Identification of Transferrin-Binding Domains in TbpB Expressed by
            <i>Neisseria gonorrhoeae</i>

DeRocco, Amanda J.; Cornelissen, Cynthia Nau

doi:10.1128/iai.00072-07

Cited by 24 publications

(27 citation statements)

References 52 publications

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“…Tf-iron uptake assays were performed as described previously (32)(33)(34)(35). Briefly, apo-human Tf (Sigma) was saturated to 20% with 55 Fe (Perkin-Elmer).…”

Section: Figmentioning

confidence: 99%

Beyond the Crystal Structure: Insight into the Function and Vaccine Potential of TbpA Expressed by Neisseria gonorrhoeae

et al. 2015

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bNeisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, is not preventable by vaccination and is rapidly developing resistance to antibiotics. However, the transferrin (Tf) receptor system, composed of TbpA and TbpB, is an ideal target for novel therapeutics and vaccine development. Using a three-dimensional structure of gonococcal TbpA, we investigated two hypotheses, i.e., that loop-derived antibodies can interrupt ligand-receptor interactions in the native bacterium and that the loop 3 helix is a critical functional domain. Preliminary loop-derived antibodies, as well as optimized second-generation antibodies, demonstrated similar modest ligand-blocking effects on the gonococcal surface but different effects in Escherichia coli. Mutagenesis of loop 3 helix residues was employed, generating 11 mutants. We separately analyzed the mutants' abilities to (i) bind Tf and (ii) internalize Tf-bound iron in the absence of the coreceptor TbpB. Single residue mutations resulted in up to 60% reductions in ligand binding and up to 85% reductions in iron utilization. All strains were capable of growing on Tf as the sole iron source. Interestingly, in the presence of TbpB, only a 30% reduction in Tf-iron utilization was observed, indicating that the coreceptor can compensate for TbpA impairment. Complete deletion of the loop 3 helix of TbpA eliminated the abilities to bind Tf, internalize iron, and grow with Tf as the sole iron source. Our studies demonstrate that while the loop 3 helix is a key functional domain, its function does not exclusively rely on any single residue. N eisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea (1), affects approximately 106 million people worldwide according to WHO estimates (2), with Ͼ300,000 cases of gonorrhea reported each year in the United States alone (3). A troubling contributor to these statistics is that infection with this bacterium does not result in any protective immunity (4). Additionally, approximately 50% of the women infected with the gonococcus are asymptomatic, resulting in increased spread and more severe clinical outcomes following infection (5). Most concerning of all is that the gonococcus has become increasingly drug resistant, with mounting evidence to suggest that current pharmacotherapies may soon be rendered obsolete (6, 7). To date, the characteristics of at least three multidrugresistant isolates have been published, all of which are fully resistant to ceftriaxone, the core component of the currently recommended combination therapy for the treatment of gonorrhea (7-11). With dwindling treatment options and no vaccine, gonorrhea is a serious public health concern that warrants further research.One approach to the development of therapeutics has been to study how the gonococcus acquires iron, an essential nutrient for nearly all microorganisms (12). During human infection, microorganisms are confronted with the challenge of obtaining iron in an environment that has evolved to specifically...

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“…Tf-iron uptake assays were performed as described previously (32)(33)(34)(35). Briefly, apo-human Tf (Sigma) was saturated to 20% with 55 Fe (Perkin-Elmer).…”

Section: Figmentioning

confidence: 99%

Beyond the Crystal Structure: Insight into the Function and Vaccine Potential of TbpA Expressed by Neisseria gonorrhoeae

et al. 2015

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“…Apo-and holo-Tf forms have different affinities for the bacterial Tbp receptors. Unlike TbpA, the TbpBs have a strong preference for binding to the holo-form of Tf (20 -23), which suggests that TbpB may play a role in efficient capture of the holo-form of Tf (24,25). For the Actinobacillus spp., in vitro studies have shown that a TbpBdeficient strain could utilize Tf-bound iron, but was avirulent (26).…”

mentioning

confidence: 99%

“…This potential for diverse receptor-Tf recognition mechanisms provides a rationale for characterizing Tf-TbpB interactions across multiple variants. The sequence heterogeneity for TbpB proteins in particular is considerable, with 47-82% sequence identity observed in variants from a cross-section of pathogenic strains (25,27). Therefore, this study seeks to determine the impact of such heterogeneity on the interaction with Tf, from the perspective of Tf.…”

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confidence: 99%

Conserved Interaction between Transferrin and Transferrin-binding Proteins from Porcine Pathogens

Silva

Calmettes

et al. 2011

Journal of Biological Chemistry

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Gram-negative porcine pathogens from the Pasteurellaceae family possess a surface receptor complex capable of acquiring iron from porcine transferrin (pTf). This receptor consists of transferrin-binding protein A (TbpA), a transmembrane iron transporter, and TbpB, a surface-exposed lipoprotein. Questions remain as to how the receptor complex engages pTf in such a way that iron is positioned for release, and whether divergent strains present distinct recognition sites on Tf. In this study, the TbpB-pTf interface was mapped using a combination of mass shift analysis and molecular docking simulations, localizing binding uniquely to the pTf C lobe for multiple divergent strains of Actinobacillus plueropneumoniae and suis. The interface was further characterized and validated with site-directed mutagenesis. Although targeting a common lobe, variants differ in preference for the two sublobes comprising the iron coordination site. Sublobes C1 and C2 participate in high affinity binding, but sublobe C1 contributes in a minor fashion to the overall affinity. Further, the TbpB-pTf complex does not release iron independent of other mediators, based on competitive iron binding studies. Together, our findings support a model whereby TbpB efficiently captures and presents iron-loaded pTf to other elements of the uptake pathway, even under low iron conditions. Bacteria are dependent upon effective and efficient iron acquisition mechanisms to survive and proliferate in the ironlimited environment of the host (1-3). Pathogenic Gram-negative bacteria within the Neisseriaceae and Pasteurellaceae families rely on specialized uptake systems to acquire iron directly from host iron-binding proteins (2-5). These bacteria can acquire iron from host transferrin (Tf) 3 in particular (6), but in some cases also lactoferrin (7, 8) and hemogobulin/haptoglobulin (9). To understand such uptake mechanisms, a detailed characterization of relevant receptor-host protein interactions is required.The bacterial Tf receptors are composed of two iron-repressible surface components, transferrin binding protein A (TbpA), a TonB-dependent integral outer membrane protein of ϳ100 kDa, and transferrin-binding protein B (TbpB), a lipoprotein varying in size from 60 to 100 kDa (2, 3, 5, 10, 11). They have been found in clinical isolates of the Neisseriaceae, Pasteurellaceae, as well as the Moraxellaceae families (12) and constitute the outer membrane receptor complex responsible for binding Tf and transporting iron across the outer membrane into the periplasmic space (4, 13). These receptors exhibit a strict host specificity. For example, receptors of porcine pathogens will specifically bind porcine transferrin (pTf) but not human, avian, bovine, or ovine Tf (14).Tf is a glycoprotein of ϳ80 kDa, composed of two highly homologous N and C lobes (15). Each lobe contains two domains, connected by two antiparallel ␤-strands in a clamshell-like fold, producing a cleft that coordinates Fe 3ϩ in the binding pocket, along with a synergistic anion (CO 3 2Ϫ or C 2 O 4 ...

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“…TbpB was previously demonstrated to be lipidated (16) and fully surface exposed (35), similar to other neisserial lipoproteins, as well as lipoproteins of several other Gram-negative bacteria, such as Klebsiella, Bordetella, and Borrelia spp. (36)(37)(38).…”

Section: Discussionmentioning

confidence: 99%

“…In E. coli, lipoprotein transport has been well characterized, with the periplasmic Lol system shuttling lipoproteins destined for the outer membrane to their proper location (39). However, unlike the majority of E. coli lipoproteins, which tend to be oriented toward the periplasm, many lipoproteins produced by the gonococcus and other related species are oriented toward the extracellular milieu (35,38). In addition, while N. gonorrhoeae possesses homologues of some of the Lol proteins, the chromosome does not encode a LolE homologue.…”

Section: Discussionmentioning

confidence: 99%

Conserved Regions of Gonococcal TbpB Are Critical for Surface Exposure and Transferrin Iron Utilization

et al. 2013

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The transferrin-binding proteins TbpA and TbpB enable Neisseria gonorrhoeae to obtain iron from human transferrin. The lipoprotein TbpB facilitates, but is not strictly required for, TbpA-mediated iron acquisition. The goal of the current study was to determine the contribution of two conserved regions within TbpB to the function of this protein. Using site-directed mutagenesis, the first mutation we constructed replaced the lipobox (LSAC) of TbpB with a signal I peptidase cleavage site (LAAA), while the second mutation deleted a conserved stretch of glycine residues immediately downstream of the lipobox. We then evaluated the resulting mutants for effects on TbpB expression, surface exposure, and transferrin iron utilization. Western blot analysis and palmitate labeling indicated that the lipobox, but not the glycine-rich motif, is required for lipidation of TbpB and tethering to the outer membrane. TbpB was released into the supernatant by the mutant that produces TbpB LSAC. Neither mutation disrupted the transport of TbpB across the bacterial cell envelope. When these mutant TbpB proteins were produced in a strain expressing a form of TbpA that requires TbpB for iron acquisition, growth on transferrin was either abrogated or dramatically diminished. We conclude that surface tethering of TbpB is required for optimal performance of the transferrin iron acquisition system, while the presence of the polyglycine stretch near the amino terminus of TbpB contributes significantly to transferrin iron transport function. Overall, these results provide important insights into the functional roles of two conserved motifs of TbpB, enhancing our understanding of this critical iron uptake system.

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Identification of Transferrin-Binding Domains in TbpB Expressed by Neisseria gonorrhoeae

Cited by 24 publications

References 52 publications

Beyond the Crystal Structure: Insight into the Function and Vaccine Potential of TbpA Expressed by Neisseria gonorrhoeae

Beyond the Crystal Structure: Insight into the Function and Vaccine Potential of TbpA Expressed by Neisseria gonorrhoeae

Conserved Interaction between Transferrin and Transferrin-binding Proteins from Porcine Pathogens

Conserved Regions of Gonococcal TbpB Are Critical for Surface Exposure and Transferrin Iron Utilization

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