Novel roles for pirin proteins and a 2-ketoglutarate:ferredoxin oxidoreductase ortholog in Bacteroides fragilis central metabolism and comparison of metabolic mutant susceptibility to metronidazole and amixicile
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The regulation of the central metabolism and fermentation pathways and its effect on antimicrobial susceptibility in the anaerobic pathogen Bacteroides fragilis is not completely understood. In this study, we show that B. fragilis encodes for two iron-dependent redox-sensitive regulatory pirin protein genes, pir1 and pir2, whose mRNA expression is upregulated following oxygen exposure and growth in iron-limiting conditions. Pir1 and Pir2 modulate short-chain fatty acids production and alter susceptibility to metronidazole (MTZ), and to amixicile (AMIX), a novel inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobes. Consistent with this, we showed that Pir1 forms direct protein-protein interactions with PFOR as determined by two-hybrid system assays. In addition, AlphaFold2-based structural analysis predicts that Pir1 and Pir2 form stable interactions with several enzymes of the central metabolism including the 2-ketoglutarate:ferredoxin oxidoreductases Kor1AB and Kor2CDAEBG. A series of metabolic mutants and electron transport chain inhibitors were used to show a wide-ranging effect of bacterial metabolism on MTZ and AMIX susceptibility. Furthermore, we show that AMIX is an effective antimicrobial against B. fragilis in an experimental model of intra-abdominal infection. This investigation led to the discovery that the kor2AEBG genes are essential for growth, and we present evidence that kor2AEBG genes have dual functions including the reductive synthesis of 2-ketoglutarate via reverse TCA cycle. However, the metabolic activity that bypasses KorAEBG function remains to be defined. Collectively our investigation reveals new information on B. fragilis central metabolism and its modulatory control by pirin proteins which may be leveraged for the future development of new narrow-spectrum antimicrobials