Liora Wittle

Nat Commun. 2026 Feb 26. doi: 10.1038/s41467-026-69967-z. Online ahead of print.

ABSTRACT

The global burden of microbial infections and antimicrobial resistance, coupled with the absence of precise bacterial recognition modalities, demands innovative breakthroughs in antibacterial treatment. Here, we report a chirality-specific biomimic-D-alanine-conjugated peptidoglycan mimics (D-PM)-designed for bacterial recognition. D-PM exhibits broad-spectrum, effective recognition across ESKAPE pathogens, antibiotic-resistant strains, and clinical isolates, while displaying minimal interaction with eukaryotic cells. We elucidate the bacterial recognition mechanism, wherein D-PM-act as a biosynthetic substrates-become incorporated into peptidoglycan biosynthesis. This reveals a mechanism by which macromolecular mimetics are assimilated into bacterial biosynthesis, providing insights into bacterial recognition. Beyond recognition, D-PM enables the construction of pathogen-specific imaging agents and antibiotic-targeted delivery systems. In localized and systemic infection models, D-PM achieves efficient pathogen localization, tissue penetration, and enhanced therapeutic outcomes. This work presents a molecularly engineered strategy for bacterial recognition and intervention, offering a translational approach to address the escalating threat of infectious diseases.

PMID:41748621 | DOI:10.1038/s41467-026-69967-z

Microorganisms. 2026 Jan 23;14(2):271. doi: 10.3390/microorganisms14020271.

ABSTRACT

Helicobacter pylori is a prevalent gastric pathogen that establishes chronic infection and contributes to gastritis, peptic ulcer disease, and gastric cancer. Its persistence depends on immune evasion strategies that promote sustained low-grade inflammation in the gastric mucosa. Nucleotide-binding oligomerization domain-like receptors (NLRs) are cytosolic pattern recognition receptors that play key roles in innate immune responses against H. pylori. Nod1 and Nod2 detect bacterial peptidoglycan delivered via the type IV secretion system or outer membrane vesicles, activating NF-κB, MAPK, and interferon signaling pathways that regulate inflammatory cytokine production, epithelial barrier function, autophagy, and antimicrobial defense. The NLRP3 inflammasome mediates the maturation of IL-1β and IL-18 primarily in myeloid cells, thereby shaping inflammatory and immunoregulatory responses during infection. In contrast, NLRC4 functions in a context-dependent manner in epithelial cells and is largely dispensable for myeloid IL-1β production. Emerging evidence also implicates noncanonical NLRs, including NLRP6, NLRP9, NLRP12, NLRX1, and NLRC5, in regulating inflammation, epithelial homeostasis, and gastric tumorigenesis. In addition, genetic polymorphisms in NLR genes influence host susceptibility to H. pylori-associated diseases. This review highlights the interplay between NLR signaling, bacterial virulence, and host immunity and identifies potential therapeutic targets.

PMID:41753558 | PMC:PMC12943089 | DOI:10.3390/microorganisms14020271

Cell Chem Biol. 2026 Feb 19;33(2):145-146. doi: 10.1016/j.chembiol.2026.01.012.

ABSTRACT

Pattern recognition receptors detect peptidoglycan and trigger inflammatory responses promoting bacterial clearance. So, how do our gut bacteria survive? In this issue of Cell Chemical Biology, Liang et al.¹ determine the structures of peptidoglycan fragments released by beneficial bacteria that mediate a NOD2-dependent reduction in inflammatory responses to bacterial products.

PMID:41720077 | DOI:10.1016/j.chembiol.2026.01.012

Diagnostics (Basel). 2026 Feb 1;16(3):433. doi: 10.3390/diagnostics16030433.

ABSTRACT

Background/Objectives: Response to neoadjuvant chemotherapy (NAC) varies substantially among breast cancer patients and is only partially explained by tumor-intrinsic factors. The gut microbiome has emerged as a potential modulator of chemotherapy efficacy, yet its role in breast cancer remains underexplored. This study aimed to characterize gut microbial composition, functional potential, and microbially derived metabolites in breast cancer patients undergoing NAC. Methods: baseline stool samples from 39 chemotherapy-naïve breast cancer patients undergoing NAC were analyzed using shotgun metagenomic sequencing and targeted metabolomics. Patients were stratified by pathological complete response (pCR, n = 17; no pCR, n = 22). Microbial taxonomic and functional profiles, short-chain fatty acids (SCFAs) and bile acids were assessed, with subgroup analysis performed in triple-negative breast cancer (TNBC). Results: Patients achieving pCR exhibited significantly higher baseline microbial richness compared to non-responders (p = 0.040). Differential abundance analysis revealed enrichment of Dialister, Kineothrix, and Jutongia in responders, whereas Rothia, Leuconostoc, Klebsiella, Jingyaoa, Cuneatibacter, Youxingia, and Bittarella were enriched in non-responders. SCFAs (acetate, propionate and butyrate) positively correlated with microbial glucose catabolic pathways, while caproate was negatively associated with multiple amino acid, lipid, vitamin, and cell wall biosynthesis pathways, including peptidoglycan maturation. Metabolomic analysis identified higher deoxycholic acid (DCA) levels in non-responders and increased C6 levels in responders, although these associations did not remain significant after multiple testing correction. Similar trends were observed in the TNBC subgroup (n = 15). Conclusions: Baseline gut microbiome diversity, taxonomic composition, and functional metabolic potential are associated with response to neoadjuvant chemotherapy in breast cancer, supporting the gut microbiome and its produced metabolites as a potential biomarker of treatment efficacy.

PMID:41681750 | PMC:PMC12896730 | DOI:10.3390/diagnostics16030433

Bio Protoc. 2026 Feb 5;16(3):e5592. doi: 10.21769/BioProtoc.5592. eCollection 2026 Feb 5.

ABSTRACT

Plasma membrane-associated condensates driven by liquid-liquid phase separation represent a novel mechanism of receptor-mediated signaling transduction, serving as mesoscale platforms that concentrate signaling molecules and modulate reaction kinetics. Condensate formation is a highly dynamic process that occurs within seconds to minutes following receptor activation. Here, we present methods for de novo reconstituting liquid-like condensates on supported lipid bilayers and assessing the condensate fluidity using fluorescence recovery after photobleaching (FRAP). This protocol encompasses supported lipid bilayer preparation, condensation imaging, and FRAP analysis using total internal reflection fluorescence (TIRF) microscopy. Supported lipid bilayers provide a membrane-mimicking environment for receptor signaling cascades, offering mechanistic insights into protein-protein and lipid-protein interactions amid micron-scale condensates. The protocol can also be adapted to study condensates associated with the internal membranes of the Golgi apparatus, mitochondria, and other organelles. Key features • Real-time imaging of condensate formation and FRAP analysis using TIRF microscopy reveals a spatiotemporal profile of signaling transduction. • Supported lipid bilayers provide a fluidic membrane environment that is critical for the biochemical reconstitution of condensates at physiological protein concentrations. • The lipid and protein components within the reconstituted condensate system can be readily manipulated to accommodate specific experimental objectives and assay designs.

PMID:41675989 | PMC:PMC12887864 | DOI:10.21769/BioProtoc.5592

Membranes (Basel). 2026 Jan 4;16(1):33. doi: 10.3390/membranes16010033.

ABSTRACT

The permeation of different chemical substances across the membrane is of utmost importance to the life and health of a living cell. Depending on the nature of the permeant, the process is mediated by either the protein (e.g., membrane channels) or lipid phases of the membrane, or both. In the case of small and physiologically important gas molecules, namely O₂ and CO₂, the literature supports the involvement of both pathways in their transport. The extent of involvement of the lipid phase, however, is directly dependent on the nature of the lipid constituents of the membrane that determine its various structural and physicochemical properties. In this study, we use molecular dynamics simulation, as a method with sufficient spatial and temporal resolutions, to analyze these properties in heterogeneous lipid bilayers, composed of phospholipids with varied tails, sphingomyelin, and cholesterol, to different degrees. Together with the calculation of the free energy profiles, diffusion constants, and gas diffusivity, the results shed light on the importance of the lipid phase of membranes in gas transport rate and how they can be modulated by their lipid composition.

PMID:41590586 | PMC:PMC12843641 | DOI:10.3390/membranes16010033

Gut Microbes. 2026 Dec 31;18(1):2615490. doi: 10.1080/19490976.2026.2615490. Epub 2026 Jan 28.

ABSTRACT

The gut microbiome plays a critical role in health, disease and immunity. To date, we have access to large datasets describing how the microbial diversity present in the gut correlates with many clinical conditions. However, the microbiome composition is taxonomically complex; influenced by many environmental factors; and variable between individuals and communities, thereby limiting functional and mechanistic insights into the microbiota‒host interactions. We are still unsure of the molecular mechanisms by which gut commensal microbes intrinsically possess to interact with the immune system and induce beneficial responses. This study has addressed this important question by revealing that only certain members of Lactobacillaceae, a bacterial family very well known for its probiotic properties, interact very intimately with macrophages because of their ability to simultaneously overexpress adhesive cell wall proteins and to self-aggregate, leading to significant production of type I interferon (IFN-I) cytokines. IFN-I cytokines are essential to confer protection against viral infections and auto-immune disorders. Specifically, we have proved that this enhanced IFN-I feature is strain-dependent and predominantly driven by cGAS, a molecule that activates the cytosolic sensor STING upon the recognition of bacterial DNA. Furthermore, another cytosolic sensor, NOD2, seems to be an additional stimulus to amplify IFN-I production, suggesting the involvement of successive molecular events for a prominent probiotic response. Our findings provide insight into how specific molecules of probiotic bacteria modulate or stimulate host responses, providing a better understanding of the molecular crosstalk between the microbiome and immune cells.

PMID:41605865 | PMC:PMC12854382 | DOI:10.1080/19490976.2026.2615490

J Mol Biol. 2026 Jan 30:169672. doi: 10.1016/j.jmb.2026.169672. Online ahead of print.

ABSTRACT

Molecular modeling and simulation play a crucial role in advancing our understanding of protein function at the molecular level, offering insights that complement experimental approaches. In particular, molecular dynamics (MD) simulations with explicit lipid bilayers have become essential for a molecular level understanding of protein-lipid interactions that regulate the structure, dynamics, and function of membrane proteins. CHARMM-GUI (http://www.charmm-gui.org) is a web-based graphical user interface designed to generate MD simulation systems and input files for various simulation engines. Here, we introduce Quick Bilayer, a new CHARMM-GUI module, which provides a streamlined and efficient one-stop platform for assembling protein structures with a diverse set of biologically relevant membrane environments. It features advanced search capabilities that allow users to identify specific lipid types and design bilayers with customized lipid compositions to meet specific research needs. To further enhance usability and scalability, Quick Bilayer now supports a REST-like API that enables seamless integration with backend services. This newly implemented command-line interface allows users to programmatically access the module, facilitating automated workflows and large-scale system generation.

PMID:41621783 | DOI:10.1016/j.jmb.2026.169672

bioRxiv [Preprint]. 2025 Dec 7:2025.12.03.692219. doi: 10.64898/2025.12.03.692219.

ABSTRACT

Excessive production of reactive oxygen species (ROS) in cells results in oxidative stress, which can promote lipid peroxidation in cellular membranes. This oxidation of membrane lipids accompanies various diseases and can even result in cell death through processes such as ferroptosis. The complex compositions and diverse morphologies of cellular membranes make understanding the mechanisms of lipid peroxidation challenging, especially when attempting to investigate membrane composition and curvature simultaneously. Here, we utilize reconstituted lipid membranes and the fluorescent oxidation probe C11-BODIPY to quantify lipid oxidation as functions of both lipid composition and membrane curvature. By tethering synthetic lipid vesicles to glass substrates, we were able to monitor lipid oxidation on a per vesicle basis using fluorescence microscopy. Our results demonstrate that highly curved membranes markedly increase both the rate and extent of lipid peroxidation across diverse membrane compositions. This effect arises from greater exposure of lipid tails to the aqueous environment, which allows more efficient transport of ROS into the hydrophobic core of the bilayer. Compositional effects on lipid peroxidation are most pronounced in membranes with low curvature (i.e., greater than 100 nm diameter) and become progressively weaker as curvature increases. We found that low to moderate cholesterol levels (i.e., 10-25 mol%) suppress curvature-dependent oxidation by tightening lipid packing, whereas high cholesterol content (i.e., 50 mol%) restores curvature sensitivity by influencing lateral lipid mobility. Together, these findings establish membrane curvature and lipid composition as interdependent determinants of oxidative susceptibility, offering new insight into how cells regulate or resist oxidative stress.

STATEMENT OF SIGNIFICANCE: Oxidative stress drives lipid peroxidation in cellular membranes, but the combined influence of membrane curvature and composition remains poorly defined. Using reconstituted lipid vesicles and a fluorescent oxidation probe, we show that lipid peroxidation is enhanced in smaller vesicles (i.e., highly curved membranes). The oxidation rate increases with unsaturated lipids, while cholesterol suppresses this effect. Measurements of membrane packing and diffusivity support these findings, demonstrating how curvature and composition together govern membrane susceptibility to oxidative damage. These results provide new insight into the physicochemical basis of membrane stability under oxidative stress and have broad implications for understanding the vulnerability of curved cellular membranes.

PMID:41573950 | PMC:PMC12822622 | DOI:10.64898/2025.12.03.692219

bioRxiv [Preprint]. 2025 Nov 19:2025.11.19.689327. doi: 10.1101/2025.11.19.689327.

ABSTRACT

Mitochondrial dysfunction is a hallmark of neurodegenerative and neuroinflammatory disorders, including hypertension and cardiovascular disease, yet strategies for safe and precise mitochondrial-targeted delivery remain limited. Here, we establish strain-promoted azide-alkyne cycloaddition (SPAAC) as a biocompatible, high-fidelity chemical platform for engineering mitochondria-targeted brain-derived exosomes (BR-EVs). Copper-free click conjugation of a mitochondrial-targeting ligand (e.g. Cy5-DBCO) under mild aqueous conditions preserved vesicle morphology (30-150 nm core; 120-200 nm hydrodynamic), proteomic composition, and uptake dynamics. Time-course imaging and fluorescence recovery after photobleaching (FRAP) revealed unaltered endocytic kinetics, >75 % mitochondrial colocalization, and intact organelle architecture. In vivo neuroinflammation and biodistribution analyses demonstrated immunological neutrality, strong central nervous system retention, and minimal peripheral dispersion following intracerebroventricular administration. Proteomic profiling of unlabeled Sprague-Dawley (SD) and hypertensive Dahl salt-sensitive (DSS) BR-EVs uncovered hypertension-driven enrichment of oxidative and complement pathways correlating with mitochondrial fragmentation and reactive oxygen species generation in neuronal cultures. These findings establish SPAAC-mediated ligand conjugation as a biocompatible and chemically precise approach for generating mitochondria-targeted exosomes that preserve exosomal identity, biodistribution, and signaling fidelity-advancing a foundational platform for organelle-specific delivery and mechanistic imaging in the central nervous system.

PMID:41332759 | PMC:PMC12667881 | DOI:10.1101/2025.11.19.689327

Adv Sci (Weinh). 2026 Mar;13(14):e20154. doi: 10.1002/advs.202520154. Epub 2026 Jan 4.

ABSTRACT

Antibacterial orthopedic implants that simultaneously promote osteointegration remain an unmet clinical challenge. Conventional enzyme-responsive antibacterial surfaces often suffer from irreversible loss of osteogenic motifs upon activation, limiting their regenerative capacity post-infection. Herein, we report a dual-anchored peptide design engineered on titanium implants (Ti-Dual) that addresses this limitation by retaining biofunctional motifs after pathogen-triggered activation. The peptide construct integrates an antimicrobial sequence (HHC36) and a cell-adhesive RGD motif connected via a gelatinase-cleavable spacer (GPLGV). Terminal azide groups enable stable dual-point grafting through SPAAC chemistry, overcoming the low grafting efficiency associated with mixed RGD grafting systems. Under physiological conditions, the constrained conformation suppresses antibacterial activity, favoring osteogenesis. Upon infection, bacterial gelatinase cleaves the linker, activating rapid and potent antibacterial effects-eliminating 99.37% of P. aeruginosa within 10 min, and 99.73% of S. aureus and 99.99% of P. aeruginosa within 120 min-while the RGD motif remains anchored, ensuring continuous cell adhesion and tissue integration. In vivo, Ti-Dual effectively eradicated multidrug-resistant P. aeruginosa, suppressed inflammatory responses, mitigated bone resorption, and enhanced osteogenesis in a rat femoral infection model. This design resolves the critical trade-off between infection responsiveness and sustained pro-regenerative function, offering a robust and adaptive strategy for infected bone defect repair.

PMID:41486549 | PMC:PMC12970275 | DOI:10.1002/advs.202520154

J Med Chem. 2025 Dec 25;68(24):26206-26217. doi: 10.1021/acs.jmedchem.5c02254. Epub 2025 Dec 15.

ABSTRACT

Using antimicrobial peptides as a template, triazolium-based peptoids were designed with strong and selective antibacterial activities. To probe their mechanism, eight distinct peptoids were investigated using biophysical methods with lipid bilayers modeling bacterial or eukaryotic membranes. Calcein leakage experiments closely parallel antibacterial assays testing activities against Gram-negative or Gram-positive bacteria and toxicity for human red blood cells. This excellent correlation shows that the membrane-association underlies these peptoids' biological activities. While circular dichroism spectroscopy confirms their designed PPI (polyproline I) helical fold, fluorescence assays quantitatively evaluate membrane association and indicate localization at the membrane interface. In the presence of peptoids, a significant reduction in lipid order parameters is observed by solid-state NMR spectroscopy. Collectively, these findings support a membrane-mediated mechanism of action for the triazolium-based peptoids similar to that for linear cationic antimicrobial peptides. Furthermore, the physicochemical and structural features of the peptoids explain their different degrees of biological activities.

PMID:41396478 | DOI:10.1021/acs.jmedchem.5c02254

J Clin Invest. 2025 Oct 2;135(23):e190851. doi: 10.1172/JCI190851. eCollection 2025 Dec 1.

ABSTRACT

Single-cell studies have revealed that intestinal macrophages maintain gut homeostasis through the balanced actions of reactive (inflammatory) and tolerant (noninflammatory) subpopulations. How such balance is impaired in inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC), remains unresolved. Here, we define colon-specific macrophage states and reveal the critical role of noninflammatory colon-associated macrophages (niColAMs) in IBD recovery. Through trans-scale analyses-integrating computational transcriptomics, proteomics, and in vivo interventional studies-we identified GIV (CCDC88A) as a key regulator of niColAMs. GIV emerged as the top-ranked gene in niColAMs that physically and functionally interacts with NOD2, an innate immune sensor implicated in CD and UC. Myeloid-specific GIV depletion exacerbates infectious colitis, prolongs disease, and abolishes the protective effects of the NOD2 ligand muramyl dipeptide in colitis and sepsis models. Mechanistically, GIV's C-terminus binds the terminal leucine-rich repeat 10 (LRR 10) of NOD2 and is required for NOD2 to dampen inflammation and clear microbes. The CD-associated 1007fs NOD2 variant, which lacks LRR 10, cannot bind GIV, which provides critical insights into how this clinically relevant variant impairs microbial sensing and clearance. These findings illuminate a critical GIV•NOD2 axis essential for gut homeostasis and highlight its disruption as a driver of dysbiosis and inflammation in IBD.

PMID:41321314 | PMC:PMC12646664 | DOI:10.1172/JCI190851

Microbiol Immunol. 2026 Jan;70(1):47-51. doi: 10.1111/1348-0421.70025. Epub 2025 Nov 26.

ABSTRACT

To develop vaccine adjuvants from bacterial peptidoglycan (PG) the immunostimulatory activity of lysozyme-solubilized PG derived from Levilactobacillus brevis and Lactiplantibacillus plantarum was investigated. Solubilized PG from both bacteria induced IL-8 in THP-1 cells, and periodate oxidation of L. plantarum PG reduced the activity, suggesting that muramyl dipeptide was partially destroyed. Periodate-oxidized L. plantarum PG showed reduced IL-8 inducing activity in NOD2-expressing cells, while it remained in NOD1-expressing cells, suggesting that γ-d-glutamyl-meso-diaminopimelic acid structure was maintained. All PG preparations stimulated RAW264.7 cells to proliferate, suggesting that they could be potent candidates for vaccine adjuvants.

PMID:41305941 | DOI:10.1111/1348-0421.70025

Adv Sci (Weinh). 2025 Nov 10:e08589. doi: 10.1002/advs.202508589. Online ahead of print.

ABSTRACT

In recent years, organic biomimetic electronic devices have gained attention for their potential applications in healthcare, as they emulate the natural functions of biological components that mediate communication between external signals and internal cellular processes. These devices integrate semi-biological components such as synthetic membranes with organic electronics. For instance, Supported Lipid Bilayers (SLBs) offer a promising substrate as biomimetic membranes, by providing a stable and controlled interface for bioelectronic and biomimetic applications. However, challenges remain in SLB formation, particularly in achieving consistent transmembrane ionic resistance due to packing defects. This work reports a framework of the dielectric properties of a SLB dielectric stack, and investigates the impact of defects on the membrane resistance variations. According to the model, lipid packing non-idealities lead to the partial hydration of the inner part of the membrane and thus to transmembrane resistance variations. These findings offer new insights into the dielectric and transmembrane barrier characteristics of SLBs by introducing a quantitative assessment method that transcends qualitative experimental observations, paving the way for a systematic approach to designing controllable membranes and biointerfaces with customizable biomimetic properties.

PMID:41214873 | DOI:10.1002/advs.202508589

bioRxiv [Preprint]. 2025 Oct 14:2025.10.13.682232. doi: 10.1101/2025.10.13.682232.

ABSTRACT

Cell membranes are composed of both bilayer-supporting and non-bilayer phospholipids, with the latter's negative intrinsic curvature aiding in membrane trafficking and the dynamics of membrane proteins. Phospholipid metabolism has long been recognized to maintain membrane fluidity, but whether it also acts to maintain the function of high-curvature lipids is not resolved. Here, we find that cells grown under hydrostatic pressure - used to artificially reduce lipid curvature - maintain lipidome curvature through metabolic acclimation. We first observed that manipulation of the lipidome curvature via the phosphatidylethanolamine (PE) to phosphatidylcholine (PC) ratio affects high-pressure growth and viability of yeast independently of membrane fluidity. In wild-type cells, X-ray scattering measurements revealed an increased propensity for lipid extracts to form non-lamellar phases after extended pressure incubations. Unexpectedly, this change in phase behavior was not due to increased levels of PE, but of phosphatidylinositol (PI), the only major phospholipid class whose curvature had not been previously characterized. We found that PI is a non-bilayer lipid, with a negative curvature intermediate to that of PE and PC. Accounting for PI, mean lipidome curvature was defended in response to pressure by two distantly related yeasts. Lipidome curvature also responded to pressure in a human cancer cell line through ether phospholipid metabolism and chain remodeling, but not in bacterial cells. These findings indicate that eukaryotic phospholipid metabolism uses diverse mechanisms to maintain curvature frustration in cell membranes.

PMID:41279765 | PMC:PMC12632798 | DOI:10.1101/2025.10.13.682232

ACS Appl Mater Interfaces. 2025 Dec 10;17(49):66235-66248. doi: 10.1021/acsami.5c14290. Epub 2025 Nov 24.

ABSTRACT

Antimicrobial peptides are widely investigated in the literature, but their mechanism of action and effects on lipid membranes are not completely understood from a physicochemical perspective. In this study, we employed a bioinspired mastoparan from wasp venom, mast-MO, and characterized its interactions with model lipid membranes, either as a supported lipid bilayer or as free-standing vesicles in solution. An array of complementary physicochemical characterization techniques was employed to study the surface activity of the peptide alone and how its adsorption affects lipid membrane properties in terms of lateral organization and integrity. We found that peptide action is related to its intrinsic surface activity, resulting in disrupted lipid packing of supported membranes and vesicles via a concentration-dependent mechanism. Changing solution conditions, e.g., ionic strength and pH, altered the electrostatic interactions between the membrane and mast-MO, resulting in less significant adsorption. This mechanism of action was also validated in vitro for Gram-negative E. coli bacteria, demonstrating rapid action (within 15 min) and potent antimicrobial activity. These results provide new information on the molecular effects of mastoparan's interactions with membranes.

PMID:41283220 | DOI:10.1021/acsami.5c14290