Construction and characterization of Moraxella catarrhalis mutants defective in expression of transferrin receptors

We have previously reported the construction of an isogenic mutant defectiv e in expression of OmpB1, the TbpB homologue, in Moraxella catarrhalis 7169 , In this report, we have extended these studies by constructing and charac terizing two new isogenic mutants in this clinical isolate. One mutant is d efective in expression of TbpA, and the other mutant is defective in expres sion of both TbpA and TbpB. These isogenic mutants were confirmed by using PCR analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an d sequencing. In vitro growth studies, comparing all three mutants, demonst rated that the tbpA mutant and the tbpAB mutant were severely limited in th eir ability to grow with human holotransferrin as the sole source of iron. In contrast, the ompB1 (tbpB) mutant was capable of utilizing iron from hum an transferrin, although not to the extent of the parental strain. While af finity chromatography with human holotransferrin showed that each Tbp was c apable of binding independently to transferrin, solid-phase transferrin bin ding studies using whole cells demonstrated that the tbpA mutant exhibited binding characteristics similar to those seen with the wild-type bacteria. However, the ompB1 (tbpB) mutant exhibited a diminished capacity for bindin g transferrin, and no binding was detected with the double mutant. These da ta suggest that the M. catarrhalis TbpA is necessary for the acquisition of iron from transferrin. In contrast, TbpB is not essential but may serve as a facilitory protein that functions to optimize this process. Together the se mutants are essential to provide a more thorough understanding of iron a cquisition mechanisms utilized by M. catarrhalis.