Alford, J. | Search bioRxiv

bioRxiv2026

Pathogenome and Plasmid-Borne Antimicrobial Resistance Phenotypes in a Multidrug-Resistant O111:H8 Shiga Toxin-Producing Escherichia coli Strain

Plasmids contribute to virulence and antimicrobial resistance in Shiga toxin-producing Escherichia coli (STEC). Here, the mobility, gene content, and evolutionary context of four plasmids carried by an O111:H8 STEC strain designated UTAK-22: pUTAK-22.1-MDR, pUTAK-22.2-MDR, pUTAK-22.3-P1, and pUTAK-22.4-pO111 were characterized. Conjugation experiments, phenotype-based selective readouts, and comparative genomics together showed that pUTAK-22.1-MDR is self-transmissible, whereas pUTAK-22.2-MDR and pUTAK-22.4-pO111 are most consistent with mobilization in trans, making use of the strain's native helper plasmid background, while pUTAK-22.3-P1 represents a conjugation-deficient IncY phage-derived replicon. Genome annotation and comparative analyses further highlighted the structural diversity and mosaic composition of these plasmids, including P1-like phage remnants, virulence loci, and distinct antimicrobial resistance modules. Phenotypic profiling of the native wild-type plasmid complement and selected transconjugants in the recipient E. coli strain WG5 further showed that the presence of co-resident plasmids and redundant resistance determinants results in dosage-dependent streptomycin tolerance. Together, these findings expand our understanding of the mobility landscape, evolutionary dynamics, and resistance potential of STEC plasmids, while underscoring the importance of interpreting resistance phenotypes in the context of a strain's natural plasmid composition.

microbiology↗

bioRxiv2023

Reinstating targeted protein degradation with DCAF1 PROTACs in CRBN PROTAC resistant settings

Targeted protein degradation (TPD) of neo-substrates with proteolysis targeting chimeras (PROTACs) or molecular glues has emerged as a key modality in exploring new biology as well as designing new drug candidates where catalytic inhibition is neither efficacious nor an option. TPD is mediated through harnessing E3 ligases and redirecting them to ubiquitinate de novo target proteins for subsequent proteasomal degradation. Until recently, E3 ligase chemical matter available for mediating TPD has been limited to a relatively low number of ligases, considering that over 600 E3 ligases are encoded by the human genome. In addition, the most utilized ligase for TPD approaches, CRBN, has been observed to be downregulated in settings of acquired resistance to immunomodulatory inhibitory drugs (IMiDs). IMiDs are molecular glues that target IKZF transcription factors to CRBN for degradation. Resistance is potentially accelerated by non-essentiality of CRBN for cell viability. Here we investigated if the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a potent, non-covalent DCAF1 binder. We show that this binder, selective for the CRL4DCAF1 E3 ligase complex, can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments confirm specific degradation via the CRL4DCAF1 E3 ligase. We further highlight the versatility of DCAF1 for TPD by developing a DCAF1-dasatininb PROTAC targeting multiple cytosolic and membrane bound tyrosine kinases. We expand these findings towards Brutons tyrosine kinase (BTK) selective PROTACs and through extensive optimization and characterization efforts share key observations that led to a potent and selective DCAF1-BTK PROTAC (DBt-10). Finally, with this PROTAC DBt-10, we show rescue of BTK degradation in a BTK-dependent, CRBN-degradation-resistant cell line and provide a rationale for E3 ligase swap to overcome CRBN mediated resistance.

biochemistry↗