Transferrin binding protein B and Transferrin binding protein A 2 expand the transferrin recognition range of Histophilus somni

Anastassia K. Pogoutse; Trevor F. Moraes

We concluded that H. somni H191 does not exhibit strict specificity for bovine transferrin. 153 However, it does display a preference for bovine transferrin while also having affinity for 154 transferrins of other bovids. To determine which transferrin binding protein(s) was responsible 155 for this binding profile, we went on to investigate each Tbp separately in a heterologous system. To address the binding contribution of each of the transferrin binding proteins (TbpA, TbpA2 160 and TbpB), we expressed His-tagged TbpAs, TbpA2, and GST-tagged HsTbpB independently in 161 E. coli, and performed displacement ELISAs (Figure 2 ). E. coli does not encode any of these 162 three receptors and so this served as an appropriate heterologous system. TbpA assays were 163 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/730739 doi: bioRxiv preprint 9 performed with whole cells while TbpB assays were performed with the soluble lysate fraction 164 (TbpB was expressed without its N-terminal cysteine and was therefore not lipidated). An 165 interaction was denoted as "specific" when unlabeled bovine transferrin lowered the absorbance 166 signal to 10% or less of the maximum and other unlabeled competitor transferrins did not 167 decrease the signal significantly. TbpA specifically bound bovine transferrin ( Figure 2B ). 168