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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
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Evidence for a Constrained Mutational Pathway to High-Level Spectinomycin Resistance in Neisseria: RpsE Loop 2

Dmitry V Kravtsov1, Dmitry A Gryadunov1, Anastasia A Anashkina1

  • 1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia.

International Journal of Molecular Sciences
|July 15, 2026
PubMed
Summary

Spectinomycin resistance in Neisseria gonorrhoeae arises from mutations in ribosomal protein S5 (RpsE). These mutations confer high resistance but reduce bacterial growth, explaining why resistance declines without drug pressure.

Keywords:
Neisseria gonorrhoeaeNeisseria spp.compensatory evolutionfitness costgrowth costribosomal protein S5rpsEspectinomycin resistance

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Area of Science:

  • Microbiology
  • Genetics
  • Molecular Biology

Background:

  • Antimicrobial resistance in Neisseria gonorrhoeae is a significant global health issue.
  • Spectinomycin resistance in N. gonorrhoeae can emerge rapidly but often disappears after drug withdrawal, suggesting a fitness cost.

Purpose of the Study:

  • To investigate the evolutionary pathways leading to spectinomycin resistance in N. gonorrhoeae and related species.
  • To understand the molecular mechanisms underlying spectinomycin resistance and its associated fitness costs.

Main Methods:

  • In vitro selection experiments were conducted on N. gonorrhoeae and commensal Neisseria species.
  • Resistant isolates were characterized using minimum inhibitory concentration (MIC) determination, whole-genome sequencing, and growth-kinetics analysis.
  • Molecular modeling was used to analyze the interaction between mutated RpsE and the ribosome.

Main Results:

  • High-level spectinomycin resistance (MIC > 2048 mg/L) was consistently associated with mutations (substitutions or deletions) in loop 2 of ribosomal protein S5 (RpsE).
  • These RpsE mutations altered the spectinomycin-binding site by perturbing interactions with 16S rRNA, leading to high resistance.
  • Mutations conferring resistance resulted in reduced bacterial growth rates, with the magnitude of the cost varying based on the specific mutation.

Conclusions:

  • Convergent evolution towards specific RpsE loop 2 mutations suggests a constrained mutational pathway to high-level spectinomycin resistance.
  • The associated growth cost of these mutations likely explains the rapid decline of resistant N. gonorrhoeae strains when spectinomycin is withdrawn.
  • Genomic surveillance is crucial for monitoring the emergence and spread of antimicrobial resistance.