 affects the ability of Borrelia burgdorferi to cross an endothelial monolayer of cells. HMEC-1 human microvascular endothelial cells were plated in 3 μm pore size Transwell inserts and grown to confluence in antibiotic free medium. B31-A3 (wild-type), Δp66, p66cc, BD205A,D207A or BDel202–208 bacteria were added to the insert at an MOI of 20 and the ability of bacteria to cross the monolayer of cells was assessed over 48 h by taking an aliquot from the insert and from the well below. Bacteria were counted manually using a Petroff-Hausser counting chamber under dark-field microscopy, and the percentage of bacteria that crossed the cell monolayer was calculated. Inserts without a cell monolayer were assessed as controls for bacterial crossing of the membrane alone. A two-way repeated measures ANOVA with Bonferroni’s post-test was performed. Asterisks indicate comparison to wild-type bacteria, plus signs indicate comparison to p66cc bacteria. **/++P < 0.01, ***P < 0.001, n = 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4478124&req=5

Figure 5: Mutation of P66 affects the ability of Borrelia burgdorferi to cross an endothelial monolayer of cells. HMEC-1 human microvascular endothelial cells were plated in 3 μm pore size Transwell inserts and grown to confluence in antibiotic free medium. B31-A3 (wild-type), Δp66, p66cc, BD205A,D207A or BDel202–208 bacteria were added to the insert at an MOI of 20 and the ability of bacteria to cross the monolayer of cells was assessed over 48 h by taking an aliquot from the insert and from the well below. Bacteria were counted manually using a Petroff-Hausser counting chamber under dark-field microscopy, and the percentage of bacteria that crossed the cell monolayer was calculated. Inserts without a cell monolayer were assessed as controls for bacterial crossing of the membrane alone. A two-way repeated measures ANOVA with Bonferroni’s post-test was performed. Asterisks indicate comparison to wild-type bacteria, plus signs indicate comparison to p66cc bacteria. **/++P < 0.01, ***P < 0.001, n = 5.
Mentions: The effect of the Del202–208 mutation on B. burgdorferi burdens in some tissues suggests a potential defect in at least some steps in the complex dissemination process. The spread of B. burgdorferi to tissues distant from the site of inoculation may occur by migration through tissues without entry into the vasculature, but is likely to be facilitated by entry into and dissemination through the vasculature. Colonization of distal sites would then involve crossing endothelial barriers while the bacteria are entering and exiting the vascular system. The potential role of the integrin-binding function of P66 in the ability to cross endothelial cell monolayers was therefore investigated in vitro based on the Boyden chamber assay (Boyden, 1962). A microvascular endothelial cell line (HMEC-1) was used to represent the cells present in small vessels or capillary beds in vivo, where B. burgdorferi can successfully exit the vasculature (Moriarty et al., 2008; 2012). Bacterial transmigration was assessed using confluent layers of HMEC-1 cells in Transwell inserts. Transwells without cells were used as controls for bacteria crossing the membrane alone. The role of P66 in this activity was quantified by comparing the ability of B31-A3 (WT), Δp66, p66cc, BD205A,D207A and BDel202–208 bacteria to cross the cell monolayer at a multiplicity of infection (MOI) of 20. Migration of all B. burgdorferi strains in wells without cells was significantly higher than that of any strain crossing wells with a HMEC-1 cell monolayer at time points ≥24 h (data not shown). By 48 h, there was a significant defect in the ability of Δp66 bacteria to cross the cell monolayer compared with WT and p66cc bacteria (Fig. 5). A similar defect was observed in BDel202–208 bacterial transmigration of the cell monolayer compared with WT and p66cc bacteria. The ability of the BD205A,D207A bacteria to cross the cell layers was not significantly different from that of WT bacteria, consistent with the decreased severity of phenotypic effects in vivo in this mutant as compared with the BDel202–208 bacteria and with the in vitro integrin-binding results. Transmigration was analysed up to 72 h post-inoculation, but the health of all B. burgdorferi strains diminished after 48 h in the cell growth medium, which is relatively nutrient poor compared with the bacterial culture medium, and results were not reproducible after 48 h.

Bottom Line: Neither change affected surface localization or channel-forming activity of P66, but both significantly reduced binding to αv β3 .The delay in tissue colonization correlated with reduced migration of the Del202-208 strains across microvascular endothelial cells, similar to Δp66 bacteria.These results indicate that integrin binding by P66 is important to efficient dissemination of B. burgdorferi, which is critical to its ability to cause disease manifestations in incidental hosts and to its maintenance in the enzootic cycle.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Microbiology, Immunology, and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI, USA.

No MeSH data available.


Related in: MedlinePlus