Biology subjects

Tresco, B. I. C.

Publications and source records attributed to Tresco, B. I. C..

4 recordsLinked to original sources

bioRxiv2023

Structural basis of Cfr-mediated antimicrobial resistance and mechanisms for its evasion

The ribosome is an essential drug target as many classes of clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent mechanisms of resistance to PTC-acting drugs is C8-methylation of the universally conserved adenine residue 2503 (A2503) of the 23S rRNA by the methyltransferase Cfr. Despite its clinical significance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. In this work, we developed a method to express a functionally-active Cfr-methyltransferase in the thermophilic bacterium Thermus thermophilus and report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-tRNAs. Our structures reveal that an allosteric rearrangement of nucleotide A2062 upon Cfr-methylation of A2503 is likely responsible for the inability of some PTC inhibitors to bind to the ribosome, providing additional insights into the Cfr resistance mechanism. Lastly, by determining the structures of the Cfr-methylated ribosome in complex with the antibiotics iboxamycin and tylosin, we provide the structural bases behind two distinct mechanisms of evading Cfr-mediated resistance.

molecular biology↗

bioRxiv2022

Genome-encoded ABCF factors implicated in intrinsic antibiotic resistance in Gram-positive bacteria: VmlR2, Ard1 and CplR

Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 from Streptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacterium Neobacillus vireti) and Clostridia (CplR from Clostridium perfringens, Clostridium sporogenes and Clostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate that C. difficile CplR (CDIF630_02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to the C. difficile 630 clinical isolate. Finally, assisted by our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction of cplR expression upon an antibiotic challenge.

microbiology↗

bioRxiv2022

Diversity-oriented synthesis encoded by deoxyoligonucleotides

Diversity-oriented synthesis (DOS)is a powerful strategy to prepare molecules with underrepresented features in commercial screening collections, resulting in the elucidation of novel biological mechanisms. In parallel to the development of DOS, DNA-encoded libraries (DELs) have emerged as an effective, efficient screening strategy to identify protein binders. Despite recent advancements in this field, most DEL syntheses are limited by the presence of sensitive DNA-based constructs. Here, we describe the design, synthesis, and validation experiments performed for a 3.7 million-member DEL, generated using diverse skeleton architectures with varying exit vectors, derived from DOS, to achieve structural diversity beyond what is possible by varying appendages alone. We will make this DEL available to the academic scientific community to increase access to novel structural features and accelerate early-phase drug discovery.

biochemistry↗

bioRxiv2022

Synthetic oxepanoprolinamide iboxamycin is highly active against human pathogen Listeria monocytogenes

Listeriosis is a dangerous food-borne bacterial disease caused by the Gram-positive Bacillota (Firmicute) bacterium Listeria monocytogenes. In this report, we show that the synthetic lincosamide iboxamycin is highly active against L. monocytogenes and can overcome the intrinsic lincosamide resistance mediated by VgaL/Lmo0919, a member of ABCF ATPase resistance determinants that act by directly removing the antibiotic from the ribosome. While iboxamycin is not bactericidal against L. monocytogenes, it displays a pronounced postantibiotic effect, which is a valuable pharmacokinetic feature. Experiments in L. monocytogenes infection models are necessary to further assess the potential of iboxamycin as a novel drug for treatment of listeriosis. We demonstrate that VmlR ARE ABCF of Bacillota bacterium Bacillus subtilis grants significant (33-fold increase in MIC) protection from iboxamycin, while LsaA ABCF of Enterococcus faecalis grants an 8-fold protective effect. Furthermore, the VmlR-mediated iboxamycin resistance is cooperative with that mediated by the Cfr 23S rRNA methyltransferase resistance determinant, resulting in up to a 512-fold increase in MIC. Therefore, emergence and spread of ABCF ARE variants capable of defeating next-generation lincosamides in the clinic is possible and should be closely monitored.

microbiology↗