Analysis of agar diffusion experiments using the new model allows significantly more accurate interpretation of experimental results and determination of MICs. The model has more general validity and is applicable to analysis of other dissipative processes, for example to antigen diffusion and to calculations of substrate load in affinity purification.
Nisin is a small cationic lanthionine antibiotic produced by Lactococcus lactis. During its antimicrobial action, it targets intermediates in the bacterial cell-wall biosynthesis, lipid II, and undecaprenyl pyrophosphate. Here, we report results from electron microscopic investigations of the effects of lethal nisin doses on Bacillus subtilis cell morphology. Bacterial membranes were permeabilized shortly after B. subtilis was incubated with nisin, but this did not lead to immediate cell death. Cell division, as well as other life functions, persisted over at least half an hour after nisin was added. Slower bacterial elongation, consistent with cell envelope inhibition and accelerated division, resulted in cell-length reduction. Abnormal morphogenesis near the division site suggests this to be the primary site of nisin action. Morphological changes are characteristic of deregulation of a filamentous cell envelope protein, Mbl, and the division-inhibiting Min system. We propose a previously undescribed model, in which the lethal action of nisin against B. subtilis starts with membrane permeabilization and is followed by accelerated cell division, cell envelope inhibition, and aberrant cell morphogenesis.cell wall ͉ electron microscopy ͉ bacterial morphology ͉ lantibiotic ͉ cell division
The increasing resistance of human pathogens to conventional antibiotics presents a growing threat to the chemotherapeutic management of infectious diseases. The lanthionine antibiotics, still unused as therapeutic agents, have recently attracted significant scientific interest as models for targeting and management of bacterial infections. We investigated the action of one member of this class, subtilin, which permeabilizes lipid membranes in a lipid II-dependent manner and binds bactoprenyl pyrophosphate, akin to nisin. The role the C and N termini play in target recognition was investigated in vivo and in vitro by using the natural N-terminally succinylated subtilin as well as enzymatically truncated subtilin variants. Fluorescence dequenching experiments show that subtilin induces leakage in membranes in a lipid II-dependent manner and that N-succinylated subtilin is roughly 75-fold less active. Solid-state nuclear magnetic resonance was used to show that subtilin forms complexes with membrane isoprenyl pyrophosphates. Activity assays in vivo show that the N terminus of subtilin plays a critical role in its activity. Succinylation of the N terminus resulted in a 20-fold decrease in its activity, whereas deletion of N-terminal Trp abolished activity altogether.
Surface Plasmon Resonance imaging (SPRi) was used to determine the presence and strength of binding of IgG, IgM and IgA against the Receptor Binding Domain (RBD) of SARS-CoV-2 in sera of 119 CoViD-19 patients. The SPRi assay measures the antibody isotype levels and the strength of binding to the RBD of ultimate 384 patient samples in one run. It turns out that during the course of the disease, the IgG levels and strength of binding increased while generally the IgM and IgA levels go down. Recovered patients all show high strength of binding of the IgG type to the RBD protein. The anti-RBD immunoglobulins SPRi assay provides additional insights in the immune status of patients recovering from CoViD-19 and this new method can furthermore be applied for the assessment of the quality of the immune reaction of healthy individuals to SARS-CoV-2 in vaccination programs.
In an attempt to exploit the large geometry changes associated with azobenzene photo-isomerization for the modulation of antibody-antigen interaction, we introduced in the backbone of the FLAG peptide (DYKDDDDK), an azobenzene unit to photo-modulate its conformational states and consequently its interaction with the monoclonal anti-FLAG-tag antibody M1. The FLAG-tag system is an established technique for purifying and detecting the corresponding fusion proteins. In this context, conflicting evidence has been presented regarding the necessity of calcium for stable binding. Using surface plasmon resonance, we showed that not the initial recognition but certainly the stability of the complex improves in the presence of calcium. Subsequently, we substituted two or three of the central aspartate residues for an artificial, azobenzene-based, photo-responsive amino acid. Four structural isomers of the artificial amino acid were considered, in total twelve FLAG-tag analogues were synthesized. Two showed significant differences in their ability to bind to the antibody in their cis versus their trans state. Interestingly, these two peptides are the two shortest of the twelve photo-peptides investigated. Finally, it was shown that for these two FLAG-analogues switching between cis and trans states is possible in the presence of the antibody.
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