Fig 1.
Expression of the two allelic variants of Tp0136 on B. burgdorferi strain B314 surface.
Treatment of B314 strain expressing Tp0136 alleles from T. pallidum SS14 and Nichols strains with antiserum obtained from a secondary syphilis (SS) patient followed by visualization using AlexaFluor-488 conjugated anti-human IgG antibodies showed punctate green spirochetes surface staining (panels b and c in the 2nd row). The SS serum did not react with other targets on the B. burgdorferi B314 control strain transformed with an empty shuttle vector (V) in the panel a in the 2nd row. DNA staining by DAPI in the panels in rows 1, 3, and 5 show all bacteria present in the respective microscopic fields. A monoclonal antibody against FlaB followed by TRITC-labeled secondary anti-mouse antibodies stained spirochetes only after permeabilization with methanol (Permeabilized, panels a, b, and c in the 6th row) but not intact spirochetes (Unpermeabilized, panels a, b, and c in the 4th row) showing that bacterial outer membrane remained unperturbed during the IFA procedure. Bar represents 16 μm.
Fig 2.
B. burgdorferi B314 strain expressing Tp0136 allelic variants bind to HEK293 more efficiently than to Vero cells but fail to adhere to HEp-2 cells.
Average binding of radiolabeled B. burgdorferi B314 expressing Tp0136 variants of the SS14 and Nichols strains to Vero cells was 6% and 8%, respectively. Percent of binding mediated by Nichols strain Tp0136 to HEK293 cells was significantly higher than binding by SS14 strain Tp0136. Both variants of Tp0136 bound poorly to the HEp-2 cells, which do not produce fibronectin. Statistical analysis was conducted using a two-tailed unpaired student t test for unequal variance to determine significant difference between the paired groups and p values calculated (*p<0.05).
Fig 3.
Tp0136 facilitated B314 binding to glioma, placental, and endothelial cell lines.
Binding of B314 expressing Tp0136 to placental (BeWo), glioma (C6), and endothelial (Ea.Hy926) cell lines were significantly higher compared to B314 control strain. Allelic variants of the Tp0136 protein did not display significant differences in binding to any of these three cell lines. Statistical analysis was conducted using a two-tailed unpaired student t test for unequal variance to determine significant difference between the paired groups and p values calculated (*p <0.05, **p<0.01, and ***p<0.001).
Fig 4.
B314 expressing Tp0136 variants from Nichols and SS14 strains bind to human fibronectin and laminin.
Binding of Tp0136-expressing B314 to wells coated with human tissue fibronectin was significantly higher than that of the control B314 (V) strain; however, binding facilitated by two alleles of Tp0136 was comparable to this ECM component. Binding of B314 expressing the SS14 Tp0136 to laminin was significantly higher than B314 expressing the Nichols variant. Statistical analysis was conducted using a two-tailed unpaired student t test for unequal variance to determine significant difference between the paired groups and p values calculated (*p<0.05, **p <0.01, ****p<0.0001).
Fig 5.
Both purified tissue fibronectin and laminin bind to Tp0136 alleles expressed on B314 strain.
Spirochetes on coverglasses were incubated with purified tissue fibronectin or laminin and binding was detected using anti-fibronectin (antiFn-FITC), 2nd row from top, and anti-laminin (DyLight 410) using YFP filter (antiLm-YFP) antibodies, bottom row. DAPI stained DNA in B314 transformants depict all spirochetes present in the respective microscopic fields. Top row shows all spirochetes in the field of view depicting fibronectin binding (2nd row) while 3rd row shows all transformed B314 in the field of view showing laminin binding (bottom row). Both allelic variants of Tp0136 showed similar efficiency in binding to fibronectin. Overall, binding of laminin to both Tp0136-expressing strains was observed with more pronounced binding to B314 expressing the SS14 Tp0136, compared to the strain expressing the Nichols variant. No laminin binding to B314(V) control was detected. Bars represent 16 μm.
Fig 6.
Fibronectin binding peptide FnbA-2 of S. aureus reduces attachment of Tp0136 –expressing B314 strain to purified fibronectin, and to C6 glioma and HEK293 cell lines.
(A) Significant reduction in binding of both Tp0136 variants to immobilized tissue fibronectin was observed upon preincubation of fibronectin with FnbA-2 (light color bars) as compared to the mock treated samples (buffer only treatment, dark color bars). (B) Significant reduction in binding of both Tp0136 alleles expressing B314 to HEK293 cells by the FnbA-2 peptide was detected as marked by underlined numbers above the bars; however, binding mediated by two Tp0136 alleles was not significantly different on these cells (p = 0.748). (C) Significant inhibition of binding to C6 glioma cells by the FnbA-2 peptide was also observed. Percent inhibition of binding by FnbA-2 peptide is indicated by the underlined numbers above the bars. Statistical analysis was conducted using a two-tailed unpaired student t test for unequal variance to determine significant difference between the paired groups and p values calculated (NS-not significant, *p<0.05, **p<0.01, ***p<0.001).