A Structurally Characterized Staphylococcus aureus Evolutionary Escape Route from Treatment with the Antibiotic Linezolid.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen that presents great health concerns. Treatment requires the use of last-line antibiotics, such as members of the oxazolidinone family, of which linezolid is the first member to see regular use in the clinic. Here, we report a short time scale selection experiment in which strains of MRSA were subjected to linezolid treatment. Clonal isolates which had evolved a linezolid-resistant phenotype were characterized by whole-genome sequencing. Linezolid-resistant mutants were identified which had accumulated mutations in the ribosomal protein uL3. Multiple clones which had two mutations in uL3 exhibited resistance to linezolid, 2-fold higher than the clinical breakpoint. Ribosomes from this strain were isolated and subjected to single-particle cryo-electron microscopic analysis and compared to the ribosomes from the parent strain. We found that the mutations in uL3 lead to a rearrangement of a loop that makes contact with Helix 90, propagating a structural change over 15 Å away. This distal change swings nucleotide U2504 into the binding site of the antibiotic, causing linezolid resistance. IMPORTANCE Antibiotic resistance poses a critical problem to human health and decreases the utility of these lifesaving drugs. Of particular concern is the "superbug" methicillin-resistant Staphylococcus aureus (MRSA), for which treatment of infection requires the use of last-line antibiotics, including linezolid. In this paper, we characterize the atomic rearrangements which the ribosome, the target of linezolid, undergoes during its evolutionary journey toward becoming drug resistant. Using cryo-electron microscopy, we describe a particular molecular mechanism which MRSA uses to become resistant to linezolid.

Echinacea purpurea Root Extract Increases Tumor Necrosis Factor Production by Concanavalin A-Activated Murine Splenocytes.

Echinacea purpurea is a common herbal remedy used to treat a variety of illnesses, though its health benefits and effects on the immune system have not been fully elucidated. In this study, we investigated the effect of E. purpurea root extract on the survival of concanavalin A (ConA)-activated murine splenocytes and the production of the cytokines interferon-gamma (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor (TNF) by these cells. We found that E. purpurea root extract did not affect splenocyte survival or IL-2 production and increased IFN-γ cytokine levels only modestly. However, TNF cytokine production by ConA-activated splenocytes increased in response to E. purpurea root extract treatment in a dose-dependent manner, suggesting that E. purpurea root extract does have immunomodulatory effects.

In Silico Fragment-Based Design Identifies Subfamily B1 Metallo-ß-lactamase Inhibitors.

Zinc ion-dependent ß-lactamases (MBLs) catalyze the hydrolysis of almost all ß-lactam antibiotics and resist the action of clinically available ß-lactamase inhibitors. We report how application of in silico fragment-based molecular design employing thiol-mediated metal anchorage leads to potent MBL inhibitors. The new inhibitors manifest potent inhibition of clinically important B1 subfamily MBLs, including the widespread NDM-1, IMP-1, and VIM-2 enzymes; with lower potency, some of them also inhibit clinically relevant Class A and D serine-ß-lactamases. The inhibitors show selectivity for bacterial MBL enzymes compared to that for human MBL fold nucleases. Cocrystallization of one inhibitor, which shows potentiation of Meropenem activity against MBL-expressing Enterobacteriaceae, with VIM-2 reveals an unexpected binding mode, involving interactions with residues from conserved active site bordering loops.