Structural basis of prokaryotic ubiquitin-like protein engagement and translocation by the mycobacterial Mpa-proteasome complex

AbstractProteasomes are present in eukaryotes, archaea and Actinobacteria, including the human pathogen Mycobacterium tuberculosis, where proteasomal degradation supports persistence inside the host. In mycobacteria and other members of Actinobacteria, prokaryotic ubiquitin-like protein (Pup) serves as a degradation tag post-translationally conjugated to target proteins for their recruitment to the mycobacterial proteasome ATPase (Mpa). Here, we use single-particle cryo-electron microscopy to determine the structure of Mpa in complex with the 20S core particle at an early stage of pupylated substrate recruitment, shedding light on the mechanism of substrate translocation. Two conformational states of Mpa show how substrate is translocated stepwise towards the degradation chamber of the proteasome core particle. We also demonstrate, in vitro and in vivo, the importance of a structural feature in Mpa that allows formation of alternating charge-complementary interactions with the proteasome resulting in radial, rail-guided movements during the ATPase conformational cycle.

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Cryo-EM structure and polymorphism of Aβ amyloid fibrils purified from Alzheimer’s brain tissue

Nature Communications ◽

10.1038/s41467-019-12683-8 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 85

Author(s):

Marius Kollmer ◽

William Close ◽

Leonie Funk ◽

Jay Rasmussen ◽

Aref Bsoul ◽

...

Keyword(s):

Electron Microscopy ◽

Brain Tissue ◽

Amyloid Fibrils ◽

Structural Basis ◽

Amyloid Angiopathy ◽

Cryo Electron Microscopy ◽

Aβ Amyloid ◽

Cerebral Amyloid ◽

Aβ Fibrils

Abstract The formation of Aβ amyloid fibrils is a neuropathological hallmark of Alzheimer’s disease and cerebral amyloid angiopathy. However, the structure of Aβ amyloid fibrils from brain tissue is poorly understood. Here we report the purification of Aβ amyloid fibrils from meningeal Alzheimer’s brain tissue and their structural analysis with cryo-electron microscopy. We show that these fibrils are polymorphic but consist of similarly structured protofilaments. Brain derived Aβ amyloid fibrils are right-hand twisted and their peptide fold differs sharply from previously analyzed Aβ fibrils that were formed in vitro. These data underscore the importance to use patient-derived amyloid fibrils when investigating the structural basis of the disease.

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The Copper-Responsive RicR Regulon Contributes to Mycobacterium tuberculosis Virulence

mBio ◽

10.1128/mbio.00876-13 ◽

2014 ◽

Vol 5 (1) ◽

Cited By ~ 34

Author(s):

Xiaoshan Shi ◽

Richard A. Festa ◽

Thomas R. Ioerger ◽

Susan Butler-Wu ◽

James C. Sacchettini ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Microbial Growth ◽

Human Pathogen ◽

Content Type ◽

Cu Toxicity ◽

Cu Homeostasis ◽

Cu Resistance ◽

Life On Earth

ABSTRACTAs with most life on Earth, the transition metal copper (Cu) is essential for the viability of the human pathogenMycobacterium tuberculosis. However, infected hosts can also use Cu to control microbial growth. Several Cu-responsive pathways are present inM. tuberculosis, including the regulated in copper repressor (RicR) regulon, which is unique to pathogenic mycobacteria. In this work, we describe the contribution of each RicR-regulated gene to Cu resistancein vitroand to virulence in animals. We found that the deletion or disruption of individual RicR-regulated genes had no impact on virulence in mice, although several mutants had Cu hypersensitivity. In contrast, a mutant unable to activate the RicR regulon was not only highly susceptible to Cu but also attenuated in mice. Thus, these data suggest that several genes of the RicR regulon are required simultaneously to combat Cu toxicityin vivoor that this regulon is also important for resistance against Cu-independent mechanisms of host defense.IMPORTANCEMycobacterium tuberculosisis the causative agent of tuberculosis, killing millions of people every year. Therefore, understanding the biology ofM. tuberculosisis crucial for the development of new therapies to treat this devastating disease. Our studies reveal that although host-supplied Cu can suppress bacterial growth,M. tuberculosishas a unique pathway, the RicR regulon, to defend against Cu toxicity. These findings suggest that Cu homeostasis pathways in both the host and the pathogen could be exploited for the treatment of tuberculosis.

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A microtubule RELION-based pipeline for cryo-EM image processing

10.1101/673566 ◽

2019 ◽

Author(s):

Alexander D. Cook ◽

Szymon W. Manka ◽

Su Wang ◽

Carolyn A. Moores ◽

Joseph Atherton

Keyword(s):

Electron Microscopy ◽

Image Processing ◽

Structural Similarity ◽

Geometric Lattice ◽

Contrast Transfer Function ◽

Biologically Relevant ◽

Cryo Electron Microscopy

AbstractMicrotubules are polar filaments built from αβ-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of α- and β-tubulin. The determination of the αβ-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins.AbbreviationsMiRP, Microtubule RELION-based Pipeline; cryo-EM, cryo-electron microscopy; MT, microtubule; CTF, contrast transfer function; PF, protofilament.

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HIV-Infected Patients Developing Tuberculosis Disease Show Early Changes in the Immune Response to Novel Mycobacterium tuberculosis Antigens

Frontiers in Immunology ◽

10.3389/fimmu.2021.620622 ◽

2021 ◽

Vol 12 ◽

Author(s):

Noemi Rebecca Meier ◽

Manuel Battegay ◽

Tom H. M. Ottenhoff ◽

Hansjakob Furrer ◽

Johannes Nemeth ◽

...

Keyword(s):

Immune Response ◽

Mycobacterium Tuberculosis ◽

Early Diagnosis ◽

Early Stage ◽

Control Group ◽

Rapid Progression ◽

Operating Characteristics ◽

Tnf Α

Background: In individuals living with HIV infection the development of tuberculosis (TB) is associated with rapid progression from asymptomatic TB infection to active TB disease. Sputum-based diagnostic tests for TB have low sensitivity in minimal and subclinical TB precluding early diagnosis. The immune response to novel Mycobacterium tuberculosis in-vivo expressed and latency associated antigens may help to measure the early stages of infection and disease progression and thereby improve early diagnosis of active TB disease.Methods: Serial prospectively sampled cryopreserved lymphocytes from patients of the Swiss HIV Cohort Study developing TB disease (“cases”) and matched patients with no TB disease (“controls”) were stimulated with 10 novel Mycobacterium tuberculosis antigens. Cytokine concentrations were measured in cases and controls at four time points prior to diagnosis of TB: T1-T4 with T4 being the closest time point to diagnosis.Results: 50 samples from nine cases and nine controls were included. Median CD4 cell count at T4 was 289/ul for the TB-group and 456/ul for the control group. Viral loads were suppressed in both groups. At T4 Rv2431c-induced and Rv3614/15c-induced interferon gamma-induced protein (IP)-10 responses and Rv2031c-induced and Rv2346/Rv2347c-induced tumor necrosis factor (TNF)-α responses were significantly higher in cases compared to controls (p < 0.004). At T3 - being up to 2 years prior to TB diagnosis - Rv2031c-induced TNF-α was significantly higher in cases compared to controls (p < 0.004). Area under the receiver operating characteristics (AUROC) curves resulted in an AUC > 0.92 for all four antigen-cytokine pairs.Conclusion: The in vitro Mycobacterium tuberculosis-specific immune response in HIV-infected individuals that progress toward developing TB disease is different from those in HIV-infected individuals that do not progress to developing TB. These differences precede the clinical diagnosis of active TB up to 2 years, paving the way for the development of immune based diagnostics to predict TB disease at an early stage.

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A nucleotidyltransferase toxin inhibits growth of Mycobacterium tuberculosis through inactivation of tRNA acceptor stems

Science Advances ◽

10.1126/sciadv.abb6651 ◽

2020 ◽

Vol 6 (31) ◽

pp. eabb6651 ◽

Cited By ~ 2

Author(s):

Yiming Cai ◽

Ben Usher ◽

Claude Gutierrez ◽

Anastasia Tolcan ◽

Moise Mansour ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Bacterial Toxins ◽

Human Pathogen ◽

Biochemical Activity ◽

Unknown Mechanism ◽

In Vitro Protein Expression ◽

Block Protein Synthesis ◽

Vitro Protein

Toxin-antitoxin systems are widespread stress-responsive elements, many of whose functions remain largely unknown. Here, we characterize the four DUF1814-family nucleotidyltransferase-like toxins (MenT1–4) encoded by the human pathogen Mycobacterium tuberculosis. Toxin MenT3 inhibited growth of M. tuberculosis when not antagonized by its cognate antitoxin, MenA3. We solved the structures of toxins MenT3 and MenT4 to 1.6 and 1.2 Å resolution, respectively, and identified the biochemical activity and target of MenT3. MenT3 blocked in vitro protein expression and prevented tRNA charging in vivo. MenT3 added pyrimidines (C or U) to the 3′-CCA acceptor stems of uncharged tRNAs and exhibited strong substrate specificity in vitro, preferentially targeting tRNASer from among the 45 M. tuberculosis tRNAs. Our study identifies a previously unknown mechanism that expands the range of enzymatic activities used by bacterial toxins, uncovering a new way to block protein synthesis and potentially treat tuberculosis and other infections.

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Mapping of TNTs using Correlative Cryo-Electron Microscopy Reveals a Novel Structure

10.1101/342469 ◽

2018 ◽

Cited By ~ 2

Author(s):

Anna Sartori-Rupp ◽

Diégo Cordero Cervantes ◽

Anna Pepe ◽

Elise Delage ◽

Karine Gousset ◽

...

Keyword(s):

Electron Microscopy ◽

Tunneling Nanotubes ◽

Cryo Electron Microscopy ◽

Novel Structure ◽

Organelle Transfer ◽

Structural Identity ◽

Membrane Bound ◽

Multicellular Organisms

AbstractThe harmonious orchestration of intercellular communication is essential for multicellular organisms. One mechanism by which cells communicate is through long, actin-rich membranous protrusions, called tunneling nanotubes, that allow for the intercellular transport of various cargoes, including viruses, organelles, and proteins between the cytoplasm of distant cells in vitro and in vivo. Over the last decade, studies have focused on their functional role but information regarding their structure and the differences with other cellular protrusions such as filopodia, is still lacking. Here, we report the structural characterization of tunneling nanotubes using correlative light- and cryo-electron microscopy approaches. We demonstrate their structural identity compared to filopodia by showing that they are comprised of a bundle of functional individual Tunneling Nanotubes containing membrane-bound compartments and allowing organelle transfer.

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Structural basis of dynamic P5CS filaments

10.1101/2021.12.02.470899 ◽

2021 ◽

Author(s):

Jiale Zhong ◽

Chen-Jun Guo ◽

Xian Zhou ◽

Chia-Chun Chang ◽

Boqi Yin ◽

...

Keyword(s):

Conformational Changes ◽

Essential Role ◽

Point Mutations ◽

Underlying Mechanism ◽

Structural Basis ◽

Multiple Interfaces ◽

Cryo Electron Microscopy ◽

Neurocutaneous Syndrome

AbstractThe bifunctional enzyme Δ1-pyrroline-5-carboxylate synthase (P5CS) is central to the synthesis of proline and ornithine. Pathogenic mutations in P5CS gene (ALDH18A1) lead to neurocutaneous syndrome and skin relaxation connective tissue disease in humans, and P5CS deficiency seriously damages the ability to resist adversity in plants, which has an essential role in agriculture and human health. Recently, P5CS has been demonstrated forming the cytoophidium in vivo and filaments in vitro. However, the underlying mechanism for the function of P5CS filamentation and catalyze the synthesis of P5C is hardly accessible without structural basis. Here, we have succeeded in determining the full-length structures of Drosophila P5CS filament in three states at resolution from 3.1 to 4.3 Å under cryo-electron microscopy, we observed the distinct ligand-binding states and conformational changes for GK and GPR domain separately. These structures show the distinctive spiral filament is assembled by P5CS tetramers and stabilized by multiple interfaces. Point mutations that deplete such interactions disturb P5CS filamentation and greatly reduce the activity. Our findings reveal a previously undescribed mechanism that filamentation is crucial for the coordination between GK and GPR domains, and provide insights into structural basis for catalysis function of P5CS filament.

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Supramolecular assembly of the E. coli LdcI upon acid stress

10.1101/2020.05.12.090381 ◽

2020 ◽

Author(s):

Matthew Jessop ◽

Clarissa Liesche ◽

Jan Felix ◽

Ambroise Desfosses ◽

Megghane Baulard ◽

...

Keyword(s):

Electron Microscopy ◽

Acid Stress ◽

Super Resolution ◽

Low Ph ◽

Lysine Decarboxylase ◽

E Coli ◽

Cryo Electron Microscopy ◽

Supramolecular Organisation

AbstractPathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid-stress response regulation of E. coli LdcI by combining biochemical and biophysical characterisation with negative stain and cryo-electron microscopy, and wide-field and super-resolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localisation of nanobody-labelled endogenous wild-type LdcI in acid-stressed E. coli cells, and show that it organises into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerisation as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-electron microscopy, and reveal the molecular determinants of LdcI polymerisation, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organisation in the acid stress response.Significance statementBacteria possess a sophisticated arsenal of defence mechanisms that allow them to survive in adverse conditions. Adaptation to acid stress and hypoxia is crucial for the enterobacterial transmission in the gastrointestinal tract of their human host. When subjected to low pH, E. coli and many other enterobacteria activate a proton-consuming resistance system based on the acid-stress inducible lysine decarboxylase LdcI. Here we develop generally-applicable tools to uncover the spatial localisation of LdcI inside the cell by super-resolution fluorescence microscopy, and investigate the in vitro supramolecular organisation of this enzyme by cryo-EM. We build on these results to propose a mechanistic model for LdcI function and offer tools for further in vivo investigations.

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In vitro and in vivo activity of oxazolidinone candidate OTB-658 against Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00974-21 ◽

2021 ◽

Author(s):

Shaochen Guo ◽

Bin Wang ◽

Lei Fu ◽

Xi Chen ◽

Weiyan Zhang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Inhibitory Concentration ◽

Early Stage ◽

Drug Resistant ◽

Lower Minimum Inhibitory Concentration ◽

Time Kill ◽

Combination Regimens ◽

Tuberculosis Activity

In this work, we assess anti-tuberculosis activity of OTB-658 in vitro and in vivo . In vitro , OTB-658 showed bacteriostatic effectiveness with a lower minimum inhibitory concentration than linezolid against Mycobacterium tuberculosis . The minimal bactericidal concentrations and time-kill curves for OTB-658 indicated similar inhibition activity to that of linezolid. OTB-658 entered macrophages to inhibit of M. tuberculosis growth. OTB-658 had a low mutant frequency (10 −8 ), which would prevent drug-resistant mutations from emerging in combination regimens. In vivo , OTB-658 reduced colony-forming unit counts in the lungs and slightly inhibited bacterial growth in the spleen in the early stage and steady state in acute and chronic murine TB models. These results support the preclinical evaluation of OTB-658 and further clinical trials in China.

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Tuberkulose - Screening

Therapeutische Umschau ◽

10.1024/0040-5930/a000181 ◽

2011 ◽

Vol 68 (7) ◽

pp. 381-387

Author(s):

Otto Schoch

Keyword(s):

Mycobacterium Tuberculosis ◽

Interferon Gamma ◽

Wird Eine ◽

Interferon Gamma Release Assays

Das primäre Ziel der Aktivitäten zur bevölkerungsbezogenen Tuberkulosekontrolle ist die Identifizierung von Patienten mit sputummikroskopisch positiver Lungentuberkulose. Wenn diese Patienten umgehend therapiert werden, haben sie nicht nur eine optimale Heilungschance, sondern übertragen auch den Krankheitserreger nicht weiter auf andere Personen. Das Screening, die systematische Suche nach Tuberkulose, erfolgt in der Regel radiologisch bei der Suche nach Erkrankten, während immunologische Teste bei der Suche nach einer Infektion mit Mycobacterium tuberculosis zur Anwendung kommen. Diese Infektion, die ein erhöhtes Risiko für die Entwicklung einer Tuberkulose-Erkrankung mit sich bringt, wird im Rahmen der Umgebungsuntersuchungen oder bei Hochrisikogruppen gesucht. Neben dem traditionellen in vivo Mantoux Hauttest stehen heute die neueren in vitro Blutteste, die sogenannten Interferon Gamma Release Assays (IGRA) zur Verfügung, die unter anderem den Vorteil einer höheren Spezifität mit sich bringen, weil die verwendeten Antigene der Mykobakterien-Wand beim Impfstamm Bacille Calmitte Guerin (BCG) und bei den meisten atypischen Mykobakterien nicht vorhanden sind. Zudem kann bei Immunsupprimierten dank einer mitgeführten Positivkontrolle eine Aussage über die Wahrscheinlichkeit eines falsch negativen Testresultates gemacht werden. Bei neu diagnostizierter Infektion mit Mycobacterium tuberculosis wird eine präventive Chemotherapie mit Isoniazid während 9 Monaten durchgeführt.

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