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New antibiotics are urgently needed to address the mounting resistance challenge. In early drug discovery, one of the bottlenecks is the elucidation of targets and mechanisms. To accelerate antibiotic research, we provide a proteomic approach for the rapid classification of compounds into those with precedented and unprecedented modes of action. We established a proteomic response library of Bacillus subtilis covering 91 antibiotics and comparator compounds, and a mathematical approach was developed to aid data analysis. Comparison of proteomic responses (CoPR) allows the rapid identification of antibiotics with dual mechanisms of action as shown for atypical tetracyclines. It also aids in generating hypotheses on mechanisms of action as presented for salvarsan (arsphenamine) and the antirheumatic agent auranofin, which is under consideration for repurposing. Proteomic profiling also provides insights into the impact of antibiotics on bacterial physiology through analysis of marker proteins indicative of the impairment of cellular processes and structures. As demonstrated for trans-translation, a promising target not yet exploited clinically, proteomic profiling supports chemical biology approaches to investigating bacterial physiology.

The article discusses the importance of fermented foods with beneficial microbes as part of man's diet. Topics mentioned include an overview of the process of fermentation which involves the breakdown of substances such as plant-based carbohydrates by bacteria or yeast, the role of the Industrial Revolution in the production of large quantities of foods and transporting them to long distances, and fermented foods as natural antibiotics.

The article discusses the importance of professional learning communities (PLCs) for teacher practice and student learning. It cites characteristics of PLCs including shared views and values on learning ability of students, focus on collaboration and reflective conversations on student development and curriculum. It also provides insights on how to successfully implement PLCs.

Non-equilibrium atmospheric-pressure plasmas are an alternative means to sterilize and disinfect. Plasma-mediated protein aggregation has been identified as one of the mechanisms responsible for the antibacterial features of plasma. Heat shock protein 33 (Hsp33) is a chaperone with holdase function that is activated when oxidative stress and unfolding conditions coincide. In its active form, it binds unfolded proteins and prevents their aggregation. Here we analyse the influence of plasma on the structure and function of Hsp33 of Escherichia coli using a dielectric barrier discharge plasma. While most other proteins studied so far were rapidly inactivated by atmospheric-pressure plasma, exposure to plasma activated Hsp33. Both, oxidation of cysteine residues and partial unfolding of Hsp33 were observed after plasma treatment. Plasma-mediated activation of Hsp33 was reversible by reducing agents, indicating that cysteine residues critical for regulation of Hsp33 activity were not irreversibly oxidized. However, the reduction yielded a protein that did not regain its original fold. Nevertheless, a second round of plasma treatment resulted again in a fully active protein that was unfolded to an even higher degree. These conformational states were not previously observed after chemical activation with HOCl. Thus, although we could detect the formation of HOCl in the liquid phase during plasma treatment, we conclude that other species must be involved in plasma activation of Hsp33. E. coli cells over-expressing the Hsp33-encoding gene hslO from a plasmid showed increased survival rates when treated with plasma while an hslO deletion mutant was hypersensitive emphasizing the importance of protein aggregation as an inactivation mechanism of plasma.

Purpose: Trimethoprim is a folate biosynthesis inhibitor. Tetrahydrofolates are essential for the transfer of C 1 units in several biochemical pathways including purine, thymine, methionine, and glycine biosynthesis. This study addressed the effects of folate biosynthesis inhibition on bacterial physiology., Experimental Design: Two complementary proteomic approaches were employed to analyze the response of Bacillus subtilis to trimethoprim. Acute changes in protein synthesis rates were monitored by radioactive pulse labeling of newly synthesized proteins and subsequent 2DE analysis. Changes in protein levels were detected using gel-free quantitative MS., Results: Proteins involved in purine and histidine biosynthesis, the σ B -dependent general stress response, and sporulation were upregulated. Most prominently, the PurR-regulon required for de novo purine biosynthesis was derepressed indicating purine depletion. The general stress response was activated energy dependently and in a subpopulation of treated cultures an early onset of sporulation was observed, most likely triggered by low guanosine triphosphate levels. Supplementation of adenosine triphosphate, adenosine, and guanosine to the medium substantially decreased antibacterial activity, showing that purine depletion becomes the bottleneck in trimethoprim-treated B. subtilis., Conclusions and Clinical Relevance: The frequently prescribed antibiotic trimethoprim causes purine depletion in B. subtilis, which can be complemented by supplementing purines to the medium., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

RWRWRW-NH2 (MP196) is an amphipathic hexapeptide that targets the bacterial cytoplasmic membrane and inhibits cellular respiration and cell wall synthesis. In previous studies it showed promising activity against Gram-positive bacteria and no significant cytotoxicity or hemolysis. MP196 is therefore used as lead structure for developing more potent antibiotic derivatives. Here we present a more comprehensive study on the parent peptide MP196 with regard to clinically relevant parameters. We found that MP196 acts rapidly bactericidal killing 97% of initial CFU within 10 min at two times MIC. We were unable to detect resistance in standard 24 and 48 h resistance frequency assays. However, MP196 was effective against some but not all MRSA and VISA strains. Serum binding of MP196 was intermediate and we confirmed its low toxicity against mammalian cell lines. MP196 did neither induce NFκB activation nor cause an increase in IL8 levels at 250 μg/mL, and no IgE-dependent activation of basophil granulocytes was detected at 500 μg/mL. Yet, MP196 demonstrated acute toxicity in mice upon injection into the blood stream. Phase contrast microscopy of mouse blood treated with MP196 revealed a shrinking of erythrocytes at 250 μg/mL and severe morphological changes and lysis of erythrocytes at 500 μg/mL. These data suggest that MP196 derivatization directed at further lowering hemolysis could be instrumental in overcoming acute toxicity. The assessment of hemolysis is a critical step in the evaluation of the clinical potential of promising antimicrobial peptides and should be accompanied by microscopy-based morphological analysis of blood cells.

Two hetero-tri-organometallic compounds with potent activity against Gram-positive bacteria including multi-resistant Staphylococcus aureus (MRSA) were identified. The compounds consist of a peptide nucleic acid backbone with an alkyne side chain, substituted with a cymantrene, a (dipicolyl)Re(CO)3 moiety, and either a ferrocene (FcPNA) or a ruthenocene (RcPNA). Comparative proteomic analysis indicates the bacterial membrane as antibiotic target structure. FcPNA accumulation in the membrane was confirmed by manganese tracing with atomic absorption spectroscopy. Both organometallics disturbed several essential cellular processes taking place at the membrane such as respiration and cell wall biosynthesis, suggesting that the compounds affect membrane architecture. Correlating with enhanced antibacterial activity, oxidative stress was induced only by the ferrocene-substituted compound. The organometallics described here target the cytoplasmic membrane, a clinically proven antibacterial target structure, feature a bactericidal but non-bacteriolytic mode of action and limited cytotoxicity within the limits of solubility. Thus, FcPNA represents a promising lead structure for the development of a new synthetic class of antibiotics.