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

Iron is a critical element essential for the growth of many organisms, and there is little free iron available in the human body (5, 11, 14, 19, 29). The sequestering of iron provides an important line of defense against pathogenic microorganisms. Most of the iron present in the body is intracellular in the ferrous form as hemoglobin, heme, ferritin, or hemosiderin. In addition, extracellular iron is complexed to the high-affinity iron-binding glycoproteins transferrin (Tf) and lactoferrin (5, 14, 19, 29). Tf-bound iron is found predominantly in serum, while it is thought that lactoferrin is the primary carrier of iron on most mucosal surfaces. To survive in and colonize the human host, many mammalian pathogens have evolved numerous mechanisms that allow them to obtain iron in vivo. Both direct iron-binding compounds and specific receptors for iron carrier proteins have been described for many of these bacteria (11, 13, 19, 28). Previous studies have demonstrated that Moraxella catarrhalis expresses proteins that bind human Tf and lactoferrin (Lf) similar to those receptors described for the pathogenic members of the families Neisseriaceae and Pasteurellaceae (10, 15, 26, 27, 31). The Tf-binding proteins (Tbps) were identified by using affinity chromatography with human holotransferrin coupled to a Sepharose matrix. These proteins are iron repressible and have molecular sizes in the range of 80 to 84 kDa (TbpB) and 115 to 120 kDa (TbpA) (31). Our previous studies identified OmpB1 as an M. catarrhalis Tf receptor (6, 20). This outer membrane protein appears to be the homologue of TbpB, which was described by Myers et al. (for clarity, OmpB1 will be referred to as TbpB in this report) (22). tbpA and tbpB have been cloned and sequenced from various strains of M. catarrhalis, and the proteins encoded by these genes have been compared (22). Similar to the tbpB of other bacteria, the M. catarrhalis tbpB exhibits marked heterogeneity between strains while tbpA appears to be highly conserved. Although the Tbps of M. catarrhalis specifically bind human Tf, similar to the Tbps expressed by other human mucosal pathogens, there are some differences observed in the arrangement of the genes that code for these proteins. Investigators have previously reported that the M. catarrhalis tbpA gene precedes tbpB, unlike other known tbp operons, and there is a third open reading frame (termed orf3) located between these genes (22). In addition, the identification of three potential promoter sequences located upstream of each open reading frame suggests that the genes are independently transcribed. Despite these observations, the lack of specific mutants, similar to the tbp mutants constructed for the pathogenic Neisseriaceae, have precluded studies designed to evaluate the relationship between the regulation and expression of the M. catarrhalis genes which code for the Tbps. In this report, we constructed isogenic mutants in M. catarrhalis 7169 that are deficient in the expression of either TbpB (previously described), TbpA, or both of these receptors. Comparative studies were performed to characterize each mutant for the ability to bind Tf at the bacterial cell surface, to utilize iron from Tf for in vitro growth, and for the affinity isolation of each receptor from a holotransferrin matrix. These Tbp mutants provide important tools that will be critical in studies designed to provide a more thorough understanding of the Tf system utilized by M. catarrhalis.