Biology subjects

Hurdle, J. G.

Publications and source records attributed to Hurdle, J. G..

5 recordsLinked to original sources

bioRxiv2023

In vivo evaluation of Clostridioides difficile enoyl-ACP reductase II (FabK) Inhibition by phenylimidazole unveils a promising narrow-spectrum antimicrobial strategy

Clostridioides difficile infection (CDI) is a leading cause of hospital-acquired diarrhea, which often stem from disruption of the gut microbiota by broad-spectrum antibiotics. The increasing prevalence of antibiotic-resistant C. difficile strains, combined with disappointing clinical trials results for recent antibiotic candidates, underscore the urgent need for novel CDI antibiotics. To this end, we investigated C. difficile enoyl ACP reductase (CdFabK), a crucial enzyme in de novo fatty acid synthesis, as a drug target for microbiome-sparing antibiotics. To test this concept, we evaluated the efficacy and in vivo spectrum of activity of the phenylimidazole analog 296, which is validated to inhibit intracellular CdFabK. Against major CDI-associated ribotypes 296 had an MIC90 of 2 {micro}g/ml, which was comparable to vancomycin (1 {micro}g/ml), a standard of care antibiotic. In addition, 296 achieved high colonic concentrations and displayed dosed-dependent efficacy in mice with colitis CDI. Mice that were given 296 retained colonization resistance to C. difficile and had microbiomes that resembled the untreated mice. Conversely, both vancomycin and fidaxomicin induced significant changes to mice microbiomes, in a manner consistent with prior reports. CdFabK therefore represents a potential target for microbiome-sparing CDI antibiotics, with phenylimidazoles providing a good chemical starting point for designing such agents.

microbiology↗

bioRxiv2022

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

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 PnimBGmutation 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.

microbiology↗

bioRxiv2022

The licorice metabolite enoxolone attenuates Clostridioides difficile pathophysiology by corrupting its metabolic and toxin production networks

Toxins TcdA and TcdB are the main virulence factors of Clostridioides difficile, a leading cause of hospital-acquired diarrhea. We investigated the therapeutic potential of inhibiting the biosynthesis of TcdA and TcdB. Accordingly, screening of structurally diverse phytochemicals with medicinal properties identified 18{beta}-glycyrrhetinic acid (enoxolone) as an inhibitor of TcdA and TcdB biosynthesis. Enoxolone also inhibited sporulation. In a CDI colitis model, enoxolone when combined with vancomycin protected mice from becoming moribund and the combination was more effective than vancomycin alone, a standard of care antibiotic for CDI. While enoxolone alone reduced the in vivo load of toxins, the monotherapy did not protect mice from CDI. Affinity based proteomics identified ATP synthase subunit alpha (AtpA) and adenine deaminase (Ade) as possible molecular targets for enoxolone. Silencing of mRNA for Ade and AtpA also reduced toxin biosynthesis, while molecular interaction analysis showed that enoxolone directly bound to Ade. Ade converts adenine to hypoxanthine as an early step in the purine salvage pathway. Metabolomics revealed enoxolone caused cells to accumulate adenosine and deplete hypoxanthine and ATP. Accordingly, supplementation with hypoxanthine partly restored toxin production. Enoxolone also impacted phosphate metabolism by reducing the amounts of cellular phosphate. Thus, supplementation with triethyl phosphate as a source of phosphate also partly restored toxin production. When hypoxanthine and triethyl phosphate were combined, toxin production was fully restored in the presence of enoxolone. Taken together, studies with enoxolone revealed metabolic pathways that affect C. difficile toxin production and could represent potential anti-virulence drug targets. IMPORTANCEClostridioides difficile, a leading cause of hospital-acquired diarrhea, produces two co-regulated toxins (TcdA and TcdB) that are the focus of most anti-virulence discovery efforts for C. difficile infection (CDI). Exploration of an alternate anti-virulence strategy led to the discovery that the licorice metabolite enoxolone inhibits C. difficile virulence by blocking the cellular biosynthesis of TcdA and TcdB. Blockage of toxin production by enoxolone was associated with multiple effects on cells, including inhibiting adenine deaminase and ATP synthase leading to disruption of purine biosynthesis and phosphate metabolism. In mice infected with C. difficile, the efficacy of enoxolone in combination with vancomycin was superior to vancomycin alone. These findings contribute to establishing toxin biosynthesis inhibition as a newer therapeutic concept for CDI.

microbiology↗

bioRxiv2020

Ebselen Exhibits Antimicrobial Activity Against Clostridioides difficile By Disrupting Redox Associated Metabolism

High recurrence rates and spread of antibiotic-resistant strains necessitate the need for alternative therapeutics for Clostridioides difficile infections (CDIs). Ebselen, a reactive organoselenium compound inhibits C. difficile virulence toxins TcdA and TcdB, by covalently binding to their cysteine protease domains. Ebselen is thought to lack antibacterial activity against C. difficile cells and its anti-toxin action is suggested to be solely responsible for its efficacy. However, C. difficile has several essential cysteine-containing enzymes that could be potential sites for covalent modification by ebselen; hence, we re-evaluated its anti-C. difficile properties. In BHI agar, ebselen inhibited almost all C. difficile strains (MICs of 2-8 {micro}g/ml), with ribotype 078 being intrinsically resistant (MIC>64 {micro}g/ml). Wilkins-Chalgren and Brucella agars are recommended media for anaerobic susceptibility testing. Ebselen was either slightly attenuated by pyruvate found in Wilkins-Chalgren agar or obliterated by blood in Brucella agars. Transcriptome analysis showed ebselen altered redox-associated processes, cysteine metabolism and significantly enhanced the expression of Stickland proline metabolism to likely regenerate NAD+ from NADH. Intracellularly cells increased the uptake of cysteines, depleted non-protein thiols and disrupted NAD+/NADH redox ratio. Growth inhibitory concentrations of ebselen also reduced toxin and spore production. Taken together, ebselen has bactericidal activity against C. difficile, with multiple mechanisms of action that negatively impacts toxin production and sporulation. To harness the polypharmacological properties of ebselen, chemical optimization is warranted, especially to obtain derivatives that could be effective in severe CDI, where intestinal bleeding could occur.

microbiology↗

bioRxiv2020

Chromosomal Resistance to Metronidazole in Clostridioides difficile can be Mediated By Epistasis Between Iron Homeostasis and Oxidoreductases

Chromosomal resistance to metronidazole has emerged in clinical Clostridioides difficile, but the genetic mechanisms remain unclear. This is further hindered by the inability to generate spontaneous metronidazole-resistant mutants in the lab to aid genetic studies. We therefore constructed a mismatch repair mutator, in non-toxigenic ATCC 700057, to unbiasedly survey the mutational landscape for de novo resistance mechanisms. In separate experimental evolutions, the mutator adopted a deterministic path to resistance, with truncation of ferrous iron transporter FeoB1 as a first-step mechanism of low level resistance. Allelic deletion of feoB1 in ATCC 700057 reduced intracellular iron content, appearing to shift cells toward flavodoxin-mediated oxidoreductase reactions, which are less favorable for metronidazoles cellular action. Higher level resistance evolved from sequential acquisition of mutations to catalytic domains of pyruvate-ferredoxin oxidoreductase (PFOR encoded by nifJ); a synonymous codon change to xdhA1 (xanthine dehydrogenase subunit A), likely affecting its translation; and lastly, frameshift and point mutations that inactivated the iron-sulfur cluster regulator (IscR). Gene silencing of nifJ, xdhA1 or iscR with catalytically dead Cas9 revealed that resistance involving these genes only occurred when feoB1 was inactivated i.e. resistance was only seen in an feoB1-deletion mutant and not the isogenic wild-type parent. These findings show that metronidazole resistance in C. difficile is complex, involving multi-genetic mechanisms that could intersect with iron-dependent metabolic pathways.

microbiology↗