Zoophysiology - Research Topics

A major concern of zoophysiology is to compare the complex mechanisms that various species from different parts of the animal phylogeny use to function – from the level of the organism down to the level of the molecule. Our research is concerned with comparative immunobiology and molecular parasitology.

We study the evolution of the immune systems by comparing model systems including the free-living amoeboid protozoon Dictyostelium discoideum and the nematode Caenorhabditis elegans, Drosophila and in addition marine invertebrates. We analyze the biological function of immune effector proteins such as antimicrobial peptides, pore-forming proteins and lysozymes from various animal taxa, including their activity spectrum, mode of action and structure-function relationships. Based on the comprehensive characterization of these ancient defensive weapons, we aim to identify natural templates for the design of new antibiotics.

We are elucidating the other side of host-pathogen interactions by investigating molecularly pathogenicity mechanisms of eukaryotic parasites and medically important human pathogens such as the causative agent of malaria, Plasmodium, and free-living and enteric amoebic parasites. Using a wide variety of methods, we particularly focus on the quantitative identification of proteins from cellular compartments (proteomics) and perform fundamental research on novel anti-infectives.

• Ancient weapons – cytolytic and antimicrobial polypeptides as defence effector molecules of animals

Antimicrobial systems in animals have been characterized at the molecular level primarily for vertebrates and arthropods. A variety of active peptides have been found and they possess highly diverse structures. The majority of them share the common feature of amphipathicity and appear to act by physical disruption of the membranes of their targets. As the mode of action suggests that their application will not create resistant strains of pathogens, such peptides are currently used as natural templates to design new antibiotics.

Among the several groups of membrane-permeabilizing peptides classified so far, the one to which the subjects of our studies belong is extraordinary; its members are relatively large polypeptides and are characterized by a compact alpha-helical and disulfide-bonded fold. Such polypeptides can be found in species of amoeboid protozoa (e.g. amoebapores), organisms which may be viewed primarily as insatiable phagocytic cells that uses bacteria as a nutrient source, in invertebrates and in vertebrates. Porcine and human cytotoxic lymphocytes contain similar peptides, termed NK-lysin and granulysin, respectively, which appear to be an important constituents of the internal defence against pathogens, e.g. intracellular bacteria. We are comparing the structures of the various antimicrobial and/or cytotoxic polypeptides and monitor their biological activities to extract the similarities and differences of effector molecules from evolutionarily highly divergent animals.

We also are characterizing antimicrobial and cytolytic proteins from body fluids of invertebrate species, such as bacteria-degrading lysozymes or various cytolysins, some of which create electron microscopically visible ring-like lesions on target cell membranes reminiscent of bacterial pore-forming toxins and of the complement system of mammals. These proteins may be viewed as broad spectrum defensive weapons against prokaryotic and eukaryotic pathogens.

Moreover, with the nematode Caenorhabditis elegans a multicellular model organism has been introduced in our project. As the entire genome of the worm has been elucidated and sophisticated techniques to manipulate the organism have been established, it is possible to analyze the antimicrobial system at the molecular and organismal level using DNA array technology and functional knock-out mutants. We are focussing on the molecular basis of the epithelial defense in C. elegans. Here, we are characterizing the molecular mechanisms which combat infections of epithels that are constantly exposed to potential pathogens, from target recognition to signal transduction and eventually synthesis and secretion of effector molecules.
(Supported by Deutsche Forschungsgemeinschaft)

Ursprüngliche Waffen im Tierreich: Membran-durchlöchernde Peptide für Verteidigung und Angriff [PDF]

• Comparative and quantitative proteomics of microbe-challenged versus unchallenged Caenorhabditis elegans, an invertebrate model organism for innate immunity and inflammation

The consensus view is that the intestinal epithelium of animals forms a physical barrier to limit access of enteric microbes to the inner milieu of the host and contributes to innate host defense by producing effector molecules, e.g. antimicrobial peptides and enzymes, against particular luminal microbes. Classical studies on processes involved in innate immunity, inflammation, and pathology are often time-consuming, costly, and ethically problematic. In recent years, invertebrate model organisms have been employed to get out of this dilemma.
C. elegans can be infected by a plethora of pathogens, most of them are pathogenic also for humans. Consequently, the nematode has emerged as a powerful surrogate host for the study of innate immunity and host-pathogen interactions and to model microbial human infectious diseases in a non-vertebrate. Signalling cascades are well investigated that face bacterial or fungal pathogens. In our project we want to focus on the downstream processes of these cascades, i. e. the differential expression of effector and regulatory molecules due to a microbial challenge. Here, comparative and quantitative proteomic techniques including tandem mass spectrometric methods are introduced into the molecular analysis of infection. Recently, we have identified a series of different lysozymes in C. elegans total extracts by a proteomic approach as a snapshot of the physiological condition of the worm at a specific time-point. It is unknown what are the effector molecules in the intestine responsible for nutrition on microbes and for defense against pathogens that may became extraintestinal and how the antimicrobial armamentarium, e. g. lysozymes, is regulated at the proteinaceous level upon infection with different pathogens. It can be hypothesized that the lysozymes, comprising several subclasses, are differentially regulated upon infection with different potential pathogens. This may also hold true for the members of other antimicrobial peptides, e. g. the family of saposin-like peptides. We are analyzing proteins and peptides of the proteome that are down- or upregulated upon microbial infection by a set of microbial pathogens for humans and C. elegans. The output of these analyses will describe the “C. elegans infectome”. The project is unique in concerns of analyzing the global proteome of the nematode as a response to the challenge of the innate immune system. The venture is supposed to decipher the response pattern to different pathogens and will bring yet uncharacterized proteins to their functional annotation.

• Invertebrate model organisms for the elucidation of basic principles in gp130-mediated signaling pathways and inflammation

Chronic inflammatory diseases are driven by dysregulated cytokine networks. In humans the gp130 cytokines are centrally involved in chronic inflammatory diseases like inflammatory bowel disease, rheumatoid arthritis and peritonitis and in colon cancer.
Invertebrate model organisms have become valuable tools in biomedical research, e.g. as surrogate hosts for infections caused by human pathogens. The use of these relatively simple organisms (C. elegans, Drosophila) allows us to focus on basic aspects of the gp130-mediated pathways, which appear to be less complex compared to those of mammals. Combined with the inherent advantages of these genetically tractable model organisms, it is feasible to unravel the core transcriptional network that is activated via gp130-like receptors. Therefore, basic molecular events associated with the initiation of inflammation and maybe even with the development of cancer can be elucidated and the information obtained will be useful to understand the much more complex processes in the corresponding human situation.

Molecular analysis of pathogenicity of amoeboid parasites and Plasmodium falciparum

• Structure and biological function of pore-forming proteins (amoebapores) of Entamoeba histolytica

As the name implies, lysis of cells and tissues is a prominent activity of the protozoon and human pathogen Entamoeba histolytica. Accumulated evidence indicates that small proteins named amoebapores are essential elements of the cytolytic machinery of the parasite. Three amoebapore isoforms exist in amoebic cytoplasmic granules and all exert lytic activity towards mammalian nucleated cells and bacteria by forming pores inside the target cell membranes. We are analyzing the biological role and the structure-function relationships of these amoebic pathogenicity factors. (Supported by DFG).

This collage shows a model of an amoebapore dimer in front of a scanning micrograph of Entamoeba histolytica which phagocytozes a human host cell. This parasite releases pore forming proteins named amoebapores which dimerize in solution representing the active molecules. The structure of the monomeric natural protein has been solved by NMR spectroscopy in a "joint venture" at the University of Kiel (Hecht et al., J. Biol. Chem. 2004).

• Archaic cytolytic and antimicrobial mechanisms of free-living and pathogenic protozoa compared to those of higher eukaryotes

Many natural cytotoxic and antimicrobial polypeptides act by permeabilizing the target cell membranes. Presumably the most ancient phylogenetic location of such membrane-active polypeptides in eukaryotes has been found with amoebapore (see above). We found cytotoxic and antimicrobial polypeptides also in free-living amoeboid protozoa (Naegleria, Acanthamoeba, Balamuthia) which are potentially highly pathogenic for humans. We are analyzing the structures of these amoebic proteins and determine their biological activities to elucidate the similarities and differences of these effector molecules. As an intensively studied cellular model system, we are using the free-living and non-pathogenic amoeboid protozoon Dictyostelium discoideum to study the molecular armament which such a primitive phagocyte may use to combat growth of phagocytozed bacteria inside its digestive vacuoles.

Entamoeba histolytica represented as a bacteria-phagocytozing (at the left) and cytolytic effector cell (at the right). In both scenarios, granule proteins such as the amoebapores are considered instrumental in killing the target cell.

• Proteases of the malaria parasite Plasmodium falciparum as potential targets for novel antiinfectives

Plasmodium falciparum is the protozoon that invades and lives within human red blood cells, causing the most deadly malaria in humans. Falciparum malaria is an ever burgeoning disease which affects millions of people annually, preferentially children in tropical and subtropical areas of the world.
Papain-like cysteine proteases of plasmodia are known to play pivotal roles in the different life stages of the parasites. Most studies employing protease inhibitors and in vitro-cultured parasites support roles for these proteases in hemoglobin hydrolysis and erythrocyte rupture. Additionally, a role in host cell invasion of this class of enzymes is under discussion. Thus, these enzymes are promising targets for new antimalarial drugs.

Intraerythrocytic P. falciparum parasites were incubated in absence (A) or presence (B) of E-64 - a potent cysteine proteinase inhibitor. Note the swollen digestive vacuole and the small hemozoin crystal of the parasite in presence of E-64 due to inhibited globin hydrolysis (red arrow, only the digestive vacuole of one parasites in a doubly infected erythrocyte is marked). The bar indicates 10 µm.

In cooperation with the group of Prof. Dr. T. Schirmeister, Pharmaceutical Chemistry, University of Würzburg, we are analysing the antiplasmodial effect of aziridine-based cysteine protease inhibitors at the cellular and molecular level. We are using proteomic (two-dimensional polyacrylamide gel electrophoresis, 2-D PAGE, and mass spectrometry), microscopic (light, fluorescence, and electron microscopy), and biochemical techniques to investigate the influence of a broad variety of cysteine protease inhibitors on the viability of the parasites, the process of red blood cell invasion and the egress of the parasites from the host cell.

See article in CAU-Unizeit (in German) (Nachdrucke der unizeit-Artikel sind - auch auszugsweise - nur mit Genehmigung der Christian-Albrechts-Universität möglich. Bitte wenden Sie sich dazu an die Pressestelle der Universität, presse@uv.uni-kiel.de)

• Proteomic analysis of the plasma membrane of human erythrocytes infected with the malarial agent P. falciparum as candidates to mediate cytoadherence

The fatality of falciparum malaria is mediated by plasmodial proteins exported to the plasma membrane of the infected erythrocyte interacting essentially with endothelial receptors leading to occlusion of capillaries and deep organ failure. The proteinaceous inventory at the interface between the parasitzed host cell and host tissues is hardly known and needs to be investigated in more detail and in totality. However, exploited techniques to analyse membrane or membrane associated proteins were not applied to this subproteome. Moreover, there is a lack of sensitivity and resolution in the few reports that adress the topic of plasmodial surface proteins. Here, we quantitatively identifiy the proteins of this compartment consisting of surface proteins of P. falciparum that are of particular relevance in context of malaria vaccine targets. For that issue we utilize the existing facilities of the Institute of Immunology and the Zoological Institute of the University of Kiel, i.e. the DIGE workstation and a MALDI-TOF/TOF tandem mass spectrometer, respectively. Furthermore, we want to go beyond the proteomic approach in order to assay putative adhesive properties of the newly identified surface proteins to endothelial receptors by cytoadherence assays and rosetting assays and by analyzing molecular interactions. The project may help to understand the molecular basis of the variable pathologies observed in malaria diesease. (Supported by Forschungsförderung der Med. Fakultät der CAU; Forschungsschwerpunkt Entzündung)

2-D PAGE images of membrane fractions of infected erythrocytes. Left, conventional 2-D PAGE; right, 16-BAC/SDS-PAGE

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	Head of Department Prof. Dr. Matthias Leippe ++49-(0)431-880-4196 - Secretary - Heidrun Wegner ++49-(0)431-880-4180 ++49-(0)431-880-4197 - Postal address - Zoologisches Institut, Abteilung Zoophysiologie Christian-Albrechts-Universität Olshausenstraße 40 D-24098 Kiel, Germany - Shipping address - Zoologisches Institut, Abteilung Zoophysiologie Christian-Albrechts-Universität Am Botanischen Garten 3-9 D-24118 Kiel, Germany		A major concern of zoophysiology is to compare the complex mechanisms that various species from different parts of the animal phylogeny use to function – from the level of the organism down to the level of the molecule. Our research is concerned with comparative immunobiology and molecular parasitology. We study the evolution of the immune systems by comparing model systems including the free-living amoeboid protozoon Dictyostelium discoideum and the nematode Caenorhabditis elegans, Drosophila and in addition marine invertebrates. We analyze the biological function of immune effector proteins such as antimicrobial peptides, pore-forming proteins and lysozymes from various animal taxa, including their activity spectrum, mode of action and structure-function relationships. Based on the comprehensive characterization of these ancient defensive weapons, we aim to identify natural templates for the design of new antibiotics. We are elucidating the other side of host-pathogen interactions by investigating molecularly pathogenicity mechanisms of eukaryotic parasites and medically important human pathogens such as the causative agent of malaria, Plasmodium, and free-living and enteric amoebic parasites. Using a wide variety of methods, we particularly focus on the quantitative identification of proteins from cellular compartments (proteomics) and perform fundamental research on novel anti-infectives. Comparative Immunology: • Ancient weapons – cytolytic and antimicrobial polypeptides as defence effector molecules of animals Antimicrobial systems in animals have been characterized at the molecular level primarily for vertebrates and arthropods. A variety of active peptides have been found and they possess highly diverse structures. The majority of them share the common feature of amphipathicity and appear to act by physical disruption of the membranes of their targets. As the mode of action suggests that their application will not create resistant strains of pathogens, such peptides are currently used as natural templates to design new antibiotics. Among the several groups of membrane-permeabilizing peptides classified so far, the one to which the subjects of our studies belong is extraordinary; its members are relatively large polypeptides and are characterized by a compact alpha-helical and disulfide-bonded fold. Such polypeptides can be found in species of amoeboid protozoa (e.g. amoebapores), organisms which may be viewed primarily as insatiable phagocytic cells that uses bacteria as a nutrient source, in invertebrates and in vertebrates. Porcine and human cytotoxic lymphocytes contain similar peptides, termed NK-lysin and granulysin, respectively, which appear to be an important constituents of the internal defence against pathogens, e.g. intracellular bacteria. We are comparing the structures of the various antimicrobial and/or cytotoxic polypeptides and monitor their biological activities to extract the similarities and differences of effector molecules from evolutionarily highly divergent animals. We also are characterizing antimicrobial and cytolytic proteins from body fluids of invertebrate species, such as bacteria-degrading lysozymes or various cytolysins, some of which create electron microscopically visible ring-like lesions on target cell membranes reminiscent of bacterial pore-forming toxins and of the complement system of mammals. These proteins may be viewed as broad spectrum defensive weapons against prokaryotic and eukaryotic pathogens. Moreover, with the nematode Caenorhabditis elegans a multicellular model organism has been introduced in our project. As the entire genome of the worm has been elucidated and sophisticated techniques to manipulate the organism have been established, it is possible to analyze the antimicrobial system at the molecular and organismal level using DNA array technology and functional knock-out mutants. We are focussing on the molecular basis of the epithelial defense in C. elegans. Here, we are characterizing the molecular mechanisms which combat infections of epithels that are constantly exposed to potential pathogens, from target recognition to signal transduction and eventually synthesis and secretion of effector molecules. (Supported by Deutsche Forschungsgemeinschaft) Caenorhabditis elegans Ursprüngliche Waffen im Tierreich: Membran-durchlöchernde Peptide für Verteidigung und Angriff [PDF] back • Comparative and quantitative proteomics of microbe-challenged versus unchallenged Caenorhabditis elegans, an invertebrate model organism for innate immunity and inflammation The consensus view is that the intestinal epithelium of animals forms a physical barrier to limit access of enteric microbes to the inner milieu of the host and contributes to innate host defense by producing effector molecules, e.g. antimicrobial peptides and enzymes, against particular luminal microbes. Classical studies on processes involved in innate immunity, inflammation, and pathology are often time-consuming, costly, and ethically problematic. In recent years, invertebrate model organisms have been employed to get out of this dilemma. C. elegans can be infected by a plethora of pathogens, most of them are pathogenic also for humans. Consequently, the nematode has emerged as a powerful surrogate host for the study of innate immunity and host-pathogen interactions and to model microbial human infectious diseases in a non-vertebrate. Signalling cascades are well investigated that face bacterial or fungal pathogens. In our project we want to focus on the downstream processes of these cascades, i. e. the differential expression of effector and regulatory molecules due to a microbial challenge. Here, comparative and quantitative proteomic techniques including tandem mass spectrometric methods are introduced into the molecular analysis of infection. Recently, we have identified a series of different lysozymes in C. elegans total extracts by a proteomic approach as a snapshot of the physiological condition of the worm at a specific time-point. It is unknown what are the effector molecules in the intestine responsible for nutrition on microbes and for defense against pathogens that may became extraintestinal and how the antimicrobial armamentarium, e. g. lysozymes, is regulated at the proteinaceous level upon infection with different pathogens. It can be hypothesized that the lysozymes, comprising several subclasses, are differentially regulated upon infection with different potential pathogens. This may also hold true for the members of other antimicrobial peptides, e. g. the family of saposin-like peptides. We are analyzing proteins and peptides of the proteome that are down- or upregulated upon microbial infection by a set of microbial pathogens for humans and C. elegans. The output of these analyses will describe the “C. elegans infectome”. The project is unique in concerns of analyzing the global proteome of the nematode as a response to the challenge of the innate immune system. The venture is supposed to decipher the response pattern to different pathogens and will bring yet uncharacterized proteins to their functional annotation. Funded by back • Invertebrate model organisms for the elucidation of basic principles in gp130-mediated signaling pathways and inflammation Chronic inflammatory diseases are driven by dysregulated cytokine networks. In humans the gp130 cytokines are centrally involved in chronic inflammatory diseases like inflammatory bowel disease, rheumatoid arthritis and peritonitis and in colon cancer. Invertebrate model organisms have become valuable tools in biomedical research, e.g. as surrogate hosts for infections caused by human pathogens. The use of these relatively simple organisms (C. elegans, Drosophila) allows us to focus on basic aspects of the gp130-mediated pathways, which appear to be less complex compared to those of mammals. Combined with the inherent advantages of these genetically tractable model organisms, it is feasible to unravel the core transcriptional network that is activated via gp130-like receptors. Therefore, basic molecular events associated with the initiation of inflammation and maybe even with the development of cancer can be elucidated and the information obtained will be useful to understand the much more complex processes in the corresponding human situation. Funded by back Molecular Parasitology: Molecular analysis of pathogenicity of amoeboid parasites and Plasmodium falciparum • Structure and biological function of pore-forming proteins (amoebapores) of Entamoeba histolytica As the name implies, lysis of cells and tissues is a prominent activity of the protozoon and human pathogen Entamoeba histolytica. Accumulated evidence indicates that small proteins named amoebapores are essential elements of the cytolytic machinery of the parasite. Three amoebapore isoforms exist in amoebic cytoplasmic granules and all exert lytic activity towards mammalian nucleated cells and bacteria by forming pores inside the target cell membranes. We are analyzing the biological role and the structure-function relationships of these amoebic pathogenicity factors. (Supported by DFG). This collage shows a model of an amoebapore dimer in front of a scanning micrograph of Entamoeba histolytica which phagocytozes a human host cell. This parasite releases pore forming proteins named amoebapores which dimerize in solution representing the active molecules. The structure of the monomeric natural protein has been solved by NMR spectroscopy in a "joint venture" at the University of Kiel (Hecht et al., J. Biol. Chem. 2004). back • Archaic cytolytic and antimicrobial mechanisms of free-living and pathogenic protozoa compared to those of higher eukaryotes Many natural cytotoxic and antimicrobial polypeptides act by permeabilizing the target cell membranes. Presumably the most ancient phylogenetic location of such membrane-active polypeptides in eukaryotes has been found with amoebapore (see above). We found cytotoxic and antimicrobial polypeptides also in free-living amoeboid protozoa (Naegleria, Acanthamoeba, Balamuthia) which are potentially highly pathogenic for humans. We are analyzing the structures of these amoebic proteins and determine their biological activities to elucidate the similarities and differences of these effector molecules. As an intensively studied cellular model system, we are using the free-living and non-pathogenic amoeboid protozoon Dictyostelium discoideum to study the molecular armament which such a primitive phagocyte may use to combat growth of phagocytozed bacteria inside its digestive vacuoles. Entamoeba histolytica represented as a bacteria-phagocytozing (at the left) and cytolytic effector cell (at the right). In both scenarios, granule proteins such as the amoebapores are considered instrumental in killing the target cell. Dictyostelium discoideum fruiting bodies back *• Proteases of the malaria parasite Plasmodium falciparum* as potential targets for novel antiinfectives** Plasmodium falciparum is the protozoon that invades and lives within human red blood cells, causing the most deadly malaria in humans. Falciparum malaria is an ever burgeoning disease which affects millions of people annually, preferentially children in tropical and subtropical areas of the world. Papain-like cysteine proteases of plasmodia are known to play pivotal roles in the different life stages of the parasites. Most studies employing protease inhibitors and in vitro-cultured parasites support roles for these proteases in hemoglobin hydrolysis and erythrocyte rupture. Additionally, a role in host cell invasion of this class of enzymes is under discussion. Thus, these enzymes are promising targets for new antimalarial drugs. Intraerythrocytic P. falciparum parasites were incubated in absence (A) or presence (B) of E-64 - a potent cysteine proteinase inhibitor. Note the swollen digestive vacuole and the small hemozoin crystal of the parasite in presence of E-64 due to inhibited globin hydrolysis (red arrow, only the digestive vacuole of one parasites in a doubly infected erythrocyte is marked). The bar indicates 10 µm. In cooperation with the group of Prof. Dr. T. Schirmeister, Pharmaceutical Chemistry, University of Würzburg, we are analysing the antiplasmodial effect of aziridine-based cysteine protease inhibitors at the cellular and molecular level. We are using proteomic (two-dimensional polyacrylamide gel electrophoresis, 2-D PAGE, and mass spectrometry), microscopic (light, fluorescence, and electron microscopy), and biochemical techniques to investigate the influence of a broad variety of cysteine protease inhibitors on the viability of the parasites, the process of red blood cell invasion and the egress of the parasites from the host cell. See article in CAU-Unizeit (in German) (Nachdrucke der unizeit-Artikel sind - auch auszugsweise - nur mit Genehmigung der Christian-Albrechts-Universität möglich. Bitte wenden Sie sich dazu an die Pressestelle der Universität, presse@uv.uni-kiel.de) *• Proteomic analysis of the plasma membrane of human erythrocytes infected with the malarial agent P. falciparum* as candidates to mediate cytoadherence** The fatality of falciparum malaria is mediated by plasmodial proteins exported to the plasma membrane of the infected erythrocyte interacting essentially with endothelial receptors leading to occlusion of capillaries and deep organ failure. The proteinaceous inventory at the interface between the parasitzed host cell and host tissues is hardly known and needs to be investigated in more detail and in totality. However, exploited techniques to analyse membrane or membrane associated proteins were not applied to this subproteome. Moreover, there is a lack of sensitivity and resolution in the few reports that adress the topic of plasmodial surface proteins. Here, we quantitatively identifiy the proteins of this compartment consisting of surface proteins of P. falciparum that are of particular relevance in context of malaria vaccine targets. For that issue we utilize the existing facilities of the Institute of Immunology and the Zoological Institute of the University of Kiel, i.e. the DIGE workstation and a MALDI-TOF/TOF tandem mass spectrometer, respectively. Furthermore, we want to go beyond the proteomic approach in order to assay putative adhesive properties of the newly identified surface proteins to endothelial receptors by cytoadherence assays and rosetting assays and by analyzing molecular interactions. The project may help to understand the molecular basis of the variable pathologies observed in malaria diesease. (Supported by Forschungsförderung der Med. Fakultät der CAU; Forschungsschwerpunkt Entzündung) 2-D PAGE images of membrane fractions of infected erythrocytes. Left, conventional 2-D PAGE; right, 16-BAC/SDS-PAGE back CAU-Kiel > Home Zoophysiology > Projects