Methods for Assessment of Interactions of Proteins with Heme: Application for Complement Proteins and Immunoglobulins

Revel, Margot; Dimitrov, Jordan D.

doi:10.1007/978-1-0716-1016-9_20

Cited by 2 publications

(3 citation statements)

References 28 publications

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“…The ability of Nim to bind heme was determined by absorption spectroscopy 72 in which the protein is titrated with heme. To determine heme binding, protein (10 µM) in buffer (20 mM Tris-HCl [pH 8.0], 0.25 M NaCl, 18% w/v glycerol and 5 mM β- mercaptoethanol) was used to collect the absorbance spectra between 350 and 700 nm at 2 nm increments in polystyrene 24-well plates in the dark.…”

Section: Methodsmentioning

confidence: 99%

Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile

et al. 2023

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Severe outbreaks and deaths have been linked to the emergence and global spread of fluoroquinolone-resistant Clostridioides difficile over the past two decades. At the same time, metronidazole, a nitro-containing antibiotic, has shown decreasing clinical efficacy in treating C. difficile infection (CDI). Most metronidazole-resistant C. difficile exhibit an unusual resistance phenotype that can only be detected in susceptibility tests using molecularly intact heme. Here, we describe the mechanism underlying this trait. We find that most metronidazole-resistant C. difficile strains carry a T-to-G mutation (which we term PnimBG) in the promoter of gene nimB, resulting in constitutive transcription. Silencing or deleting nimB eliminates metronidazole resistance. NimB is related to Nim proteins that are known to confer resistance to nitroimidazoles. We show that NimB is a heme-dependent flavin enzyme that degrades nitroimidazoles to amines lacking antimicrobial activity. Furthermore, occurrence of the PnimBG mutation is associated with a Thr82Ile substitution in DNA gyrase that confers fluoroquinolone resistance in epidemic strains. Our findings suggest that the pandemic of fluoroquinolone-resistant C. difficile occurring over the past few decades has also been characterized by widespread resistance to metronidazole.

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Section: Methodsmentioning

confidence: 99%

Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile

et al. 2023

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“…Point mutations were generated in pRPF185-PnimB by standard DpnI mediated site-directed mutagenesis, variants were confirmed by Sanger Analysis of heme binding by absorption spectroscopy. The ability of Nim to bind heme was determined using an established method 61 in which the protein is titrated with heme. Nim proteins were purified as above, without hemin being added to the E. coli culture and lysis buffer.…”

Section: Structural Modeling Ofmentioning

confidence: 99%

“…Analysis of heme binding by absorption spectroscopy. The ability of Nim to bind heme was determined using an established method 61 in which the protein is titrated with heme. Nim proteins were purified as above, without hemin being added to the E. coli culture and lysis buffer.…”

Section: Generation Of Knock Out Strainmentioning

confidence: 99%

Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile

Olaitan

Dureja

Youngblom

et al. 2022

Preprint

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Severe outbreaks and deaths have been linked to the emergence and global spread of fluoroquinolone-resistant Clostridioides difficile over the past two decades. At the same time, metronidazole, a nitro-containing antibiotic, has shown decreasing clinical efficacy in treating C. difficile infection (CDI). Most metronidazole-resistant C. difficile exhibit an unusual resistance phenotype that can only be detected in susceptibility tests utilizing molecularly intact heme. Here we describe the mechanism underlying this trait, which we discovered using molecular genetics, phylogenetics, and population analyses. Most metronidazole-resistant strains evolved a T to G mutation, we term PnimBG, in the -10 regulatory promoter of the 5-nitroimidazole reductase nimB, resulting in the gene being constitutively transcribed. Silencing or deleting nimB eliminated metronidazole resistance. We identified the protein as a heme-dependent nitroreductase that degraded nitro-drugs to an amine lacking antimicrobial activity. We further discovered that the metronidazole-resistant PnimBG mutation was strongly associated with the Thr82Ile substitution conferring fluoroquinolone resistance in epidemic strains. Re-analysis of published genomes from global isolates confirmed that all but one encoding PnimBG also carried the Thr82Ile mutation. Our findings suggest that fluoroquinolone and metronidazole resistance co-mediated the pandemic of healthcare-associated C. difficile that are associated with poorer treatment outcomes in CDI patients receiving metronidazole.

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Methods for Assessment of Interactions of Proteins with Heme: Application for Complement Proteins and Immunoglobulins

Cited by 2 publications

References 28 publications

Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile

Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile

Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile

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