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1. Potentiating antibiotic efficacy via perturbation of non-essential gene expression

   https://doi.org/10.1038/s42003-021-02783-x  

   Otoupal, Peter B. ; Eller, Kristen A. ; Erickson, Keesha E. ; Campos, Jocelyn ; Aunins, Thomas R. ; Chatterjee, Anushree ( November 2021 , Communications Biology)

   Proliferation of multidrug-resistant (MDR) bacteria poses a threat to human health, requiring new strategies. Here we propose using fitness neutral gene expression perturbations to potentiate antibiotics. We systematically explored 270 gene knockout-antibiotic combinations inEscherichia coli, identifying 90 synergistic interactions. Identified gene targets were subsequently tested for antibiotic synergy on the transcriptomic level via multiplexed CRISPR-dCas9 and showed successful sensitization ofE. coliwithout a separate fitness cost. These fitness neutral gene perturbations worked as co-therapies in reducing aSalmonella entericaintracellular infection in HeLa. Finally, these results informed the design of four antisense peptide nucleic acid (PNA) co-therapies,csgD,fnr,recAandacrA, against four MDR, clinically isolated bacteria. PNA combined with sub-minimal inhibitory concentrations of trimethoprim against two isolates ofKlebsiella pneumoniaeandE. colishowed three cases of re-sensitization with minimal fitness impacts. Our results highlight a promising approach for extending the utility of current antibiotics.
2. The Prebiotic Kitchen: A Guide to Composing Prebiotic Soup Recipes to Test Origins of Life Hypotheses

   https://doi.org/10.3390/life11111221  

   Vincent, Lena ; Colón-Santos, Stephanie ; Cleaves, H. James ; Baum, David A. ; Maurer, Sarah E. ( November 2021 , Life)

   “Prebiotic soup” often features in discussions of origins of life research, both as a theoretical concept when discussing abiological pathways to modern biochemical building blocks and, more recently, as a feedstock in prebiotic chemistry experiments focused on discovering emergent, systems-level processes such as polymerization, encapsulation, and evolution. However, until now, little systematic analysis has gone into the design of well-justified prebiotic mixtures, which are needed to facilitate experimental replicability and comparison among researchers. This paper explores principles that should be considered in choosing chemical mixtures for prebiotic chemistry experiments by reviewing the natural environmental conditions that might have created such mixtures and then suggests reasonable guidelines for designing recipes. We discuss both “assembled” mixtures, which are made by mixing reagent grade chemicals, and “synthesized” mixtures, which are generated directly from diversity-generating primary prebiotic syntheses. We discuss different practical concerns including how to navigate the tremendous uncertainty in the chemistry of the early Earth and how to balance the desire for using prebiotically realistic mixtures with experimental tractability and replicability. Examples of two assembled mixtures, one based on materials likely delivered by carbonaceous meteorites and one based on spark discharge synthesis, are presented to illustrate these challenges. We explore alternative proceduresmore »for making synthesized mixtures using recursive chemical reaction systems whose outputs attempt to mimic atmospheric and geochemical synthesis. Other experimental conditions such as pH and ionic strength are also considered. We argue that developing a handful of standardized prebiotic recipes may facilitate coordination among researchers and enable the identification of the most promising mechanisms by which complex prebiotic mixtures were “tamed” during the origin of life to give rise to key living processes such as self-propagation, information processing, and adaptive evolution. We end by advocating for the development of a public prebiotic chemistry database containing experimental methods (including soup recipes), results, and analytical pipelines for analyzing complex prebiotic mixtures.« less
3. SurA is a cryptically grooved chaperone that expands unfolded outer membrane proteins

   https://doi.org/10.1073/pnas.2008175117  

   Marx, Dagan C. ; Plummer, Ashlee M. ; Faustino, Anneliese M. ; Devlin, Taylor ; Roskopf, Michaela A. ; Leblanc, Mathis J. ; Lessen, Henry J. ; Amann, Barbara T. ; Fleming, Patrick J. ; Krueger, Susan ; et al ( October 2020 , Proceedings of the National Academy of Sciences)

   The periplasmic chaperone network ensures the biogenesis of bacterial outer membrane proteins (OMPs) and has recently been identified as a promising target for antibiotics. SurA is the most important member of this network, both due to its genetic interaction with the β-barrel assembly machinery complex as well as its ability to prevent unfolded OMP (uOMP) aggregation. Using only binding energy, the mechanism by which SurA carries out these two functions is not well-understood. Here, we use a combination of photo-crosslinking, mass spectrometry, solution scattering, and molecular modeling techniques to elucidate the key structural features that define how SurA solubilizes uOMPs. Our experimental data support a model in which SurA binds uOMPs in a groove formed between the core and P1 domains. This binding event results in a drastic expansion of the rest of the uOMP, which has many biological implications. Using these experimental data as restraints, we adopted an integrative modeling approach to create a sparse ensemble of models of a SurA•uOMP complex. We validated key structural features of the SurA•uOMP ensemble using independent scattering and chemical crosslinking data. Our data suggest that SurA utilizes three distinct binding modes to interact with uOMPs and that more than one SurA canmore »bind a uOMP at a time. This work demonstrates that SurA operates in a distinct fashion compared to other chaperones in the OMP biogenesis network.

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4. Benziodoxole-Derived Organosulfonates: The Strongest Hypervalent Iodine Electrophiles and Oxidants

   https://doi.org/10.1055/s-0039-1690761  

   Yusubov, Mekhman S. ; Postnikov, Pavel ; Yoshimura, Akira ; Zhdankin, Viktor V. ( February 2020 , Synlett)

   This account describes the development of organosulfonyloxy-substituted iodine(III) and iodine(V) benziodoxole derived reagents, which are thermally stable compounds with useful reactivity patterns. Iodine(III) benziodoxoles and pseudobenziodoxoles are powerful electrophiles and mild oxidants toward various unsaturated compounds. In particular, pseudocyclic benziodoxole-derived triflate (IBA-OTf) is an efficient reagent for oxidative heteroannulation reactions. Aldoximes react with nitriles in the presence of IBA-OTf at room temperature to give 1,2,4-oxadiazoles in high yields. Moreover, IBA-triflate is used as a catalyst in oxidative heteroannulations with m-chloroperoxybenzoic acid as the terminal oxidant. The iodine(V) benziodoxole derived tosylates, DMP-tosylate and IBX-tosylate, are superior oxidants for the oxidation of structurally diverse, synthetically useful alcohols, utilized as key precursors in the total syntheses of polyketide antibiotics and terpenes. And finally, the most powerful hypervalent iodine(V) oxidant, 2-iodoxybenzoic acid ditriflate (IBX·2HOTf), is prepared by treatment of IBX with trifluoromethanesulfonic acid. According to the X-ray data, the I–OTf bonds in IBX-ditriflate have ionic character, leading to the high reactivity of this reagent in various oxidations. In particular, IBX-ditriflate can oxidize polyfluorinated primary alcohols, which are generally extremely resistant to oxidation. 1 Introduction 2 Iodine(III) Benziodoxole Based Organosulfonates 3 Pseudocyclic Iodine(III) Benziodoxole Triflate (IBA-triflate) 4 Pseudocyclic Iodine(III) Benziodoxole Tosylates 5 Iodine(V) Benziodoxole Derivedmore »Tosylates 6 Iodine(V) Benziodoxole Derived Triflate (IBX-ditriflate) 7 Conclusions« less
5. Activity-based ATP analog probes for bacterial histidine kinases

   https://doi.org/bs.mie.2022.01.003  

   Lembke, Hannah K. ; Carlson, Erin E. ( January 2022 , Methods in enzymology)

   Histidine kinases (HKs) are sensor proteins found ubiquitously in prokaryotes. They are the first protein in two-component systems (TCSs), signaling pathways that respond to a myriad of environmental stimuli. TCSs are typically comprised of a HK and its cognate response regulator (RR) which often acts as a transcription factor. RRs will bind DNA and ultimately lead to a cellular response. These cellular outputs vary widely, but HKs are particularly interesting as they are tied to antibiotic resistance and virulence pathways in pathogenic bacteria, making them promising drug targets. We anticipate that HK inhibitors could serve as either standalone antibiotics or antivirulence therapies. Additionally, while the cellular response mediated by the HKs is often well-characterized, very little is known about which stimuli trigger the sensor kinase to begin the phosphorylation cascade. Studying HK activity and enrichment of active HKs through activity-based protein profiling will enable these stimuli to be elucidated, filling this fundamental gap in knowledge. Here, we describe methods to evaluate the potency of putative HK inhibitors in addition to methods to calculate kinetic parameters of various activity-based probes designed for the HKs.