Effective plant disease phenotyping is crucial for resistance breeding, but traditional visual assessment is often inaccurate and inefficient. This is particularly challenging when breeding lettuce (Lactuca sativa) for resistance to downy mildew, given the obligate biotrophic lifestyle of the causal pathogen Bremia lactucae. We discovered that B. lactucae-infected lettuce exhibits patches of increased blue-green fluorescence (BGF) under UV-A excitation from 6 days post-inoculation, preceding visible symptoms. Co-localization of BGF with hyphae, visualized with trypan blue, indicates that BGF is induced by downy mildew colonization. We therefore investigated its potential for non-invasive disease detection and quantification, as well as the underlying physiological changes. Using a custom imaging system, we demonstrate that BGF leaf area correlates with downy mildew severity and can be automatically quantified via a U-Net-based convolutional neural network, enabling early, objective disease assessment. Exploring transcriptomic and metabolomic changes associated with BGF, we found that induction of the phenylpropanoid pathway led to accumulation of caffeoylquinic acids, whose fluorescence spectra overlap with that of BGF tissue, supporting the hypothesis that these compounds contribute to the fluorescence signal. BGF imaging offers a powerful tool for phenotyping in lettuce breeding and for identifying quantitative resistance traits that support durable downy mildew resistance.

NZ2114 is a triple-mutant of the natural peptide antibiotic plectasin that targets the peptidoglycan precursor molecule Lipid II using a supramolecular action, involving assembly of plectasin-Lipid II complexes in a large oligomeric structure, that is enhanced by calcium ions. Due to its superior potency against Staphylococcus aureus strains, NZ2114 was the candidate that was advanced to clinical trials, and it has become the standard-template for the development of improved plectasin derivatives. However, the molecular underpinning for NZ2114’s improved potency remains opaque, with biochemical data pointing to chemical modification of the Lipid II target in Staphylococci that would impair the target binding capacity of plectasin but not the one of NZ2114. Here, using an integrative structural biology approach based on solid-state NMR, high-speed atomic force microscopy, and affinity assays, we demonstrate that both NZ2114 and plectasin bind effectively to Staphylococcal Lipid II variants, which means that NZ2114’s greater potency against S. aureus does not result from a difference in target binding. Instead, we show that the three residue substitutions in NZ2114 change its N-terminal fold, markedly increasing its sensitivity to calcium ions, which results in a different supramolecular action on the membrane surface. Altogether, our study provides new insights for the design of superior drug candidates.

Lactic acid bacteria (LAB) represent a category of probiotics commonly found in the gastrointestinal tracts of both humans and animals. The bacteriocins synthesized by LAB strains are believed to play a crucial role in promoting intestinal health. Bacteriocin ZFM216-ab, comprised of two distinct components, was purified from the cell-free supernant of Lacticaseibacillus rhamnosus ZFM216 (CCTCC NO: M2020325). The molecular weights of these components were determined to be 991.58 Da and 1150.63 Da. Bacteriocin ZFM216-ab exhibits broadspectrum antibacterial activity, exhibiting minimum inhibitory concentrations (MICs) of 7.50 mu g/mL and 3.75 mu g/mL against Listeria monocytogenes and Salmonella paratyphi B, respectively, in a concentration-dependent manner. In an in vitro human intestinal microbiota model, an increase in the population of Lacticaseibacillus was observed following intervention with L. rhamnosus ZFM216. In contrast, direct intervention with Bacteriocin ZFM216-ab led to a significant enhancement of Lactobacillus, Streptococcus and Faecalibacterium abundance. In both intervention groups, a reduction in Clostridium_sensu_stricto_1 and Paraclostridium was noted. Furthermore, the metabolic levels of short-chain fatty acids (SCFAs) in fecal samples were significantly elevated, particularly acetic acid and isobutyric acid. These findings highlight the potential of L. rhamnosus ZFM216 and its bacteriocin as promising candidates for the modulation of gut microbiota homeostasis.

Peptidoglycan hydrolases facilitate bacterial cell wall growth by creating space for insertion of new material and allowing physical separation of daughter cells. In Escherichia coli, three peptidoglycan amidases, AmiA, AmiB and AmiC, cleave septal peptidoglycan during cell division. The LytM-domain proteins EnvC, NlpD and ActS activate these amidases either from inside the cell or the outer membrane: EnvC binds to the cytoplasmic membrane-anchored divisome components FtsEX, while NlpD and ActS are outer membrane-anchored lipoproteins. Here we report the identification of a novel periplasmic deacetylase called SddA that removes acetyl groups from denuded peptidoglycan glycan strands, the products of amidases. The sddA gene is co-expressed with the gene encoding EnvC, linking SddA function to amidase activation. Consistent with this link, the deletion of sddA alleviates phenotypes associated with lack of amidase activation, while overexpression of sddA alleviates phenotypes related to a defective Tol-Pal system and causes cell chaining due to reduced septum peptidoglycan cleavage. We present a model according to which SddA modulates the activation of the septum-splitting amidases during cell division.

Membrane lipid oxidation is a universal process that occurs in situations of oxidative stress and is encountered in numerous physiological and pathological situations. Oxidized truncated phospholipids make up a large part of the oxidation products and alter the membrane properties in a way that can lead to cell death. However, the underlying mechanisms are not well understood nor is it clear whether environmental factors, such as pH, can modulate these effects. Using model membranes, we investigate how individual lipid aldehydes and carboxylic acids with truncated acyl chains alter the membrane structure. Our data shows that lipid aldehydes and carboxylic acids have different permeabilization efficiencies towards molecules of varying charge and size and that ΔC9 truncated lipids are usually more efficient in permeabilizing membranes than ΔC5. In terms of physical mechanisms, the ΔC9 truncated lipid carboxylic acid induces permeabilization and membrane curvature in a pH-dependent fashion. This is explained by ionization-dependent exposure of the carboxyl group to the water-bilayer interface, which increases the intrinsic molecular curvature of the oxidized lipid. Conversely, ΔC9 truncated lipid aldehydes and nonionized carboxyls do not induce curved structures but are more efficient in increasing permeability toward larger molecules. We further show that truncated lipids can escape the bilayer and accumulate at interfaces, implying that they might act on neighboring cells. This study indicates that oxidized phospholipids with truncated acyl chains disrupt membrane structure, depending on their specific molecular structure and the pH of the environment, opening a possible route for the design of lipid nanoparticles with pH-dependent drug release.

Gram-negative bacteria possess an asymmetric outer membrane (OM) primarily composed of lipopolysaccharides (LPS) on the outer leaflet and phospholipids on the inner leaflet. The outer membrane functions as an effective permeability barrier to compounds such as antibiotics. Studying LPS biosynthesis is therefore helpful to explore novel strategies for new antibiotic development. Metabolic glycan labeling of the bacterial surface has emerged as a powerful method to investigate LPS biosynthesis. However, the previously reported methods of labeling LPS are based on radioactivity or difficult-to-produce analogs of bacterial sugars. In this study, we report on the incorporation of azido galactose into the LPS of the Gram-negative bacteria Escherichia coli and Salmonella typhi via metabolic labeling. As a common sugar analog, azido galactose successfully labeled both O-antigen and core of Salmonella LPS, but not E. coli LPS. This labeling of Salmonella LPS, as shown by SDS-PAGE analysis and fluorescence microscopy, differs from the previously reported labeling of either O-antigen or core of LPS. Our findings are useful for studying LPS biogenesis pathways in Gram-negative bacteria like Salmonella. In addition, our approach is helpful for screening for agents that target LPS biosynthesis as it allows for the detection of newly synthesized LPS that appears in the OM. Furthermore, this approach may also aid in isolating chemically modified LPS for vaccine development or immunotherapy.

Lipids adhere to membrane proteins to stimulate or suppress molecular and ionic transport and signal transduction. Yet, the molecular details of lipid-protein interaction and their functional impact are poorly characterized. Here we combine NMR, coarse-grained molecular dynamics (CGMD), and functional assays to reveal classic cooperativity in the binding and subsequent activation of a bacterial inward rectifier potassium (Kir) channel by phosphatidylglycerol (PG), a common component of many membranes. Past studies of lipid activation of Kir channels focused primarily on phosphatidylinositol bisphosphate, a relatively rare signaling lipid that is tightly regulated in space and time. We use solid-state NMR to quantify the binding of unmodified C-13-PG to the K+ channel KirBac1.1 in liposomes. This specific lipid-protein interaction has a dissociation constant (K-d) of similar to 7 mol percentage PG (Chi(PG)) with positive cooperativity (n = 3.8) and approaches saturation near 20% Chi(PG). Liposomal flux assays show that K+ flux also increases with PG in a cooperative manner with an EC50 of similar to 20% Chi(PG), within the physiological range. Further quantitative fitting of these data reveals that PG acts as a partial (80%) agonist with fivefold K+ flux amplification. Comparisons of NMR chemical shift perturbation and CGMD simulations at different Chi(PG) confirm the direct interaction of PG with key residues, several of which would not be accessible to lipid headgroups in the closed state of the channel. Allosteric regulation by a common lipid is directly relevant to the activation mechanisms of several human ion channels. This study highlights the role of concentration-dependent lipid-protein interactions and tightly controlled protein allostery in the activation and regulation of ion channels.

Inward-rectifier K+ (Kir) channels are tetrameric ligand-gated ion channels. These Kir channels conduct inward K+ current in response to this anionic lipid association, regulating the resting membrane potential in excitable cells. Noted channelopathies include forms of heart disease, psychiatric disorders, and alcoholism. The prokaryotic channel KirBac1.1 shares its fold and regulatory mechanisms with homologous human channels. Using solid-state NMR (SSNMR), K+ efflux assays, SSNMR-driven simulated annealing structure calculations, and coarse-grain molecular dynamics (CG-MD) we resolved an intricate allosteric network between the inner gate, outer gate, and intracellular Kir domain.

Antimicrobial resistance is a leading cause of mortality, calling for the development of new antibiotics. The fungal antibiotic plectasin is a eukaryotic host defence peptide that blocks bacterial cell wall synthesis. Here, using a combination of solid-state nuclear magnetic resonance, atomic force microscopy and activity assays, we show that plectasin uses a calcium-sensitive supramolecular killing mechanism. Efficient and selective binding of the target lipid II, a cell wall precursor with an irreplaceable pyrophosphate, is achieved by the oligomerization of plectasin into dense supra-structures that only form on bacterial membranes that comprise lipid II. Oligomerization and target binding of plectasin are interdependent and are enhanced by the coordination of calcium ions to plectasin's prominent anionic patch, causing allosteric changes that markedly improve the activity of the antibiotic. Structural knowledge of how host defence peptides impair cell wall synthesis will likely enable the development of superior drug candidates. Plectasin, a natural host defence peptide of fungal origin, uses a calcium-sensitive supramolecular killing mechanism to block bacterial cell wall synthesis.

Lipid oxidation is a universal process encountered in many pathophysiological situations. Several oxidized lipids are signalling molecules while others alter cell membrane properties which leads to a loss of membrane function. However, it is not known how a complex mixture of oxidized lipids would alter membrane properties, and how the initial presence of unsaturated lipids would influence this process. We further do not know how certain oxidized lipids can leave the membrane and become signalling molecules.

Oxidative stress in combination with acid stress has been shown to inactivate a wide spectrum of microorganisms, including multi-resistant bacteria. This occurs e.g. in phagolysosomes or during treatment by cold atmospheric pressure plasmas (CAP) and possibly depends on the cell membrane. We therefore explored the effects of CAP-generated reactive oxygen and nitrogen species (RONS) on bacterial growth inhibition and membranes in neutral and acidic suspensions. We observed that growth inhibition was most efficient when bacteria were treated by a mix of short and long-lived RONS in an acidic environment. Membrane packing was affected mainly upon contact with short-lived RONS, while also acidity strongly modulated packing. Under these conditions, Gram-negative bacteria displayed large potassium release while SYTOX Green influx remained marginal. Growth inhibition of Gram-negative bacteria correlated well with outer membrane (OM) permeabilization that occurred upon contact with short and/or long-lived RONS in synergy with acidity. In Gram-positive bacteria, CAP impaired membrane potential possibly through pore formation upon contact with short-lived RONS while formation of membrane protein hydroperoxides was probably involved in these effects. In summary, our study provides a wide perspective on understanding inactivation mechanisms of bacteria by RONS in combination with acidity.

Epilancin 15X is a lantibiotic that has an antimicrobial activity in the nanomolar concentration range towards Staphylococcus simulans. Such low MICs usually imply that these peptides employ a mechanism of action (MoA) involving high affinity targets. Here we studied this MoA by using epilancin 15X's ability to dissipate the membrane potential of intact S. simulans cells. These membrane depolarization assays showed that treatment of the bacteria by antibiotics known to affect the bacterial cell wall synthesis pathway decreased the membrane depolarization effects of epilancin 15X. Disruption of the Lipid II cycle in intact bacteria using several methods led to a decrease in the activity of epilancin 15X. Antagonism-based experiments on 96-well plate and agar diffusion plate pointed towards a possible interaction between epilancin 15X and Lipid II and this was confirmed by Circular Dichroism (CD) based experiments. However, this interaction did not lead to a detectable effect on either carboxyfluorescein (CF) leakage or proton permeability. All experiments point to the involvement of a phosphodiester-containing target within a polyisoprene-based biosynthesis pathway, yet the exact identity of the target remains obscure so far.

Physical properties of biological membranes directly or indirectly govern biological processes. Yet, the interplay between membrane and integral membrane proteins is difficult to assess due to reciprocal effects between membrane proteins, individual lipids, and membrane architecture. Using solid-state NMR (SSNMR) we previously showed that KirBac1.1, a bacterial Inward-Rectifier K+ channel, nucleates bilayer ordering and microdomain formation through tethering anionic lipids. Conversely, these lipids cooperatively bind cationic residues to activate the channel and initiate K+ flux. The mechanistic details governing the relationship between cooperative lipid loading and bilayer ordering are, however unknown. To investigate, we generated KirBac1.1 samples with different concentrations of 13C-lableded phosphatidyl glycerol (PG) lipids and acquired a full suite of SSNMR 1D temperature series experiments using the ordered all-trans (AT) and disordered trans-gauche (TG) acyl conformations as markers of bilayer dynamics. We observed increased AT ordered signal, decreased TG disordered signal, and increased bilayer melting temperature with increased PG concentration. Further, we identified cooperativity between ordering and direct binding of PG lipids, indicating KirBac1.1-driven bilayer ordering and microdomain formation is a classically cooperative Hill-type process driven by and predicated upon direct binding of PG lipids. Our results provide unique mechanistic insight into how proteins and lipids in tandem contribute to supramolecular bilayer heterogeneity in the lipid membrane.

Bacterial infections present a major global health threat, often displaying resistance to various antibiotics. Lipoteichoic acid (LTA) is a vital component of bacterial cell envelopes of Gram‐positive bacteria, crucial for cell integrity, cell division, and host inflammation. Due to its essential role for bacteria, LTA and its biosynthesis are also attractive drug targets, however, there is only scant molecular knowledge on LTA and its precursor molecules in membranes. Here, we report the isolation and molecular characterization of diglucosyldiacylglycerol (Glc2‐DAG), the glycolipid precursor molecule that anchors LTA in the bacterial plasma‐membrane. Using a tailored growth medium and purification protocols, we isolated 13C‐isotope labelled Glc2‐DAG from bacteria, which can then be used for high‐resolution NMR studies. Using solution‐state and solid‐state NMR, we show an in‐depth molecular characterization of Glc2‐DAG, including in native‐like membranes. Our approach may help to identify antibiotics that directly target LTA precursor molecules, and it offers a tool for future investigations into the role of Glc2‐DAG in bacterial physiology.

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug -resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, lipid II, and lipid IIIWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid -anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.

Antimicrobial peptides (AMPs) commonly target bacterial membranes and show broad-spectrum activity against microorganisms. In this research we used three AMPs (nisin, epilancin 15×, [R4L10]-teixobactin) and tested their membrane effects towards three strains (Staphylococcus simulans, Micrococcus flavus, Bacillus megaterium) in relation with their antibacterial activity. We describe fluorescence and luminescence-based assays to measure effects on membrane potential, intracellular pH, membrane permeabilization and intracellular ATP levels. The results show that our control peptide, nisin, performed mostly as expected in view of its targeted pore-forming activity, with fast killing kinetics that coincided with severe membrane permeabilization in all three strains. However, the mechanisms of action of both Epilancin 15× as well as [R4L10]-teixobactin appeared to depend strongly on the bacterium tested. In certain specific combinations of assay, peptide and bacterium, deviations from the general picture were observed. This was even the case for nisin, indicating the importance of using multiple assays and bacteria for mode of action studies to be able to draw proper conclusions on the mode of action of AMPs.

Lipid oxidation is a universal degradative process of cell membrane lipids that is induced by oxidative stress and reactive oxygen and nitrogen species (RONS) in multiple pathophysiological situations. It has been shown that certain oxidized lipids alter membrane properties, leading to a loss of membrane function. Alteration of membrane properties is thought to depend on the initial membrane lipid composition, such as the number of acyl chain unsaturations. However, it is unclear how oxidative damage is related to biophysical properties of membranes. We therefore set out to quantify lipid oxidation through various analytical methods and determine key biophysical membrane parameters using model membranes containing lipids with different degrees of lipid unsaturation. As source for RONS, we used cold plasma, which is currently developed as treatment for infections and cancer. Our data revealed complex lipid oxidation that can lead to two main permeabilization mechanisms. The first one appears upon direct contact of membranes with RONS and depends on the formation of truncated oxidized phospholipids. These lipids seem to be partly released from the bilayer, implying that they are likely to interact with other membranes and potentially act as signaling molecules. This mechanism is independent of lipid unsaturation, does not rely on large variations in lipid packing, and is most probably mediated via short-living RONS. The second mechanism takes over after longer incubation periods and probably depends on the continued formation of lipid oxygen adducts such as lipid hydroperoxides or ketones. This mechanism depends on lipid unsaturation and involves large variations in lipid packing. This study indicates that polyunsaturated lipids, which are present in mammalian membranes rather than in bacteria, do not sensitize membranes to instant permeabilization by RONS but could promote long-term damage.

Teixobactin is a cyclic undecadepsipeptide that has shown excellent potency against multidrug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). In this article, we present the design, synthesis, and antibacterial evaluations of 16 different teixobactin analogues. These simplified analogues contain commercially available hydrophobic, non-proteogenic amino acid residues instead of synthetically challenging expensive L-allo-enduracididine amino acid residue at position 10 together with different combinations of arginines at positions 3, 4 and 9. The new teixobactin analogues showed potent antibacterial activity against a broad panel of Gram-positive bacteria, including MRSA and VRE strains. Our work also presents the first demonstration of the potent antibiofilm activity of teixobactin analogoues against Staphylococcus species associated with serious chronic infections. Our results suggest that the use of hydrophobic, non-proteogenic amino acids at position 10 in combination with arginine at positions 3, 4 and 9 holds the key to synthesising a new generation of highly potent teixobactin analogues to tackle resistant bacterial infections and biofilms.

The rapid rise of antimicrobial resistance calls for the development of new antibiotics. Ideal templates for next-generation antibiotics could be drugs that target the cell wall precursor, Lipid II, with vancomycin, nisin, or teixobactin being well-known examples. In this chapter the authors review a selection of NMR studies on the mode of action of Lipid II-binding antibiotics with an emphasis on solid-state NMR findings in lipid membranes that are vital to obtain physiologically relevant drug-binding modes.