Caroline A. Evans

Spore-forming bacteria produce two distinct cell types: vegetative cells and resilient spores. While antibiotic resistance is typically associated with vegetative cells, spores play a critical role in disseminating resistance genes due to their durability and transmissibility. We previously demonstrated that cephamycin antibiotics target the conserved spore-specific protein SpoVD, significantly reducing spore formation in pathogens including Clostridioides difficile . Here, we show that when C. difficile acquires CdmecA , a homologue of Staphylococcus aureus mecA , one of the most globally burdensome resistance genes, the anti-sporulation effect of cephamycins is bypassed. Cd MecA functionally replaces Cd SpoVD, restoring sporulation and producing phenotypically distinct spores. We further show that mecA is prevalent across C. difficile strains and other pathogenic, gut, and environmental spore-formers. Since SpoVD is conserved, MecA may broadly co-opt sporulation; we confirm this in Clostridium perfringens . This work reveals an unusual resistance mechanism with unexpected physiological consequences, reshaping our understanding of antibiotic resistance within the context of sporulation and microbial adaptation.

Nearly all bacteria are surrounded by a mesh-like macromolecule called peptidoglycan that gives them their shape and helps them resist turgor pressure. To grow and maintain their peptidoglycan, bacteria produce a wide range of enzymes, including the relatively understudied ʟ,ᴅ-transpeptidase (LDT) family. LDTs can catalyse several different reactions and vary widely in copy number: some bacteria have none, whilst others have more than twenty. To better understand why some bacteria have so many LDTs, we examined 18 putative ones from Rhizobium johnstonii , a nitrogen-fixing, symbiotic bacterium. Heterologous expression revealed several highly active enzymes, one of which, Ldt _Rj8 , we further characterized in detail. In vitro assays showed that Ldt _Rj8 was capable of ʟ,ᴅ-transpeptidation, carboxypeptidation, substitution, and endopeptidation, but that its preferred activity differed at different pHs. Ldt _Rj8 particularly excelled at ʟ,ᴅ-substitution, utilizing all of the tested ᴅ-amino acids, and, surprisingly, most of the ʟ-amino acids as well. Ldt _Rj8 ’s pH-modulated activity could help R. johnstonii respond to acidic conditions encountered throughout the rhizobium-legume symbiosis, and its ʟ-amino acid substitution activity, which we show to be a more general property of LDTs, may regulate ʟ,ᴅ-transpeptidation and explain the existence of isomeric muropeptides often reported in the literature.

Peptidoglycan is an essential component of the bacterial cell envelope—a mesh-like macromolecule that protects the bacterium from osmotic stress and its internal turgor pressure. The composition and architecture of peptidoglycan is heterogeneous and changes as bacteria grow, divide, and respond to their environment. Though peptidoglycan has long been studied via LC-MS/MS, the analysis of this data remains challenging as peptidoglycan’s unusual composition and branching can’t be handled by proteomics software. Here we describe user-friendly open-source tools and a web interface for building peptidoglycan databases, performing MS searches, and predicting the MS/MS fragmentation of muropeptides. We then use Rhizobium leguminosarum to describe a step-by-step strategy for the high-resolution analysis of peptidoglycan. The unprecedented detail of R. leguminosarum’s peptidoglycan composition (>250 muropeptides) reveals even the subtlest remodelling between growth conditions. These new and easier to use tools enable more systematic analyses of peptidoglycan dynamics.

Bespoke visualization tools were developed to enhance the analysis of complementary partial RNase digests via LC-MS. These tools enable efficient identity testing of mRNA through comprehensive sequence mapping.

Background: The epithelial-to-mesenchymal transition (EMT) is a common feature in early cancer invasion. Increased vimentin is a canonical marker of the EMT; however, the role of vimentin in EMT remains unknown. Methods: To clarify this, we induced EMT in lung cancer cells with TGF-β1, followed by treatment with the vimentin-targeting drug ALD-R491, live-cell imaging, and quantitative proteomics. Results: We identified 838 proteins in the intermediate filament fraction of cells. TGF-β1 treatment increased the proportion of vimentin in this fraction and the levels of 24 proteins. Variants of fibronectin showed the most pronounced increase (137-fold), followed by regulators of the cytoskeleton, cell motility, and division, such as the mRNA-splicing protein SON. TGF-β1 increased cell spreading and cell migration speed, and changed a positive correlation between cell migration speed and persistence to negative. ALD-R491 reversed these mesenchymal phenotypes to epithelial and the binding of RNA-binding proteins, including SON. Conclusions: These findings present many new interactors of intermediate filaments, describe how EMT and vimentin filament dynamics influence the intermediate filament interactome, and present ALD-R491 as a possible EMT-inhibitor. The observations support the hypothesis that the dynamic turnover of vimentin filaments and their interacting proteins govern mesenchymal cell migration, EMT, cell invasion, and cancer metastasis.

The Epithelial-to-mesenchymal transition (EMT) is a common feature in early cancer invasion. Increased vimentin is a canonical marker of the EMT, however, the role of vimentin in EMT remains unknown. To clarify this, we induced EMT in lung cancer cells with TGF-β1, followed by treatment with the vimentin-targeting drug ALD-R491, live-cell imaging and quantitative proteomics. We identified 838 proteins in the intermediate filament fraction of cells. TGF-β1-treatment increased the proportion of vimentin in this fraction, and the levels of 24 proteins. Variants of fibronectin showed the most pronounced, 137-fold increase, followed by regulators of the cytoskeleton, cell motility and division, such as the mRNA-splicing protein SON. TGF-β1 increased cell spreading, cell migration speed and changed a positive correlation between cell migration speed and persistence to negative. ALD-R491 reversed these mesenchymal phenotypes to epithelial and the binding of RNA-binding proteins, including SON. These findings present ALD-R491 as a possible EMT-inhibitor and describe how EMT and vimentin dynamics influence the interactome of intermediate filaments. The observations support the hypothesis that the dynamic turnover of vimentin filaments and their interacting proteins govern mesenchymal cell migration, EMT, cell invasion and cancer metastasis.