Finally, we tested the VapD toxin for ribonuclease activity in vitro. The current work is aimed at uncovering the contributions of vapBC-1 and vapXD to NTHi-caused otitis
media, which could lead to new vaccine or pharmaceutical targets for the prophylaxis and therapy of this disease. Results Interactions of the Vap proteins in vivo To Cilengitide chemical structure detect the ability of VapB-1 and VapC-1 to form heterodimers in vivo, β-galactosidase activity assays were carried out using an E. coli-based LexA protein-protein interaction reporter system as previously described . In this system, with no protein fused to the LexA DNA binding domain (DBD) of either plasmid pSR658 or pSR659 in strain SU202, the repressor
KPT-8602 mw cannot form a dimer, and the expression of the lacZ reporter gene is constitutive. However, a reconstituted repressor formed by heterodimerization of fused proteins can bind to the engineered operator region, decreasing transcription INK1197 of the reporter gene, but a homodimer, if formed, cannot bind to the operator. Since the LexA DBD plasmids have different copy numbers (pSR658 has a higher copy number than pSR659), we constructed reciprocal fusions and analyzed each set as an internal control for heterodimerization. When we fused VapC-1 to the LexA DBD in pSR658 (pDD859) and VapB-1 to pSR659 (pDD867), the reporter gene expression was decreased to 458 ± 47 Miller units, whereas the unfused LexA DBD in the vectors pSR658 and pSR659 allowed constitutive transcription of the reporter gene at 1,611 ± 138 Miller units (Figure 1). This indicated
a strong protein:protein interaction. When the fusions were reversed, with VapB-1 in pSR658 (pDD866) and VapC-1 in pSR659 (pDD868), the pair heterodimerized and repressed lacZ expression to 682 ± 61 Miller units. Interestingly, there was a significant difference between the reciprocal fusions, with the pDD859/pDD867 pair being the most efficient at repressing the reporter gene. Figure 1 VapB-1 Tryptophan synthase and VapC-1 heterodimerize in vivo . 86-028NP vapB-1 or vapC-1 was fused to the LexA DNA binding domain (DBD) in the vectors pSR658 or pSR659, resulting in pDD866 or pDD868, respectively. Reciprocally, vapC-1 or vapB-1 was also fused to the LexA DBD in the vectors pSR658 or pSR659, resulting in pDD859 or pDD867, respectively. Each pair was co-transformed into the reporter strain SU202 and the amount of heterodimerization was quantitated by β-galactosidase activity assays (n = 3 in triplicate). Data are expressed as mean ± SD. To investigate the hetero-interactions between VapX and VapD, the same reporter system was used as above. With VapX in pSR658 (pDD882) and VapD in pSR659 (pDD884), the reporter gene expression was decreased to 162 ± 27 Miller units, compared to the expression in the presence of the control vectors of 1,783 ± 85 Miller units (Figure 2).