Novel roles for pirin proteins and a 2-ketoglutarate: ferredoxin oxidoreductase ortholog in Bacteroides fragilis central metabolism and comparison of metabolic mutants susceptibility to metronidazole and amixicile

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 are upregulated following oxygen exposure and growth in iron-limiting conditions. Δpir1 and Δpir2 mutants showed altered short-chain fatty acids production compared to the parent strain. Overexpression of Pir1 and Pir2 increased susceptibility to metronidazole (MTZ), and to amixicile (AMIX), a novel inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobes. We hypothesized these observations were a result of a modulatory effect of Pir1 and Pir2 proteins. Consistent with this, we showed that Pir1 forms direct protein-protein interactions with PFOR. In addition, AlphaFold2-based structural analysis predicts that Pir1 and Pir2 form stable interactions with several enzymes of the central metabolism including the 2-ketoglutate:ferredoxin oxidoreductases (KFOR), 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. There was no cross-resistance between MTZ resistant strains with 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. Support for a novel Kor2AEBG activity comes from the findings showing that addition of compounds containing oleic acid stimulated growth of the Δkor2AEBG mutant. 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. These data provide new genetic and metabolic knowledge which may be leveraged for the future development of new narrow-spectrum antimicrobials.