Search

Clostridium difficile causes life-threatening diarrhea and is the leading cause of healthcare-associated bacterial infections in the United States. TcdA and TcdB bacterial toxins are primary determinants of disease pathogenesis and are attractive therapeutic targets. TcdA and TcdB contain domains that use UDP-glucose to glucosylate and inactivate host Rho GTPases, resulting in cytoskeletal changes causing cell rounding and loss of intestinal integrity. Transition state analysis revealed glucocationic character for the TcdA and TcdB transition states. We identified transition state analogue inhibitors and characterized them by kinetic, thermodynamic and structural analysis. Iminosugars, isofagomine and noeuromycin mimic the transition state and inhibit both TcdA and TcdB by forming ternary complexes with Tcd and UDP, a product of the TcdA- and TcdB-catalyzed reactions. Both iminosugars prevent TcdA- and TcdB-induced cytotoxicity in cultured mammalian cells by preventing glucosylation of Rho GTPases. Iminosugar transition state analogues of the Tcd toxins show potential as therapeutics for C. difficile pathology., (© 2021. The Author(s).)

Clostridium difficile 630 possesses a cryptic but functional gene cluster vanG Cd homologous to the vanG operon of Enterococcus faecalis . Expression of vanG Cd in the presence of subinhibitory concentrations of vancomycin is accompanied by peptidoglycan amidation on the meso -DAP residue. In this paper, we report the presence of two potential asparagine synthetase genes named asnB and asnB2 in the C. difficile genome whose products were potentially involved in this peptidoglycan structure modification. We found that asnB expression was only induced when C. difficile was grown in the presence of vancomycin, yet independently from the vanG Cd resistance and regulation operons. In addition, peptidoglycan precursors were not amidated when asnB was inactivated. No change in vancomycin MIC was observed in the asnB mutant strain. In contrast, overexpression of asnB resulted in the amidation of most of the C. difficile peptidoglycan precursors and in a weak increase of vancomycin susceptibility. AsnB activity was confirmed in E. coli . In contrast, the expression of the second asparagine synthetase, AsnB2, was not induced in the presence of vancomycin. In summary, our results demonstrate that AsnB is responsible for peptidoglycan amidation of C. difficile in the presence of vancomycin.

Vancomycin and related glycopeptides are drugs of last resort for the treatment of severe infections caused by Gram-positive bacteria such as Enterococcus species, Staphylococcus aureus, and Clostridium difficile. Vancomycin was long considered immune to resistance due to its bactericidal activity based on binding to the bacterial cell envelope rather than to a protein target as is the case for most antibiotics. However, two types of complex resistance mechanisms, each comprised of a multi-enzyme pathway, emerged and are now widely disseminated in pathogenic species, thus threatening the clinical efficiency of vancomycin. Vancomycin forms an intricate network of hydrogen bonds with the d-Ala-d-Ala region of Lipid II, interfering with the peptidoglycan layer maturation process. Resistance to vancomycin involves degradation of this natural precursor and its replacement with d-Ala-d-lac or d-Ala-d-Ser alternatives to which vancomycin has low affinity. Through extensive research over 30 years after the initial discovery of vancomycin resistance, remarkable progress has been made in molecular understanding of the enzymatic cascades responsible. Progress has been driven by structural studies of the key components of the resistance mechanisms which provided important molecular understanding such as, for example, the ability of this cascade to discriminate between vancomycin sensitive and resistant peptidoglycan precursors. Important structural insights have been also made into the molecular evolution of vancomycin resistance enzymes. Altogether this molecular data can accelerate inhibitor discovery and optimization efforts to reverse vancomycin resistance. Here, we overview our current understanding of this complex resistance mechanism with a focus on the structural and molecular aspects., (© 2020 The Protein Society.)

Sporulation during Clostridioides difficile infection (CDI) contributes to recurrent disease. Cell division and sporulation both require peptidoglycan biosynthesis. We show C. difficile growth and sporulation is attenuated by antisenses to murA and murC or the MurA inhibitor fosfomycin. Thus, targeting the early steps of peptidoglycan biosynthesis might reduce the onset of recurrent CDI., Competing Interests: Declaration of competing interest None to declare., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

CRS3123 is a novel small molecule that potently inhibits methionyl-tRNA synthetase of Clostridioides difficile , inhibiting C. difficile toxin production and spore formation. CRS3123 has been evaluated in a multiple-ascending-dose placebo-controlled phase 1 trial. Thirty healthy subjects, ages 18 to 45 years, were randomized into three cohorts of 10 subjects each, receiving either 200, 400, or 600 mg of CRS3123 (8 subjects per cohort) or placebo (2 subjects per cohort) by oral administration twice daily for 10 days. CRS3123 was generally safe and well tolerated, with no serious adverse events (SAEs) or severe treatment-emergent adverse events (TEAEs) reported. All subjects completed their assigned treatment and follow-up visits, and there were no trends in systemic, vital sign, or laboratory TEAEs. There were no QTcF interval changes or any clinically significant changes in other electrocardiogram (ECG) intervals or morphology. CRS3123 showed limited but detectable systemic uptake; although absorption increased with increasing dose, the increase was less than dose proportional. Importantly, the bulk of the oral dose was not absorbed, and fecal concentrations were substantially above the MIC 90 value of 1 μg/ml at all dosages tested. Subjects receiving either of the two lower doses of CRS3123 exhibited minimal disruption of normal gut microbiota after 10 days of twice-daily dosing. CRS3123 was inactive against important commensal anaerobes, including Bacteroides , bifidobacteria, and commensal clostridia. Microbiome data showed favorable differentiation compared to other CDI therapeutics. These results support further development of CRS3123 as an oral agent for the treatment of CDI. (This study has been registered at Clinicaltrials.gov under identifier NCT02106338.)., (Copyright © 2019 Lomeli et al.)

Clostridioides difficile infection (CDI) is a leading cause of significant morbidity, mortality, and healthcare-related costs in the United States. After standard therapy, recurrence rates remain high, and multiple recurrences are not uncommon. Causes include treatments employing broad-spectrum agents that disrupt the normal host microbiota, as well as treatment-resistant spore formation by C. difficile . Thus, novel druggable anti- C. difficile targets that promote narrow-spectrum eradication and inhibition of sporulation are desired. As a critical rate-limiting step within the FAS-II bacterial fatty acid synthesis pathway, which supplies precursory component phospholipids found in bacterial cytoplasmic and spore-mediated membranes, enoyl-acyl carrier protein (ACP) reductase II (FabK) represents such a target. FabK is essential in C. difficile ( Cd FabK) and is structurally and mechanistically distinct from other isozymes found in gut microbiota species, making Cd FabK an attractive narrow-spectrum target. We report here the kinetic evaluation of Cd FabK, the biochemical activity of a series of phenylimidazole analogues, and microbiological data suggesting these compounds' selective antibacterial activity against C. difficile versus several other prominent gut organisms. The compounds display promising, selective, low micromolar Cd FabK inhibitory activity without significantly affecting the growth of other gut organisms, and the series prototype ( 1b ) is shown to be competitive for the Cd FabK cofactor and uncompetitive for the substrate. A series analogue ( 1g ) shows maintained inhibitory activity while also possessing increased solubility. These findings represent the basis for future drug discovery efforts by characterizing the Cd FabK enzyme while demonstrating its druggability and potential role as a narrow-spectrum antidifficile target.

Clostridium difficile infection (CDI) is an antibiotic-induced microbiota shift disease of the large bowel. While there is a need for narrow-spectrum CDI antibiotics, it is unclear which cellular proteins are appropriate drug targets to specifically inhibit C. difficile. We evaluated the enoyl-acyl carrier protein (ACP) reductase II (FabK), which catalyzes the final step of bacterial fatty acid biosynthesis. Bioinformatics showed that C. difficile uses FabK as its sole enoyl-ACP reductase, unlike several major microbiota species. The essentiality of fabK for C. difficile growth was confirmed by failure to delete this gene using ClosTron mutagenesis and by growth inhibition upon gene silencing with CRISPR interference antisense to fabK transcription or by blocking protein translation. Inhibition of C. difficile's FASII pathway could not be circumvented by supply of exogenous fatty acids, either during fabK's gene silencing or upon inhibition of the enzyme with a phenylimidazole-derived inhibitor (1). The inability of fatty acids to bypass FASII inhibition is likely due to the function of the transcriptional repressor FapR. Inhibition of FabK also inhibited spore formation, reflecting the enzyme's role in de novo fatty acid biosynthesis for the formation of spore membrane lipids. Compound 1 did not inhibit growth of key microbiota species. These findings suggest that C. difficile FabK is a druggable target for discovering narrow-spectrum anti- C. difficile drugs that treat CDI but avoid collateral damage to the gut microbiota.

Objectives: The identification and characterization of clinical Clostridioides difficile isolates with reduced fidaxomicin susceptibility., Methods: Agar dilution assays were used to determine fidaxomicin MICs. Genome sequence data were obtained by single-molecule real-time (SMRT) sequencing in addition to amplicon sequencing of rpoB and rpoC alleles. Allelic exchange was used to introduce the identified mutation into C. difficile 630Δerm. Replication rates, toxin A/B production and spore formation were determined from the strain with reduced fidaxomicin susceptibility., Results: Out of 50 clinical C. difficile isolates, isolate Goe-91 revealed markedly reduced fidaxomicin susceptibility (MIC >64 mg/L). A V1143D mutation was identified in rpoB of Goe-91. When introduced into C. difficile 630Δerm, this mutation decreased fidaxomicin susceptibility (MIC >64 mg/L), but was also associated with a reduced replication rate, low toxin A/B production and markedly reduced spore formation. In contrast, Goe-91, although also reduced in toxin production, showed normal growth rates and only moderately reduced spore formation capacities. This indicates that the rpoBV1143D allele-associated fitness defect is less pronounced in the clinical isolate., Conclusions: To the best of our knowledge, this is the first description of a pathogenic clinical C. difficile isolate with markedly reduced fidaxomicin susceptibility. The lower-than-expected fitness burden of the resistance-mediating rpoBV1143D allele might be an indication for compensatory mechanisms that take place during in vivo selection of mutants.

The rise of antibiotic resistance has created a mounting crisis across the globe and an unmet medical need for new antibiotics. As part of our efforts to develop new antibiotics to target the uncharted surface bacterial transglycosylase, we report an affinity-based ligand screen method using penicillin-binding proteins immobilized on beads to selectively isolate the binders from complex natural products. In combination with mass spectrometry and assays with moenomycin A and salicylanilide analogues (1-10) as reference inhibitors, we isolated four potent antibacterials confirmed to be benastatin derivatives (11-13) and albofungin (14). Compounds 11 and 14 were effective antibiotics against a broad-spectrum of Gram-positive and Gram-negative bacteria, including Acinetobacter baumannii, Clostridium difficile, Staphylococcus aureus, and drug-resistant strains with minimum inhibitory concentrations in the submicromolar to nanomolar range.

Background and Aim: Clostridium difficile is a major cause of health care-associated infection, but disagreement between diagnostic tests is an ongoing barrier to clinical decision-making. Conventional enzyme immunoassay (EIA) for toxin detection is currently the most frequently used technique for C. difficile infection (CDI) diagnosis, but its low sensitivity makes the development of an alternative strategy necessary for improving the diagnosis in developing countries., Methods: Between years 2011 and 2015, 154 stool samples from patients with antibiotic-associated diarrhea were examined by toxigenic culture and EIA for the diagnosis of CDI. In the year 2015, when glutamate dehydrogenase (GDH) test was first available in Brazil, 53 of those fecal specimens were also tested by the C. diff Quik Chek Complete rapid immunoassay. At this time, we prospectively assessed the impact of this test on CDI treatment rates before and after it was introduced in clinical practice., Results: The GDH component of C. diff Quik Chek Complete test had a sensitivity of 100% and specificity of 95.1% compared with toxigenic culture, with 89.8% concordance. The Tox A/B II EIA and the toxin portion of C. diff Quik Chek Complete yielded sensitivities between values of 50-58.3%, with 100% specificities. The introduction of GDH test increased the number of treated patients with CDI from 57.7% to 100%., Conclusions: Glutamate dehydrogenase test is a reliable method for the diagnosis of CDI and greatly increases the number of properly treated patients with CDI. Therefore, this exam should be considered the mainstay for the laboratory diagnosis of CDI in developing countries., (© 2017 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

In most bacteria, the essential targets of β-lactam antibiotics are the d,d-transpeptidases that catalyze the last step of peptidoglycan polymerization by forming 4→3 cross-links. The peptidoglycan of Clostridium difficile is unusual since it mainly contains 3→3 cross-links generated by l,d-transpeptidases. To gain insight into the characteristics of C. difficile peptidoglycan cross-linking enzymes, we purified the three putative C. difficile l,d-transpeptidase paralogues Ldt Cd1 , Ldt Cd2 , and Ldt Cd3 , which were previously identified by sequence analysis. The catalytic activities of the three proteins were assayed with a disaccharide-tetrapeptide purified from the C. difficile cell wall. Ldt Cd2 and Ldt Cd3 catalyzed the formation of 3→3 cross-links (l,d-transpeptidase activity), the hydrolysis of the C-terminal d-Ala residue of the disaccharide-tetrapeptide substrate (l,d-carboxypeptidase activity), and the exchange of the C-terminal d-Ala for d-Met. Ldt Cd1 displayed only l,d-carboxypeptidase activity. Mass spectrometry analyses indicated that Ldt Cd1 and Ldt Cd2 were acylated by β-lactams belonging to the carbapenem (imipenem, meropenem, and ertapenem), cephalosporin (ceftriaxone), and penicillin (ampicillin) classes. Acylation of Ldt Cd3 by these β-lactams was not detected. The acylation efficacy of Ldt Cd1 and Ldt Cd2 was higher for the carbapenems (480 to 6,600 M -1 s -1 ) than for ampicillin and ceftriaxone (3.9 to 82 M -1 s -1 ). In contrast, the efficacy of the hydrolysis of β-lactams by Ldt Cd1 and Ldt Cd2 was higher for ampicillin and ceftriaxone than for imipenem. These observations indicate that Ldt Cd1 and Ldt Cd2 are inactivated only by β-lactams of the carbapenem class due to a combination of rapid acylation and the stability of the resulting covalent adducts., (Copyright © 2017 American Society for Microbiology.)

Clostridium difficile is a clinically significant pathogen that causes mild-to-severe (and often recurrent) colon infections. Disease symptoms stem from the activities of two large, multidomain toxins known as TcdA and TcdB. The toxins can bind, enter, and perturb host cell function through a multistep mechanism of receptor binding, endocytosis, pore formation, autoproteolysis, and glucosyltransferase-mediated modification of host substrates. Monoclonal antibodies that neutralize toxin activity provide a survival benefit in preclinical animal models and prevent recurrent infections in human clinical trials. However, the molecular mechanisms involved in these neutralizing activities are unclear. To this end, we performed structural studies on a neutralizing monoclonal antibody, PA50, a humanized mAb with both potent and broad-spectrum neutralizing activity, in complex with TcdA. Electron microscopy imaging and multiangle light-scattering analysis revealed that PA50 binds multiple sites on the TcdA C-terminal combined repetitive oligopeptides (CROPs) domain. A crystal structure of two PA50 Fabs bound to a segment of the TcdA CROPs helped define a conserved epitope that is distinct from previously identified carbohydrate-binding sites. Binding of TcdA to the host cell surface was directly blocked by either PA50 mAb or Fab and suggested that receptor blockade is the mechanism by which PA50 neutralizes TcdA. These findings highlight the importance of the CROPs C terminus in cell-surface binding and a role for neutralizing antibodies in defining structural features critical to a pathogen's mechanism of action. We conclude that PA50 protects host cells by blocking the binding of TcdA to cell surfaces.

In 2013, the Centers for Disease Control highlighted Clostridium difficile as an urgent threat for antibiotic-resistant infections, in part due to the emergence of highly virulent fluoroquinolone-resistant strains. Limited therapeutic options currently exist, many of which result in disease relapse. We sought to identify molecules specifically targeting C. difficile in high-throughput screens of our diversity-oriented synthesis compound collection. We identified two scaffolds with apparently novel mechanisms of action that selectively target C. difficile while having little to no activity against other intestinal anaerobes; preliminary evidence suggests that compounds from one of these scaffolds target the glutamate racemase. In vivo efficacy data suggest that both compound series may provide lead optimization candidates.

Kibdelomycin is a complex novel antibiotic, discovered by applying a highly sophisticated chemical-genetic Staphylococcus aureus Fitness Test (SaFT) approach, that inhibits the clinically established bacterial targets, gyrase and topoisomerase IV. It exhibits broad-spectrum antibacterial activity against aerobic bacteria including MRSA and Acinetobacter baumannii. It is slowly bactericidal and has a low frequency of resistance. In an anaerobic environment, it exhibits narrow-spectrum activity and inhibits the growth of gut bacteria Clostridium difficile (MIC 0.125μg/mL) without affecting the growth of commensal Gram-negative organisms particularly, Bacteroides sp. It is highly efficacious in the hamster model of C. difficile infection providing 100% protection at >6mg/kg and 80% protection at 1.56mg/kg by oral dosing without systemic exposure. X-ray co-crystal structures of kibdelomycin bound to GyrB and ParE showed a unique dual arm 'U shaped' multisite binding never encountered with any other gyrase inhibitors. Kibdelomycin is poised for preclinical development for C. difficile treatment, and most importantly, the co-crystal structures of kibdelomycin provide unique insight for structure-guided structure modification, which could lead to better broader-spectrum systemic antibiotic potentially covering many ESKAPE pathogens., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Clostridium difficile infection (CDI) causes moderate to severe disease, resulting in diarrhea and pseudomembranous colitis. CDI is difficult to treat due to production of inflammation-inducing toxins, resistance development, and high probability of recurrence. Only two antibiotics are approved for the treatment of CDI, and the pipeline for therapeutic agents contains few new drugs. MBX-500 is a hybrid antibacterial, composed of an anilinouracil DNA polymerase inhibitor linked to a fluoroquinolone DNA gyrase/topoisomerase inhibitor, with potential as a new therapeutic for CDI treatment. Since MBX-500 inhibits three bacterial targets, it has been previously shown to be minimally susceptible to resistance development. In the present study, the in vitro and in vivo efficacies of MBX-500 were explored against the Gram-positive anaerobe, C. difficile. MBX-500 displayed potency across nearly 50 isolates, including those of the fluoroquinolone-resistant, toxin-overproducing NAP1/027 ribotype, performing as well as comparator antibiotics vancomycin and metronidazole. Furthermore, MBX-500 was a narrow-spectrum agent, displaying poor activity against many other gut anaerobes. MBX-500 was active in acute and recurrent infections in a toxigenic hamster model of CDI, exhibiting full protection against acute infections and prevention of recurrence in 70% of the animals. Hamsters treated with MBX-500 displayed significantly greater weight gain than did those treated with vancomycin. Finally, MBX-500 was efficacious in a murine model of CDI, again demonstrating a fully protective effect and permitting near-normal weight gain in the treated animals. These selective anti-CDI features support the further development of MBX 500 for the treatment of CDI.

Clostridium difficile infection (CDI) is a serious diarrheal disease that often develops following prior antibiotic usage. One of the major problems with current therapies (oral vancomycin and metronidazole) is the high rate of recurrence. Nitazoxanide (NTZ), an inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobic bacteria, parasites, Helicobacter pylori, and Campylobacter jejuni, also shows clinical efficacy against CDI. From a library of ∼250 analogues of NTZ, we identified leads with increased potency for PFOR. MIC screens indicated in vitro activity in the 0.05- to 2-μg/ml range against C. difficile. To improve solubility, we replaced the 2-acetoxy group with propylamine, producing amixicile, a soluble (10 mg/ml), nontoxic (cell-based assay) lead that produced no adverse effects in mice by oral or intraperitoneal (i.p.) routes at 200 mg/kg of body weight/day. In initial efficacy testing in mice treated (20 mg/kg/day, 5 days each) 1 day after receiving a lethal inoculum of C. difficile, amixicile showed slightly less protection than did vancomycin by day 5. However, in an optimized CDI model, amixicile showed equivalence to vancomycin and fidaxomicin at day 5 and there was significantly greater survival produced by amixicile than by the other drugs on day 12. All three drugs were comparable by measures of weight loss/gain and severity of disease. Recurrence of CDI was common for mice treated with vancomycin or fidaxomicin but not for mice receiving amixicile or NTZ. These results suggest that gut repopulation with beneficial (non-PFOR) bacteria, considered essential for protection against CDI, rebounds much sooner with amixicile therapy than with vancomycin or fidaxomicin. If the mouse model is indeed predictive of human CDI disease, then amixicile, a novel PFOR inhibitor, appears to be a very promising new candidate for treatment of CDI.

Clostridium difficile causes severe hospital-acquired antibiotic-associated diarrhea due to the activity of two large protein toxins. Current treatments suffer from a high relapse rate and are generating resistant strains; thus new methods of dealing with these infections that target the virulence factors directly are of interest. Phage display was used to identify peptides that bind to the catalytic domain of C. difficile Toxin A. Library screening and subsequent quantitative binding and inhibition studies showed that several of these peptides are potent inhibitors. Fragment-based computational docking of these peptides elucidated the binding modes within the active site. These antitoxin peptides may serve as potential lead compounds to further engineer peptidomimetic inhibitors of the clostridial toxins.

Clostridium difficile is a leading cause of nosocomial infections. The major virulence factors of this pathogen are the multi-domain toxins TcdA and TcdB. These toxins contain a cysteine protease domain (CPD) that autoproteolytically releases a cytotoxic effector domain upon binding intracellular inositol hexakisphosphate. Currently, there are no known inhibitors of this protease. Here, we describe the rational design of covalent small molecule inhibitors of TcdB CPD. We identified compounds that inactivate TcdB holotoxin function in cells and solved the structure of inhibitor-bound protease to 2.0 Å. This structure reveals the molecular basis of CPD substrate recognition and informed the synthesis of activity-based probes for this enzyme. The inhibitors presented will guide the development of therapeutics targeting C. difficile, and the probes will serve as tools for studying the unique activation mechanism of bacterial toxin CPDs., (Copyright © 2010 Elsevier Ltd. All rights reserved.)