LipidII:Just Another Brick in the Wall?

Nearly all bacteria contain a peptidoglycan cell wall. The peptidoglycan precursor molecule is LipidII, containing the basic peptidoglycan building block attached to a lipid. Although the suitability of LipidII as an antibacterial target has long been recognized, progress on elucidating the role(s) of LipidII in bacterial cell biology has been slow. The focus of this review is on exciting new developments, both with respect to antibacterials targeting LipidII as well as the emerging role of LipidII in organizing the membrane and cell wall synthesis. It appears that on both sides of the membrane, LipidII plays crucial roles in organizing cytoskeletal proteins and peptidoglycan synthesis machineries. Finally, the recent discovery of no less than three different categories of LipidII flippases will be discussed

In vivo cluster formation of nisin and Lipid II is correlated with membrane depolarization

Nisin and related lantibiotics kill bacteria by pore formation, or by sequestering Lipid II. Some lantibiotics sequester Lipid II into clusters, which were suggested to kill cells through delocalized peptidoglycan synthesis. Here, we show that cluster formation is always concomitant with (i) membrane pore formation and (ii) membrane depolarization. Nisin variants that cluster Lipid II kill L-form bacteria with similar efficiency, suggesting that delocalization of peptidoglycan synthesis is not the primary killing mechanism of these lantibiotics

Bicyclic enol cyclocarbamates inhibit penicillin-binding proteins

Natural products form attractive leads for the development of chemical probes and drugs. The antibacterial lipopeptide Brabantamide A contains an unusual enol cyclocarbamate and we used this scaffold as inspiration for the synthesis of a panel of enol cyclocarbamate containing compounds. By equipping the scaffold with different groups, we identified structural features that are essential for antibacterial activity. Some of the derivatives block incorporation of hydroxycoumarin carboxylic acid-amino d-alanine into the newly synthesized peptidoglycan. Activity-based protein-profiling experiments revealed that the enol carbamates inhibit a specific subset of penicillin-binding proteins in B. subtilis and S. pneumoniae

LipidII regulates membrane association of MreB.

<p>Using Total Internal Reflection Fluorescence (TIRF) microscopy, association and dissociation of MreB-GFP with the membrane can be followed. Upon depletion of MurG and subsequent halt of conversion of LipidI to LipidII, MreB-GFP is released from the membrane (upper row); after induction of MurG expression, LipidII production is resumed and MreB-GFP is re-localized on the membrane (lower row). Shown are snapshots (A, B, D, E) of single TIRF images at the respective time points and an analysis of the variance in intensity over time (C, F), with red indicating regions of high protein mobility and blue denoting low mobility. (Adapted with permission from Macmillan Publishers Ltd.: Nature Chemical Biology; K. Schirner et al., <i>Nat Chem Biol</i> 11, 38–45 [2015], Macmillan Publishers Ltd. 2015.)</p

In Vivo

Nisin and related lantibiotics kill bacteria by pore formation, or by sequestering Lipid II. Some lantibiotics sequester Lipid II into clusters, which were suggested to kill cells through delocalized peptidoglycan synthesis. Here, we show that cluster formation is always concomitant with (i) membrane pore formation and (ii) membrane depolarization. Nisin variants that cluster Lipid II kill L-form bacteria with similar efficiency, suggesting that delocalization of peptidoglycan synthesis is not the primary killing mechanism of these lantibiotics

Flavin Binding to the High Affinity Riboflavin Transporter RibU

The first biochemical and spectroscopic characterization of a purified membrane transporter for riboflavin (vitamin B2) is presented. The riboflavin transporter RibU from the bacterium Lactococcus lactis was overexpressed, solubilized, and purified. The purified transporter was bright yellow when the cells had been cultured in rich medium. We used a detergent-compatible matrix-assisted laser desorption ionization time-of-flight mass spectrometry method to show that the source of the yellow color was riboflavin that had been co-purified with the transporter. The method appears generally applicable for substrate identification of purified membrane proteins. Substrate-free RibU was produced by expressing the protein in cells cultured in chemically defined medium. Riboflavin, FMN, and roseoflavin bound to RibU with high affinity and 1:1 stoichiometry (Kd for riboflavin is 0.6 nM), but FAD did not bind to the transporter. The absorption spectrum of riboflavin changed dramatically when the substrate bound to RibU. Well resolved bands appeared at 441, 464, and 486 nm, indicating a hydrophobic binding pocket. The fluorescence of riboflavin was almost completely quenched upon binding to RibU, and also the tryptophan fluorescence of the transporter was quenched when flavins bound. The results indicate that riboflavin is stacked with one or more tryptophan residues in the binding pocket of RibU. Mutagenesis experiments showed that Trp-68 was involved directly in the riboflavin binding. The structural properties of the binding site and mechanistic consequences of the exceptionally high affinity of RibU for its substrate are discussed in relation to soluble riboflavin-binding proteins of known structure.

X-ray structure of the mouse serotonin 5-HT3 receptor

International audienceNeurotransmitter-gated ion channels of the Cys-loop receptor family mediate fast neurotransmission throughout the nervous system. The molecular processes of neurotransmitter binding, subsequent opening of the ion channel and ion permeation remain poorly understood. Here we present the X-ray structure of a mammalian Cys-loop receptor, the mouse serotonin 5-HT3 receptor, at 3.5 Å resolution. The structure of the proteolysed receptor, made up of two fragments and comprising part of the intracellular domain, was determined in complex with stabilizing nanobodies. The extracellular domain reveals the detailed anatomy of the neurotransmitter binding site capped by a nanobody. The membrane domain delimits an aqueous pore with a 4.6 Å constriction. In the intracellular domain, a bundle of five intracellular helices creates a closed vestibule where lateral portals are obstructed by loops. This 5-HT3 receptor structure, revealing part of the intracellular domain, expands the structural basis for understanding the operating mechanism of mammalian Cys-loop receptors

Author Correction: c-Kit-positive ILC2s exhibit an ILC3-like signature that may contribute to IL-17-mediated pathologies (Nature Immunology, (2019), 20, 8, (992-1003), 10.1038/s41590-019-0423-0)

In the version of this article initially published, the cell conversion was stated incorrectly (‘ILC2s to ILC1-like cells’) in the first sentence of the second paragraph in the second Results subsection; the correct description is ‘ILC1s to ILC2-like cells’. Also, the far left plot in Fig. 1d was missing the vertical axis label; this should be ‘IL-13−APC’. Finally, in the legend to Fig. 3a,b, the human ILCs were identified incorrectly (CD45+CD56‒CD127‒); the correct phenotype is CD45+CD56‒CD127+. The errors have been corrected in the HTML and PDF versions of the article

c-Kit-positive ILC2s exhibit an ILC3-like signature that may contribute to IL-17-mediated pathologies

Here we identify a group 2 innate lymphoid cell (ILC2) subpopulation that can convert into interleukin-17 (IL-17)-producing NKp44− ILC3-like cells. c-Kit and CCR6 define this ILC2 subpopulation that exhibits ILC3 features, including RORγt, enabling the conversion into IL-17-producing cells in response to IL-1β and IL-23. We also report a role for transforming growth factor-β in promoting the conversion of c-Kit− ILC2s into RORγt-expressing cells by inducing the upregulation of IL23R, CCR6 and KIT messenger RNA in these cells. This switch was dependent on RORγt and the downregulation of GATA-3. IL-4 was able to reverse this event, supporting a role for this cytokine in maintaining ILC2 identity. Notably, this plasticity has physiological relevance because a subset of RORγt+ ILC2s express the skin-homing receptor CCR10, and the frequencies of IL-17-producing ILC3s are increased at the expense of ILC2s within the lesional skin of patients with psoriasis