Publications (17)163.36 Total impact
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Article: Atomic model of the type III secretion system needle.
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ABSTRACT: Pathogenic bacteria using a type III secretion system (T3SS) to manipulate host cells cause many different infections including Shigella dysentery, typhoid fever, enterohaemorrhagic colitis and bubonic plague. An essential part of the T3SS is a hollow needle-like protein filament through which effector proteins are injected into eukaryotic host cells. Currently, the three-dimensional structure of the needle is unknown because it is not amenable to X-ray crystallography and solution NMR, as a result of its inherent non-crystallinity and insolubility. Cryo-electron microscopy combined with crystal or solution NMR subunit structures has recently provided a powerful hybrid approach for studying supramolecular assemblies, resulting in low-resolution and medium-resolution models. However, such approaches cannot deliver atomic details, especially of the crucial subunit-subunit interfaces, because of the limited cryo-electron microscopic resolution obtained in these studies. Here we report an alternative approach combining recombinant wild-type needle production, solid-state NMR, electron microscopy and Rosetta modelling to reveal the supramolecular interfaces and ultimately the complete atomic structure of the Salmonella typhimurium T3SS needle. We show that the 80-residue subunits form a right-handed helical assembly with roughly 11 subunits per two turns, similar to that of the flagellar filament of S. typhimurium. In contrast to established models of the needle in which the amino terminus of the protein subunit was assumed to be α-helical and positioned inside the needle, our model reveals an extended amino-terminal domain that is positioned on the surface of the needle, while the highly conserved carboxy terminus points towards the lumen.Nature 06/2012; 486(7402):276-9. · 36.28 Impact Factor -
Article: Sam50 functions in mitochondrial intermembrane space bridging and biogenesis of respiratory complexes.
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ABSTRACT: Mitochondria possess an outer membrane (OMM) and an inner membrane (IMM), which folds into invaginations called cristae. Lipid composition, membrane potential, and proteins in the IMM influence organization of cristae. Here we show an essential role of the OMM protein Sam50 in the maintenance of the structure of cristae. Sam50 is a part of the sorting and assembly machinery (SAM) necessary for the assembly of β-barrel proteins in the OMM. We provide evidence that the SAM components exist in a large protein complex together with the IMM proteins mitofilin and CHCHD3, which we term the mitochondrial intermembrane space bridging (MIB) complex. Interactions between OMM and IMM components of the MIB complex are crucial for the preservation of cristae. After destabilization of the MIB complex, we observed deficiency in the assembly of respiratory chain complexes. Long-term depletion of Sam50 influences the amounts of proteins from all large respiratory complexes that contain mitochondrially encoded subunits, pointing to a connection between the structural integrity of cristae, assembly of respiratory complexes, and/or the maintenance of mitochondrial DNA (mtDNA).Molecular and cellular biology 03/2012; 32(6):1173-88. · 6.06 Impact Factor -
Article: Automatic quantification of in vitro NET formation.
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ABSTRACT: Neutrophil Extracellular Traps (NETs) consist of decondensed chromatin studded with granular and some cytoplasmic proteins. They are formed by activated neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) as the result of an active cell death program, named NETosis. NETosis can be induced by a wide range of stimuli including coculture of neutrophils with pathogens (bacteria, fungi, parasites, virus particles), activated platelets, or pathogen components. The first step of the NETotic cascade is stimulation of one or several receptors followed by activation of the Raf/MEK/ERK pathway that culminates in the assembly of the multimeric NADPH oxidase complex and the production of reactive oxygen species (ROS). Later, intracellular membranes disintegrate, the granular protein Neutrophil Elastase enters the nucleus and processes core histones that also get hypercitrullinated. This leads to decondensation and mobilization of chromatin. The amount of NET formation varies with the degree of stimulation, and this is dependent on the type and concentration of the stimulus. NETs can be quantified using various methods including fluorescence microscopy or measuring DNA release. Each of these methods have specific drawbacks: analysis of fluorescence microscopy is prone to subjective variations, and DNA release does not differentiate between DNA that has been released by NETosis or by other forms of cell death. Here we present a protocol to semi-automatically quantify NET formation. It relies on the observation that anti-chromatin antibodies bind more readily to decondensed chromatin present in the nuclei of cells undergoing NETosis and in the NETs. Relating the fluorescence signals of the anti-chromatin antibody to the signals of a DNA-binding dye allows the automatic calculation of the percentage of netting neutrophils. This method does not require sophisticated microscopic equipment, and the images are quantified with a public-domain software package.Frontiers in immunology. 01/2012; 3:413. -
Article: Critical role for heat shock protein 20 (HSP20) in migration of malarial sporozoites.
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ABSTRACT: Plasmodium sporozoites, single cell eukaryotic pathogens, use their own actin/myosin-based motor machinery for life cycle progression, which includes forward locomotion, penetration of cellular barriers, and invasion of target cells. To display fast gliding motility, the parasite uses a high turnover of actin polymerization and adhesion sites. Paradoxically, only a few classic actin regulatory proteins appear to be encoded in the Plasmodium genome. Small heat shock proteins have been associated with cytoskeleton modulation in various biological processes. In this study, we identify HSP20 as a novel player in Plasmodium motility and provide molecular genetics evidence for a critical role of a small heat shock protein in cell traction and motility. We demonstrate that HSP20 ablation profoundly affects sporozoite-substrate adhesion, which translates into aberrant speed and directionality in vitro. Loss of HSP20 function impairs migration in the host, an important sporozoite trait required to find a blood vessel and reach the liver after being deposited in the skin by the mosquito. Our study also shows that fast locomotion of sporozoites is crucial during natural malaria transmission.Journal of Biological Chemistry 12/2011; 287(4):2410-22. · 4.77 Impact Factor -
Article: Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases.
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ABSTRACT: Blood neutrophils provide the first line of defense against pathogens but have also been implicated in thrombotic processes. This dual function of neutrophils could reflect an evolutionarily conserved association between blood coagulation and antimicrobial defense, although the molecular determinants and in vivo significance of this association remain unclear. Here we show that major microbicidal effectors of neutrophils, the serine proteases neutrophil elastase and cathepsin G, together with externalized nucleosomes, promote coagulation and intravascular thrombus growth in vivo. The serine proteases and extracellular nucleosomes enhance tissue factor- and factor XII-dependent coagulation in a process involving local proteolysis of the coagulation suppressor tissue factor pathway inhibitor. During systemic infection, activation of coagulation fosters compartmentalization of bacteria in liver microvessels and reduces bacterial invasion into tissue. In the absence of a pathogen challenge, neutrophil-derived serine proteases and nucleosomes can contribute to large-vessel thrombosis, the main trigger of myocardial infarction and stroke. The ability of coagulation to suppress pathogen dissemination indicates that microvessel thrombosis represents a physiological tool of host defense.Nature medicine 08/2010; 16(8):887-96. · 27.14 Impact Factor -
Article: Protein refolding is required for assembly of the type three secretion needle.
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ABSTRACT: Pathogenic Gram-negative bacteria use a type three secretion system (TTSS) to deliver virulence factors into host cells. Although the order in which proteins incorporate into the growing TTSS is well described, the underlying assembly mechanisms are still unclear. Here we show that the TTSS needle protomer refolds spontaneously to extend the needle from the distal end. We developed a functional mutant of the needle protomer from Shigella flexneri and Salmonella typhimurium to study its assembly in vitro. We show that the protomer partially refolds from alpha-helix into beta-strand conformation to form the TTSS needle. Reconstitution experiments show that needle growth does not require ATP. Thus, like the structurally related flagellar systems, the needle elongates by subunit polymerization at the distal end but requires protomer refolding. Our studies provide a starting point to understand the molecular assembly mechanisms and the structure of the TTSS at atomic level.Nature Structural & Molecular Biology 07/2010; 17(7):788-92. · 12.71 Impact Factor -
Article: Neutrophil extracellular traps: how to generate and visualize them.
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ABSTRACT: Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria, fungi and parasites. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase. Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility. We describe methods to isolate Neutrophil Granulocytes from peripheral human blood and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.Journal of Visualized Experiments 01/2010; -
Article: Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans.
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ABSTRACT: Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.PLoS Pathogens 10/2009; 5(10):e1000639. · 9.13 Impact Factor -
Article: Mouse neutrophil extracellular traps in microbial infections.
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ABSTRACT: Neutrophil extracellular traps (NETs) play an important role in innate immunity to microbial infections. NETs have been described in several species, but the molecular details of NET formation and their role in infection has not been addressed, partly because we lack optimal experimental models. Here we describe tools to investigate NET formation in neutrophils isolated from mice. Upon in vitro stimulation of wild-type mouse neutrophils with PMA, we analyzed 3 important steps in the process of NET formation: reactive oxygen species (ROS) production, NET cell death and NET release. As expected, neutrophils from NADPH oxidase-deficient mice failed to produce ROS and did not die nor release NETs upon stimulation. We found that neutrophils from several mouse strains produced NETs with different efficiency and that NET formation correlated with the amount of ROS produced. Activation with Candida albicans also resulted in ROS production and NET cell death. The hyphal form of this fungus induced NETs more effectively than the yeast form. With this work, we provide tools to study in vitro NET assembly in the mouse system.Journal of Innate Immunity 01/2009; 1(3):181-93. · 4.21 Impact Factor -
Article: Infection at the cellular level.
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ABSTRACT: Fine structural analysis of the infection process is indispensable for understanding the relation between microorganisms and host cells. This chapter focuses on standard techniques for transmission as well as scanning electron microscopy that will be of benefit even to researchers new to the field.Methods in cell biology 02/2008; 88:477-96. · 2.05 Impact Factor -
Article: Novel cell death program leads to neutrophil extracellular traps.
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ABSTRACT: Neutrophil extracellular traps (NETs) are extracellular structures composed of chromatin and granule proteins that bind and kill microorganisms. We show that upon stimulation, the nuclei of neutrophils lose their shape, and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate, allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death process is distinct from apoptosis and necrosis and depends on the generation of reactive oxygen species (ROS) by NADPH oxidase. Patients with chronic granulomatous disease carry mutations in NADPH oxidase and cannot activate this cell-death pathway or make NETs. This novel ROS-dependent death allows neutrophils to fulfill their antimicrobial function, even beyond their lifespan.The Journal of Cell Biology 02/2007; 176(2):231-41. · 10.26 Impact Factor -
Article: Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guérin mutants that secrete listeriolysin.
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ABSTRACT: The tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG) was equipped with the membrane-perforating listeriolysin (Hly) of Listeria monocytogenes, which was shown to improve protection against Mycobacterium tuberculosis. Following aerosol challenge, the Hly-secreting recombinant BCG (hly+ rBCG) vaccine was shown to protect significantly better against aerosol infection with M. tuberculosis than did the parental BCG strain. The isogenic, urease C-deficient hly+ rBCG (DeltaureC hly+ rBCG) vaccine, providing an intraphagosomal pH closer to the acidic pH optimum for Hly activity, exhibited still higher vaccine efficacy than parental BCG. DeltaureC hly+ rBCG also induced profound protection against a member of the M. tuberculosis Beijing/W genotype family while parental BCG failed to do so consistently. Hly not only promoted antigen translocation into the cytoplasm but also apoptosis of infected macrophages. We concluded that superior vaccine efficacy of DeltaureC hly+ rBCG as compared with parental BCG is primarily based on improved cross-priming, which causes enhanced T cell-mediated immunity.Journal of Clinical Investigation 10/2005; 115(9):2472-9. · 15.39 Impact Factor -
Article: Lack of stage-specific proteins in coccoid Helicobacter pylori cells.
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ABSTRACT: Helicobacter pylori exists in two distinct forms, rod shaped or coccoid, in stomachs of infected patients. Based on in vitro proteome comparisons, there are no detectable coccoid-specific proteins, which argues against the specific adaptation of coccoid Helicobacter to distinct biological functions, such as enhanced persistence or transmission to other hosts.Infection and Immunity 12/2004; 72(11):6738-42. · 4.16 Impact Factor -
Article: Neutrophil extracellular traps kill bacteria.
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ABSTRACT: Neutrophils engulf and kill bacteria when their antimicrobial granules fuse with the phagosome. Here, we describe that, upon activation, neutrophils release granule proteins and chromatin that together form extracellular fibers that bind Gram-positive and -negative bacteria. These neutrophil extracellular traps (NETs) degrade virulence factors and kill bacteria. NETs are abundant in vivo in experimental dysentery and spontaneous human appendicitis, two examples of acute inflammation. NETs appear to be a form of innate response that binds microorganisms, prevents them from spreading, and ensures a high local concentration of antimicrobial agents to degrade virulence factors and kill bacteria.Science 04/2004; 303(5663):1532-5. · 31.20 Impact Factor -
Article: Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases
Nature Medicine, v.16, 887-U87 (2010). -
Article: Protein refolding is required for assembly of the type three secretion needle
Nature Structural & Molecular Biology, v.17, 788-792 (2010). -
Article: Neutrophil extracellular traps: how to generate and visualize them.
Journal of Visualized Experiments, v.36.
Top co-authors
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Institutions
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2007–2012
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Max Planck Institute for Infection Biology
- Department of Cellular Microbiology
Berlin, Land Berlin, Germany
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