المؤلفون: Reva, Oleg, N, La Cono, Violetta, Crisafi, Francesca, Smedile, Francesco, Mudaliyar, Manasi, Ghosal, Debnath, Giuliano, Laura, Krupovic, Mart, Yakimov, Michail, M

المساهمون: University of Pretoria South Africa, Istituto di Scienze Polari / Institute of Polar Sciences Messina (ISP-CNR), University of Melbourne, The Mediterranean Science Commission (CIESM), Virologie des archées - Archaeal Virology, Université Paris Cité (UPCité)-Microbiologie Intégrative et Moléculaire (UMR6047), Institut Pasteur Paris (IP)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur Paris (IP)-Centre National de la Recherche Scientifique (CNRS), This study was partially supported by a grant from the FUTURENZYMES Project (Contract 101000327), funded by the European Union's Horizon 2020 Research Program. MK was supported by Agence Nationale de la Recherche (grant ANR-20-CE20-0009). This project was funded by an NHMRC grant (APP1196924 to DG), and an Human Frontier Science Program (HFSP) grant (RGEC33/2023) to DG., ANR-20-CE20-0009,VIROMET,Devoiler le virome des archées methanogenes(2020), European Project: 101000327,H2020-FNR-2020-2,FUTURENZYMES(2021)

المصدر: ISSN: 1758-2229 ; Environmental Microbiology Reports ; https://pasteur.hal.science/pasteur-04539987Test ; Environmental Microbiology Reports, 2024, 16 (2), pp.e13258. ⟨10.1111/1758-2229.13258⟩.

مصطلحات موضوعية: [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology

الوصف: International audience ; DNA methylation serves a variety of functions across all life domains. In this study, we investigated archaeal methylomics within a tripartite xylanolytic halophilic consortium. This consortium includes Haloferax lucertense SVX82, Halorhabdus sp. SVX81, and an ectosymbiotic Candidatus Nanohalococcus occultus SVXNc, a nano‐sized archaeon from the DPANN superphylum. We utilized PacBio SMRT and Illumina cDNA sequencing to analyse samples from consortia of different compositions for methylomics and transcriptomics. Endogenous c TAG methylation, typical of Haloferax , was accompanied in this strain by methylation at four other motifs, including GDG c HC methylation, which is specific to the ectosymbiont. Our analysis of the distribution of methylated and unmethylated motifs suggests that autochthonous cTAG methylation may influence gene regulation. The frequency of GRAGA a G methylation increased in highly expressed genes, while C c TTG and GTCG a GG methylation could be linked to restriction‐modification (RM) activity. Generally, the RM activity might have been reduced during the evolution of this archaeon to balance the protection of cells from intruders, the reduction of DNA damage due to self‐restriction in stressful environments, and the benefits of DNA exchange under extreme conditions. Our methylomics, transcriptomics and complementary electron cryotomography (cryo‐ET) data suggest that the nanohaloarchaeon exports its methyltransferase to methylate the Haloferax genome, unveiling a new aspect of the interaction between the symbiont and its host.

العلاقة: info:eu-repo/semantics/altIdentifier/pmid/38589217; info:eu-repo/grantAgreement//101000327/EU/Technologies of the Future for Low-Cost Enzymes for Environment-Friendly Products/FUTURENZYMES; pasteur-04539987; https://pasteur.hal.science/pasteur-04539987Test; https://pasteur.hal.science/pasteur-04539987/documentTest; https://pasteur.hal.science/pasteur-04539987/file/Reva2024EnvironMicrobiolRep.pdfTest; PUBMED: 38589217

الإتاحة: https://doi.org/10.1111/1758-2229.13258Test
https://pasteur.hal.science/pasteur-04539987Test
https://pasteur.hal.science/pasteur-04539987/documentTest
https://pasteur.hal.science/pasteur-04539987/file/Reva2024EnvironMicrobiolRep.pdfTest

المؤلفون: Ye, Xi, Paul, Bindusmita, Mo, Joyce, Reynolds, Eric C., Ghosal, Debnath, Veith, Paul D.

المساهمون: Australia-India Strategic Research Fund, Human Frontier Science Program, National Health and Medical Research Council, Australian Research Council

المصدر: MicrobiologyOpen ; volume 13, issue 2 ; ISSN 2045-8827 2045-8827

الوصف: Prevotella intermedia , a Gram‐negative bacterium from the Bacteroidota phylum, is associated with periodontitis. Other species within this phylum are known to possess the general O ‐glycosylation system. The O ‐glycoproteome has been characterized in several species, including Tannerella forsythia , Porphyromonas gingivalis , and Flavobacterium johnsoniae . In our study, we used electron cryotomography (cryoET) and glycoproteomics to reveal the ultrastructure of P. intermedia and characterize its O ‐glycoproteome. Our cryoET analysis unveiled the ultrastructural details of the cell envelope and outer membrane vesicles (OMVs) of P. intermedia . We observed an electron‐dense surface layer surrounding both cells and OMVs. The OMVs were often large (>200 nm) and presented two types, with lumens being either electron‐dense or translucent. LC‐MS/MS analyses of P. intermedia fractions led to the identification of 1655 proteins, which included 62 predicted T9SS cargo proteins. Within the glycoproteome, we identified 443 unique O ‐glycosylation sites within 224 glycoproteins. Interestingly, the O ‐glycosylation motif exhibited a broader range than reported in other species, with O ‐glycosylation found at D(S/T)(A/I/L/M/T/V/S/C/G/F/N/E/Q/D/P). We identified a single O ‐glycan with a delta mass of 1531.48 Da. Its sequence was determined by MS2 and MS3 analyses using both collision‐induced dissociation and high‐energy collisional dissociation fragmentation modes. After partial deglycosylation with trifluoromethanesulfonic acid, the O ‐glycan sequence was confirmed to be dHex‐dHex‐HexNAc (HPO 3 ‐C 6 H 12 O 5 )‐dHex‐Hex‐HexA‐Hex(dHex). Bioinformatic analyses predicted the localization of O ‐glycoproteins, with 73 periplasmic proteins, 53 inner membrane proteins, 52 lipoproteins, 26 outer membrane proteins, and 14 proteins secreted by the T9SS.

الإتاحة: https://doi.org/10.1002/mbo3.1401Test

المؤلفون: Puls, Jan-Samuel, Winnerling, Benjamin, Power, Jeffrey J, Krüger, Annika M, Brajtenbach, Dominik, Johnson, Matthew, Bilici, Kevser, Camus, Laura, Fließwasser, Thomas, Schneider, Tanja, Sahl, Hans-Georg, Ghosal, Debnath, Kubitscheck, Ulrich, Heilbronner, Simon, Grein, Fabian

المصدر: The ISME Journal ; volume 18, issue 1 ; ISSN 1751-7362 1751-7370

الوصف: Many bacteria produce antimicrobial compounds such as lantibiotics to gain advantage in the competitive natural environments of microbiomes. Epilancins constitute an until now underexplored family of lantibiotics with an unknown ecological role and unresolved mode of action. We discovered production of an epilancin in the nasal isolate Staphylococcus epidermidis A37. Using bioinformatic tools, we found that epilancins are frequently encoded within staphylococcal genomes, highlighting their ecological relevance. We demonstrate that production of epilancin A37 contributes to Staphylococcus epidermidis competition specifically against natural corynebacterial competitors. Combining microbiological approaches with quantitative in vivo and in vitro fluorescence microscopy and cryo-electron tomography, we show that A37 enters the corynebacterial cytoplasm through a partially transmembrane-potential-driven uptake without impairing the cell membrane function. Upon intracellular aggregation, A37 induces the formation of intracellular membrane vesicles, which are heavily loaded with the compound and are essential for the antibacterial activity of the epilancin. Our work sheds light on the ecological role of epilancins for staphylococci mediated by a mode of action previously unknown for lantibiotics.

الإتاحة: https://doi.org/10.1093/ismejo/wrae044Test
https://academic.oup.com/ismej/article-pdf/18/1/wrae044/57139102/wrae044.pdfTest

المؤلفون: Pakharukova, Natalia, Malmi, Henri, Tuittila, Minna, Dahlberg, Tobias, 1990, Ghosal, Debnath, Chang, Yi-Wei, Myint, Si Lhyam, Paavilainen, Sari, Knight, Stefan David, Lamminmäki, Urpo, Uhlin, Bernt Eric, Andersson, Magnus, Jensen, Grant, Zavialov, Anton V.

المصدر: Nature. 609(7926):335-340

مصطلحات موضوعية: mikrobiologi, Microbiology

الوصف: Adhesive pili assembled via the chaperone-usher pathway (CUP) are hair-like appendages that mediate host tissue colonization and biofilm formation of Gram-negative bacteria 1-3. Archaic CUP pili, the most diverse and widespread CUP adhesins, are promising vaccine and drug targets due to their prevalence in the most troublesome multidrug-resistant (MDR) pathogens 1,4,5. However, their architecture and assembly-secretion process remain unknown. Here, we present the 3.4 Å resolution cryo-electron microscopy structure of the prototypical archaic Csu pilus that mediates biofilm formation of Acinetobacter baumannii, a notorious MDR nosocomial pathogen. In contrast to the thick helical tubes of the classical type 1 and P pili, archaic pili assemble into a conceptually novel ultrathin zigzag architecture secured by an elegant clinch mechanism. The molecular clinch provides the pilus with high mechanical stability as well as superelasticity, a property observed now for the first time in biomolecules, while enabling a more economical and faster pilus production. Furthermore, we demonstrate that clinch formation at the cell surface drives pilus secretion through the outer membrane. These findings suggest that clinch-formation inhibitors might represent a new strategy to fight MDR bacterial infections.

وصف الملف: electronic

الوصول الحر: https://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-198528Test
https://doi.org/10.1038/s41586-022-05095-0Test
https://umu.diva-portal.org/smash/get/diva2:1686020/FULLTEXT01.pdfTest

المؤلفون: Kreida, Stefan, Narita, Akihiro, Johnson, Matthew D., Tocheva, Elitza I., Das, Anath, Ghosal, Debnath, Jensen, Grant J.

المصدر: Structure, 31(4), 385-394, (2023-04-06)

مصطلحات موضوعية: Molecular Biology, Structural Biology

الوصف: Agrobacterium tumefaciens causes crown gall disease in plants by the horizontal transfer of oncogenic DNA. The conjugation is mediated by the VirB/D4 type 4 secretion system (T4SS) that assembles an extracellular filament, the T-pilus, and is involved in mating pair formation between A. tumefaciens and the recipient plant cell. Here, we present a 3 Ã… cryoelectron microscopy (cryo-EM) structure of the T-pilus solved by helical reconstruction. Our structure reveals that the T-pilus is a stoichiometric assembly of the VirB2 major pilin and phosphatidylglycerol (PG) phospholipid with 5-start helical symmetry. We show that PG head groups and the positively charged Arg 91 residues of VirB2 protomers form extensive electrostatic interactions in the lumen of the T-pilus. Mutagenesis of Arg 91 abolished pilus formation. While our T-pilus structure is architecturally similar to previously published conjugative pili structures, the T-pilus lumen is narrower and positively charged, raising questions of whether the T-pilus is a conduit for ssDNA transfer. ; © 2023 Elsevier Ltd. We thank Dr. Songye Chen, Caltech cryo-EM facility, for assistance during data collection; Dr. Tsui-Fen Chou, Dr. Brett Lomenick, and Dr. Jeff Jones from the Caltech Proteome Exploration Laboratory for conducting the protein mass spectrometry analysis. We also thank Dr. Kevin Williams and UCLA Lipidomics for performing lipid extraction and lipid data collection and giving valuable advice in lipidomic experimental design and data interpretation. This project was funded by a National Institutes of Health grant (R01 AI127401 to G.J.J.), a National Health and Medical Research Council grant (APP1196924 to D.G.), and a Natural Sciences and Engineering Research Council of Canada Discovery grant (RGPIN 04345 to E.I.T.). S.K. is supported by the Swedish Research Council (2019-06293). Author contributions: Conceptualization, G.J.J. and D.G.; methodology, S.K., A.N., A.D., E.I.T., M.D.J., and D.G.; investigation, S.K., A.N., AD., M.D.J., and D.G.; formal ...

العلاقة: https://resolver.caltech.edu/CaltechAUTHORS:20230322-366935000.8Test; oai:authors.library.caltech.edu:fw66h-vj713; eprintid:120363; resolverid:CaltechAUTHORS:20230323-670848000.1; https://www.ncbi.nlm.nih.gov/pmc/PMC10168017Test; https://doi.org/10.1016/j.str.2023.02.005Test

الإتاحة: https://doi.org/10.1016/j.str.2023.02.005Test
https://www.ncbi.nlm.nih.gov/pmc/PMC10168017Test

المؤلفون: Shepherd, Doulin C., Kaplan, Mohammed, Vankadari, Naveen, Kim, Ki Woo, Larson, Charles L., Dutka, PrzemysÅ‚aw, Beare, Paul A., Krzymowski, Edward, Heinzen, Robert A., Jensen, Grant J., Ghosal, Debnath

المصدر: iScience, 26(7), 107210, (2023-07-21)

مصطلحات موضوعية: Multidisciplinary

الوصف: Coxiella burnetii is an obligate zoonotic bacterium that targets macrophages causing a disease called Q fever. It has a biphasic developmental life cycle where the extracellular and metabolically inactive small cell variant (SCV) transforms inside the host into the vegetative large cell variant (LCV). However, details about the morphological and structural changes of this transition are still lacking. Here, we used cryo-electron tomography to image both SCV and LCV variants grown either under axenic conditions or purified directly from host cells. We show that SCVs are characterized by equidistant stacks of inner membrane that presumably facilitate the transition to LCV, a transition coupled with the expression of the Dot/Icm type IVB secretion system (T4BSS). A class of T4BSS particles were associated with extracellular densities possibly involved in host infection. Also, SCVs contained spherical multilayered membrane structures of different sizes and locations suggesting no connection to sporulation as once assumed. ; © 2023 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0Test/). ; This project was funded by the National Institutes of Health (grant R01 AI127401 to G.J.J.), an NHMRC grant (APP1196924 to D.G.), and the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases (Grant number AI000931-20 to R.A.H. and C.L.L.). D.C.S. is supported by the Melbourne Research Scholarship. We are grateful to Prof. Elitza Tocheva for insightful discussions, and Somavally Dalvi for her help with figure preparation. ; Conceptualization, D.G., G.J.J., and M.K.; Methodology, D.G., M.K., D.C.S., C.L.L., P.D., P.A.B., and E.K.; Investigation, M.K., D.G., D.C.S., C.L.L., P.D., and P.A.B.; Formal Analysis, M.K., D.G., D.C.S., C.L.L., P.D., P.A.B., K.W.K., N.V., E.K., and R.A.H.; Writing– Original Draft, D.G., M.K., and N.V.; Writing– Review and Editing, M.K., D.G., D.C.S., C.L.L., P.D., ...

العلاقة: https://ars.els-cdn.com/content/image/1-s2.0-S2589004223012877-mmc1.pdfTest; https://doi.org/10.1016/j.isci.2023.107210Test; oai:authors.library.caltech.edu:w9khr-5r449; https://www.ncbi.nlm.nih.gov/pmc/PMC10362272Test

الإتاحة: https://doi.org/10.1016/j.isci.2023.107210Test
https://www.ncbi.nlm.nih.gov/pmc/PMC10362272Test

المؤلفون: Kreida, Stefan, Narita, Akihiro, Johnson, Matthew D., Tocheva, Elitza I., Das, Anath, Ghosal, Debnath, Jensen, Grant J.

الوصف: Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease by the horizontal transfer of oncogenic DNA that is integrated into the host's genome. The conjugation is mediated by the conjugative VirB/D4 type 4 secretion system (T4SS). A. tumefaciens T4SS assembles an extracellular filament, the T-pilus, that is involved in the formation of a mating pair between A. tumefaciens and the recipient plant cell by a not fully understood mechanism. Here, we present a 3 Ã… cryo-EM structure of the T-pilus, solved by helical reconstruction. Our structure reveals that the T-pilus comprises the major pilin protein VirB2 and phosphatidylglycerol (PG) phospholipid at a 1:1 stoichiometric ratio with 5-start helical symmetry. We further show that PG-headgroups and the positively charged Arg 91 residues of VirB2 protomers form extensive electrostatic interactions in the lumen of the T-pilus. Mutagenesis of Arg 91 destabilized the VirB2 protein and completely abolished pilus formation. While our T-pilus structure shows architectural similarity with previously published conjugative pili structures, positively charged sidechains protrude into the lumen and the lumen is narrower, raising questions whether the T-pilus is a conduit for ssDNA transfer. We also show that the VirB2 subunits in T-pilus filament are not cyclic, as previously thought. ; The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. We thank Dr. Songye Chen, Caltech cryo-EM facility for assistance during data collection; Dr. Tsui-Fen Chou, Dr. Brett Lomenick and Dr. Jeff Jones from Caltech Proteome Exploration Laboratory for conducting the protein mass-spec analysis. We also thank Dr. Kevin Williams and UCLA Lipidomics for performing lipid extraction, lipid data collection and giving valuable advice in lipidomic experimental design and data interpretation. This project was funded by a National Institutes of Health grant (R01 AI127401 to G.J.J), a National Health and ...

العلاقة: https://resolver.caltech.edu/CaltechAUTHORS:20230323-670848000.1Test; https://doi.org/10.1101/2022.09.25.509369Test; oai:authors.library.caltech.edu:fmx62-ehp59; eprintid:120324; resolverid:CaltechAUTHORS:20230322-366935000.8

الإتاحة: https://doi.org/10.1101/2022.09.25.509369Test

المؤلفون: Dutka, PrzemysÅ‚aw, Liu, Yuxi, Maggi, Stefano, Ghosal, Debnath, Wang, Jue, Carter, Stephen D., Zhao, Wei, Vijayrajratnam, Sukhithasri, Vogel, Joseph P., Jensen, Grant J.

الوصف: The Legionella pneumophila Dot/Icm type IV secretion system (T4SS) delivers effector proteins into host cells during infection. Despite its significance as a potential drug target, our current understanding of its atomic structure is limited to isolated subcomplexes. In this study, we used subtomogram averaging and integrative modeling to construct a nearly-complete model of the Dot/Icm T4SS accounting for seventeen protein components. We locate and provide insights into the structure and function of six new components including DotI, DotJ, DotU, IcmF, IcmT, and IcmX. We find that the cytosolic N-terminal domain of IcmF, a key protein forming a central hollow cylinder, interacts with DotU, providing insight into previously uncharacterized density. Furthermore, our model, in combination with analyses of compositional heterogeneity, explains how the cytoplasmic ATPase DotO is connected to the periplasmic complex via interactions with membrane-bound DotI/DotJ proteins. Coupled with in situ infection data, our model offers new insights into the T4SS-mediated secretion mechanism. ; The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license. Electron microscopy was performed in the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech and Pacific Northwest Center for Cryo-EM (PNCC). We thank the Liu lab for sharing data and expertise in using I3 to resolve the DotO hexamer of dimers. Y.L. was supported by a Jane Coffin Childs Memorial Fund for Medical Research post-doctoral fellowship. This work was supported by the National Institutes of Health (grant R01-AI127401 to G.J.J.). AUTHOR CONTRIBUTIONS. P.D. prepared samples, reconstructed tomograms and picked particles for a subset of data, performed subtomogram averaging, performed data exploration, located proteins forming the system, participated in building the model, drafted the manuscript, and prepared ...

العلاقة: https://doi.org/10.1101/2023.03.22.533729Test; oai:authors.library.caltech.edu:47yv1-y5250; eprintid:120378; resolverid:CaltechAUTHORS:20230324-517798000.1

الإتاحة: https://doi.org/10.1101/2023.03.22.533729Test

المؤلفون: Kaplan, Mohammed, Shepherd, Doulin C., Vankadari, Naveen, Kim, Ki Woo, Larson, Charles L., Dutka, PrzemysÅ‚aw, Beare, Paul A., Krzymowski, Edward, Heinzen, Robert A., Jensen, Grant J., Ghosal, Debnath

الوصف: Coxiella burnetii is an obligate zoonotic bacterium that targets macrophages to cause a disease known as Q fever. It has a biphasic developmental lifecycle where the extracellular and metabolically inactive small cell variant (SCV) transforms, under host acidic environment, into the vegetative large cell variant (LCV). However, the details about the morphological and structural changes that accompany this biphasic cycle are still lacking. Here, we used cryo-electron tomography to image the different cell variants of C. burnetii grown either under axenic conditions in different pH or purified directly from host cells revealing the major developmental, morphological and structural transitions. We show that SCVs are characterized by equidistant stacks of inner membrane that presumably allow a smooth transition to LCV, a transition coupled with the expression of the Dot/Icm type IVB secretion system (T4BSS). A class of T4BSS particles were associated with extracellular densities including a tubular structure possibly involved in host interaction or effector delivery. Also, SCVs and cells in the transition state contained spherical multilayered membrane structures of different sizes and locations suggesting that they are not related to a sporulation process as once assumed. ; The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. This project was funded by the National Institutes of Health (grant R01 AI127401 to G.J.J), an NHMRC grant (APP1196924 to DG), and the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases (Grant number AI000931-20 to R.A.H. and C.L.L.). DCS is supported by the Melbourne Research Scholarship. We are grateful to Prof. Hayley Newton for critically reading the manuscript, Prof. Elitza Tocheva for insightful discussions, and Somavally Dalvi for her help with figure preparation. The authors have declared no competing interest. ; Submitted - ...

العلاقة: https://doi.org/10.1101/2022.08.23.505044Test; oai:authors.library.caltech.edu:18mys-mvh12; eprintid:120354; resolverid:CaltechAUTHORS:20230322-368470000.41

الإتاحة: https://doi.org/10.1101/2022.08.23.505044Test

المؤلفون: Ghosal, Debnath

مصطلحات موضوعية: 610

الإتاحة: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608006Test