New functions of pirin proteins and a 2‐ketoglutarate: Ferredoxin oxidoreductase ortholog in Bacteroides fragilis metabolism and their impact on antimicrobial susceptibility to metronidazole and amixicile

• Published in: MicrobiologyOpen (Weinheim) Vol. 13; no. 4; pp. e1429 - n/a
• Main Authors: Gough, Andrea M., Parker, Anita C., O'Bryan, Patricia J., Whitehead, Terence R., Roy, Sourav, Garcia, Brandon L., Hoffman, Paul S., Jeffrey Smith, C., Rocha, Edson R.
• Format: Journal Article
• Language: English
• Published: England John Wiley & Sons, Inc 01-08-2024; John Wiley and Sons Inc; Wiley
• Subjects: amixicile; Anaerobes; anaerobic bacteria; Anaerobic conditions; Anti-Bacterial Agents - metabolism; Anti-Bacterial Agents - pharmacology; Antibiotics; antimicrobial; Antimicrobial agents; B. fragilis; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteroides fragilis; Bacteroides fragilis - drug effects; Bacteroides fragilis - enzymology; Bacteroides fragilis - genetics; Bacteroides fragilis - metabolism; Bioavailability; Carbon; Dehydrogenases; Drug resistance; Electron transport; Electron transport chain; Enzymes; Fatty acids; Fermentation; Ferredoxin; Gene expression; Gene Expression Regulation, Bacterial; Genes; Glucose; Hybrid systems; Iron; Metabolism; Metronidazole; Metronidazole - metabolism; Metronidazole - pharmacology; Microbial Sensitivity Tests; Original; Oxidoreductase; Pathogens; Phospholipids; pirin‐protein interactions; Plasmids; Prokaryotes; Protein turnover; Proteins; Pyruvic acid; Structural analysis; Susceptibility; Tricarboxylic acid cycle; antimicrobial; pirin‐protein interactions; metronidazole; B. fragilis; amixicile; anaerobic bacteria
• ISSN: 2045-8827; 2045-8827
• DOI: 10.1002/mbo3.1429

The understanding of how central metabolism and fermentation pathways regulate antimicrobial susceptibility in the anaerobic pathogen Bacteroides fragilis is still incomplete. Our study reveals that B. fragilis encodes two iron‐dependent, redox‐sensitive regulatory pirin protein genes, pir1 and pir2. The mRNA expression of these genes increases when exposed to oxygen and during growth in iron‐limiting conditions. These proteins, Pir1 and Pir2, influence the production of short‐chain fatty acids and modify the susceptibility to metronidazole and amixicile, a new inhibitor of pyruvate: ferredoxin oxidoreductase in anaerobes. We have demonstrated that Pir1 and Pir2 interact directly with this oxidoreductase, as confirmed by two‐hybrid system assays. Furthermore, structural analysis using AlphaFold2 predicts that Pir1 and Pir2 interact stably with several central metabolism enzymes, including the 2‐ketoglutarate:ferredoxin oxidoreductases Kor1AB and Kor2CDAEBG. We used a series of metabolic mutants and electron transport chain inhibitors to demonstrate the extensive impact of bacterial metabolism on metronidazole and amixicile susceptibility. We also show that amixicile is an effective antimicrobial against B. fragilis in an experimental model of intra‐abdominal infection. Our investigation led to the discovery that the kor2AEBG genes are essential for growth and have dual functions, including the formation of 2‐ketoglutarate via the reverse TCA cycle. However, the metabolic activity that bypasses the function of Kor2AEBG following the addition of phospholipids or fatty acids remains undefined. Overall, our study provides new insights into the central metabolism of B. fragilis and its regulation by pirin proteins, which could be exploited for the development of new narrow‐spectrum antimicrobials in the future.
This study reveals that several enzymes involved in the central metabolism of Bacteroides fragilis are regulated by protein‐protein interactions with pirin proteins. We observed changes in susceptibility to the antimicrobials metronidazole and amixicile in various metabolic mutants. Amixicile, a novel inhibitor that binds to thiamine−diphosphate dependent enzymes, has proven effective in eliminating B. fragilis in a model of intra‐abdominal infection. Furthermore, we identified a 2‐ketoacid: ferredoxin oxidoreductase as essential for growth and proposed its multifunctional roles.