Mutagenesis of the Loop 3 α-Helix of Neisseria gonorrhoeae TdfJ Inhibits S100A7 Binding and Utilization

Maurakis, Stavros; Stoudenmire, Julie L.; Rymer, Jeffrey K.; Chazin, Walter J.; Cornelissen, Cynthia Nau

doi:10.1128/mbio.01670-22

Cited by 5 publications

(4 citation statements)

References 58 publications

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“…This region could have a significant role in the interaction of HpuB with hHb, and in ensuring heme extraction from Hb as seen previously with loop 3 helices of both TbpA and TdfJ (43,62,63). Therefore, acquiring a highresolution structure of HpuB, either by protein x-ray crystallography or cryo-electron microcopy, will help better inform the choice of future mutational targets on the extracellular loops as in other studies (43,62,63). As previously published, Transferrin-Binding Protein B (TbpB) and factor H binding protein (fHbp) mutants that are unable to bind host ligands demonstrated better vaccine potential (33)(34)(35).…”

Section: Discussionmentioning

confidence: 80%

“…We demonstrated the importance of residues 538-544 which constitute a hypothetical α-helical region in loop 7. This region could have a significant role in the interaction of HpuB with hHb, and in ensuring heme extraction from Hb as seen previously with loop 3 helices of both TbpA and TdfJ (43,62,63). Therefore, acquiring a highresolution structure of HpuB, either by protein x-ray crystallography or cryo-electron microcopy, will help better inform the choice of future mutational targets on the extracellular loops as in other studies (43,62,63).…”

Section: Discussionmentioning

confidence: 84%

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Investigating the importance of surface exposed loops in the gonococcal HpuB transporter for hemoglobin binding and utilization

Awate,

Ng,

Stoudenmire

et al. 2023

Preprint

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Neisseria gonorrhoeaeis the etiological agent of the sexually-transmitted infection gonorrhea and a global health challenge since no protective immunity results from infection and far fewer treatment options are available with increasing antimicrobial resistance. With no efficacious vaccines, researchers are exploring new targets for vaccine development and innovative therapeutics. The outer membrane TonB-dependent transporters (TdTs) produced byN. gonorrhoeaeare considered promising antigen targets as they are highly conserved and play crucial roles in overcoming nutritional immunity. One of these TdTs, the hemoglobin transport system comprised of HpuA and HpuB, allowsN. gonorrhoeaeto acquire iron from hemoglobin (hHb). In the current study, mutations in thehpuBgene were generated to better understand the structure-function relationships in HpuB. This study is one of the first to demonstrate thatN. gonorrhoeaecan bind to and utilize hemoglobin produced by animals other than humans. This study also determined that when HpuA is absent, mutations targeting extracellular loop 7 of HpuB led to defective hHb binding and utilization. However, when the lipoprotein HpuA is present, these loop 7 mutants recovered their ability to bind hHB, although their growth phenotype remained significantly impaired. Interestingly, loop 7 contains putative heme binding motifs and a hypothetical α-helical region. Taken together, these results highlight the importance of loop 7 in the functionality of HpuB in binding hHb, and extracting and internalizing iron.

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Section: Discussionmentioning

confidence: 80%

Section: Discussionmentioning

confidence: 84%

Investigating the importance of surface exposed loops in the gonococcal HpuB transporter for hemoglobin binding and utilization

Awate,

Ng,

Stoudenmire

et al. 2023

Preprint

Self Cite

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“…Therefore, the strength of gene regulation by Ngo Zur is dependent on the accuracy and length of the palindromic consensus sequence. Further, TdfJ has been identified as an ideal vaccine antigen for the prevention of gonorrhea (10,35). Its, tight transcriptional regulation by Zur in response to zinc, demonstrate its potential to be a molecular tool that will allow further investigation of its role during infection and enhance the understanding behind creating an efficacious vaccine.…”

Section: Discussionmentioning

confidence: 99%

“…Cell pellets were harvested by centrifugation and stored at -80C before lysis. Cell pellets were re-suspended in lysis buffer (20 mM HEPES, 250 mM NaCl, pH 8.0, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 U/µL Pierce Universal Nuclease for Cell lysis) and lysed using a Dounce homogenizer, with 10 mL of buffer used per gram of cell pellet (35). The resulting cell suspension was mechanically lysed by sonication at 50% amplitude at Pulse-time mode, with sonication at 30 second intervals for 10 minutes.…”

Section: Expression and Purification Of Recombinant His-ngfurmentioning

confidence: 99%

Characterizing the dual regulation ofNeisseria gonorrhoeae tdfJin response to zinc and iron

Padmanabhan,

Stoudenmire,

Branch

et al. 2024

Preprint

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Gonorrhea is a sexually transmitted infection, caused by the bacterial pathogenNeisseria gonorrhea(Ngo) and affects millions of individuals of all age groups across the globe every year. Infection withNgodoes not result in protection and no effective vaccine has been developed, leaving antibiotics as the only treatment option. With the emergence of strains showing high levels of antibiotic resistance, there is an urgent need for development of novel therapeutics for disease prevention. During pathogenesis the host employs nutritional immunity, to restrict important transition metals such as zinc away fromNgo. This process is counteracted inNgoby the production of highly efficient zinc import TonB-dependent Transporters (TDTs) which are promising vaccine antigens and zinc shuttle ABC transporters found to be important for intracellular survival. InNgozinc homeostasis and transport proteins are regulated by the Zinc uptake regulator (Zur) which represses transcription in the presence of zinc and activates transcription in the absence of zinc. In this study, characterize the promoter elements of the zinc import TDT,tdfJ, which results in dual regulation by zinc and iron. We characterize specificity and binding affinities for regulation oftdfJby a second regulator, Ferric uptake regulator (Fur) in response to iron. The response oftdfJto both iron and zinc and its potential to be an important invasin, makes it an attractive candidate to investigate female genital tract infections. The female genital tract is a conglomerate of these conditions and infections here are often asymptomatic. Taken together, this research provides important knowledge on the regulation of virulence mechanisms in response to zinc, which will aid in the development of therapeutics and an efficacious vaccine against a gonococcal infection.

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Investigating the importance of selected surface-exposed loops in HpuB for hemoglobin binding and utilization by Neisseria gonorrhoeae

Awate,

Ng,

Stoudenmire

et al. 2024

Infect Immun

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Neisseria gonorrhoeae is the etiological agent of the sexually transmitted infection gonorrhea. The pathogen is a global health challenge since no protective immunity results from infection, and far fewer treatment options are available with increasing antimicrobial resistance. With no efficacious vaccines, researchers are exploring new targets for vaccine development and innovative therapeutics. The outer membrane TonB-dependent transporters (TdTs) produced by N. gonorrhoeae are considered promising vaccine antigens as they are highly conserved and play crucial roles in overcoming nutritional immunity. One of these TdTs is part of the hemoglobin transport system comprised of HpuA and HpuB. This system allows N. gonorrhoeae to acquire iron from hemoglobin (hHb). In the current study, mutations in the hpuB gene were generated to better understand the structure–function relationships in HpuB. This study is one of the first to demonstrate that N. gonorrhoeae can bind to and utilize hemoglobin produced by animals other than humans. This study also determined that when HpuA is absent, mutations targeting extracellular loop 7 of HpuB led to defective hHb binding and utilization. However, when the lipoprotein HpuA is present, these loop 7 mutants recovered their ability to bind hHb, although the growth phenotype remained significantly impaired. Interestingly, loop 7 contains putative heme-binding motifs and a hypothetical α-helical region, both of which may be important for the use of hHb. Taken together, these results highlight the importance of loop 7 in the functionality of HpuB in binding hHb and extracting and internalizing iron.

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Mutagenesis of the Loop 3 α-Helix of Neisseria gonorrhoeae TdfJ Inhibits S100A7 Binding and Utilization

Cited by 5 publications

References 58 publications

Investigating the importance of surface exposed loops in the gonococcal HpuB transporter for hemoglobin binding and utilization

Investigating the importance of surface exposed loops in the gonococcal HpuB transporter for hemoglobin binding and utilization

Characterizing the dual regulation ofNeisseria gonorrhoeae tdfJin response to zinc and iron

Investigating the importance of selected surface-exposed loops in HpuB for hemoglobin binding and utilization by Neisseria gonorrhoeae

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