scholarly journals Characterization of a clinical Clostridioides difficile isolate with markedly reduced fidaxomicin susceptibility and a V1143D mutation in rpoB

2018 ◽  
Vol 74 (1) ◽  
pp. 6-10 ◽  
Author(s):  
Julian Schwanbeck ◽  
Thomas Riedel ◽  
Friederike Laukien ◽  
Isabel Schober ◽  
Ines Oehmig ◽  
...  
Keyword(s):  
Single Molecule ◽  
Sequence Data ◽  
Normal Growth ◽  
Amplicon Sequencing ◽  
Spore Formation ◽  
Compensatory Mechanisms ◽  
Toxin A ◽  
Clostridioides Difficile

Abstract 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.

scholarly journals In vivo assembly and single-molecule characterization of the transcription machinery from Shewanella oneidensis MR-1

2009 ◽  
Vol 65 (1) ◽  
pp. 66-76 ◽  
Author(s):  
Natalie R. Gassman ◽  
Sam On Ho ◽  
You Korlann ◽  
Janet Chiang ◽  
Yim Wu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Shewanella Oneidensis ◽  
Transcription Machinery

scholarly journals Watching cellular machinery in action, one molecule at a time

2016 ◽  
Vol 216 (1) ◽  
pp. 41-51 ◽  
Author(s):  
Enrico Monachino ◽  
Lisanne M. Spenkelink ◽  
Antoine M. van Oijen
Keyword(s):  
Dynamic Behavior ◽  
Single Molecule ◽  
Protein Complexes ◽  
Imaging Techniques ◽  
Ensemble Averaging ◽  
Cellular Processes ◽  
Cellular Machinery

Single-molecule manipulation and imaging techniques have become important elements of the biologist’s toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of individual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication. We also discuss how single-molecule approaches have increased our understanding of the dynamic behavior of complex multiprotein systems. These methods have shown that the behavior of multicomponent protein complexes is highly stochastic and less linear and deterministic than previously thought. Further development of single-molecule tools will help to elucidate the molecular dynamics of these complex systems both inside the cell and in solutions with purified components.

scholarly journals Nanomechanics and co-transcriptional folding of Spinach and Mango

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Jaba Mitra ◽  
Taekjip Ha
Keyword(s):  
Single Molecule ◽  
Rna Aptamers ◽  
Biophysical Characterization ◽  
Mechanical Responses ◽  
Discrete Step ◽  
Force Relaxation ◽  
G Quadruplex ◽  
And Function

Abstract Recent advances in fluorogen-binding “light-up” RNA aptamers have enabled protein-free detection of RNA in cells. Detailed biophysical characterization of folding of G-Quadruplex (GQ)-based light-up aptamers such as Spinach, Mango and Corn is still lacking despite the potential implications on their folding and function. In this work we employ single-molecule fluorescence-force spectroscopy to examine mechanical responses of Spinach2, iMangoIII and MangoIV. Spinach2 unfolds in four discrete steps as force is increased to 7 pN and refolds in reciprocal steps upon force relaxation. In contrast, GQ-core unfolding in iMangoIII and MangoIV occurs in one discrete step at forces >10 pN and refolding occurred at lower forces showing hysteresis. Co-transcriptional folding using superhelicases shows reduced misfolding propensity and allowed a folding pathway different from refolding. Under physiologically relevant pico-Newton levels of force, these aptamers may unfold in vivo and subsequently misfold. Understanding of the dynamics of RNA aptamers will aid engineering of improved fluorogenic modules for cellular applications.

A new animal model for studies of in vivo folate kinetics

1981 ◽  
Vol 241 (6) ◽  
pp. G516-G519
Author(s):  
S. E. Steinberg ◽  
C. Campbell ◽  
R. S. Hillman
Keyword(s):  
Animal Model ◽  
Local Excision ◽  
Present Report ◽  
Normal Growth ◽  
Target Tissue ◽  
Growth And Nutrition ◽  
Intracellular Metabolism ◽  
Uptake And Metabolism

Characterization of transport kinetics involved in tissue folate supply has been limited by the inability to accurately measure the uptake and metabolism of folate by rapidly proliferating tissues. The present report describes a new animal model that uses subcutaneous fibrosarcoma implants as a target tissue for studies of folate supply. The fibrosarcoma grows rapidly as a well-encapsulated, nonmetastasizing nodule(s) and does not disrupt the animal's normal growth and nutrition. Moreover, the tumor nodules avidly take up labeled folate and can be removed easily by local excision for measurements of uptake and intracellular metabolism of folate.

scholarly journals SMRT and Illumina RNA Sequencing and Characterization of a Key NAC Gene LoNAC29 during the Flower Senescence in Lilium oriental ‘Siberia’

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 869
Author(s):  
Jing Luo ◽  
Ruirui Li ◽  
Xintong Xu ◽  
Hairui Niu ◽  
Yujie Zhang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Sequence Data ◽  
Economic Loss ◽  
Flower Senescence ◽  
Cut Flower ◽  
Transient Overexpression ◽  
Flower Quality ◽  
New Evidence ◽  
Senescence Associated Genes

Lily (Lilium spp.) is an important cut flower around the world. Flower senescence in lilies is characterized by the wilting and abscission of tepals, which results in a decrease in flower quality and huge economic loss. However, the mechanism underlying flower senescence in lilies is largely unknown. In this study, single-molecule, real-time (SMRT) and Illumina sequencing were carried out in L. oriental ‘Siberia’. Sequencing yielded 73,218 non-redundant transcripts, with an N50 of 3792 bp. These data were further integrated with three published transcriptomes through cogent analysis, which yielded 62,960 transcripts, with an increase in N50 of 3935 bp. Analysis of differentially expressed genes showed that 319 transcription factors were highly upregulated during flower senescence. The expression of twelve NAC genes and eleven senescence-associated genes (SAGs) showed that LoNAC29 and LoSAG39 were highly expressed in senescent flowers. Transient overexpression of LoNAC29 and LoSAG39 in tepals of lily notably accelerated flower senescence, and the promoter activity of LoSAG39 was strongly induced by LoNAC29. This work supported new evidence for the molecular mechanism of flower senescence and provided better sequence data for further study in lilies.

scholarly journals Towards characterization of DNA structure under physiological conditions in vivo at the single-molecule level using single-pair FRET

2012 ◽  
Vol 40 (16) ◽  
pp. e121-e121 ◽  
Author(s):  
Tomáš Fessl ◽  
František Adamec ◽  
Tomáš Polívka ◽  
Silvie Foldynová-Trantírková ◽  
František Vácha ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Structure ◽  
Single Pair ◽  
Single Molecule Level ◽  
Physiological Conditions

scholarly journals Toxin A–Predominant Pathogenic Clostridioides difficile: A Novel Clinical Phenotype

2019 ◽  
Vol 70 (12) ◽  
pp. 2628-2633 ◽  
Author(s):  
Qianyun Lin ◽  
Nira R Pollock ◽  
Alice Banz ◽  
Aude Lantz ◽  
Hua Xu ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Toxin Production ◽  
Toxin A ◽  
Toxin B ◽  
Clostridioides Difficile ◽  
Quantitative Immunoassay ◽  
Clinical Detection ◽  
Stool Samples

Abstract Background Most Clostridioides difficile toxinogenic strains produce both toxins A and B (A+B+), but toxin A–negative, toxin B–positive (A−B+) variants also cause disease. We report the identification of a series of pathogenic clinical C. difficile isolates that produce high amounts of toxin A with low or nondetectable toxin B. Methods An ultrasensitive, quantitative immunoassay was used to measure toxins A and B in stool samples from 187 C. difficile infection (CDI) patients and 44 carriers. Isolates were cultured and assessed for in vitro toxin production and in vivo phenotypes (mouse CDI model). Results There were 7 CDI patients and 6 carriers who had stools with detectable toxin A (TcdA, range 23–17 422 pg/mL; 5.6% of samples overall) but toxin B (TcdB) below the clinical detection limit (<20 pg/mL; median TcdA:B ratio 17.93). Concentrations of toxin A far exceeded B in in vitro cultures of all 12 recovered isolates (median TcdA:B ratio 26). Of 8 toxin A>>B isolates tested in mice, 4 caused diarrhea, and 3 of those 4 caused lethal disease. Ribotyping demonstrated strain diversity. TcdA-predominant samples were also identified at 2 other centers, with similar frequencies (7.5% and 6.8%). Conclusions We report the discovery of clinical pathogenic C. difficile strains that produce high levels of toxin A but minimal or no toxin B. This pattern of toxin production is not rare (>5% of isolates) and is consistently observed in vitro and in vivo in humans and mice. Our study highlights the significance of toxin A in human CDI pathogenesis and has important implications for CDI diagnosis, treatment, and vaccine development.

Ultrasensitive and quantitative toxin measurement correlates with baseline severity, severe outcomes, and recurrence among hospitalized patients with Clostridioides difficile infection

2021 ◽  
Author(s):  
Carolyn D Alonso ◽  
Ciarán P Kelly ◽  
Kevin W Garey ◽  
Anne J Gonzales-Luna ◽  
David Williams ◽  
...  
Keyword(s):  
Single Molecule ◽  
Scoring Systems ◽  
Toxin A ◽  
Baseline Severity ◽  
Clostridioides Difficile ◽  
Positive Stool ◽  
Outcome Group ◽  
Clostridioides Difficile Infection ◽  
Group 2 ◽  
Group 1

Abstract Background Stool toxin concentrations may impact Clostridioides difficile infection (CDI) severity and outcomes. We correlated fecal C. difficile toxin concentrations, measured by an ultrasensitive and quantitative assay, with CDI baseline severity, attributable outcomes, and recurrence. Methods We enrolled 615 hospitalized adults (≥ 18y) with CDI (acute diarrhea, positive stool NAAT, and decision to treat). Baseline stool toxin A and B concentrations were measured by Single Molecule Array. Subjects were classified by baseline CDI severity (four scoring methods) and outcomes within 40 days (death, ICU stay, colectomy, and recurrence). Results Among 615 patients (median 68.0 years), in all scoring systems, subjects with severe baseline disease had higher stool toxin A+B concentrations than those without (P<0.01). Nineteen subjects (3.1%) had a severe outcome primarily-attributed to CDI (group 1). This group had higher median toxin A+B [14,303 pg/mL (IQR 416.0, 141,967)] than subjects in whom CDI only contributed to the outcome [group 2, 163.2 pg/mL(0.0, 8423.3)], subjects with severe outcome unrelated to CDI [group 3, 158.6 pg/mL (0.0, 1795.2)], or no severe outcome [group 4, 209.5 pg/mL (0.0, 8566.3)](P=0.003). Group 1 was more likely to have detectable toxin (94.7%) than groups 2-4 (60.5-66.1%)(P=0.02). Individuals with recurrence had higher toxin A+B [2266.8 pg/mL(188.8, 29411)] than those without [154.0 pg/mL(0.0, 5864.3)](P<0.001) and higher rates of detectable toxin (85.7% versus 64.0%, P=0.004). Conclusions In CDI patients, ultrasensitive stool toxin detection and concentration correlated with severe baseline disease, severe CDI-attributable outcomes, and recurrence, confirming the contribution of toxin quantity to disease presentation and clinical course.

scholarly journals The Hyperthermophilic Restriction-Modification Systems of Thermococcus kodakarensis Protect Genome Integrity

2021 ◽  
Vol 12 ◽  
Author(s):  
Kelly M. Zatopek ◽  
Brett W. Burkhart ◽  
Richard D. Morgan ◽  
Alexandra M. Gehring ◽  
Kristin A. Scott ◽  
...  
Keyword(s):  
Single Molecule ◽  
Biochemical Characterization ◽  
Genome Integrity ◽  
Thermococcus Kodakarensis ◽  
Recognition Sites ◽  
Mtase Activity ◽  
Restriction Modification

Thermococcus kodakarensis (T. kodakarensis), a hyperthermophilic, genetically accessible model archaeon, encodes two putative restriction modification (R-M) defense systems, TkoI and TkoII. TkoI is encoded by TK1460 while TkoII is encoded by TK1158. Bioinformative analysis suggests both R-M enzymes are large, fused methyltransferase (MTase)-endonuclease polypeptides that contain both restriction endonuclease (REase) activity to degrade foreign invading DNA and MTase activity to methylate host genomic DNA at specific recognition sites. In this work, we demonsrate T. kodakarensis strains deleted for either or both R-M enzymes grow more slowly but display significantly increased competency compared to strains with intact R-M systems, suggesting that both TkoI and TkoII assist in maintenance of genomic integrity in vivo and likely protect against viral- or plasmid-based DNA transfers. Pacific Biosciences single molecule real-time (SMRT) sequencing of T. kodakarensis strains containing both, one or neither R-M systems permitted assignment of the recognition sites for TkoI and TkoII and demonstrated that both R-M enzymes are TypeIIL; TkoI and TkoII methylate the N6 position of adenine on one strand of the recognition sequences GTGAAG and TTCAAG, respectively. Further in vitro biochemical characterization of the REase activities reveal TkoI and TkoII cleave the DNA backbone GTGAAG(N)20/(N)18 and TTCAAG(N)10/(N)8, respectively, away from the recognition sequences, while in vitro characterization of the MTase activities reveal transfer of tritiated S-adenosyl methionine by TkoI and TkoII to their respective recognition sites. Together these results demonstrate TkoI and TkoII restriction systems are important for protecting T. kodakarensis genome integrity from invading foreign DNA.

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