B cell genomics behind cross-neutralization of SARS-CoV-2 variants and SARS-CoV

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Thiostrepton, an Inhibitor of 50S Ribosome Subunit Function

Thiostrepton inhibits (14)C-leucine incorporation by intact cells of Bacillus megaterium as well as (14)C-phenylalanine incorporation by a poly U-directed extract of Escherichia coli. Extracts of E. coli which are pretreated by incubation with thiostrepton cannot be reactivated by dialysis to more than 5% of their former activity. The 50S ribosome subunit appears to be the site of thiostrepton action, since protein-synthesizing activity can be restored to dialyzed pretreated extracts by supplementation with 50S ribosome subunits but not with 30S ribosome subunits. This technique also provides a simple sensitive method for detection of the biological activity of very small amounts of 50S ribosome subunits.     

Differential assay for high-throughput screening of antibacterial compounds

The previously described Bacillus subtilis reporter strain BAU-102 is capable of detecting cell wall synthesis inhibitors that act at all stages of the cell wall synthesis pathway. In addition, this strain is capable of detecting compounds with hydrophobic/surfactant activity and alternative mechanisms of cell wall disruption. BAU-102 sequesters preformed beta-gal in the periplasm, suggesting leakage of beta-gal as the means by which this assay detects compound activities. A model is proposed according to which beta-gal release by BAU-102 reflects activation of pathways leading to autolysis. The authors also report a simplified high-throughput assay using BAU-102 combined with the fluorogenic substrate N-methylumbelliferyl-beta-D-galactoside as a single reagent. Cell wall inhibitors release beta-gal consistently only after 60 min of incubation, whereas compounds with surfactant activity show an almost immediate release. A high-throughput screen of a 480-compound library of known bioactives yielded 8 compounds that cause beta-gal release. These results validate the BAU-102 assay as an effective tool in antimicrobial drug discovery.     

C-terminal functionalization of nylon-3 polymers: effects of C-terminal groups on antibacterial and hemolytic activities

Nylon-3 polymers contain β-amino-acid-derived subunits and can be viewed as higher homologues of poly(α-amino acids). This structural relationship raises the possibility that nylon-3 polymers offer a platform for development of new materials with a variety of biological activities, a prospect that has recently begun to receive experimental support. Nylon-3 homo- and copolymers can be prepared via anionic ring-opening polymerization of β-lactams, and use of an N-acyl-β-lactam as coinitiator in the polymerization reaction allows placement of a specific functional group, borne by the N-acyl-β-lactam, at the N-terminus of each polymer chain. Controlling the unit at the C-termini of nylon-3 polymer chains, however, has been problematic. Here we describe a strategy for specifying C-terminal functionality that is based on the polymerization mechanism. After the anionic ring-opening polymerization is complete, we introduce a new β-lactam, approximately 1 equiv relative to the expected number of polymer chains. Because the polymer chains bear a reactive imide group at their C-termini, this new β-lactam should become attached at this position. If the terminating β-lactam bears a distinctive functional group, that functionality should be affixed to most or all C-termini in the reaction mixture. We use the new technique to compare the impact of N- and C-terminal placement of a critical hydrophobic fragment on the biological activity profile of nylon-3 copolymers. The synthetic advance described here should prove to be generally useful for tailoring the properties of nylon-3 materials.     

Peptide analogues of the VanS catalytic center inhibit VanR binding to its cognate promoter

The dodecamer peptide SLCHDSVIGWEC, named E12, was selected from a combinatorial peptide library on the basis of its ability to bind to VanR, the two-component signal transduction response regulator which controls expression of vancomycin resistance in Enterococcus faecium. The binding of E12 was localized to the N-terminal, regulatory domain of VanR which contains Asp-55, the residue which accepts the phosphoryl group from His-164 in the activated VanS sensor kinase. E12, along with a related sequence SLAHDSIIGYLS, named E12.1, was found to inhibit the binding of VanR approximately P to a DNA segment which corresponds to its cognate promoter PvanH. With a single gap, both E12 and E12.1 could be aligned with the octadecamer sequence YLAHDIKTPLTSIIGYLS, comprising Tyr-161 through Ser-178, of the catalytic center dimerization domain of VanS, a sequence with which VanR also normally interacts. Alanine substitution analysis of E12.1 identified six amino acids as indispensable for its ability to inhibit VanR approximately P-PvanH DNA complex formation. A similar analysis of the corresponding amino acids in VanS showed a parallel dependence except for the substitutions Leu-162 --> Ala and Gly-175 --> Ala which interfered with the ability of E12.1 to compete with protein-DNA complex formation, but did not inhibit the ability of VanS to bind VanR. Our findings support a model in which E12 mimics the VanS phosphorylatable sequence with which the regulatory domain of VanR interacts, and thus functions as a "minimalist" analogue of VanS. Our results also indicate the usefulness of phage-displayed peptides as a general tool for mimicking the interacting faces of interacting proteins.     

The control region for erythromycin resistance: Free energy changes related to induction and mutation to constitutive expression

Summary The mRNA sequence in plasmid pE194 encoding the gene responsible for erythromycin resistance has been deduced from the corresponding DNA sequence. The putative control region of this gene located at the 5 end of the messenger transcribed along with the coding sequence comprises a series of inverted complementary repeat sequences that can redistribute and assume alternative double stranded conformations. Activation of the messenger depends on the degree to which ribosomes inhibited (stalled) by erythromycin disrupt the secondary structure in a part of the control region that simultaneously codes for a small peptide and overlaps two of the inverted repeat sequences. In this model, ribosome stall is produced by erythromycin, the inducer, and the result of inverted repeat sequence redistribution is the unmasking of a ribosome binding site for synthesis of the protein that mediates the resistance phenotype. Free energy calculations have been made for the postulated active and inactive conformations of the control region as well as for 4 classes of mutants that have been mapped in the control region. In addition, calculations have been made for hypothetical sequence alterations corresponding to mutants not yet found. Two modes of induction have been defined and their associated free energy changes calculated; these are (i) preemptive induction, in which ribosome stall resulting in disruption of stem sequence conformation causes one inverted repeat sequence to preempt a second. In the resultant redistribution of complementary inverted repeat sequences, the control region assumes the active conformation owing to unmasking of a sequestered ribosome binding site, and (ii) direct dissociation of the critical stem sequences to unmask the sequenstered ribosome binding site, a reaction that bypasses the preemptive association mechanism. The base change found in one of the mutants is consistent with the preemptive mode of induction according to which ribosome stall to an extent that destabilizes the firt three of 13 possible paired bases in the inactive form of the messenger suffices to induce expression of the resistance phenotype. The single-base-change mutations to constitutive expression most commonly found are precisely those that produce the greatest loss of negative free energy; this majority group affects the control region sequence further downstream and appears to act by the direct dissociation mode. Finally, a pair of weakly interacting inverted repeat sequences flanking the more strongly interacting repeats is present; it is postulated that this outer pair serves during the initial phase of induction, as well as later, to turn off expression of the induced messenger after removal of an inductive stimulus. The wide variety of possible mutations capable of disrupting stability of the control region suggests a model to explain the diversity of partial constitutive phenotypes found in earlier studies.     

23S ribosomal ribonucleic acid of macrolide-producing streptomycetes contains methylated adenine.

Coresistance to macrolide, lincosamide, and streptogramin B-type (MLS) antibiotics by a common biochemical mechanism characterizes clinically resistant pathogens. Of 10 streptomycetes tested for resistance to macrolide, lincosamide, and streptogramin B-type antibiotics, only 1, Streptomyces erythreus, the organism used for production of erythromycin, was found resistant to all three classes; moreover, it was the only streptomycete in the series tested found to contain N6-dimethyladenine (m62A) in 23S ribosomal ribonucleic acid, the structural alteration of ribosomal ribonucleic acid associated with clinical resistance. Of the seven streptomycetes tested for the presence of m62A and N6-methyladenine (m6A), two, S. fradiae and S. cirratus, which produce the macrolide antibiotics tylosin and cirramycin, respectively, were found to contain m6A, but not m62A. The remaining strains tested, including strains which produce lincomycin and streptogramins, contained neither m6A nor m62A.     

Roles of salt and conformation in the biological and physicochemical behavior of protegrin-1 and designed analogues: correlation of antimicrobial, hemolytic, and lipid bilayer-perturbing activities

Protegrins are short (16-18 residues) cationic peptides from porcine leukocytes that display potent, broad-spectrum antimicrobial activity. Protegrin-1 (PG-1), one of five natural homologues, adopts a rigid beta-hairpin structure that is stabilized by two disulfide bonds. We have previously employed the principles of beta-hairpin design to develop PG-1 variants that lack disulfide bonds but nevertheless display potent antimicrobial activity [Lai, J. R., Huck, B. R., Weisblum, B., and Gellman, S. H. (2002) Biochemistry 41, 12835-12842.]. The activity of these disulfide-free variants, however, is attenuated in the presence of salt, and the activity of PG-1 itself is not. Salt-induced inactivation of host-defense peptides, such as human defensins, is thought to be important in some pathological situations (e.g., cystic fibrosis), and the variation in salt-sensitivity among our PG-1 analogues offers a model system with which to explore the origins of these salt effects. We find that the variations in antimicrobial activity among our peptides are correlated with the folding propensities of these molecules and with the extent to which the peptides induce leakage of contents from synthetic liposomes. Comparable correlations were observed between folding and hemolytic activity. The extent to which added salt reduces antimicrobial activity parallels salt effects on vesicle perturbation, which suggests that the biological effects of high salt concentrations arise from modulation of peptide-membrane interactions.