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The molecular mechanism of outer membrane protein insertion by BamA and its role as a target for novel antibiotics

English title The molecular mechanism of outer membrane protein insertion by BamA and its role as a target for novel antibiotics
Applicant Hiller Sebastian
Number 167125
Funding scheme NRP 72 Antimicrobial Resistance
Research institution Abteilung Strukturbiologie und Biophysik Biozentrum Universität Basel
Institution of higher education University of Basel - BS
Main discipline Molecular Biology
Start/End 01.01.2017 - 30.06.2021
Approved amount 396'151.00
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All Disciplines (3)

Discipline
Molecular Biology
Biophysics
Biochemistry

Keywords (7)

Membrane protein structure; Novel Antibiotics; Membrane protein function; Bacterial outer membrane; BamA; Omp85 insertase; Nuclear Magnetic Resonance (NMR)

Lay Summary (German)

Lead
Viele besonders gefährliche Bakterien schützen sich mit einer äusseren Membran. Wir wollen die Entstehungsmechanismen dieser Membran verstehen, um Angriffsziele für neue Wirkstoffe zu finden.
Lay summary

Infektionen mit sogenannten Gram-negativen antibiotikaresistenten Bakterien sind schwierig zu behandeln, da diese Bakterien eine äussere Membran besitzen, die sie schützt. Doch auch diese Membran bietet Angriffspunkte für Wirkstoffe. Wir untersuchen einen dieser Punkte, das Protein BamA. Dieses steuert den Prozess, mit dem andere Proteine in die Membran eingebaut werden. Wird BamA ausgeschaltet, ist das tödlich für das Bakterium. Wir beleuchten die Funktion von BamA und den Einbau von Proteinen in atomarer Auflösung, mit Hilfe von NMR-Spektroskopie und Röntgenkristallographie. Das macht die Wechselwirkungen von BamA mit anderen Molekülen sichtbar. So können wir die Mechanismen besser verstehen und nach Wirkstoffen suchen, die sie unterbrechen.

Hintergrund
Von den Bakterien der Gram-negativen Klasse treten immer häufiger resistente Stämme auf, gegen die alle bekannten Antibiotika unwirksam sind. Neue Wirkstoffe gegen diese Bakterien werden dringend benötigt.

Ziel
Ziel unseres Projekts ist es, den Mechanismus zu verstehen, den Gram-negative Bakterien benutzen, um Membranproteine in ihre äussere Membran einzubauen.

Bedeutung
Unsere Erkenntnisse können neue mögliche Angriffsziele für antibiotische Wirkstoffe zeigen. Diese lassen sich damit gezielt suchen und anpassen. Wir werden auch bestehende Antibiotikakandidaten entsprechend optimieren.

Direct link to Lay Summary Last update: 05.12.2017

Lay Summary (French)

Lead
De nombreuses bactéries particulièrement dangereuses se protègent à l’aide d’une membrane extérieure. Nous souhaitons comprendre les mécanismes qui permettent le développement de cette membrane afin de découvrir des cibles pour de nouveaux principes actifs.
Lay summary

Les bactéries Gram-négatives résistantes aux antibiotiques sont responsables d’infections difficiles à traiter car elles possèdent une membrane protectrice externe. Cependant, cette membrane présente des points faibles pouvant être exploités pour de nouveaux traitements. Nous étudions l’un d’entre eux, la protéine BamA. Celle-ci contrôle le processus d’intégration d’autres protéines à la membrane et sa désactivation est mortelle pour la bactérie. Nous analysons la fonction de BamA et l’intégration des protéines à une résolution atomique par spectroscopie RMN et cristallographie aux rayons X. La mise en évidence des interactions existant entre la protéine BamA et d’autres molécules nous permet de mieux comprendre son fonctionnement et de rechercher les substances capables de l’interrompre.

Contexte
Des souches résistantes de bactéries de la classe Gram-négative, contre lesquelles tous les antibiotiques connus sont inefficaces, apparaissent de plus en plus fréquemment. Il n’existe actuellement aucune nouvelle génération de médicaments pour lutter contre ces micro-organismes.

Objectif
L’objectif de notre projet est de comprendre le mécanisme utilisé par les bactéries Gram-négatives pour intégrer des protéines membranaires à leur membrane externe.

Importance
Nos résultats visent à mettre en évidence d’éventuelles nouvelles cibles pour les substances antibiotiques qui pourront être développées et adaptées de manière spécifique. D’une manière similaire, nous pourrons également optimiser des antibiotique déjà existants et potentiellement utilisables.


Direct link to Lay Summary Last update: 05.12.2017

Lay Summary (English)

Lead
Many particularly dangerous bacteria are protected by an outer membrane. We want to understand the mechanism by which this membrane develops in order to find targets for new active substances.
Lay summary

Infections by so-called Gram-negative antibiotic-resistant bacteria are difficult to treat because these bacteria are protected by two membranes. Yet there are points in the outer membrane that are open to attack by active substances. We are characterizing one of these points, the protein BamA. BamA controls the process of incorporating other proteins into the membrane. If BamA is knocked out, this is fatal for the bacterium. We are studying the function of BamA and the incorporation of proteins at the atomic level, using NMR spectroscopy and X-ray crystallography. This makes the interactions between BamA and other molecules visible. In this way we can gain a better understanding of the mechanisms and look for active substances that interrupt them.

Background
Resistant strains of Gram-negative bacteria against which all known antibiotics are ineffective occur with increasing frequency. There is an urgent need for new active substances to control them.

Aim
The aim of our project is to understand the mechanisms that Gram-negative bacteria use to incorporate membrane proteins into their outer membrane.

Relevance
Our findings can point to possible new targets for antibiotic substances that can then be sought and adapted specifically. We will also optimise existing antibiotic candidates in the same way.


Direct link to Lay Summary Last update: 05.12.2017

Responsible applicant and co-applicants

Employees

Publications

Publication
The antibiotic darobactin mimics a β-strand to inhibit outer membrane insertase
Kaur Hundeep, Jakob Roman P., Marzinek Jan K., Green Robert, Imai Yu, Bolla Jani Reddy, Agustoni Elia, Robinson Carol V., Bond Peter J., Lewis Kim, Maier Timm, Hiller Sebastian (2021), The antibiotic darobactin mimics a β-strand to inhibit outer membrane insertase, in Nature, 593(7857), 125-129.
Regulation of chaperone function by coupled folding and oligomerization
Mas Guillaume, Burmann Björn M., Sharpe Timothy, Claudi Beatrice, Bumann Dirk, Hiller Sebastian (2020), Regulation of chaperone function by coupled folding and oligomerization, in Science Advances, 6(43), eabc5822.
Expression, Purification, and Structural Biology of Membrane Proteins
Kaur Hundeep, Grahl Anne, Hartmann Jean-Baptiste, Hiller Sebastian (2020), Expression, Purification, and Structural Biology of Membrane Proteins, in Methods Molecular Biology, 373-396.
The electrostatic core of the outer membrane protein X from E. coli
Rath Parthasarathi, Sharpe Timothy, Hiller Sebastian (2020), The electrostatic core of the outer membrane protein X from E. coli, in Biochimica et Biophysica Acta (BBA) - Biomembranes, 1862(1), 183031-183031.
Chimeric peptidomimetic antibiotics against Gram-negative bacteria
Luther Anatol, Urfer Matthias, Zahn Michael, Müller Maik, Wang Shuang-Yan, Mondal Milon, Vitale Alessandra, Hartmann Jean-Baptiste, Sharpe Timothy, Monte Fabio Lo, Kocherla Harsha, Cline Elizabeth, Pessi Gabriella, Rath Parthasarathi, Modaresi Seyed Majed, Chiquet Petra, Stiegeler Sarah, Verbree Carolin, Remus Tobias, Schmitt Michel, Kolopp Caroline, Westwood Marie-Anne, Desjonquères Nicolas, Brabet Emile, et al. (2019), Chimeric peptidomimetic antibiotics against Gram-negative bacteria, in Nature, 576(7787), 452-458.
A new antibiotic selectively kills Gram-negative pathogens
Imai Yu, Meyer Kirsten J., Iinishi Akira, Favre-Godal Quentin, Green Robert, Manuse Sylvie, Caboni Mariaelena, Mori Miho, Niles Samantha, Ghiglieri Meghan, Honrao Chandrashekhar, Ma Xiaoyu, Guo Jason J., Makriyannis Alexandros, Linares-Otoya Luis, Böhringer Nils, Wuisan Zerlina G., Kaur Hundeep, Wu Runrun, Mateus André, Typas Athanasios, Savitski Mikhail M., Espinoza Josh L., O’Rourke Aubrie, et al. (2019), A new antibiotic selectively kills Gram-negative pathogens, in Nature, 576(7787), 459-464.
Identification of conformation-selective nanobodies against the membrane protein insertase BamA by an integrated structural biology approach
Kaur Hundeep, Hartmann Jean-Baptiste, Jakob Roman P., Zahn Michael, Zimmermann Iwan, Maier Timm, Seeger Markus A., Hiller Sebastian (2019), Identification of conformation-selective nanobodies against the membrane protein insertase BamA by an integrated structural biology approach, in Journal of Biomolecular NMR, 73(6-7), 375-384.
A guide to quantifying membrane protein dynamics in lipids and other native‐like environments by solution‐state NMR spectroscopy
Bibow Stefan, Hiller Sebastian (2019), A guide to quantifying membrane protein dynamics in lipids and other native‐like environments by solution‐state NMR spectroscopy, in The FEBS Journal, 286(9), 1610-1623.
Two-State Folding of the Outer Membrane Protein X into a Lipid Bilayer Membrane
Rath Parthasarathi, Sharpe Timothy, Kohl Bastian, Hiller Sebastian (2019), Two-State Folding of the Outer Membrane Protein X into a Lipid Bilayer Membrane, in Angewandte Chemie International Edition, 1.
Sequence-Specific Solution NMR Assignments of the β-Barrel Insertase BamA to Monitor Its Conformational Ensemble at the Atomic Level
Hartmann Jean-Baptiste, Zahn Michael, Burmann Irena Matečko, Bibow Stefan, Hiller Sebastian (2018), Sequence-Specific Solution NMR Assignments of the β-Barrel Insertase BamA to Monitor Its Conformational Ensemble at the Atomic Level, in Journal of the American Chemical Society, 140, 11252.

Datasets

Structure of the darobactin-bound E. coli BAM complex (BamABCDE)

Author Jakob, Roman; Maier, Timm; Hiller, Sebastian; et, al
Persistent Identifier (PID) 10.2210/pdb7NRI/pdb
Repository PDB


Collaboration

Group / person Country
Types of collaboration
Prof. Dr. Markus Seeger, University of Zurich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Dr. Peter Bond Singapore (Asia)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Polyphor AG Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Industry/business/other use-inspired collaboration
Roche Pharma AG Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel
- Industry/business/other use-inspired collaboration
Prof. Dr. Timm Maier, Biozentrum, University of Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Dr. Kim Lewis, Northeastern University United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
2nd Nanobodies Conference, Brussels, Belgium Talk given at a conference Targeting the outer membrane insertase with novel antibiotics 10.09.2021 Brüssel, Belgium Hiller Sebastian;
17th EUROMAR Conference Talk given at a conference The outer membrane insertase as an antibiotic target 07.07.2021 Portoroz, Slovenia Hiller Sebastian;
International ICMRBS Web Seminar Series Individual talk The outer membrane insertase as an antibiotic target 29.04.2021 Internet, Switzerland Hiller Sebastian;
EMBO Young Investigator Sectorial Meeting Structural Biology Talk given at a conference Outer membrane insertase targeted by novel antibiotics 04.09.2020 Internet, Switzerland Hiller Sebastian;
International online seminar series on antibiotics Individual talk The outer membrane protein insertase BamA as a target for novel antibiotics 01.07.2020 Internet, Switzerland Hiller Sebastian;
Structural Biology Lecture Series, Dana Farber Research Institute Individual talk Dynamic Chaperone-Client Interactions in Outer Membrane Protein Biogenesis and Parkinson’s Disease 10.03.2020 Boston, United States of America Hiller Sebastian;
Colloquium Microbiology, Northeastern University Individual talk Dynamic Chaperone-Client Interactions in Outer Membrane Protein Biogenesis and Parkinson’s Disease 09.03.2020 Boston, United States of America Hiller Sebastian;
2nd Alpine Conference Talk given at a conference The mechanism of outer membrane protein insertion by BamA and its role as a target for novel antibiotics 19.01.2020 St. Anton, Austria Hiller Sebastian;
Lecture series ETH Zurich Individual talk Dynamic chaperone–client-interactions studied by solution NMR spectroscopy 04.12.2019 Zürich, Switzerland Hiller Sebastian;
5th SFB 803 Symposium Talk given at a conference Mechanisms in Outer Membrane Protein Folding and Biogenesis 23.09.2018 Göttingen, Germany Hiller Sebastian;
FASEB conference “Molecular Biophysics of Membranes” Talk given at a conference Mechanisms in Outer Membrane Protein Folding and Biogenesis 21.06.2018 Olean NY, United States of America Hiller Sebastian;
Basel Infectious Diseases Study Club Individual talk The mechanism of outer membrane protein insertion and its role as a target for novel antibiotics 07.12.2017 Basel, Switzerland Hiller Sebastian;
GRC membrane protein folding Poster Outer membrane protein folding by the BAM-complex 04.06.2017 Easton MA USA, United States of America Zahn Michael;
NRP 72 Kick-off meeting Talk given at a conference The molecular mechanism of outer membrane protein insertion by BamA and its role as a target for new antibiotics 15.03.2017 Basel, Switzerland Hiller Sebastian;


Communication with the public

Communication Title Media Place Year
Media relations: radio, television Beitrag über Antibiotika in 3Sat Nano SRF / 3Sat International 2020

Associated projects

Number Title Start Funding scheme
152989 Structure, Function and Folding of Membrane Proteins and their Complexes 01.08.2014 SNSF Professorships
187170 A versatile technology platform for identification and development of novel bio-antibiotics 01.04.2020 Bridge - Discovery

Abstract

In recent years, multidrug resistance (MDR) of microorganisms against antibiotics is worldwide on the rise. The currently most threatening species include the Gram-negative Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. In these Gram-negative bacteria, the outer membrane presumably provides an ideal target for novel antibiotics. Among the typically around 80 outer membrane proteins (Omps), only two - BamA and LptD - are generally essential and highly conserved across Gram-negative bacteria. LptD is efficiently targeted by the protein epitope mimic (PEM) POL7080, a cyclic peptide with potent antimicrobial activity against P. aeruginosa that is currently in clinical trials (Polyphor AG, Allschwil BL). With increased spreading of MDR-strains, the identification of additional new targets is urgently needed.BamA is an integral membrane protein, forming the central unit of the Bam complex. Deletion of BamA and even certain single-point mutations are lethal to E. coli, underscoring the relevance as an outer membrane antibiotic target. BamA inserts a broad range of different substrates into the membrane, but despite knowledge of the structure, the functional mechanism of BamA has so far remained unclear. Since the interaction with its substrates is only semi-specific, there is a high potential for specific high-affinity binders that may block BamA function. A description of the BamA functional mechanism at the atomic level would thus provide essential insights, how such new antibiotics may be developed. In collaboration with our industry partner Polyphor AG (Allschwil BL), we want to investigate the mechanism of action of a new family of PEM antibiotics that have been shown to be highly active against all Gram-negative ESKAPE pathogens. The new family of PEMs is presumably targeting other members of the ß-barrel outer membrane protein family such as the insertase BamA.We will pursue the following objectives:1.) An atomic level description of the functional mechanism of substrate insertion by BamA.2.) An atomic level structural and functional description of its inhibition by PEM antibiotics and newly identified natural inhibitors.3.) Establishment of a quantitative assay suitable for high-throughput screening of new BamA inhibitors.To achieve these goals, we will use solution NMR spectroscopy, as well as X-ray crystallography, and biochemical and biophysical methods. We base our work on strong expertise in these techniques, as well as on BamA and Bam complex biochemistry.
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