Impact of Single Substitution Mutations in Conserved N-Terminal Residues of FtsI on Penicillin Binding and FtsW Interaction in Neisseria gonorrhoeae
Date
2025-05-09
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
0000-0002-3726-3045
Type
Thesis
Degree Level
Masters
Abstract
Background: Penicillin-binding protein 2 of Neisseria gonorrhoeae (PBP2/FtsINg) is an essential cell division protein and the major target of β-lactam antibiotics. As a transpeptidase involved in bacterial cell wall synthesis, its C-terminal domain mutations are well-known for their role in chromosomally-mediated β-lactam resistance. However, the function of the N-terminal domain remains poorly understood. This study investigates the conservation and functional roles of N-terminal FtsI residues across proteobacterial species, their impact on penicillin-binding in N. gonorrhoeae, and their effect on FtsWNg interaction to determine its role in the functionality of FtsINg.
Methods: FtsINg protein sequences and homologs from proteobacterial species were retrieved from the NCBI database and aligned to identify conserved residues. Site-directed mutagenesis was performed on FtsINg to introduce single amino acid substitutions at the identified conserved residues. Recombinant histidine-tagged wild-type and mutant FtsINg were analyzed for structural integrity using size exclusion chromatography multi-angle light scattering (SEC-MALS), dynamic light scattering (DLS), and circular dichroism (CD). The effects of mutations on penicillin-binding were ascertained by isothermal titration calorimetry (ITC). Bacterial two-hybrid (B2H) assays evaluated the impact of these mutations on FtsINg-FtsWNg interaction.
Results: Four amino acid residues (R75, R167, G180, and E193) were 100% conserved in the N-terminus of FtsI from 2132 Proteobacteria species and 19 Neisseria species. DLS and SEC-MALS methods demonstrated that wild-type and mutant FtsINg were monomeric, monodisperse, and free from aggregation. CD analysis confirmed the structural integrity of these proteins. ITC revealed increased penicillin-binding affinity (apparent Kd) for R75A, R75G, R167A, R167G, and by 2.6-fold, 4-fold, 2.9-fold, 2.1-fold, and 2-fold, respectively, compared to wild-type FtsINg (p < 0.05). B2H assays showed that alanine and glycine substitutions at R75, R167, and E193 disrupted FtsINg-FtsWNg interaction, with residual β-galactosidase interaction levels ranging from 56% to 95%.
Conclusions: This study identifies four conserved N-terminal residues in FtsINg as potential allosteric modulators of penicillin binding and FtsWNg interaction, highlighting their roles in antibiotic resistance and cell division. Conservation of these residues across Proteobacteria raises intriguing possibilities about horizontal gene transfer and provides potential targets for novel antimicrobial strategies.
Description
Keywords
Neisseria gonorrhoeae, conserved FtsI N-terminal residues, single substitution mutations, FtsI–FtsW interaction, penicillin binding, potential allosteric modulators, proteobacterial species.
Citation
Degree
Master of Science (M.Sc.)
Department
Biochemistry, Microbiology and Immunology
Program
Microbiology and Immunology