Novel roles for pirin proteins and a 2-ketoglutarate: ferredoxin oxidoreductase ortholog inBacteroides fragiliscentral metabolism and comparison of metabolic mutants susceptibility to metronidazole and amixicile

Abstract

ABSTRACTThe regulation of the central metabolism and fermentation pathways and its effect on antimicrobial susceptibility in the anaerobic pathogenBacteroides fragilisis not completely understood. In this study we show thatB. fragilisencodes for two iron-dependent redox-sensitive regulatory pirin protein genes,pir1andpir2,whose mRNA expression are upregulated following oxygen exposure and growth in iron-limiting conditions.Δpir1andΔpir2mutants 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 againstB. fragilisin an experimental model of intra-abdominal infection. This investigation led to the discovery that thekor2AEBGgenes are essential for growth, and we present evidence thatkor2AEBGgenes 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 theΔkor2AEBGmutant. However, the metabolic activity that bypasses KorAEBG function remains to be defined. Collectively our investigation reveals new information onB. fragiliscentral 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.