125 research outputs found
The Fascinating Coat Surrounding Mycobacteria
No full textThe mycobacterial cell envelope is fascinating in several ways. First, its composition is unique by the exceptional lipid content, which consists of very long-chain (up to C90) fatty acids, the so-called mycolic acids, and a variety of exotic compounds. Second, these lipids are atypically organized into a Gram-negative-like outer membrane (mycomembrane) in these Gram-positive bacteria, as recently revealed by CEMOVIS, and this mycomembrane also contains pore-forming proteins. Third, the mycolic acids esterified a holistic heteropolysaccharide (arabinogalacan), which in turn is linked to the peptidoglycan to form the cell wall skeleton (CWS). In slow-growing pathogenic mycobacterial species, this giant structure is surrounded by a capsular layer composed mainly of polysaccharides, primarily a glycogen-like glucan. The CWS is separated from the plasma membrane by a periplasmic space. A challenging research avenue for the next decade comprises the identification of the components of the uptake and secretion machineries and the isolation and biochemical characterization of the mycomembrane
Covariate identification in the human gut microbiome.
No full textSummary
More than eighty years after the isolation of the first gastrointestinal bacterium in 1885, research on the microbes inside our body finally took off. Since then, improvements in culturing techniques paved the way for the exploration of the complex microbial community inside our guts. In the last decades, the rapid development of sequencing approaches, allowing the identification of our microbial companions based on their DNA, has speeded up the field of gut microbiome research substantially. Sequencing-based assessment of microbial communities in human fecal material in medium-scale (N<400), cross-sectional studies have now linked alterations in gut microbiota composition to disease, as well as chronically suboptimal health and wellbeing. The discovery of these associations has stimulated the search for specific microbiome-based biomarkers for a wide range of pathologies. However, major challenges still hamper the once assumed imminent translation of microbiome monitoring into diagnostic and clinical practice. One such hurdle is the lack of knowledge on the impact of host and environmental factors on microbiota variation within a healthy population, which is essential for robust disease marker identification.
In this thesis we applied a two fold strategy to improve knowledge about the functioning and population-level variability of the gut microbial-human ecosystem in healthy individuals. First, we aimed to facilitate the scale up of faecal sampling initiatives by upgrading time and cost-efficiency of the ‘golden standard’ of fecal sampling procedures, while maintaining or improving user-experience, such that the large numbers necessary to detect a presumed signal in a background of multiple confounders can be obtained faster and with fewer resources. Second, we characterized the effect of specific parameters on the gut-ecosystem, thereby contributing to a better understanding of the factors driving it, and consequently aiding future gut microbiome studies through improved study design, confounder analyses and interpretation of results.
For the first objective, facilitating the scale up of faecal sampling initiatives, we evaluated current collection and preservation techniques of fecal samples for microbiome research on various aspects, including among others userfriendliness, cost, and effect on observed microbiota compostion. Although immediate freezing of samples can still be considered the golden standard, the substantial processing costs associated to such protocols, which mostly stem from cold chain management and aliquotting frozen material under sterile conditions in the laboratory, is the main reason to opt for buffer-based solutions (chapter 3). We designed two new, improved fecal sampling devices allowing rapid aliquotting of frozen fecal material. These devices reduce processing time and cost related to frozen fecal sampling and combine this with similar or improved user-experience for study participants. With the proposed devices large-scale, high-quality sampling would be no longer cost-prohibitive (chapter 4).
Several studies were carried out in order to characterize the effect of specific parameters on the gut-ecosystem, as stated in the second objective. Building upon the rich metadata of the Flemish Gut Flora project, a large-scale (N>1000) research effort based in Belgium and an equally scaled Dutch validation cohort, we identified a set of 69 microbiota covariates with a replication rate of over 92% and a cumulative, nonredundant effect size of 7.63%. The observed small effect size suggests the influence of additional, currently unknown covariates as well as intrinsic microbial ecological processes such as founder effects, species interactions, and dynamics. Out of a total of 503 parameters, stool consistency, as measured by self-assessed Bristol stool scale (BSS) score, emerged as the top feature covarying with fecal microbiome composition. We demonstrated in several study cohorts that stool consistency strongly correlates with all known major microbiome markers. It is negatively correlated with species richness (the number of species in a sample) and linked to the relative abundance of key species Akkermansia and Methanobrevibacter. Enterotypes, which are prevalent constellations of gut microbiome composition, are distinctly distributed over the BSS-scores, with a higher prevalence of Prevotella-enterotyped samples in the looser stool categories (chapters 5 and 6). BSS score has been put forward as an indicative measure of transit time, but also reflects water availability. Both rate of passage and water activity variation are thus potential mechanisms for niche differentiation within the colon ecosystem. By demonstrating - through water activity measurements - that fecal samples provide sufficient unbound water to support the growth of most bacteria, we however showed that the observed effects of stool consistency on colon microbiota composition are unlikely to be a result of water activity variation, but rather represent differences in transit time or other, currently unassessed variables (chapter 7).
Furthermore we assessed the effect of inulin supplementation, combining ecosystem-wide microbiome and metabolome profiling techniques, throughout a cross-over intervention aimed at improving constipation in healthy individuals. We demonstrated that the effect of inulin on the fecal microbiota is mainly restricted to changes in Anaerostipes, Bilophila, and Bifidobacterium relative abundances. Regarding fecal metabolites, only dodecanal was found to increase abundance with the inulin intervention. Hence, inulin indeed selectively influences growth of a limited number of colon bacteria, meeting the most debated criterion of the definition of prebiotics. In addition, we found first indications that reduction of Bilophila, a genus containing known pathobionts, is associated with enhanced host wellbeing and could play a role in the inulin-associated prebiotic mechanism (chapter 8).
In conclusion, the fecal sampling devices designed during this thesis together with the obtained results regarding covariates of gut microbiome composition will contribute to the development of microbiome research as a clinical and diagnostic field.status: Publishe
Molecular basis of secondary multidrug transport
No full textThe Major Facilitator Superfamily groups a vast number of secondary transporters that import or export distinct substrates. Among these, multidrug antiporters constitute a peculiar class of transporters, both because of their multispecificity, recognizing structurally very diverse substrates, and because of their transport mechanism, that relies on bilayer-mediated extrusion of cytotoxic compounds. An accurate and detailed description of the conformational changes that underlie the transport cycle is still lacking and the structural basis for energetic coupling in these transporters has not been elucidated, with so far only limited crystallographic evidence available. We investigate the molecular basis of secondary multidrug transport with biochemical and biophysical studies on LmrP, a Major Facilitator Superfamily multidrug transporter from Lactococcus lactis. We used extensive continuous-wave electron paramagnetic resonance and double electron-electron resonance measurements on a library of spin-labeled LmrP mutants to uncover the conformational states involved in transport and to investigate how protons and ligands shift the equilibrium between conformers to enable transport. We find that the transporter switches between outward-open and outward-closed conformations depending on the protonation states of specific acidic residues forming a transmembrane protonation relay. We observe that substrate binding restricts the conformational freedom of LmrP and induces localized conformational changes. Our data allows to build a model of secondary multidrug transport wherein substrate binding initiates the transport cycle by opening the extracellular side to protons. Subsequent protonation of membrane-embedded acidic residues induces substrate release to the extracellular side and triggers a cascade of conformational changes that culminates in a proton release to the intracellular side. Parallel to this, we have optimized our purification and expression protocol in order to set up crystallization trials on LmrP. Through extensive screening and optimization of the lipidation state of LmrP, using ad hoc methods for sample preparation, we were able to obtain low-resolution diffracting crystals. By improving our lipidation technique and modifying the lipid composition we further improved crystal quality. Other factors such as ligand addition, the presence of secondary detergent and additives for controlling phase separation and nucleation were tested, paving the way to high resolution structure determination of LmrP.Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe
Structural basis of multidrug transport by LmrP, a secondary multidrug transporter
No full textMajor Facilitator Superfamily (MFS) transporters are ubiquitous and play an essentialrole in multiple physiological processes. Some multidrug transporters (MDR) form asub-family of these MFS transporters and are capable to recognize and extrude out ofthe cell an unusually wide range of chemically dissimilar noxious molecules thereforeconferring multidrug resistance to their host. Although they share low sequence similarity,crystal structures reveal a conserved three-dimensional fold with the rest of the family.These observations led to the proposal of a common resistance mechanism for MDR-MFStransporter. Despite intense investigation, the transport mechanism has not been yetdeciphered as only two MFS multidrug transporter have been structurally elucidated.Therefore, in order to address this lack, we decided to solve the crystal structure ofLactococcus lactis multidrug resistance Protein. LmrP is a multidrug transporter from L.lactis belongings to the Major Facilitator Superfamily (MFS). The proteins is poweredby the proton motive force and is capable to recognize and transport a wide range oflipophilic compounds including well known classes of antibiotic.During this work, we have intensively explored the crystallization space of the proteinfrom amino acid sequence through the degree of lipidation to the composition of thecrystallization drop. By honing our lipidation protocol, we were able to produce a largeamount of crystals in various conditions that diffracted X-rays up to 5 Å. We removed fourresidues in the loop connecting the two halves of the protein. Engineered LmrP crystalsdiffracted X-rays beyond 3.0 Å and they had a completely different morphology that hadnot been observed for wild type LmrP crystals. This work reports the crystal structureof LmrP at 2.9 Å in complex with a Hoechst 33342 molecule, a prototypical substrate ofmultidrug transporter. The protein was crystallized in the inward-close/outward-openconformation. The crystal structure reveals another putative conformational switch,consisting of D340 and R135, that has not yet been identifed so far by other techniques.Intriguingly, the structure also reveals an unidentifed density in the binding site thatexpands in the two lobes of the protein and that lines up with the substrate. Our nativemass spectrometry analysis indicates that the density could be due to a lipidic molecule.To our knowledge, this is the frst time that a lipid is reported in the binding cavity ofa multidrug transporter. We, therefore, propose that the lipid plays a modulation rolein the recognition of the substrate. If true, this could shed light on a completely newmechanism of secondary multidrug transport.Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe
Future perspective for potential <i>Helicobacter pylori</i> eradication therapies
No full text Helicobacter pylori infection of the human stomach causes chronic inflammation and forms a major risk factor for the development of peptic ulcer disease and gastric cancer. Current standard eradication therapies use an acid-suppressing drug and two antibiotics, now frequently supplemented with bismuth. Declining eradication efficiencies, off-target effects of lengthy broad-spectrum antibiotic treatments and the desire of a more systematic eradication in asymptomatic H. pylori carriers to suppress gastric cancer incidence spur a search for an effective vaccine and alternative therapeutic options. Here, we review the current progress in the field, with an emphasis on narrow-spectrum or nonantibiotic therapeutics. </jats:p
Conformational kinetics and dynamics of the secondary multidrug transporter LmrP
No full textLe transport sélectif d’ions ou de molécules à travers la membrane plasmique d’une cellule est assuré par des transporteurs membranaires. La Major Facilitator Superfamily (MFS) regroupe la majorité des transporteurs secondaires. Parmi les MFS, les transporteurs multidrogues constituent une classe à part entière puisqu’ils sont capables de transporter des substrats de structures variées. LmrP est un transporteur secondaire multidrogue naturellement exprimé par la bactérie à gram positif Lactococcus lactis. La compréhension du mécanisme de transport de cette protéine permettrait d’obtenir des informations sur le transport secondaire des MFS et sur le mécanisme du transport multidrogue qui joue un rôle dans la résistance bactérienne aux antibiotiques.Un modèle de transport a déjà été proposé pour LmrP grâce à des mesures de distances réalisées en détergent par DEER (Double Electron Electron Resonance). Le rôle des lipides sur l’équilibre conformationnel a également été mis en évidence grâce à cette technique, et la structure de la protéine a récemment été résolue, offrant davantage d’informations sur la liaison du substrat ainsi que la reconnaissance multidrogue. Malgré cette importante caractérisation, ce modèle reste encore incomplet puisqu’il ne tient pas compte de la dynamique de la protéine ni d’effets cinétiques. De plus, dans la membrane bactérienne, LmrP utilise un gradient de protons comme source énergétique pour le transport de substrats. Toutefois, toutes les mesures ont jusqu’à présent été réalisées à pH constant, et le modèle actuel prédit que l’état de repos ne peut être observé dans ces conditions. Afin de compléter ce modèle, nous avons utilisé une méthode de fluorescence sur molécule isolée (smFRET) qui permet, en plus des distributions de distances, d’obtenir des constantes de vitesse de transition et d’identifier des conformations transitoires. Après optimisation de la préparation des échantillons, en plus de confirmer les résultats précédemment obtenus par DEER, nous avons évalué l’impact des ligands sur la cinétique conformationnelle de la protéine par smFRET. Etonnement, leur impact est différent sur la face cytosolique et extracellulaire de la protéine, suggérant que la protéine est en réalité très flexible. Les différents ligands testés ont également différents effets sur la cinétique de la protéine. La deuxième partie de cette thèse s’est axée sur un développement méthodologique pour reconstituer LmrP en protéoliposomes pouvant supporter un gradient de protons. Le but d’un tel système serait à terme de pouvoir réaliser des tests de transport quantitatifs, ainsi que de pouvoir étudier LmrP en présence d’un gradient de protons par smFRET pour isoler les états intermédiaires manquants au modèle de transport.Ce travail démontre que le smFRET est une excellente méthode pour étudier les protéines membranaires, et surtout, qu’il s’agit d’une méthode de choix pour obtenir des informations cinétiques généralement inaccessibles via des méthodes de caractérisation classiques. De plus, ce travail a permis d’optimiser la reconstitution de LmrP en protéoliposomes capables de maintenir un gradient de pH. -----------------------------------------------------------------------------------------------------------------------------------------------------------------------Selective transport of ions or molecules across the membrane of a cell requires membrane proteins. The MajorFacilitator Superfamily (MFS) is the largest family of secondary transporters, all of them transporting substratesfollowing the alternating access model. Some of these transporters, multidrug (MDR) transporters, are particularas they can carry several structurally different substrates. LmrP is a secondary MFS MDR transporter naturallyexpressed by the Gram-positive bacteria Lactococcus lactis. Understanding its transport mechanism at themolecular level would allow the obtention of additional information on secondary and multidrug transport.A transport model has already been proposed for LmrP thanks to distance measurements realised in detergentusing DEER (Double Electron Electron Resonance). The role of the lipids on the conformational equilibrium hasalso been studied using this technique and the high-resolution structure of the protein in complex with thesubstrate Hoechst has been recently solved and offers details of substrate binding and multidrug recognition.However, despite this important characterisation, the transport model remains incomplete as it does notconsider the dynamics of the protein. It is therefore not possible to determine whether the observed functionalof structural effects are due to kinetics. Moreover, in bacterial membranes, LmrP uses the transmembraneproton gradient to energise the transport of substrates. All the biophysical characterisations have thus far onlybeen realised at a constant pH, and the current transport model predicts that the resting state cannot beobserved in such conditions.To validate and complete this model, we used FRET (Förster Resonance Energy Transfer) on single molecules(smFRET) that allows, in addition to distance distributions, the retrieval of transition rate constants and theidentification of transient and/or rare states. We monitored five distance reporters in detergent at different pHvalues, with and without ligand. One of these distance reporters was also reconstituted in nanodiscs to assessthe influence of the lipid bilayer.After the optimisation of sample preparation, despite the differences between smFRET and DEER (e.g. probesor frozen-state vs. freely diffusing molecules at room temperature), we first confirmed the DEER results, i.e. theprotein mostly adopts two conformations, inward-open and outward-open, in an equilibrium modulated by pH,substrate binding and specific mutations. We then characterised the impact of the ligands on the kinetics of thetransition between the conformations adopted by the protein. Surprisingly, the ligands affect the cytosolic andextracellular sides of the protein differently. Moreover, the different tested ligands modulate the transitionkinetics differently, and the prototypical Hoechst substrate even stabilised an extra-open state.The second part of this thesis focused on the methodological development of LmrP reconstitution inproteoliposomes able to maintain a pH gradient. In the end, the purpose of such a system would be to realisequantitative protein transport assays, as well as to study LmrP in the presence of a proton gradient using smFRETin order to isolate missing intermediate states in the transport model.This work demonstrates that smFRET is an excellent method to study membrane proteins and most importantly,is the method of choice to obtain kinetic information that is inaccessible through classical methods such as DEER,X-ray crystallography or cryo-EM. Additionally, this work has laid down the first optimisation steps towards LmrPreconstitution in proteoliposomes able to maintain a pH gradient.Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe
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