Research Projects

Molecular Machines in Action
A fundamental property of many biological processes is that they are performed by highly organized, multicomponent macromolecular assemblies, often referred to as molecular machines. The Marlovits group studies the structural basis for assembly, regulation, and function of transmembrane molecular machines. We use a multidisciplinary approach, by combining molecular biology, genetic, cellular, biochemical, and a wide-range of structural (EM, X-ray, NMR, X-linking/mass spectrometry) tools. We are developing novel imaging and modeling technologies to visualize dynamic molecular processes in unprecedented detail in situ and in action.

Microbial Pathogenesis
Gram-negative pathogens such as Yersinia, Shigella, Pseudomonas, enteropathogenic/enterohemorrhagic E. coli (EPEC/EHEC) and Salmonella are the causative agents for many diseases known to both animals and humans. These pathogens often originate as foodborne diseases and result in outcomes ranging from a mild stomach ache to death. Central to the pathogenicity are bacterial toxins ('effectors'), which are delivered via the type III secretion system, a large membrane embedded machinery, from the bacterium to its host cell. Once delivered, these translocated effector proteins have the remarkable capacity to modulate various host-cell pathways that induce membrane ruffling and subsequently make the host accessible to bacterial infection.

Type III secretion system: Unfolded protein transport across membranes
Our recent structural analysis (Schraidt & Marlovits, Science 2010) of the injectisome, the most prominent, cylindrical structure of the type III secretion system, revealed a potential secretion path through the central part of the membrane embedded complex. However, the inner diameter of this path is too small to accommodate a fully folded effector protein, suggesting that either the injectisome must undergo large conformational changes during transport or the effector proteins need to be unfolded.

To investigate the type III secretion of human pathogens, we focused (1) on determining the secretion path of injectisomes, (2) on understanding the mechanism of transport, and (3) on visualizing protein transport in situ. We discovered that substrates are inserted into the secretion path in a polar fashion - N-terminal regions first – and that they are transported in an unfolded state. To understand, whether such a behaviour is in fact observed in situ, we analszed protein transport across membranes in a near-native state using cryo electron tomography (Radics et al 2014). For the first time, we were able to visualize pathogenic type III secretion systems in action.

Technology Development - Atomic structure determination from lower resolution cryo-EM maps
Direct electron detectors are key to the recent revolution in structural biology and have made it possible to generate electron density maps at near atomic resolution using cryo electron microscopy from non-crystalline sample material. However, building accurate models into these 3-5Å maps remains a challenge. We recently developed a new modeling approach that integrates Monte Carlo optimization with local density guided moves, Rosetta all-atom refinement, and real space B-factor fitting, thus yielding accurate models from experimental maps for three different systems with resolutions as low as 4.5Å (DiMaio et al Nature Methods 2015). Based on increasing need within the scientific community, we expanded this work by developing easy-to -use modeling tools which build accurate models at the highest possible resolution from single particle electron microscopy maps.

FUTURE PROJECTS AND GOALS

By understanding the molecular mechanism of TTSS-mediated protein transport at the highest possible resolution, we hope to provide a basis for the development of novel therapeutic strategies that will either inhibit its activity or modify the system for targeted drug delivery.

Header Image: Copyright HZI

Research Team

7

Group Leader

Prof. Dr. Thomas Marlovits
Phone:+49 40 8998 87650

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Technical Assistant

Barbara Grüter
Phone:+49 40 8998 87657

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Postdoctoral Scientist

Rory Hennell James
Phone:+49 40 8998 87657

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PhD Student

Maurice Pantel
Phone:+49 40 8998 87657

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Postdoctoral Scientist

Dr. Oliver Vesper
Phone:+49 40 8998 87653

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Postdoctoral Scientist

Jirka Wald
Phone:+49 40 8998 87655

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Postdoctoral Scientist

Dr. Biao Yuan
Phone:+49 40 8998 87657

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Publications

2023

Seyfert CE, Müller AV, Walsh DJ, Birkelbach J, Kany AM, Porten C, Yuan B, Krug D, Herrmann J, Marlovits TC, Hirsch AKH, Müller R (2023) New Genetically Engineered Derivatives of Antibacterial Darobactins Underpin Their Potential for Antibiotic Development. J Med Chem. 66(23):16330-16341. doi: 10.1021/acs.jmedchem.3c01660.

Yuan B, Scholz J, Wald J, Thuenauer R, Hennell James R, Ellenberg I, Windhorst S, Faix J, Marlovits TC (2023) Structural basis for subversion of host cell actin cytoskeleton during Salmonella infection.  Sci Adv. 9(49):eadj5777. doi: 10.1126/sciadv.adj5777.

Lugmayr W, Kotov V, Goessweiner-Mohr N, Wald J, DiMaio F, Marlovits TC (2023) StarMap: a user-friendly workflow for Rosetta-driven molecular structure refinement. Nat Protoc. 18(1):239-264. doi: 10.1038/s41596-022-00757-9.

Seyfert CE, Porten C, Yuan B, Deckarm S, Panter F, Bader CD, Coetzee J, Deschner F, Tehrani KHME, Higgins PG, Seifert H, Marlovits TC, Herrmann J, Müller R (2023) Darobactins Exhibiting Superior Antibiotic Activity by Cryo-EM Structure Guided Biosynthetic Engineering. Angew Chem Int Ed Engl. 62(2):e202214094. doi: 10.1002/anie.202214094.

Bunduc CM, Ding Y, Kuijl C, Marlovits TC, Bitter W, Houben (2023) Reconstitution of a minimal ESX-5 type VII secretion system suggests a role for PPE proteins in the outer membrane transport of proteins. ENG. mSphere. 8(5):e0040223. doi: 10.1128/msphere.00402-23.

Simonis A, Kreer C, Albus A, Rox K, Yuan B, Holzmann D, Wilms JA, Zuber S, Kottege L, Winter S, Meyer M, Schmitt K, Gruell H, Theobald SJ, Hellmann AM, Meyer C, Ercanoglu MS, Cramer N, Munder A, Hallek M, Fätkenheuer G, Koch M, Seifert H, Rietschel E, Marlovits TC, van Koningsbruggen-Rietschel S, Klein F, Rybniker J (2023) Discovery of highly neutralizing human antibodies targeting Pseudomonas aeruginosa. Cell. S0092-8674(23)01084-X. doi: 10.1016/j.cell.2023.10.002.

Wald J, Marlovits TC (2023) Holliday junction branch migration driven by AAA+ ATPase motors. Curr Opin Struct Biol. 82:102650. doi: 10.1016/j.sbi.2023.102650.

Genthe E, Miletic S, Tekkali I, Hennell James R, Marlovits TC, Heuser P(2023) PickYOLO: Fast deep learning particle detector for annotation of cryo electron tomograms. J Struct Biol. 107990. doi: 10.1016/j.jsb.2023.107990.

Reggiano G, Lugmayr W, Farrell D, Marlovits TC, DiMaio F (2023) Residue-level error detection in cryoelectron microscopy models. Structure. S0969-2126(23)00158-2. doi: 10.1016/j.str.2023.05.002

2022

Lugmayr W, Kotov V, Goessweiner-Mohr N, Wald J, DiMaio F, Marlovits TC (2022) StarMap: a user-friendly workflow for Rosetta-driven molecular structure refinement. Nat Protoc. doi: 10.1038/s41596-022-00757-9.

Seyfert CE, Porten C, Yuan B, Deckarm S, Panter F, Bader C, Coetzee J, Deschner F, Tehrani K, Higgins PG, Seifert H, Marlovits T, Herrmann J, Müller R (2022) Darobactins Exhibiting Superior Antibiotic Activity by Cryo-EM Structure Guided Biosynthetic Engineering. Angew Chem Int Ed Engl. doi: 10.1002/anie.202214094.

Wald J, Fahrenkamp D, Goessweiner-Mohr N, Lugmayr W, Ciccarelli L, Vesper O, Marlovits TC (2022) Mechanism of AAA+ ATPase-mediated RuvAB-Holliday junction branch migration. Nature. doi: 10.1038/s41586-022-05121-1

Bergeron JRC, Marlovits TC (2022) Cryo-EM of the injectisome and type III secretion systems. Curr Opin Struct Biol. 75:102403. doi: 10.1016/j.sbi.2022.102403

2021

Killer M, Wald J, Pieprzyk J, Marlovits TC, Löw C (2021) PepT1 and PepT2 reveal mechanistic insights into substrate and drug transport across epithelial membranes. Sci. Adv. 7, eabk3259 doi: 10.1126/sciadv.abk3259

Yuan B, Portaliou AG, Parakra R, Smit JH, Wald J, Li Y, Srinivasu B, Loos MS, Singh Dhupar H, Fahrenkamp D, Kalodimos CG, Duong van Hoa F, Cordes T, Karamanou S, Marlovits TC, Economou A (2021) Structural dynamics of the functional nonameric Type III translocase export gate. J. Mol. Biol. 167188, https://doi.org/10.1016/j.jmb.2021.167188.

Banger A, Sindram J, Otten M, Kania J, Wilms D, Strzelczyk A, Miletic S, Marlovits TC, Karg M, Hartmann L (2021) Synthesis and self-assembly of amphiphilic precision glycomacromolecules. Polym. Chem., 2021,12, 4795-4802 https://doi.org/10.1039/D1PY00422K

Kotov V, Lunelli M, Wald J, Kolbe M, Marlovits TC (2021) Helical reconstruction of Salmonella and Shigella needle filaments attached to type 3 basal bodies. Biochemistry and Biophysics Reports 27:101039. https://doi.org/10.1016/j.bbrep.2021.101039

Bunduc CM, Fahrenkamp D, Wald J, Ummels R, Bitter W, Houben ENG, Marlovits TC (2021) Structure and dynamics of a mycobacterial type VII secretion system Nature. doi: 10.1038/s41586-021-03517-z.

Miletic S, Fahrenkamp D, Goessweiner-Mohr N, Wald J, Pantel M, Vesper O, Kotov V, Marlovits TC (2021) Substrate-engaged type III secretion system structures reveal gating mechanism for unfolded protein translocation. Nature Communications 12(1):1546. doi:10.1038/s41467-021-21143-1.

Kotov V, Mlynek G, Vesper O, Pletzer M, Wald J, Teixeira-Duarte CM, Celia H, Garcia-Alai M, Nussberger S, Buchanan SK, Morais-Cabral JH, Loew C, Djinovic-Carugo K, Marlovits TC (2021) In-depth interrogation of protein thermal unfolding data with MoltenProt. Protein Sci. 30(1):201-217. doi: 10.1002/pro.3986. PMID: 33140490

2020

Pinotsis N, Zielinska K, Babuta M, Arolas JL, Kostan J, Khan MB, Schreiner C, Salmazo A, Ciccarelli L, Puchinger M, Gkougkoulia EA, Ribeiro EA Jr, Marlovits TC, Bhattacharya A, Djinovic-Carugo K (2020) Calcium modulates the domain flexibility and function of an α-actinin similar to the ancestral α-actinin. Proc Natl Acad Sci U S A. 117(36):22101-22112. doi: 10.1073/pnas.1917269117.

Chabloz A, Schaefer J V, Kozieradzki I, Cronin S J F, Strebinger D, Macaluso F, Wald J, Rabbitts T H, Plückthun A, Marlovits TC, Penninger J M (2020). Salmonella-based platform for efficient delivery of functional binding proteins to the cytosol. Commun Biol 3, 342. doi.org/10.1038/s42003-020-1072-4

2019

Kotov V, Vesper O, Alai M G, Loew C, Marlovits TC (2019) Moltenprot: A High-Throughput Analysis Platform to Assess Thermodynamic Stability of Membrane Proteins and Complexes. Biophysical Journal 116 (3), 191a. https://doi.org/10.1016/j.bpj.2018.11.1060

Miletic S, Goessweiner-Mohr N, Marlovits TC (2019) The Structure of the Type III Secretion System Needle Complex. Current Topics in Microbiology and Immunology. Springer, Berlin, Heidelberg. doi.org/10.1007/82_2019_178

Wald J, Pasin M, Richter M, Walther CH, Mathai N, Kirchmair J, Makarov VA, Gössweiner-Mohr N, Marlovits TC, Zanella I, Real-Hohn A, Verdaguer N, Blaas D, Schmidtke M. Cryo-EM Structure of Pleconaril-Resistant Rhinovirus-B5 Complexed to the Antiviral OBR-5-340 Reveals Unexpected Binding Site. PNAS 2019 Sept 17;116(38):19109–19115. doi: 10.1073/pnas.1904732116

Kotov V, Bartels K, Veith K, Josts I, Subhramanyam UKT, Günther C, Labahn J, Marlovits TC, Moraes I, Tidow H, Löw C, Garcia-Alai MM (2019) High-throughput stability screening for detergent-solubilized membrane proteins. Sci Rep. 2019 Jul 17;9(1):10379. doi: 10.1038/s41598-019-46686-8.

Guo EZ, Desrosiers DC, Zalesak J, Tolchard J, Berbon M, Habenstein B, Marlovits T, Loquet A, Galán JE (2019) A polymorphic helix of a Salmonella needle protein relays signals defining distinct steps in type III secretion. PLoS Biol. 2019 Jul 1;17(7):e3000351. doi: 10.1371/journal.pbio.3000351

2017

Beckham KSH, Ciccarelli L., Bunduc CM, Mertens HDT, Ummels R, Lugmayr L, Mayr J, Rettel M, Savitski MM, Svergun DI, Bitter W, Wilmanns M, Marlovits TC*, Parret AHA*, Houben ENG* (2017). Structure of the mycobacterial ESX-5 type VII secretion system membrane complex by single-particle analysis. Nature Microbiology 10;2:17047. doi: 10.1038/nmicrobiol.2017.47.

Song M , Sukovich DJ, Ciccarelli L, Mayr J, Fernandez-Rodriguez J, Mirsky EA, Tucker AC, Gordon DB, Marlovits TC, Voigt CA (2017) Control of type III protein secretion using a minimal genetic system. Nature communications 9;8:14737. doi: 10.1038/ncomms14737.

2016

Dietsche T, Tesfazgi Mebrhatu M, Brunner MJ, Abrusci P, Franz-Wachtel M, Schärfe C, Zilkenat S, Grin I, Galán J E, Kohlbacher O, Lea S, Macek B, Marlovits TC, Robinson CV, Wagner S (2016). Structural and Functional Characterization of the Bacterial Type III Secretion Export Apparatus. PLOS PATHOG. 12, 12, e1006071 doi: 10.1371/journal.ppat.1006071

Smaldone G, Pirone L, Pedone E, Marlovits T, Vitagliano L, Ciccarelli L. (2016) The BTB domains of the potassium channel tetramerization domain proteins prevalently assume pentameric states. FEBS Lett. 590:1663-71. doi: 10.1002/1873-3468.12203.

2015

DiMaio F, Song Y, Li X, Brunner MJ,  Xu C, Conticello V, Egelman E, Marlovits TC, Cheng Y, Baker D* (2015) Atomic accuracy models from 4.5 Å cryo-electron microscopy data with density-guided iterative local rebuilding and refinement. Nature Methods 12:361-5. doi: 10.1038/nmeth.3286.

2014

Galan JE*,  Lara-Tejero, M, Marlovits TC, Wagner S. (2014) Bacterial type III secretion systems: specialized nanomachines for protein delivery into target cells. Ann Rev Microbiology 68:415-38. doi: 10.1146/annurev-micro-092412-155725

Hornung P, Troc P, Malvezzi F, Maier M, Demianova Z, Zimniak T, Litos G, Lampert F, Schleiffer A, Brunner M, Mechtler K, Herzog F, Marlovits TC, Westermann S.* (2014) A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A.J Cell Biol 206:509-24. doi: 10.1083/jcb.201403081

Radics J, Königsmaier L, Marlovits TC (2014) Structure of a pathogenic type 3 secretion system in action. Nature Structural & Molecular Biology 21:82-7. doi:10.1038/nsmb.2722

   - Highlighted as Cover in NSMB

Brunner MJ, Fronzes R.*, Marlovits TC* (2014). Envelope spanning secretion systems in Gram-negative bacteria. In: Bacterial Membranes: Structural and Molecular Biology Fronzes R. (eds)

2013

Simunovic M, Mim C, Marlovits TC, Resch G, Unger VM, Voth* GA (2013) Protein-mediated transformation of lipid vesicles into tubular networks. Biophys J 105(3):711-9 - Highlighted as Cover in Biophys J

2012

Fernandez-Rodriguez J, Marlovits TC* (2012) Induced heterdimerization and purification of two target proteins by a synthetic coiled-coil tag Protein Science 21(4):511-9     -  Highlighted, Protein Science, 21, (2012)

Kosarewicz A, Königsmaier L, Marlovits TC* (2012) The blueprint of the Type-3 Injectisome. Phil Trans Royal Society  B 367:1140-54

2011

Schraidt O, Marlovits TC* (2011) Three-dimensional model of Salmonella’s Needle Complex at Subnanometer Resolution. Science 331:1192-95

   - Perspectives, Science, 331:1147-1148

   - This week in Science: Science 331:1109

Khan AG, Pickl-Herk A, Gajdzik L, Marlovits TC, Fuchs R, Blaas D* (2011) Entry of a heparan sulphate-binding HRV8 variant strictly depends on dynamin but not on clathrin, caveolin, and flotillin. Virology  30;412(1):55-67

2010

Galkin VE, Schmied WH, Schraidt O, Marlovits TC*, Egelman EH* (2010) The structure of the Salmonella typhimurium type III secretion system needle shows divergence from the flagellar system. J Mol Biol. 396(5):1392-7

Khan AG, Pickl-Herk A, Gajdzik L, Marlovits TC, Fuchs R, Blaas D* (2010) Human rhinovirus 14 enters rhabdomyosarcoma cells expressing icam-1 by a clathrin-, caveolin-, and flotillin-independent pathway. J Virol. 84(8):3984-92

Marlovits TC*, Stebbins CE* (2010) Type III secretion systems shape up as they ship out. Curr Opin Microbiol. 13(1):47-52

Schraidt O, Lefebre MD, Brunner MJ, Schmied WH, Schmidt A, Radics J, Mechtler K, Galán JE, Marlovits TC* (2010) Topology and organization of the Salmonella typhimurium type III secretion needle complex components. PloS Pathog. 6(4):e1000824

Wagner S, Königsmaier L, Lara-Tejero M, Lefebre M, Marlovits TC*, Galán JE* (2010) Organization and coordinated assembly of the type III secretion export apparatus. Proc Natl Acad Sci U S A. 107(41):17745-50

2006

Marlovits TC, Kubori T, Lara-Tejero M, Thomas D, Unger VM, Galán JE* (2006) Assembly of the inner rod determines needle length in the type III secretion injectisome. Nature. 441(7093):637-40

2004

Marlovits TC, Kubori T, Sukhan A, Thomas DR, Galán JE, Unger VM* (2004) Structural insights into the assembly of the type III secretion needle complex. Science. 306(5698):1040-2 - This week in Science: Science 306:937)

2002

Marlovits TC, Haase W, Herrmann C, Aller SG, Unger VM* (2002) The membrane protein FeoB contains an intramolecular G protein essential for Fe(II) uptake in bacteria. Proc Natl Acad Sci U S A. 99(25):16243-8

   - Editor’s choice in Science Signalling Sci. Is FeoB the Missing Link in GPCR Evolution?

2000

Ronacher B, Marlovits TC, Moser R, Blaas D* (2000) Expression and folding of human very-low-density lipoprotein receptor fragments: neutralization capacity toward human rhinovirus HRV2. Virology. 278(2):541-50

Hewat EA, Neumann E, Conway JF, Moser R, Ronacher B, Marlovits TC, Blaas D* (2000) The cellular receptor to human rhinovirus 2 binds around the 5-fold axis and not in the canyon: a structural view. EMBO J. 19(23):6317-25

1999

Verdaguer N, Marlovits TC, Bravo J, Stuart DI, Blaas D, Fita I* (1999) Crystallization and preliminary X-ray analysis of human rhinovirus serotype 2 (HRV2). Acta Crystallogr D Biol Crystallogr. 55(Pt 8):1459-61

1998

Hewat EA, Marlovits TC, Blaas D* (1998) Structure of a neutralizing antibody bound monovalently to human rhinovirus 2. J Virol. 72(5):4396-402

Marlovits TC, Abrahamsberg C, Blaas D* (1998) Very-low-density lipoprotein receptor fragment shed from HeLa cells inhibits human rhinovirus infection. J Virol. 72(12):10246-50

Marlovits TC, Abrahamsberg C, Blaas D* (1998) Soluble LDL minireceptors. Minimal structure requirements for recognition of minor group human rhinovirus. J Biol Chem. 273(50):33835-40

Marlovits TC, Zechmeister T, Gruenberger M, Ronacher B, Schwihla H, Blaas D* (1998) Recombinant soluble low density lipoprotein receptor fragment inhibits minor group rhinovirus infection in vitro. FASEB J. 12(9):695-703

Marlovits TC, Zechmeister T, Schwihla H, Ronacher B, Blaas D* (1998) Recombinant soluble low-density lipoprotein receptor fragment inhibits common cold infection. J Mol Recognit. 11(1-6):49-51

1995

Ayasse M*, Marlovits T, Tengö J, Taghizadeh T, Francke W (1995) Are there pheromonal dominance signals in the bumblebee Bombus hypnorum L. (Hymenoptera, Apidae). Apidologie. 26:163-180