Determination of Surface-Exposed, Functional Domains of Gonococcal Transferrin-Binding Protein A

Yost-Daljev, Mary Kate; Cornelissen, Cynthia Nau

doi:10.1128/iai.72.3.1775-1785.2004

Cited by 28 publications

(46 citation statements)

References 52 publications

(69 reference statements)

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“…Specificity was demonstrated by reduced binding of the post-immune antisera against a mutant strain that lacks TbpA (Fig.1C). Surface exposure of the L2 epitope was demonstrated in a previous study wherein an HA epitope insertion into the L2 domain was also surface exposed in whole gonococci [35]. We have previously noted that the sequence comprising L2 of TbpA was well conserved among gonococcal strains [12] and in the current study, this observation was extended to include L2 from gonococcal strain MS11 (Fig.…”

Section: Rationale For Selection Of Epitopes For Inclusion In a Chimementioning

confidence: 75%

Gonococcal transferrin binding protein chimeras induce bactericidal and growth inhibitory antibodies in mice

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Masri

Hollander

et al. 2007

Vaccine

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We have previously demonstrated the full-length gonococcal transferrin binding proteins (TbpA and TbpB) to be promising antigens in the development of a protective vaccine against Neisseria gonorrhoeae. In the current study we employed a genetic chimera approach fusing domains from TbpA and TbpB to the A2 domain of cholera toxin, which naturally binds in a non-covalent fashion to the B subunit of cholera toxin during assembly. For one construct, the N-terminal half of TbpB (NB) was fused to the A2 subunit of cholera toxin. In a second construct, the loop 2 region (L2) of TbpA was genetically fused between the NB domain and the A2 domain, generating a double chimera. Both chimeras were immunogenic and induced serum bactericidal and vaginal growthinhibiting antibodies. This study highlights the potential of using protective epitopes instead of fulllength proteins in the development of an efficacious gonococcal vaccine.

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Section: Rationale For Selection Of Epitopes For Inclusion In a Chimementioning

confidence: 75%

Gonococcal transferrin binding protein chimeras induce bactericidal and growth inhibitory antibodies in mice

Price

Masri

Hollander

et al. 2007

Vaccine

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“…To provide a selectable marker for incorporation of the TbpA L9HA epitope insertion, a new plasmid was constructed. The previously described plasmids pVCU521 and pUNCH755 (16,27) were digested with XbaI. The pUNCH755 fragment containing am mTn3(Cm) cassette was then ligated to pVCU521.…”

Section: Methodsmentioning

confidence: 99%

“…To generate the double mutant MCV858, strain FA19 was used as the recipient strain for transformation with two genetically unlinked markers (encoding TbpA L9HA and TbpB LSAC). Congression was used to provide a selectable marker for gonococcal transformation as previously described (27). Briefly, linearized plasmid DNA encoding the TbpA loop 9 HA epitope insertion and plasmid DNA encoding the TbpB LSAC mutation were added simultaneously as donor DNAs to competent gonococci.…”

Section: Methodsmentioning

confidence: 99%

“…A previously described gonococcal strain, MCV515, contains an HA epitope tag in outer membrane loop 9 of TbpA (L9HA) (27). While TbpB acts as an accessory protein that enhances the efficiency of transferrin iron uptake when TbpA is wild type, the TbpA L9HA mutant grows on transferrin only when TbpB is also produced.…”

Section: Tbpb Is Palmitate Labeled In Mcv843 (Tbpb ⌬Gly) But Not In Mmentioning

confidence: 99%

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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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“…Previous studies suggested that the TbpA plug domain is important for iron internalization (63); however, the precise mechanism was not defined. We hypothesize that the plug domain of TbpA functions in multiple steps of transferrin-iron uptake.…”

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Identification of TbpA Residues Required for Transferrin-Iron Utilization by Neisseria gonorrhoeae

Noto

Cornelissen

2008

Infect Immun

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Neisseria gonorrhoeae requires iron for survival in the human host and therefore expresses high-affinity receptors for iron acquisition from host iron-binding proteins. The gonococcal transferrin-iron uptake system is composed of two transferrin binding proteins, TbpA and TbpB. TbpA is a TonB-dependent, outer membrane transporter critical for iron acquisition, while TbpB is a surface-exposed lipoprotein that increases the efficiency of iron uptake. The precise mechanism by which TbpA mediates iron acquisition has not been elucidated; however, the process is distinct from those of characterized siderophore transporters. Similar to these TonB-dependent transporters, TbpA is proposed to have two distinct domains, a ␤-barrel and a plug domain. We hypothesize that the TbpA plug coordinates iron and therefore potentially functions in multiple steps of transferrin-mediated iron acquisition. To test this hypothesis, we targeted a conserved motif within the TbpA plug domain and generated single, double, and triple alanine substitution mutants. Mutagenized TbpAs were expressed on the gonococcal cell surface and maintained wild-type transferrin binding affinity. Single alanine substitution mutants internalized iron at wild-type levels, while the double and triple mutants showed a significant decrease in iron uptake. Moreover, the triple alanine substitution mutant was unable to grow on transferrin as a sole iron source; however, expression of TbpB compensated for this defect. These data indicate that the conserved motif between residues 120 and 122 of the TbpA plug domain is critical for transferrin-iron utilization, suggesting that this region plays a role in iron acquisition that is shared by both TbpA and TbpB.

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Determination of Surface-Exposed, Functional Domains of Gonococcal Transferrin-Binding Protein A

Cited by 28 publications

References 52 publications

Gonococcal transferrin binding protein chimeras induce bactericidal and growth inhibitory antibodies in mice

Gonococcal transferrin binding protein chimeras induce bactericidal and growth inhibitory antibodies in mice

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

Identification of TbpA Residues Required for Transferrin-Iron Utilization by Neisseria gonorrhoeae

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