Isocitrate binds to the itaconic acid-responsive LysR-type transcriptional regulator RipR in Salmonella pathogenesis

Journal home page for Journal of Biological ChemistryAbstract

Macrophages produce itaconic acid in phagosomes in response to bacterial cell wall component lipopolysaccharide (LPS) to eliminate invading pathogenic bacteria. Itaconic acid competitively inhibits the first enzyme of the bacterial glyoxylate cycle. To overcome itaconic acid stress, bacteria employ the bacterial LysR-type transcriptional regulator RipR. However, it remains unknown which molecule activates RipR in bacterial pathogenesis. In this study, we determined the crystal structure of the regulatory domain (RD) of RipR from the intracellular pathogen Salmonella. The RipR RD structure exhibited the typical dimeric arrangement with the putative ligand binding site between the two subdomains. Our isothermal titration calorimetry experiments identified isocitrate as the physiological ligand of RipR, whose intracellular level is increased in response to itaconic acid stress. We further found that 3-phenylpropionic acid significantly decreased the resistance of the bacteria to an itaconic acid challenge. Consistently, the complex structure revealed that the compound is antagonistically bound to the RipR ligand binding site. This study provides the molecular basis of bacterial survival in itaconic acid stress from our immune systems. Further studies are required to reveal biochemical activity, which would elucidate how Salmonella survives in macrophage phagosomes by defending against itaconic acid inhibition of bacterial metabolism.

Keywords

itaconic acid

LysR-type transcriptional regulator

isocitrate

glyoxylate cycle

3-phenylpropionic acid

Abbreviations and NomenclatureGFP

Green fluorescent protein

ITC

isothermal titration calorimetry

LTTR

LysR-type transcriptional regulator

IPTG

isopropyl β-D-1 thiogalactopyranoside

EDTA

ethylene-diamine-tetraacetic acid

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

© 2022 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.

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