Novobiocin Primarily Targets ParE in Neisseria gonorrhoeae

Background

Neisseria gonorrhoeae is a Gram-negative bacterium that causes gonorrhea, one of the most common sexually transmitted infections worldwide. According to the World Health Organization (WHO), there were 82.3 million new cases of gonorrhea among people aged 15–49 in 2020. However, the emergence and spread of multidrug-resistant strains have made the treatment of gonorrhea increasingly challenging. Although the WHO currently recommends the use of ceftriaxone combined with azithromycin for treatment, some strains have shown resistance to both drugs. Therefore, the search for new anti-gonorrhea drugs has become an urgent priority.

Novobiocin is an aminocoumarin antibiotic originally isolated from Streptomyces niveus and was once used to treat severe infections caused by Staphylococcus aureus. The mechanism of action of novobiocin involves the inhibition of DNA gyrase. However, unlike in other bacteria, the primary target of novobiocin in Neisseria gonorrhoeae remains unclear. This study aims to elucidate the mechanism of action of novobiocin in Neisseria gonorrhoeae and explore its potential as a lead compound for novel anti-gonorrhea drugs.

Source of the Paper

This paper was co-authored by Yoshimasa Ishizaki, Chigusa Hayashi, Kazuaki Matoba, and Masayuki Igarashi, all from the Institute of Microbial Chemistry (Bikaken) in Tokyo, Japan. The paper was accepted on November 22, 2024, and published in The Journal of Antibiotics with the DOI 10.1038/s41429-024-00797-1.

Research Process and Results

1. Research Process

a) Bacterial Culturing and Resistant Strain Screening

The study first used the Neisseria gonorrhoeae DSM9188 strain as the research subject. Resistant mutants were screened using two methods: - Continuous Subculture Method: Bacteria were continuously subcultured for 18 generations in a medium containing sub-inhibitory concentrations (sub-MIC) of novobiocin, ultimately yielding resistant strains. - Single Colony Isolation Method: Bacteria were inoculated onto agar plates containing 16 times the MIC of novobiocin, and resistant colonies were selected.

b) Whole Genome Sequencing and Mutation Analysis

Whole genome sequencing of the resistant strains revealed that all resistant strains had mutations in the parE gene, while no mutations were found in the gyrB gene. Specific mutations included Thr169Ile and Gly75Ser. Additionally, some resistant strains also had mutations in the mtrR gene, which is associated with drug efflux pumps.

c) Genetic Engineering

To validate the impact of mutations on novobiocin resistance, the research team constructed several genetically modified strains: - parE Mutant: The Ile76 in the parE gene was mutated to Met. - gyrB Mutant: The Met82 in the gyrB gene was mutated to Ile. - Double Mutant: Both parE and gyrB mutations were introduced.

d) Minimum Inhibitory Concentration (MIC) Determination

The MIC values of novobiocin against the mutant strains were determined using the microbroth dilution method. The results showed: - parE Mutant: The MIC value significantly increased, indicating that the parE mutation enhanced novobiocin resistance. - gyrB Mutant: The MIC value showed no significant change, indicating that the gyrB mutation had no significant effect on novobiocin resistance. - Double Mutant: The MIC value was intermediate between the parE single mutant and the wild-type strain, suggesting that the gyrB mutation partially restored sensitivity to novobiocin.

e) Molecular Modeling Analysis

The research team used the molecular docking software GNINA to analyze the binding affinity of novobiocin to parE and gyrB. The results showed that Ile76 in parE and Met82 in gyrB are key residues determining novobiocin sensitivity. Molecular modeling further validated the impact of these residues on novobiocin binding.

2. Key Findings

• Novobiocin’s Primary Target is parE: Unlike in Escherichia coli and Staphylococcus aureus, the primary target of novobiocin in Neisseria gonorrhoeae is the parE subunit of DNA topoisomerase IV, rather than the gyrB subunit of DNA gyrase.
• Amino Acid Polymorphisms in parE and gyrB: Unique amino acid polymorphisms (Ile76 and Met82) in parE and gyrB of Neisseria gonorrhoeae determine novobiocin sensitivity.
• Potential of Dual-Target Inhibitors: The findings suggest that designing dual-target novobiocin derivatives that inhibit both parE and gyrB may have stronger anti-gonorrhea activity and delay the emergence of resistance.

3. Conclusions and Significance

This study reveals the unique mechanism of action of novobiocin in Neisseria gonorrhoeae, identifying parE as its primary target rather than gyrB. This discovery provides an important theoretical basis for the development of novel anti-gonorrhea drugs. By designing dual-target inhibitors that simultaneously inhibit parE and gyrB, more effective anti-gonorrhea drugs can be developed, potentially reducing the emergence of resistance.

4. Research Highlights

• Redefining Novobiocin’s Target: This study is the first to clearly identify parE as the primary target of novobiocin in Neisseria gonorrhoeae, challenging traditional understanding.
• Unique Amino Acid Polymorphisms: The unique amino acid polymorphisms in parE and gyrB of Neisseria gonorrhoeae determine novobiocin sensitivity.
• Potential of Dual-Target Inhibitors: The findings provide new insights for the development of dual-target inhibitors, offering significant application value.