An adjunctive therapy administered with an antibiotic prevents enrichment of antibiotic-resistant clones of a colonizing opportunistic pathogen.

A key challenge in antibiotic stewardship is figuring out how to use antibiotics therapeutically without promoting the evolution of antibiotic resistance. Here, we demonstrate proof of concept for an adjunctive therapy that allows intravenous antibiotic treatment without driving the evolution and onward transmission of resistance. We repurposed the FDA-approved bile acid sequestrant cholestyramine, which we show binds the antibiotic daptomycin, as an 'anti-antibiotic' to disable systemically-administered daptomycin reaching the gut. We hypothesized that adjunctive cholestyramine could enable therapeutic daptomycin treatment in the bloodstream, while preventing transmissible resistance emergence in opportunistic pathogens colonizing the gastrointestinal tract. We tested this idea in a mouse model of Enterococcus faecium gastrointestinal tract colonization. In mice treated with daptomycin, adjunctive cholestyramine therapy reduced the fecal shedding of daptomycin-resistant E. faecium by up to 80-fold. These results provide proof of concept for an approach that could reduce the spread of antibiotic resistance for important hospital pathogens.

Antibiotics are essential for treating infections. But their use can inadvertently lead to the emergence of antibiotic-resistant bacteria that do not respond to antibiotic drugs, making infections with these bacteria difficult or impossible to treat. Finding ways to prevent antibiotic resistance is critical to preserving the effectiveness of antibiotics. Many bacteria that cause infections in hospitals live in the intestines, where they are harmless. But these bacteria can cause life-threatening infections when they get into the bloodstream. When patients with bloodstream infections receive antibiotics, the bacteria in their intestines are also exposed to the drugs. This can kill off all antibiotic-susceptible bacteria, leaving behind only bacteria that have mutations that allow them to survive the drugs. These drug-resistant bacteria can then spread to other patients causing hard-to-treat infections. To stop this cycle of antibiotic treatment and antibiotic resistance, Morley et al. tested whether giving a drug called cholestyramine with intravenous antibiotics could protect the gut bacteria. In the experiments, mice were treated systemically with an antibiotic called daptomycin, which caused the growth of daptomycin-resistant strains of bacteria in the mice's intestines. In the laboratory, Morley et al. discovered that cholestyramine can inactivate daptomycin. Giving the mice cholestyramine and daptomycin together prevented the growth of antibiotic-resistant bacteria in the mice's intestines. Moreover, cholestyramine is taken orally and is not absorbed into the blood. It therefore only inactivates the antibiotic in the gut, but not in the blood. The experiments provide preliminary evidence that giving cholestyramine with antibiotics might help prevent the spread of drug resistance. Cholestyramine is already used to lower cholesterol levels in people. More studies are needed to determine if cholestyramine can protect gut bacteria and prevent antibiotic resistance in people.

The Use of 13C Direct-Detect NMR to Characterize Flexible and Disordered Proteins.

NMR spectroscopy remains the only experimental technique that provides (near) atomistic structural information for intrinsically disordered proteins (IDPs), but their sequence and structure characteristics still pose major challenges for high-resolution spectroscopy. Carbon-13 direct-detect NMR spectroscopy can overcome poor spectral dispersion and other difficulties associated with traditional 1H-detected NMR of nonaggregating disordered proteins. This chapter presents spectroscopic protocols suitable for complete characterization of IDPs that rely exclusively on 13C direct-detect experiments. The protocols described span initial characterization and chemical shift assignment; structure constraint through residual dipolar coupling and paramagnetic relaxation enhancement measurements; and assessment of intramolecular dynamics through 15N spin relaxation. The experiments described empower investigators to establish molecular mechanisms and structure-function relationships for IDPs and other proteins characterized by high internal flexibility.

Incomplete Refolding of Antibody Light Chains to Non-Native, Protease-Sensitive Conformations Leads to Aggregation: A Mechanism of Amyloidogenesis in Patients?

Genetic, biochemical, and pharmacologic evidence supports the hypothesis that conformationally altered or misfolded protein states enable aggregation and cytotoxicity in the systemic amyloid diseases. Reversible structural fluctuations of natively folded proteins are involved in the aggregation of many degenerative disease associated proteins. Herein, we use antibody light chains (LCs) that form amyloid fibrils in AL amyloidosis to consider an alternative hypothesis of amyloidogenesis: that transient unfolding and incomplete extracellular refolding of secreted proteins can lead to metastable, alternatively folded states that are more susceptible to aggregation or to endoproteolysis that can release aggregation-prone fragments. Refolding of full-length λ6a LC dimers comprising an interchain disulfide bond from heat- or chaotrope-denatured ensembles in buffers yields the native dimeric state as well as alternatively folded dimers and aggregates. LC variants lacking an interchain disulfide bond appear to refold fully reversibly to the native state. The conformation of a backbone peptidyl-proline amide in the LC constant domain, which is cis in the native state, may determine whether the LC refolds back to the native state. A proline to alanine (P147A) LC variant, which cannot form the native cis-amide conformation, forms amyloid fibrils from the alternatively folded ensemble, whereas all the full-length λ6a LCs we have studied to date do not form amyloid under analogous conditions. P147A LC variants are susceptible to endoproteolysis by thrombin, enabling amyloidogenesis of the fragments released. Thus, non-native LC structural ensembles containing a tyrosine 146-proline 147 trans-amide bond can initiate and propagate amyloid formation, either directly or after aberrant endoproteolysis.

Substrate Specificity of the Kinase P-TEFb towards the RNA Polymerase II C-Terminal Domain.

The positive transcription elongation factor b (P-TEFb) promotes transcription elongation through phosphorylation of the RNA polymerase II C-terminal domain. This process is not well understood, partly due to difficulties in determining the specificity of P-TEFb toward the various heptad repeat motifs within the C-terminal domain. A simple assay using mass spectrometry was developed to identify the substrate specificity of the Drosophila melanogaster P-TEFb (DmP-TEFb) in vitro. This assay demonstrated that DmP-TEFb preferentially phosphorylates Ser5 and, surprisingly, that pre-phosphorylation or conserved amino acid variation at the 7-position in the heptad can alter DmP-TEFb specificity, leading to the creation of distinct double-phosphorylation marks.