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Veillonella parvula, prototypical member of the oral and gut microbiota, is at times commensal yet also potentially pathogenic. The definition of the molecular basis tailoring this contrasting behavior is key for broadening our understanding of the microbiota driven pathogenic and/or tolerogenic mechanisms that take place within our body. In this study, we focused on the chemistry of the main constituent of the outer membrane of V. parvula, the lipopolysaccharide (LPS). LPS molecules indeed elicit pro-inflammatory and immunomodulatory responses depending on their chemical structures. Here we report about the structural elucidation of the LPS from two strains of V. parvula and show important and unprecedented differences in both the lipid and carbohydrate moieties, including the identification of a novel galactofuranose and mannitol-containing O-antigen repeating unit for one of the two strains. Furthermore, by harnessing computational studies, in vitro human cell models, as well as lectin binding solid phase assays, we discovered that the two chemically diverse LPS immunologically behave differently and have attempted to identify the molecular determinant(s) governing this phenomenon. Whether a pro-inflammatory potential has been evidenced for the lipid A moiety, by contrast a plausible “immune modulating” action has been proposed for the peculiar O-antigen portion.

Molecular Dynamics (MD) simulations constitute a powerful tool that provides a 3D perspective of the dynamical behavior of chemical systems. Herein the first MD study of the dynamics of a catalytic organometallic system, in micellar media, is presented. The challenging methane catalytic functionalization into ethyl propionate through a silver-catalyzed process has been targeted as the case study. The intimate nature of the micelles formed with the surfactants sodium dodecylsulfate (SDS) and potassium perfluorooctane sulfonate (PFOS) has been ascertained, as well as the relative distribution of the main actors in this transformation, namely methane, the diazo reagent and the silver catalyst, the latter in two different forms: the initial compound and a silver-carbene intermediate. Catalyst deactivation occurs with halide containing surfactants dodecyltrimethylammonium chloride (DTAC) and Triton X-100. Computed simulations allow explaining the experimental results, indicating that micelles behave differently regarding the degree of accumulation and the local distribution of the reactants and their effect in the molecular collisions leading to net reaction.

Toll-like receptors (TLRs), classified as pattern recognition receptors, have a primordial role in the activation of the innate immunity. In particular, TLR4 binds to lipopolysaccharides (LPS), a membrane constituent of Gram-negative bacteria, and, together with Myeloid Differentiation factor 2 (MD-2) protein, forms a heterodimeric complex which leads to the activation of the innate immune system response. Identification of TLRs has sparked great interest in the therapeutic manipulation of the innate immune system. In particular, TLR4 antagonists may be useful for the treatment of septic shock, certain autoimmune diseases, noninfectious inflammatory disorders, and neuropathic pain, and TLR4 agonists are under development as vaccine adjuvants in antitumoral treatments. Therefore, TLR4 has risen as a promising therapeutic target, and its modulation constitutes a highly relevant and active research area. Deep structural understanding of TLR4 signaling may help in the design and discovery of TLR4-modulating molecules with desirable therapeutic properties. Computational studies of the different independent domains composing the TLR4 were undertaken, to understand the differential domain organization of TLR4 in aqueous and membrane environments, including Liquid-disordered (Ld) and Liquid-ordered (Lo) membrane models, to account for the TLR4 recruitment in lipid rafts over activation. We modeled, by means of all-atom Molecular Dynamics (MD) simulations, the structural assembly of plausible full-length TLR4 models embedded into a realistic plasma membrane, accounting for the active (agonist) state of the TLR4, thus providing an analysis at both atomic/molecular and thermodynamic levels of the TLR4 assembly and biological activity. Our results unveil relevant molecular aspects involved in the mechanism of receptor activation, and adaptor recruitment in the innate immune pathways, and will promote the discovery of new TLR4 modulators and probes.

Background and purpose: The pathogenesis of osteoarthritis (OA) implicates a low-grade inflammation associated to the activation of the innate immune system. Toll like receptor (TLR) stimulation triggers the release of inflammatory mediators, which aggravate OA severity. The aim was to study the preventive effect of 6-shogaol (6S), a potential TLR4 inhibitor, on the treatment of experimental knee OA., Experimental approach: OA was induced in C57BL6 mice by surgical section of the medial meniscotibial ligament, which received 6S for eight weeks. Cartilage damage, inflammatory mediator presence, and disease markers were assessed in the joint tissues by immunohistochemistry. Computational modelling was used to predict binding modes of 6S into the TLR4/MD2 receptor and its permeability across cellular membranes. Employing LPS-stimulated chondrocytes and MAPK assay, we clarified 6S action mechanisms., Key results: 6S treatment was able to prevent articular cartilage lesions, synovitis, and the presence of pro-inflammatory mediators and disease markers in OA animals. Molecular modelling studies predicted 6S interaction with the TLR4/MD-2 heterodimer in an antagonist conformation through its binding into the MD-2 pocket. In cell culture, we confirmed that 6S reduced LPS-induced TLR4 inflammatory signaling pathways. Besides, MAPK assay demonstrated that 6S directly inhibits the ERK1/2 phosphorylation activity., Conclusion and implications: 6S evoked a preventive action on cartilage and synovial inflammation in OA mice. 6S effect may take place not only by hindering the interaction between TLR4 ligands and the TLR4/MD-2 complex in chondrocytes, but also through inhibition of ERK phosphorylation, implying a pleiotropic effect on different mediators activated during OA, which proposes it as an attractive drug for OA treatment.

AntiMicrobial Resistance (AMR) is a worldwide health emergency. ESKAPE pathogens include the most relevant AMR bacterial families. In particular, Gram-negative bacteria stand out due to their cell envelope complexity which exhibits strong resistance to antimicrobials. A key element for AMR is the chemical structure of lipid A, modulating the physico-chemical properties of the membrane and permeability to antibiotics. Liposomes are used as models of bacterial membrane infective vesicles. In this work, coarse-grained molecular dynamics simulations were used to model liposomes from ESKAPE Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa). We captured the role of lipid A, cardiolipin and cholesterol on liposome morphology and physico-chemical properties. Additionally, the reported antimicrobial peptides Cecropin B1, JB95, and PTCDA1-kf, were used to unveil their implications on membrane disruption. This study opens a promising starting point to understand molecular keys of bacterial membranes and to promote the discovery of new antimicrobials to overcome AMR.

Antimicrobial Resistance (AMR) is a worldwide health emergency. ESKAPE pathogens include the most relevant AMR bacterial families. In particular, Gram-negative bacteria stand out due to their cell envelope complexity, which exhibits strong resistance to antimicrobials. A key element for AMR is the chemical structure of bacterial lipopolysaccharide (LPS), and the phospholipid composition of the membrane, inflecting the membrane permeability to antibiotics. We have applied coarse-grained molecular dynamics simulations to capture the role of the phospholipid composition and lipid A structure in the membrane properties and morphology of ESKAPE Gram-negative bacterial vesicles. Moreover, the reported antimicrobial peptides Cecropin B1, JB95, and PTCDA1-kf were used to unveil their implications for membrane disruption. This study opens a promising starting point for understanding the molecular keys of bacterial membranes and promoting the discovery of new antimicrobials to overcome AMR..., La resistencia a los antimicrobianos (AMR) es una emergencia sanitaria mundial. Los patógenos ESKAPE incluyen las familias bacterianas más resistentes a antibióticos y son altamente virulentas. En particular, las bacterias Gram negativas destacan por la complejidad de su pared celular, que presenta una fuerte resistencia frente a los antibióticos. Un elemento clave para la AMR es la estructura química del lipopolisacárido bacteriano (LPS) y la composición de los fosfolípidos de la membrana bacteriana, que influyen en su permeabilidad a los antibióticos. Se han empleado simulaciones de dinámica molecular de grano grueso para captar el papel de la composición de los fosfolípidos y la estructura del LPS en las propiedades y morfología de modelos de vesículas bacterianas Gram negativas ESKAPE. Además, se han empleado los péptidos antimicrobianos Cecropin B1, JB95 y PTCDA1-kf para desvelar su mecanismo disrupción de la membrana bacteriana. Este estudio abre un prometedor punto de partida para comprender las claves moleculares de la resistencia en membranas bacterianas y acelerar el descubrimiento de nuevos antibióticos para hacer frente a la AMR...

Toll-like receptors (TLRs) are pattern recognition receptors involved in innate immunity. In particular, TLR4 binds to lipopolysaccharides (LPS), a membrane constituent of Gram-negative bacteria and, together with MD-2 protein, forms a heterodimeric complex which leads to the activation of the innate immune system response. TLR4 activation has been associated with certain autoimmune diseases, noninfectious inflammatory disorders, and neuropathic pain,suggesting a wide range of possible clinical settings for the application of TLR4 antagonists,while TLR4 agonists would be useful as adjuvants in vaccine development and in cancer immunotherapy.[1,2] Specific molecular features of extracellular, transmembrane, and cytoplasmic domains of TLR4 are crucial for coordinating the complex innate immune signaling pathway. Although structural and biochemical data is currently available for the independent TLR4 domains, this only provides a partial fragmented view, because full-length proteins are flexible entities and dynamics play a key role in their functionality. Therefore, many structural and dynamical features of the TLR4 mode of action remain largely unknown.[3]Computational studies of the different independent domains composing the TLR4 were undertaken, using ab-initio calculations, homology modeling, protein-protein docking, all-atom molecular dynamics simulations, and thermodynamics calculations, to understand the differential domain organization of TLR4. From the information gathered from our independent TLR4 domains studies, we have modeled, by all-atom MD simulations, the structural assembly of plausible full-length TLR4 models embedded into realistic plasma membranes, with different chemical compositions, accounting for the active (agonist) state of the TLR4. We have also applied computational techniques to characterize, at the atomic level, the molecular recognition processes by reported TLR4 modulators, thus proposing a mechanism for their biological activity. The

Antimicrobial resistance (AMR) represents a major threat to global public health in the 21st century, dramatically increasing the pandemic expectations in the coming years. The ongoing need to develop new antimicrobial treatments that are effective against multi-drug-resistant pathogens has led the research community to investigate innovative strategies to tackle AMR. The bacterial cell envelope has been identified as one of the key molecular players responsible for antibiotic resistance, attracting considerable interest as a potential target for novel antimicrobials effective against AMR, to be used alone or in combination with other drugs. However, the multicomponent complexity of bacterial membranes provides a heterogeneous morphology, which is typically difficult to study at the molecular level by experimental techniques, in spite of the significant development of fast and efficient experimental protocols. In recent years, computational modeling, in particular, molecular dynamics simulations, has proven to be an effective tool to reveal key aspects in the architecture and membrane organization of bacterial cell walls. Here, after a general overview about bacterial membranes, AMR mechanisms, and experimental approaches to study AMR, we review the state-of-the-art computational approaches to investigate bacterial AMR envelopes, including their limitations and challenges ahead. Representative examples illustrate how these techniques improve our understanding of bacterial membrane resistance mechanisms, hopefully leading to the development of novel antimicrobial drugs escaping from bacterial resistance strategies.

The innate immunity TLR4/MD-2 system is a membrane receptor of paramount importance as therapeutic target. Its assembly, upon binding of Gram-negative bacteria lipopolysaccharide (LPS), and also dependent on the membrane composition, finally triggers the immune response cascade. We here combine ab-initio calculations, molecular docking, all-atom molecular dynamics simulations, and thermodynamics calculations to provide the most realistic and complete 3D models of the active full TLR4 complex embedded into a realistic membrane to date. Our studies reveal functional and structural insights into the transmembrane domain behavior in different membrane environments, the ectodomain bouncing movement, and the dimerization patterns of the intracellular TIR domain. Our work provides TLR4 models as reasonable 3D structures for the (TLR4/MD-2/LPS) 2 architecture accounting for the active (agonist) state of the TLR4, and pointing to a signal transduction mechanism across cell membrane. These observations unveil relevant molecular aspects involved in the TLR4 innate immune pathways and will promote the discovery of new TLR4 modulators.

Modern adjuvants for vaccine formulations are immunostimulating agents whose action is based on the activation of pattern recognition receptors (PRRs) by well-defined ligands to boost innate and adaptive immune responses. Monophosphoryl lipid A (MPLA), a detoxified analogue of lipid A, is a clinically approved adjuvant that stimulates toll-like receptor 4 (TLR4). The synthesis of MPLA poses manufacturing and quality assessment challenges. Bridging this gap, we report here the development and preclinical testing of chemically simplified TLR4 agonists that could sustainably be produced in high purity and on a large scale. Underpinned by computational and biological experiments, we show that synthetic monosaccharide-based molecules (FP compounds) bind to the TLR4/MD-2 dimer with submicromolar affinities stabilizing the active receptor conformation. This results in the activation of MyD88- and TRIF-dependent TLR4 signaling and the NLRP3 inflammasome. FP compounds lack in vivo toxicity and exhibit adjuvant activity by stimulating antibody responses with a potency comparable to MPLA.

Antimicrobial Resistance (AMR) is a worldwide health emergency. ESKAPE pathogens include the most relevant AMR bacterial families. In particular, Gram-negative bacteria stand out due to their cell envelope complexity, which exhibits strong resistance to antimicrobials. A key element for AMR is the chemical structure of bacterial lipopolysaccharide (LPS), and the phospholipid composition of the membrane, inflecting the membrane permeability to antibiotics. We have applied coarse-grained molecular dynamics simulations to capture the role of the phospholipid composition and lipid A structure in the membrane properties and morphology of ESKAPE Gram-negative bacterial vesicles. Moreover, the reported antimicrobial peptides Cecropin B1, JB95, and PTCDA1-kf were used to unveil their implications for membrane disruption. This study opens a promising starting point for understanding the molecular keys of bacterial membranes and promoting the discovery of new antimicrobials to overcome AMR...