Point Mutations in TbpA Abrogate Human Transferrin Binding in Neisseria gonorrhoeae

Area of Science:

• Microbiology
• Structural Biology
• Vaccine Development

Background:

• TonB-dependent transporters (TDTs) are crucial for metal acquisition in pathogens like Neisseria gonorrhoeae.
• Neisseria gonorrhoeae causes gonorrhea, a disease with no current vaccine; TDTs are explored as vaccine candidates due to their conservation and in vivo expression.
• Transferrin binding protein A (TbpA) is a key virulence factor for iron acquisition from human transferrin (hTf).

Purpose of the Study:

• To identify and characterize single-point substitutions in TbpA that affect human transferrin (hTf) binding and iron acquisition.
• To investigate the role of TbpA's loop 3 helix (L3H) structure in hTf binding and iron utilization.
• To understand the functional significance of specific residues (D355 and A356) in TbpA's interaction with hTf.

Main Methods:

• Site-directed mutagenesis was used to create TbpA mutants (D355P and A356P) in Neisseria gonorrhoeae.
• hTf binding assays were performed to quantify the interaction between mutant TbpA proteins and hTf.
• Iron uptake experiments were conducted using Fe-loaded hTf as the sole iron source to assess bacterial growth and function.
• The role of TbpB in restoring TbpA function was evaluated by co-expression studies.

Main Results:

• TbpA mutants D355P and A356P showed significantly reduced hTf binding and impaired iron uptake.
• The A356P mutation allowed bacterial growth when hTf was the only iron source, indicating partial function.
• The helix structure, rather than charge, appears more critical for hTf binding and iron utilization.
• Co-expression of TbpB restored function in all tested TbpA mutants.

Conclusions:

• Residues D355 and A356 in TbpA are critical for efficient hTf binding, iron extraction, and uptake in Neisseria gonorrhoeae.
• The structural integrity of the L3H is vital for TbpA function, suggesting structural rather than charge-based interactions with hTf.
• Targeting these TbpA residues or their structural motifs could be a strategy for developing novel gonorrhea therapeutics or vaccines.