Neisseria meningitidis Expressing Transferrin Binding Proteins of Actinobacillus pleuropneumoniae Can Utilize Porcine Transferrin for Growth

Neisseria meningitidis, an important cause of bacterial meningitis worldwide (21), is exclusively a pathogen of humans (11). This is reflected in its iron acquisition systems, which exploit iron-sequestering compounds such as human transferrin (hTF), human lactoferrin, and hemoglobin (19, 28, 47). The meningococcal receptors for hTF and human lactoferrin are unable to bind these molecules from other animal species, whereas one of the hemoglobin receptors has been shown to utilize nonhuman sources of hemoglobin, although with reduced efficiencies (19, 47). The inability of N. meningitidis to use nonhuman iron sources such as TF and lactoferrin can be seen in animal models for meningococcal infection. Intraperitoneal injection of large numbers of bacteria in mice and infant rats in the absence of an accessible iron source can cause bacteremia, infection of the central nervous system, and death (31, 43). However, the large doses of organisms required and the speed with which the animals die suggest that mortality is the consequence of toxicity rather than a true infection (31). In contrast, when mice are supplemented with an accessible iron source, such as hTF or iron dextran, consistently high mortality rates are achieved with doses of bacteria several orders of magnitude lower (5, 6, 22, 44). At these doses, the survival and pathogenicity of N. meningitidis strains in mice are clearly dependent on the levels of an accessible iron source (5, 6, 22, 44). In an attempt to mimic the route of entry of N. meningitidis more realistically, infant mice have been successfully infected via the nasal route, although, as with previous models, a supplement of hTF or iron dextran is necessary (30, 40). If N. meningitidis could be engineered to utilize iron from TF from a suitable animal host, an animal model which more closely mimics the course of the human infection might be developed. N. meningitidis acquires iron from hTF by a receptor-mediated process involving two meningococcal TF binding proteins (Tbp). TbpA is an outer membrane protein that is thought to transfer the iron from hTF across the bacterial periplasmic membrane in a TonB-dependent process (11, 19). TbpB is a lipoprotein anchored in the outer membrane, and its ability to discriminate between iron-loaded and apo-TF has led to the proposition that its role is to enrich the bacterial surface with the iron source (4, 11, 19, 35, 38). Together TbpA and TbpB constitute the bacterial TF receptor; both bind exclusively to hTF (14, 23). There is evidence that both are required for meningococcal growth in vitro when hTF is the sole iron source (23), although Pintor et al. (34) have demonstrated growth when TbpB alone is expressed. There is some dispute as to whether TbpA and TbpB actually form a complex on the surface of N. meningitidis and whether they interact with a 1:1 or 2:1 stoichiometry (4, 11, 34, 35). Following its transport into the periplasm, iron is subsequently transported across the inner membrane by the ferric binding protein (Fbp) ABC transporter complex (19, 24). An analogous system of iron uptake from TF has been demonstrated in a range of species from the Neisseriaceae and Pasteurellaceae families, each restricted to the TF molecule from its particular natural host (19). TbpA and TbpB, encoded by the genes tbpA and tbpB, respectively, are expressed from a chromosomal operon in response to an iron-restricted growth environment and are probably under the control of the ferric uptake regulation repressor protein, Fur (15, 19). The tbp operons from N. meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae (all specific to hTF), Actinobacillus pleuropneumoniae (specific to porcine TF [pTF]), and Pasteurella haemolytica (specific to bovine, sheep, and goat TFs) have been cloned and sequenced (1, 17, 18, 27, 33). Recombinant TbpA, recombinant TbpB, and truncated or chimeric TbpB proteins have been expressed and purified from Escherichia coli for binding studies (8, 9, 14, 17, 26, 29, 33, 36–38). However, the expression of heterologous or chimeric Tbp in members of the Neisseriaceae or Pasteurallaceae families, which would be useful for functional studies, has not been reported. The aim of this study was to construct a meningococcal mutant strain in which the tbpA and tbpB genes were replaced with those from A. pleuropneumoniae and to test the ability of the engineered strain to bind to, and utilize iron from, pTF instead of hTF. Demonstration of the ability to change host TF specificity would enable further structure-function studies of the iron uptake system. Additionally, it would open up new possibilities for the development of improved animal models of meningococcal infection and disease.