Construction and characterization of Moraxella catarrhalis mutants defective in expression of transferrin receptors.
We have previously reported the construction of an isogenic mutant defective in expression of OmpB1, the TbpB homologue, in Moraxella catarrhalis 7169. In this report, we have extended these studies by constructing and characterizing two new isogenic mutants in this clinical isolate. One mutant is defective in expression of TbpA, and the other mutant is defective in expression of both TbpA and TbpB. These isogenic mutants were confirmed by using PCR analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and sequencing. In vitro growth studies, comparing all three mutants, demonstrated that the tbpA mutant and the tbpAB mutant were severely limited in their ability to grow with human holotransferrin as the sole source of iron. In contrast, the ompB1 (tbpB) mutant was capable of utilizing iron from human transferrin, although not to the extent of the parental strain. While affinity chromatography with human holotransferrin showed that each Tbp was capable of binding independently to transferrin, solid-phase transferrin binding 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 binding transferrin, and no binding was detected with the double mutant. These data 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 these mutants are essential to provide a more thorough understanding of iron acquisition mechanisms utilized by M. catarrhalis.
Luke
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Department of Microbiology, Center for Microbial Pathogenesis, State University of New York at Buffalo, Buffalo, New York 14214, USA.
Campagnari
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D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D033901:Bacterial Transferrin Receptor Complex; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D006801:Humans; D007501:Iron / Q000378:metabolism*; D033862:Iron-Binding Proteins; D001936:Moraxella catarrhalis / Q000378:metabolism*; D009154:Mutation; D016133:Polymerase Chain Reaction; D014168:Transferrin / Q000378:metabolism; D033863:Transferrin-Binding Proteins
D001425:Bacterial Outer Membrane Proteins; D033901:Bacterial Transferrin Receptor Complex; D002352:Carrier Proteins; D033862:Iron-Binding Proteins; D014168:Transferrin; D033863:Transferrin-Binding Proteins; C117491:ompB1 protein, Moraxella catarrhalis; D007501:Iron