Liora Wittle

bioRxiv [Preprint]. 2026 Mar 14:2026.03.13.711678. doi: 10.64898/2026.03.13.711678.

ABSTRACT

The envelope of Gram-negative bacteria like Escherichia coli is multilayered with two membranes sandwiching a peptidoglycan cell wall. The inner membrane is a typical phospholipid bilayer whereas the outer membrane is asymmetric with phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. We recently discovered that inactivation of the conserved peptidoglycan synthesis machinery responsible for cell elongation causes defects in both peptidoglycan and LPS synthesis in E. coli . This finding suggests that the isolation of suppressors that rescue the growth phenotype caused by an impaired cell elongation system is an attractive means of identifying factors involved in coordinating the biogenesis of different envelope layers. Here, we report the results of a global, transposon sequencing-based screen for such suppressors. The inactivation of a number of factors including the phospholipid synthesis enzyme PlsX was found to partially suppress the growth defects of a cell elongation mutant. Deletion of plsX also conferred increased resistance to CHIR-090, an inhibitor of the committed step of LPS synthesis catalyzed by LpxC, suggesting that loss of PlsX function stimulates LPS synthesis. Evidence is presented that increased CHIR-090 resistance is not mediated by changes in the activity of the proteolytic system (YejM-LapB-FtsH) controlling LpxC turnover. Rather, our results are consistent with a model in which the phospholipid precursor acyl-phosphate produced by PlsX serves as an inhibitor of LpxC to lower the rate of LPS synthesis when phospholipid synthesis capacity is reduced.

IMPORTANCE: Over the last several decades, most proteins essential for Gram-negative cell surface assembly have been characterized. However, relatively little is known about how the synthesis of different envelope layers is coordinated to promote uniform surface growth. Here, we report the results of a transposon sequencing-based genetic screen for mutants that suppress defects in the conserved peptidoglycan synthesis machinery responsible for cell elongation. Inactivation of the plsX gene encoding a phospholipid synthesis enzyme was found to both suppress the growth defect of a cell elongation mutant and to confer elevated resistance to an inhibitor of lipopolysaccharide synthesis. Our results suggest the attractive possibility that the product of PlsX, acyl-phosphate, may play a regulatory role in coordinating the phospholipid and lipopolysaccharide synthesis pathways.

PMID:41959078 | PMC:PMC13060810 | DOI:10.64898/2026.03.13.711678

J Appl Crystallogr. 2026 Feb 27;59(Pt 2):343-356. doi: 10.1107/S1600576726000312. eCollection 2026 Apr 1.

ABSTRACT

Supported lipid bilayers (SLBs) are crucial model membrane platforms to study the structure and dynamics of cellular membranes. Vesicle fusion (VF) is one of the most widely used approaches to forming SLBs, though it suffers from compositional limitations and substrate compatibility constraints. The solvent-assisted lipid bilayer (SALB) technique enables the possibility of forming SLBs using a wider range of membrane compositions and substrate platforms through organic-solvent-mediated bilayer assembly, yet questions remain regarding structural equivalence and potential organic solvent incorporation effects. Using neutron reflectometry (NR), we systematically compare the structure and composition of phosphatidylcholine-based SLBs formed by either VF or SALB methodologies. SALB conditions were optimized for NR solid/liquid cells, and structural characterization revealed comparable bilayer architectures between the two formation methods, although some changes in the lipid acyl chain thickness were observed. SALBs showed up to 99.2 ± 0.9% surface coverage using ultrapure water for solvent exchange, but the reproducibility of the method was poor. Enhanced-contrast NR using either deuterated lipids or solvents allowed for the quantitative detection of residual organic solvent incorporation of the SALBs, which was up to 3.3 ± 0.9 vol.% in the tail regions. Making use of 1 mM CaCl₂ during solvent exchange substantially improved SALB reproducibility, reducing coverage variability from 21-30 to 2 vol.%. Validation studies using the antimicrobial peptide melittin demonstrated that membrane-peptide interactions proceeded according to established mechanisms, with peptide incorporation of 18 vol.% for the low-coverage (69.7 ± 0.8%) SALB. The quantified solvent incorporation levels and small changes in acyl chain layer thickness in the SALBs must be considered when interpreting protein-membrane interaction studies, which suggests that validation of the SALB methodology for membrane research applications requires assessment on a case by case basis.

PMID:41959866 | PMC:PMC13060456 | DOI:10.1107/S1600576726000312

Asia Pac J Clin Oncol. 2026 Apr 9. doi: 10.1111/ajco.70114. Online ahead of print.

ABSTRACT

Cancer nanotherapeutics have demonstrated remarkable potential in improving drug solubility, targeting specificity, and pharmacokinetics; however, their clinical translation has remained disproportionately limited. A major yet insufficiently addressed contributor to this translational failure is endosomal entrapment, a process by which nanocarrier-drug complexes, despite efficient cellular internalization, become sequestered within endo-lysosomal compartments, preventing effective cytosolic or nuclear drug delivery. This paper critically examines endosomal entrapment as a central intracellular bottleneck that compromises therapeutic efficacy across multiple nanocarrier platforms, including liposomes, polymeric nanoparticles, lipid nanoparticles, and hybrid systems. The intracellular trafficking routes governing nanoparticle uptake and their convergence toward lysosomal degradation are reviewed, followed by an analysis of the mechanistic determinants of endosomal retention, such as pH-dependent ion trapping, enzymatic degradation, limited membrane permeability, and rapid endosomal maturation. The limitations of conventional nanocarrier evaluation metrics-including particle size, zeta potential, and cellular uptake assays-in predicting functional intracellular drug release are highlighted. Furthermore, the paper evaluates pharmaceutical strategies designed to overcome endosomal entrapment, including pH-responsive materials, proton sponge polymers, fusogenic lipids, membrane-disruptive peptides, ionizable lipids, and prodrug-based approaches, with emphasis on their translational feasibility and safety considerations. Finally, a pharmaceutics-oriented design framework is proposed that positions endosomal escape as a critical quality attribute alongside scalability, regulatory readiness, and clinical relevance. By shifting the focus from cellular uptake to productive intracellular drug delivery, this work provides a rational roadmap for improving the translational success of cancer nanotherapeutics.

PMID:41957529 | DOI:10.1111/ajco.70114

Elife. 2026 Apr 2;14:RP109465. doi: 10.7554/eLife.109465.

ABSTRACT

Most bacteria possess a peptidoglycan (PG) sacculus, which is continuously remodeled during cell growth and division. The turnover products generated in this process are typically imported into the cell and reused for PG biosynthesis. While the underlying pathways have been studied intensively in gammaproteobacteria, knowledge of their presence and physiological roles in other bacterial lineages remains limited. Here, we comprehensively investigate PG recycling in the alphaproteobacterial model organism Caulobacter crescentus. Characterizing the activities of key enzymes in vitro and in vivo, we show that this species contains a functional PG recycling pathway, including the MurU shunt. Our results reveal that PG recycling is critical for C. crescentus cell morphology and division and is dynamically regulated to balance the flux of metabolic intermediates toward PG biosynthesis and central carbon metabolism. Importantly, defects in PG recycling strongly impair the intrinsic ampicillin resistance of C. crescentus without changing the activity of its β-lactamase BlaA, likely by limiting PG precursor biosynthesis and thereby decreasing the activity of the cell wall biosynthetic machinery in the presence of residual antibiotic. These findings underscore the central role of PG recycling in bacterial fitness and suggest that inhibiting this process could provide a promising strategy to combat β-lactam-resistant pathogens.

PMID:41925725 | PMC:PMC13046382 | DOI:10.7554/eLife.109465

J Reprod Immunol. 2026 Apr 1:104882. doi: 10.1016/j.jri.2026.104882. Online ahead of print.

ABSTRACT

Polycystic ovary syndrome (PCOS) is increasingly recognized as a disorder of impaired immune-endocrine homeostasis. Emerging evidence indicates that intestinal dysbiosis and microbial metabolite imbalance can activate pattern recognition receptors (PRRs), forming a PRR microbiota reproductive axis that contributes to PCOS pathophysiology. This review synthesizes current insights into how gut-derived signals, including LPS, peptidoglycans, SCFAs, bile acids, and tryptophan metabolites, modulate TLR-, NLR-, and RLR-mediated pathways to disrupt ovarian, endometrial, and systemic immune regulation. We further propose a unifying framework, the Reproductive Immune Tolerance Disruption Theory, which posits that chronic PRR activation shifts reproductive tract immunity from a tolerogenic to a low-grade inflammatory state, thereby promoting hyperandrogenism, anovulation, insulin resistance, and metabolic dysfunction. We also summarize recent multi-omics and immunometabolic studies that clarify the crosstalk between gut microbial signatures and innate immune signaling. Finally, we highlight precision strategies, including PRR-selective immunomodulation, microbiota-based therapies, and epigenetic metabolic interventions that hold translational potential for redefining PCOS management. Understanding PRR-driven microbial immunomodulation provides a mechanistic framework for reconciling endocrine, metabolic, and reproductive abnormalities in PCOS, guiding the development of targeted therapeutic approaches.

PMID:41935935 | DOI:10.1016/j.jri.2026.104882

Curr Pharm Des. 2026 Mar 19. doi: 10.2174/0113816128431821251206141218. Online ahead of print.

ABSTRACT

Paraprobiotics, non-viable microbial cells or their components, are attracting interest as a safer alternative to live probiotics for immune support. This narrative review synthesizes evidence on (i) preparation methods of paraprobiotics (thermal, high-pressure, irradiation/UV, ohmic heating), (ii) mechanistic links between microbe-associated molecular patterns and host pattern-recognition receptors, and (iii) functional outcomes across in vitro, animal, and early human studies. Preparation methods influence biological activity: conditions that preserve or expose key surface structures (S-layer proteins, lipoteichoic acids, peptidoglycans, β-glucans) modulate molecular and immune signaling and cytokine profiles. Paraprobiotics frequently increased macrophage phagocytosis and nitric oxide production, altered pro- and anti-inflammatory cytokines, improved epithelial barrier markers, and enhanced resistance to pathogens. Several research gaps remain, including limited mechanistic studies, inconsistent reporting of processing kinetics and cell integrity, nonstandard dose units, and variability in immune responses. The literature indicates that pattern recognition receptor- mediated immunomodulation by paraprobiotics requires further rigorous investigation. Overall, this review underscores paraprobiotics as a safe and promising adjunct for modulating immune function, particularly for immunocompromised individuals and for use in food-based applications.

PMID:41937524 | DOI:10.2174/0113816128431821251206141218

ACS Appl Mater Interfaces. 2026 Apr 8;18(13):19946-19957. doi: 10.1021/acsami.6c02551. Epub 2026 Mar 30.

ABSTRACT

Understanding the superselective, multivalent interactions that drive immunity, infection, and biosensing requires (i) model surfaces with precisely tunable receptor density and (ii) quantitative readouts. Here, we introduce a fully automated well-plate-based platform using cell-mimicking supported lipid bilayers (SLBs) that enables both requirements with standardized, commercially available equipment. We outline the main challenges associated with integrating liquid handling with the shear and disruption-sensitive SLB system and offer practical guidelines to overcome them. The resulting versatile and scalable workflow yielded high-quality antifouling surfaces without laborious manual pipetting, while preserving the simplicity of pipet and well-plate-based liquid handling. It enables complex assays requiring more than 1000 aspiration and dispensing steps over >12 h while maintaining surface integrity. Control over the receptor density was achieved by variation of the molar fraction of a biotin-functionalized lipid inside the SLB, to which streptavidin and biotinylated receptors were bound using the strong biotin-streptavidin interaction. A quantitative readout of the receptor density employing fluorescently labeled streptavidin was statistically validated in the low pmol·cm^-2 range. The generated arrays were applied in two biorecognition studies requiring elaborate liquid-handling procedures. DNA hybridization showcases a strong, specific, and stoichiometric binding of fluorescently labeled complementary DNA. Furthermore, the platform was established as an alternative type of glycan array capable of studying the complex, multivalent binding of labeled virus particles scalable to the high-throughput processing of samples.

PMID:41910407 | PMC:PMC13067233 | DOI:10.1021/acsami.6c02551

Biophys J. 2026 Mar 31:S0006-3495(26)00238-9. doi: 10.1016/j.bpj.2026.03.055. Online ahead of print.

ABSTRACT

Lateral transport of membrane proteins is governed by the hydrodynamics of lipid bilayers and their coupling to the surrounding fluids. Protein mobility determines its diffusion rate, which can regulate important cellular functions such as cell signaling, especially when diffusion is the rate limiting process. Classic theories, such as the Saffman-Delbrück (SD) model, established the foundation for understanding protein mobility in membranes. SD theory has since been extended in many ways to understand and model the observations and measurements in living and reconstituted systems. In this review, we focus on hydrodynamic models that treat the membrane as a two-dimensional fluid continuum with embedded proteins, enabling quantitative predictions of protein mobility, correlated motion, and transport across planar and curved membranes. We summarize theoretical and computational advances for free-standing and supported membranes, discuss the role of hydrodynamic interactions in crowded systems, and discuss the extensions to active and passive protein assemblies. In tandem we also discuss the experimental studies that verified these theories or motivated their extensions. Recent advances in the rate and accuracy of acquiring microscopy data have made many of these hydrodynamic predictions experimentally accessible, highlighting the growing role of hydrodynamic modeling in connecting membrane mechanics to protein transport in cells and reconstituted systems.

PMID:41925278 | DOI:10.1016/j.bpj.2026.03.055

Diseases. 2026 Mar 7;14(3):99. doi: 10.3390/diseases14030099.

ABSTRACT

BACKGROUND/OBJECTIVES: Mycobacterial infections and autoimmune diseases affect many worldwide, and growing evidence suggests that there is a bidirectional relationship. This review examines mechanisms by which various autoimmune diseases predispose patients to mycobacterial infections, and vice versa.

METHODS: We conducted a PubMed/MEDLINE search using the keywords "mycobacterium" and the names of the autoimmune conditions to identify relevant papers.

RESULTS: Rheumatoid arthritis therapies, especially TNF-α inhibitors, raise tuberculosis (TB) and non-tuberculous mycobacteria (NTM) risk. Type 1 diabetes features impaired cell-mediated immunity and macrophage dysfunction, with evidence for Mycobacterium avium subspecies paratuberculosis (MAP) mimicry involving HSP65-GAD65. In systemic lupus erythematosus, immune dysregulation plus corticosteroids and cytotoxins elevates TB and NTM risk, amplified in endemic settings. In multiple sclerosis, heightened TLR2/4/9 signaling agents that inhibit pyrimidine synthesis may increase IL-10 and reduce antimycobacterial immunity. Crohn's disease shows genetic susceptibility (e.g., NOD2 variants) and MAP detection, supporting impaired clearance of intracellular mycobacteria.

CONCLUSIONS: Overall, evidence supports a bidirectional relationship: mycobacterial antigens can initiate or amplify autoimmunity via molecular mimicry and chronic stimulation, while autoimmune biology and iatrogenic immunosuppression increase susceptibility to infection. Implications include latent TB screening before immunosuppression, attention to local epidemiology, and vigilance for NTM. Research priorities include prospective cohorts, mechanistic studies of mimicry and NOD2-TLR pathways, safety registries, and trials of screening and prophylaxis.

PMID:41892000 | PMC:PMC13025366 | DOI:10.3390/diseases14030099

Chembiochem. 2026 Mar 27;27(6):e202500845. doi: 10.1002/cbic.202500845.

ABSTRACT

Mycobacteria, including the tuberculosis pathogen Mycobacterium tuberculosis, are enclosed by a highly complex cell envelope with an outer membrane, or mycomembrane, which provides extraordinary protection from antibiotics and other stresses. The inner leaflet of the mycomembrane consists of arabinogalactan-linked mycolate (AGM), which is an enormous glycoconjugate comprising mycolic acids esterified to terminal D-arabinofuranosyl residues of an underlying arabinogalactan-peptidoglycan complex, also referred to as the mycoloyl-arabinogalactan-peptidoglycan (mAGP) complex. Whereas AGM biosynthesis is comparatively well characterized, less is known about AGM degradation by endogenous or exogenous factors. To facilitate studies on AGM breakdown by hydrolytic enzymes, here we synthesized fluorescence resonance energy transfer (FRET)-based mono- and disaccharide probes that mimic fragments of AGM and are designed to fluoresce upon cleavage. We devised a synthetic route that established the glycolipid core with the desired regio- and stereochemistry and allowed late-stage selective functionalization of the core with a FRET pair. Our data show that the intact FRET-AGM probes exist in a fluorescence-quenched state, but when exposed to lysin B (LysB), an AGM-degrading mycobacteriophage hydrolase with therapeutic relevance, the probes were activated through lipid ester hydrolysis, thereby generating fluorescence signal. FRET-AGM probes were activated by known mycomembrane glycolipid hydrolases, but not by several other types of hydrolases, demonstrating specificity. FRET-AGM probes may be useful in the future for identifying novel AGM hydrolases and quantitatively monitoring the activity of AGM hydrolases, which could provide insights into mycomembrane degradative processes and aid in tuberculosis therapeutic development.

PMID:41879737 | DOI:10.1002/cbic.202500845

Biochemistry (Mosc). 2026 Jan;91(Suppl 1):S133-S162. doi: 10.1134/S000629792560406X.

ABSTRACT

Proteins that bind components of bacterial cell wall play a key role in innate immunity and interactions between bacteria and host organisms. They participate in the control of peptidoglycan synthesis and degradation, determine the pathogenic specificity of bacteria, affect their ability to adhere and invade, and serve as important elements of molecular recognition. The review discusses proteins of diverse origins and their recombinant analogues, their structure and binding mechanisms, and prospects for application in the diagnostics of bacterial infections and functionalization of nanomaterials.

PMID:41843876 | DOI:10.1134/S000629792560406X

Adv Healthc Mater. 2026 Mar 19:e03711. doi: 10.1002/adhm.202503711. Online ahead of print.

ABSTRACT

Bacterial infections are the second leading cause of death worldwide. In particular, the increase of bacterial resistance and the emergence of superbacteria have put forward urgent requirements for the prevention and treatment of infection. Bacterial vaccines are considered as an effective strategy to prevent infection. Bacterial outer membrane vesicles (OMVs) are nanoscale spherical membrane structures produced by bacteria, containing a variety of bioactive substances, such as lipopolysaccharides, peptidoglycans, lipoproteins, etc. Due to its inherent adjuvant properties and antigen delivery ability, it can enhance humoral and cellular immune response, thereby improving the deficiency of low immunogenicity of vaccines. Moreover, OMV surface can be engineered to carry and display a variety of antigens. These advantages make OMV an ideal platform for developing innovative vaccines. In this review, followed by description of the classification, advantages and disadvantages of existing bacterial vaccines, the principles and challenges of OMV-based bacterial vaccines are discussed, providing new ideas for the treatment of infectious diseases.

PMID:41852302 | DOI:10.1002/adhm.202503711

Front Microbiol. 2026 Feb 26;17:1761985. doi: 10.3389/fmicb.2026.1761985. eCollection 2026.

ABSTRACT

Probiotics are well recognized for their ability to modulate host immune responses; however, growing evidence indicates that many of their beneficial effects are mediated by structural components rather than by viable microorganisms. Among these components, probiotic-derived peptidoglycan has emerged as a key immunologically active molecule with a critical role in regulating both innate and adaptive immunity. Although substantial experimental data exist regarding its underlying mechanisms, the context-dependent immunomodulatory and anti-inflammatory functions of peptidoglycan have not been comprehensively integrated. In this review, we provide an up-to-date and comprehensive overview of the molecular and cellular mechanisms that govern the immunoregulatory properties of probiotic-derived peptidoglycan. We first discuss the structural diversity and processing of peptidoglycan and their implications for host recognition via pattern-recognition receptors, particularly Toll-like receptor 2 (TLR2) and nucleotide-binding oligomerization domain proteins 1 and 2 (NOD1/2). We then critically evaluate current evidence supporting the therapeutic potential of probiotic-derived peptidoglycan in infectious diseases, inflammatory bowel disease (IBD), autoimmune disorders, allergic inflammation, and cancer. Collectively, these findings suggest that peptidoglycan holds considerable promise for the development of next-generation microbiota-based immunotherapeutic strategies.

PMID:41834851 | PMC:PMC12980888 | DOI:10.3389/fmicb.2026.1761985

Colloids Surf B Biointerfaces. 2026 Mar 2;263:115584. doi: 10.1016/j.colsurfb.2026.115584. Online ahead of print.

ABSTRACT

A reliable assessment of skin permeation is essential for the rational design of transdermal formulations which effectively deliver active ingredients. In particular, a skin screening platform capable of evaluating skin permeability and elucidating the penetration mechanisms of skin penetration enhancers (SPEs), such as liposomes, is critical for advancing transdermal delivery systems. Here, we developed stratum corneum (SC) lipid membrane models which reproduce the barrier properties of human SC and enable mechanistic study of liposomal transdermal delivery. The models were prepared by annealing ceramide, free fatty acids, cholesterol, and cholesterol sulfate on polycarbonate membranes, forming orthorhombic (OR) and hexagonal (HEX) lipid packing structures representing healthy and impaired barriers, respectively. ATR-FTIR and wide-angle X-ray scattering (WAXS) analyses showed that fatty acid chain length governed lipid packing, with long chains promoting OR and short chains favoring HEX packing structure. Franz diffusion experiments demonstrated permeability trends comparable to those reported for excised human SC, with strong correlations between two systems (Pearson correlation coefficients of 0.869 for k_p and 0.943 for t_lag). When liposomes permeated through this model, liposomal niacinamide showed markedly enhanced permeation compared to both aqueous and dipropylene glycol (DPG) formulations. Post-permeation FTIR analysis revealed increased orthorhombicity of SC lipids, while complementary elemental analyses suggested that this effect was driven by cholesterol integration into the SC lipids, supporting a vesicle-lipid fusion mechanism. Collectively, this SC lipid membrane model enables quantitative permeability analysis and mechanistic studies of SPEs, supporting the rational optimization of transdermal delivery systems in dermocosmetic and pharmaceutical applications.

PMID:41795262 | DOI:10.1016/j.colsurfb.2026.115584