Structural basis of bacterial glyeans biosynthesis and processing: ímpact in human health and disease

181 p.El objetivo a largo plazo de nuestro grupo es estudiar a nivel molecular la estructura, la especificidad por sustrato y el mecanismo de acción de diferentes enzimas bacterianas involucradas en el reconocimiento o modificación de carbohidratos que sean relevantes en la interacción, tanto beneficiosa como patogénica, con el ser humano como hospedador.En este contexto, durante mi tesis, he trabajado en la comprensión a nivel estructural de la catálisis, así como del mecanismo de reconocimiento del sustrato de enzimas de bacterias tanto beneficiosas (Akkermansia muciniphila) como perjudiciales (Mycobacterium tuberculosis) para la salud del hospedador. Siguiendo este criterio, la tesis ha sido dividida en dos secciones; el estudio de la maquinaria enzimática que Akkermansia muciniphila, bacteria que forman parte de la microbiota intestinal, presenta para la digestión de los azúcares presentes en las mucinas por un lado, y el estudio de la biosíntesis de los glucolípidos (fosfatidil-myo-inositol mannósidos; PIMs, lipomannanos; LM y lipoarabinomannanos; LAM) presentes en la compleja envoltura celular de M. tuberculosis, por otro lado

Raman Spectroscopy as a Tool to Study the Pathophysiology of Brain Diseases

The Raman phenomenon is based on the spontaneous inelastic scattering of light, which depends on the molecular characteristics of the dispersant. Therefore, Raman spectroscopy and imaging allow us to obtain direct information, in a label-free manner, from the chemical composition of the sample. Since it is well established that the development of many brain diseases is associated with biochemical alterations of the affected tissue, Raman spectroscopy and imaging have emerged as promising tools for the diagnosis of ailments. A combination of Raman spectroscopy and/or imaging with tagged molecules could also help in drug delivery and tracing for treatment of brain diseases. In this review, we first describe the basics of the Raman phenomenon and spectroscopy. Then, we delve into the Raman spectroscopy and imaging modes and the Raman-compatible tags. Finally, we center on the application of Raman in the study, diagnosis, and treatment of brain diseases, by focusing on traumatic brain injury and ischemia, neurodegenerative disorders, and brain cancer.The APC was funded by grant PID2020-117405GB100, funded by MCIN/AEI/10.13039/501100011033 and, as appropriate, by “ERDF A way of making Europe” by the “European Union” or by the “European Union NextGenerationEU/PRTR”; by the Basque Government, grant numbers ELKARTEK22/86 and IT1625-22; and by Fundación Ramón Areces, grant number CIVP20S11276

Turning universal O into rare Bombay type blood

Red blood cell antigens play critical roles in blood transfusion since donor incompatibilities can be lethal. Recipients with the rare total deficiency in H antigen, the Oh Bombay phenotype, can only be transfused with group Oh blood to avoid serious transfusion reactions. We discover FucOB from the mucin-degrading bacteria Akkermansia muciniphila as an -1,2-fucosidase able to hydrolyze Type I, Type II, Type III and Type V H antigens to obtain the afucosylated Bombay phenotype in vitro. X-ray crystal structures of FucOB show a three-domain architecture, including a GH95 glycoside hydrolase. The structural data together with site-directed mutagenesis, enzymatic activity and computational methods provide molecular insights into substrate specificity and catalysis. Furthermore, using agglutination tests and flow cytometry-based techniques, we demonstrate the ability of FucOB to convert universal O type into rare Bombay type blood, providing exciting possibilities to facilitate transfusion in recipients/patients with Bombay phenotype

Dissecting the structural and chemical determinants of the "open-to-closed" motion in the mannosyltransferase PimA from Mycobacteria

The phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential peripheral membrane glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PimA undergoes functionally important conformational changes, including (i) α-helix-To-β-strand and β-strand-To-α-helix transitions and (ii) an "open-To-closed"motion between the two Rossmann-fold domains, a conformational change that is necessary to generate a catalytically competent active site. In previous work, we established that GDP-Man and GDP stabilize the enzyme and facilitate the switch to a more compact active state. To determine the structural contribution of the mannose ring in such an activation mechanism, we analyzed a series of chemical derivatives, including mannose phosphate (Man-P) and mannose pyrophosphate-ribose (Man-PP-RIB), and additional GDP derivatives, such as pyrophosphate ribose (PP-RIB) and GMP, by the combined use of X-ray crystallography, limited proteolysis, circular dichroism, isothermal titration calorimetry, and small angle X-ray scattering methods. Although the β-phosphate is present, we found that the mannose ring, covalently attached to neither phosphate (Man-P) nor PP-RIB (Man-PP-RIB), does promote the switch to the active compact form of the enzyme. Therefore, the nucleotide moiety of GDP-Man, and not the sugar ring, facilitates the "open-To-closed"motion, with the β-phosphate group providing the high-Affinity binding to PimA. Altogether, the experimental data contribute to a better understanding of the structural determinants involved in the "open-To-closed"motion not only observed in PimA but also visualized and/or predicted in other glycosyltransfeases. In addition, the experimental data might prove to be useful for the discovery and/or development of PimA and/or glycosyltransferase inhibitors

Super-Resolution Microscopy to Study Interorganelle Contact Sites

Interorganelle membrane contact sites (MCS) are areas of close vicinity between the membranes of two organelles that are maintained by protein tethers. Recently, a significant research effort has been made to study MCS, as they are implicated in a wide range of biological functions, such as organelle biogenesis and division, apoptosis, autophagy, and ion and phospholipid homeostasis. Their composition, characteristics, and dynamics can be studied by different techniques, but in recent years super-resolution fluorescence microscopy (SRFM) has emerged as a powerful tool for studying MCS. In this review, we first explore the main characteristics and biological functions of MCS and summarize the different approaches for studying them. Then, we center on SRFM techniques that have been used to study MCS. For each of the approaches, we summarize their working principle, discuss their advantages and limitations, and explore the main discoveries they have uncovered in the field of MCS

The phosphatidyl-myo-inositol dimannoside acyltransferase PatA is essential for Mycobacterium tuberculosis growth in vitro and in vivo

Mycobacterium tuberculosis comprises an unusual cell envelope dominated by unique lipids and glycans that provides a permeability barrier against hydrophilic drugs and is central for its survival and virulence. Phosphatidyl-myo-inositol mannosides (PIMs) are glycolipids considered not only key structural components of the cell envelope but also the precursors of lipomannan (LM) and lipoarabinomannan (LAM), important lipoglycans implicated in host-pathogen interactions. Here, we focus on PatA, a membrane associated acyltransferase that transfers a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM1/PIM2 We validate that the function of PatA is vital for M. tuberculosis in vitro and in vivo We constructed a patA conditional mutant and showed that silencing patA is bactericidal in batch cultures. This phenotype was associated with significant reduced levels of Ac1PIM2, an important structural component of the mycobacterial inner membrane. The requirement of PatA for viability was also demonstrated during macrophage infection and in a mouse model of infection, where a dramatic decrease in viable counts was observed upon silencing of the patA gene. This is reminiscent of the behavior of PimA, the mannosyltransferase that initiates the PIM pathway, also found to be essential for M. tuberculosis growth in vitro and in vivo Altogether, the experimental data highlights the significance of the early steps of PIM biosynthetic pathway for M. tuberculosis physiology and uncover that PatA is a novel target for drug discovery programs against this major human pathogen.IMPORTANCE TB is the leading cause of death from a single infectious agent. The emergence of drug resistance in strains of M. tuberculosis, the etiologic agent of TB, emphasizes the need of identifying new targets and antimicrobial agents. The mycobacterial cell envelope is a major factor in this intrinsic drug resistance. Here, we have focused on the biosynthesis of PIMs, key virulence factors and important components of the cell envelope. Specifically, we have determined PatA, the acyltransferase responsible for the first acylation step of the PIM synthesis pathway, is essential in M. tuberculosis These results highlight the importance of early steps of PIM biosynthetic pathway for mycobacterial physiology and the suitability of PatA as a potential new drug target

Turning universal O into rare Bombay type blood

People with the rare Bombay-type Oh blood group can only be transfused with Oh blood. Here, the authors characterize a bacterial α−1,2-fucosidase that can convert universal O type into rare Bombay type blood

Molecular ruler mechanism and interfacial catalysis of the integral membrane acyltransferase PatA

Glycolipids are prominent components of bacterial membranes that play critical roles not only in maintaining the structural integrity of the cell but also in modulating host-pathogen interactions. PatA is an essential acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. We demonstrate by electron spin resonance spectroscopy and surface plasmon resonance that PatA is an integral membrane acyltransferase tightly anchored to anionic lipid bilayers, using a two-helix structural motif and electrostatic interactions. PatA dictates the acyl chain composition of the glycolipid by using an acyl chain selectivity “ruler.” We established this by a combination of structural biology, enzymatic activity, and binding measurements on chemically synthesized nonhydrolyzable acyl–coenzyme A (CoA) derivatives. We propose an interfacial catalytic mechanism that allows PatA to acylate hydrophobic PIMs anchored in the inner membrane of mycobacteria, through the use of water-soluble acyl-CoA donors