Project acronym Anti-Virome
Project A combined evolutionary and proteomics approach to the discovery, induction and application of antiviral immunity factors
Researcher (PI) Frank Kirchhoff
Host Institution (HI) UNIVERSITAET ULM
Call Details Advanced Grant (AdG), LS6, ERC-2012-ADG_20120314
Summary "Humans are equipped with a variety of intrinsic immunity or host restriction factors. These evolved under positive selection pressure for diversification and represent a first line of defence against invading viruses. Unfortunately, however, many pathogens have evolved effective antagonists against our defences. For example, the capability of HIV-1 to counteract human restriction factors that interfere with reverse transcription, uncoating and virion release has been a prerequisite for the global spread of AIDS. We are just beginning to understand the diversity and induction of antiretroviral factors and how pandemic HIV-1 group M (major) strains evolved to counteract all of them. Here, I propose to use a genetics, proteomics and evolutionary approach to discover and define as-yet-unknown antiviral effectors and their inducers. To identify novel antiviral factors, we will examine the capability of all primate genes that are under strong positive selection pressure to inhibit HIV and its simian (SIV) precursors. This examination from the evolutionary perspective of the invading pathogen will also reveal which adaptations allowed HIV-1 to cause the AIDS pandemic. Furthermore, complex peptide-protein libraries representing essentially the entire human peptidome, will be utilized to identify novel specific inducers of antiviral restriction factors. My ultimate aim is to unravel the network of inducers and effectors of antiviral immunity - the ""Anti-Virome"" - and to use this knowledge to develop novel effective preventive and therapeutic approaches based on the induction of combinations of antiviral factors targeting different steps of the viral life cycle. The results of this innovative and interdisciplinary program will provide fundamental new insights into intrinsic immunity and may offer alternatives to conventional vaccine and therapeutic approaches because most restriction factors have broad antiviral activity and are thus effective against various pathogens."
Summary
"Humans are equipped with a variety of intrinsic immunity or host restriction factors. These evolved under positive selection pressure for diversification and represent a first line of defence against invading viruses. Unfortunately, however, many pathogens have evolved effective antagonists against our defences. For example, the capability of HIV-1 to counteract human restriction factors that interfere with reverse transcription, uncoating and virion release has been a prerequisite for the global spread of AIDS. We are just beginning to understand the diversity and induction of antiretroviral factors and how pandemic HIV-1 group M (major) strains evolved to counteract all of them. Here, I propose to use a genetics, proteomics and evolutionary approach to discover and define as-yet-unknown antiviral effectors and their inducers. To identify novel antiviral factors, we will examine the capability of all primate genes that are under strong positive selection pressure to inhibit HIV and its simian (SIV) precursors. This examination from the evolutionary perspective of the invading pathogen will also reveal which adaptations allowed HIV-1 to cause the AIDS pandemic. Furthermore, complex peptide-protein libraries representing essentially the entire human peptidome, will be utilized to identify novel specific inducers of antiviral restriction factors. My ultimate aim is to unravel the network of inducers and effectors of antiviral immunity - the ""Anti-Virome"" - and to use this knowledge to develop novel effective preventive and therapeutic approaches based on the induction of combinations of antiviral factors targeting different steps of the viral life cycle. The results of this innovative and interdisciplinary program will provide fundamental new insights into intrinsic immunity and may offer alternatives to conventional vaccine and therapeutic approaches because most restriction factors have broad antiviral activity and are thus effective against various pathogens."
Max ERC Funding
1 915 200 €
Duration
Start date: 2013-04-01, End date: 2018-03-31
Project acronym ARCHAELLUM
Project Assembly and function of the crenarchaeal flagellum
Researcher (PI) Sonja-Verena Albers
Host Institution (HI) ALBERT-LUDWIGS-UNIVERSITAET FREIBURG
Call Details Starting Grant (StG), LS6, ERC-2012-StG_20111109
Summary "Archaea constitute the third domain of life and are believed to be close to the origin of life. They comprise a diverse group of micro-organisms that combine bacterial and eukaryotic features, but also employ many novel mechanisms. They possess a unique cell envelope with a cytoplasmic membrane of ether lipids surrounded by a proteinaceous S-layer and various cell appendages such as flagella, pili and more unusual structures. Studies have shown that the archaeal flagellum is an unique structure as it functionally resembles the bacterial flagellum, but structurally it is a simple type IV pilus. Moreover, we have shown that this type IV pilus can rotate. Therefore I propose to name the archaeal flagellum, the archaellum, as it is fundamentally different from the bacterial flagellum.
In this proposal I aim to understand the assembly and mechanism of rotation of the archaellum of the thermocacidophilic crenarchaen Sulfolobus acidocaldarius by using biochemical, genetic and biophysical methods. The main milestons are:
- Biochemical and structural characterization of all archaellum subunits
- To understand the assembly pathway of the archaellum and the interactions of its different
subunits
- To understand how rotation of the filament is achieved and which subunits are important
for this movement
This work will identify a new, relatively simple motor complex that has evolved from primordial type IV pili assembly machineries and therefore uncover general principles of macromolecular assemblies at cellular surfaces and a novel mechanism to generate mechanical force that can be translated into movement."
Summary
"Archaea constitute the third domain of life and are believed to be close to the origin of life. They comprise a diverse group of micro-organisms that combine bacterial and eukaryotic features, but also employ many novel mechanisms. They possess a unique cell envelope with a cytoplasmic membrane of ether lipids surrounded by a proteinaceous S-layer and various cell appendages such as flagella, pili and more unusual structures. Studies have shown that the archaeal flagellum is an unique structure as it functionally resembles the bacterial flagellum, but structurally it is a simple type IV pilus. Moreover, we have shown that this type IV pilus can rotate. Therefore I propose to name the archaeal flagellum, the archaellum, as it is fundamentally different from the bacterial flagellum.
In this proposal I aim to understand the assembly and mechanism of rotation of the archaellum of the thermocacidophilic crenarchaen Sulfolobus acidocaldarius by using biochemical, genetic and biophysical methods. The main milestons are:
- Biochemical and structural characterization of all archaellum subunits
- To understand the assembly pathway of the archaellum and the interactions of its different
subunits
- To understand how rotation of the filament is achieved and which subunits are important
for this movement
This work will identify a new, relatively simple motor complex that has evolved from primordial type IV pili assembly machineries and therefore uncover general principles of macromolecular assemblies at cellular surfaces and a novel mechanism to generate mechanical force that can be translated into movement."
Max ERC Funding
1 464 317 €
Duration
Start date: 2013-02-01, End date: 2018-01-31
Project acronym BACNK
Project Recognition of bacteria by NK cells
Researcher (PI) Ofer Mandelboim
Host Institution (HI) THE HEBREW UNIVERSITY OF JERUSALEM
Call Details Advanced Grant (AdG), LS6, ERC-2012-ADG_20120314
Summary NK cells that are well known by their ability to recognize and eliminate virus infected and tumor cells were also implicated in the defence against bacteria. However, the recognition of bacteria by NK cells is only poorly understood. we do not know how bacteria are recognized and the functional consequences of such recognition are also weakly understood. In the current proposal we aimed at determining the “NK cell receptor-bacterial interactome”. We will examine the hypothesis that NK inhibitory and activating receptors are directly involved in bacterial recognition. This ground breaking hypothesis is based on our preliminary results in which we show that several NK cell receptors directly recognize various bacterial strains as well as on a few other publications. We will generate various mice knockouts for NCR1 (a major NK killer receptor) and determine their microbiota to understand the physiological function of NCR1 and whether certain bacterial strains affects its activity. We will use different human and mouse NK killer and inhibitory receptors fused to IgG1 to pull-down bacteria from saliva and fecal samples and then use 16S rRNA analysis and next generation sequencing to determine the nature of the bacteria species isolated. We will identify the bacterial ligands that are recognized by the relevant NK cell receptors, using bacterial random transposon insertion mutagenesis approach. We will end this research with functional assays. In the wake of the emerging threat of bacterial drug resistance and the involvement of bacteria in the pathogenesis of many different chronic diseases and in shaping the immune response, the completion of this study will open a new field of research; the direct recognition of bacteria by NK cell receptors.
Summary
NK cells that are well known by their ability to recognize and eliminate virus infected and tumor cells were also implicated in the defence against bacteria. However, the recognition of bacteria by NK cells is only poorly understood. we do not know how bacteria are recognized and the functional consequences of such recognition are also weakly understood. In the current proposal we aimed at determining the “NK cell receptor-bacterial interactome”. We will examine the hypothesis that NK inhibitory and activating receptors are directly involved in bacterial recognition. This ground breaking hypothesis is based on our preliminary results in which we show that several NK cell receptors directly recognize various bacterial strains as well as on a few other publications. We will generate various mice knockouts for NCR1 (a major NK killer receptor) and determine their microbiota to understand the physiological function of NCR1 and whether certain bacterial strains affects its activity. We will use different human and mouse NK killer and inhibitory receptors fused to IgG1 to pull-down bacteria from saliva and fecal samples and then use 16S rRNA analysis and next generation sequencing to determine the nature of the bacteria species isolated. We will identify the bacterial ligands that are recognized by the relevant NK cell receptors, using bacterial random transposon insertion mutagenesis approach. We will end this research with functional assays. In the wake of the emerging threat of bacterial drug resistance and the involvement of bacteria in the pathogenesis of many different chronic diseases and in shaping the immune response, the completion of this study will open a new field of research; the direct recognition of bacteria by NK cell receptors.
Max ERC Funding
2 499 800 €
Duration
Start date: 2013-03-01, End date: 2018-02-28
Project acronym CBM-INNATE
Project Regulation and Function of CARD9 / BCL10 / MALT1 Signalosomes in Innate Immunity and Inflammation
Researcher (PI) Jürgen Maximilian Ruland
Host Institution (HI) KLINIKUM RECHTS DER ISAR DER TECHNISCHEN UNIVERSITAT MUNCHEN
Call Details Advanced Grant (AdG), LS6, ERC-2012-ADG_20120314
Summary Acute inflammation is a response to infection or tissue damage that is critical for host protection and tissue homeostasis. However, deregulated or chronic inflammation is harmful to the host and can cause multiple diseases including inflammatory bowel disease, rheumatoid arthritis, cardiovascular diseases, neuroinflammatory disease and cancer. Cells of the innate immune system sense microbial or sterile danger via pattern recognition receptors (PRRs). Subsequently, these PRRs engage intracellular signalling modules to elicit inflammatory effector mechanisms. We have recently identified the CARD9 / BCL10 / MALT1 (CBM) signalosome as a central proinflammatory signalling complex in innate immune cells. This molecular platform responds to stimuli from transmembrane SYK-coupled C-type lectin receptors and from intracellular danger sensors such as RIG-I-like helicases, NOD2 and presumably others to robustly activate NF-κB and MAPK pathways. Innate CBM signalling is engaged upon fungal, bacterial or viral recognition and upon sterile cell injury and it is essential for host protection in humans and mice. Still, it is unclear how the CARD9 / BCL10 / MALT1 signalosome is activated on a molecular level and how CBM responses are transduced to effector cascades. Moreover, although CARD9 polymorphisms are linked to various human inflammatory diseases, the cell type- and signal-specific roles of CBM signalosomes in complex diseases in vivo are unknown. Here we aim to take an integrated genetic, biochemical and in vivo approach to comprehensively dissect the regulation of the CARD9 / BCL10 / MALT1 complex in innate immunity and to define the role of this signalosome in clinically relevant inflammatory diseases. Mechanistic in vitro studies will be combined with the in vivo analysis of CBM function in genetically defined mouse models to gain better insights into the regulation of innate immunity and to pave the way to novel therapeutics for inflammatory diseases.
Summary
Acute inflammation is a response to infection or tissue damage that is critical for host protection and tissue homeostasis. However, deregulated or chronic inflammation is harmful to the host and can cause multiple diseases including inflammatory bowel disease, rheumatoid arthritis, cardiovascular diseases, neuroinflammatory disease and cancer. Cells of the innate immune system sense microbial or sterile danger via pattern recognition receptors (PRRs). Subsequently, these PRRs engage intracellular signalling modules to elicit inflammatory effector mechanisms. We have recently identified the CARD9 / BCL10 / MALT1 (CBM) signalosome as a central proinflammatory signalling complex in innate immune cells. This molecular platform responds to stimuli from transmembrane SYK-coupled C-type lectin receptors and from intracellular danger sensors such as RIG-I-like helicases, NOD2 and presumably others to robustly activate NF-κB and MAPK pathways. Innate CBM signalling is engaged upon fungal, bacterial or viral recognition and upon sterile cell injury and it is essential for host protection in humans and mice. Still, it is unclear how the CARD9 / BCL10 / MALT1 signalosome is activated on a molecular level and how CBM responses are transduced to effector cascades. Moreover, although CARD9 polymorphisms are linked to various human inflammatory diseases, the cell type- and signal-specific roles of CBM signalosomes in complex diseases in vivo are unknown. Here we aim to take an integrated genetic, biochemical and in vivo approach to comprehensively dissect the regulation of the CARD9 / BCL10 / MALT1 complex in innate immunity and to define the role of this signalosome in clinically relevant inflammatory diseases. Mechanistic in vitro studies will be combined with the in vivo analysis of CBM function in genetically defined mouse models to gain better insights into the regulation of innate immunity and to pave the way to novel therapeutics for inflammatory diseases.
Max ERC Funding
2 440 200 €
Duration
Start date: 2013-02-01, End date: 2019-01-31
Project acronym cdGMP
Project Time, space and speed: cdGMP signaling in cell behavior and reproduction
Researcher (PI) Urs Jenal
Host Institution (HI) UNIVERSITAT BASEL
Call Details Advanced Grant (AdG), LS6, ERC-2012-ADG_20120314
Summary Bacterial biofilms are the primary cause of chronic infections and of resulting infection relapses. To be able to interfere with bacterial persistence it is vital to understand the molecular details of biofilm formation and to define how motile planktonic cells transit into surface-grown communities. The nucleotide second messenger cyclic di-guanosinemonophosphate (cdGMP) has emerged as a central regulatory factor governing bacterial surface adaptation and biofilm formation. Although cdGMP signaling may well represent the Achilles heel of bacterial communities, cdGMP networks in bacterial pathogens are exquisitely complex and an integrated cellular system to uncover the details of cdGMP dynamics is missing.
To quantitatively describe cdGMP signaling we propose to exploit Caulobacter crescentus, an organism with a simple bimodal life-style that integrates the sessile-motile switch into its asymmetric division cycle. We aim to: 1) identify the role and regulation of all diguanylate cyclases and phosphodiesterases that contribute to the asymmetric cellular program with the goal to model the temporal and spatial distribution of cdGMP during development; 2) identify and characterize cdGMP effectors, their downstream targets and cellular pathways; 3) elucidate how cdGMP coordinates cell differentiation with cell growth and propagation; 4) unravel the role of cdGMP as an allosteric regulator in mechanosensation and in rapid adaptation of bacteria to growth on surfaces; 5) develop novel tools to quantitatively describe cdGMP network dynamics as the basis for mathematical modeling that provides the predictive power to experimentally test and refine important network parameters. We propose a multidisciplinary research program at the forefront of bacterial signal transduction that will provide the molecular and conceptual framework for a rapidly growing research field of second messenger signaling in pathogenic bacteria.
Summary
Bacterial biofilms are the primary cause of chronic infections and of resulting infection relapses. To be able to interfere with bacterial persistence it is vital to understand the molecular details of biofilm formation and to define how motile planktonic cells transit into surface-grown communities. The nucleotide second messenger cyclic di-guanosinemonophosphate (cdGMP) has emerged as a central regulatory factor governing bacterial surface adaptation and biofilm formation. Although cdGMP signaling may well represent the Achilles heel of bacterial communities, cdGMP networks in bacterial pathogens are exquisitely complex and an integrated cellular system to uncover the details of cdGMP dynamics is missing.
To quantitatively describe cdGMP signaling we propose to exploit Caulobacter crescentus, an organism with a simple bimodal life-style that integrates the sessile-motile switch into its asymmetric division cycle. We aim to: 1) identify the role and regulation of all diguanylate cyclases and phosphodiesterases that contribute to the asymmetric cellular program with the goal to model the temporal and spatial distribution of cdGMP during development; 2) identify and characterize cdGMP effectors, their downstream targets and cellular pathways; 3) elucidate how cdGMP coordinates cell differentiation with cell growth and propagation; 4) unravel the role of cdGMP as an allosteric regulator in mechanosensation and in rapid adaptation of bacteria to growth on surfaces; 5) develop novel tools to quantitatively describe cdGMP network dynamics as the basis for mathematical modeling that provides the predictive power to experimentally test and refine important network parameters. We propose a multidisciplinary research program at the forefront of bacterial signal transduction that will provide the molecular and conceptual framework for a rapidly growing research field of second messenger signaling in pathogenic bacteria.
Max ERC Funding
2 496 000 €
Duration
Start date: 2013-05-01, End date: 2018-04-30
Project acronym COhABIT
Project Consequences of helminth-bacterial interactions
Researcher (PI) Nicola Harris
Host Institution (HI) ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE
Call Details Starting Grant (StG), LS6, ERC-2012-StG_20111109
Summary "Throughout evolution both intestinal helminths and commensal bacteria have inhabited our intestines. This ""ménage à trois"" situation is likely to have exerted a strong selective pressure on the development of our metabolic and immune systems. Such pressures remain in developing countries, whilst the eradication of helminths in industrialized countries has shifted this evolutionary balance—possibly underlying the increased development of chronic inflammatory diseases. We hypothesize that helminth-bacterial interactions are a key determinant of healthy homeostasis.
Preliminary findings from our laboratory indicate that helminth infection of mice alters the abundance and diversity of intestinal bacteria and impacts on the availability of immuno-modulatory metabolites; this altered environment correlates with a direct health advantage, protecting against inflammatory diseases such as asthma and rheumatoid arthritis. We intend to validate and extend these data in humans by performing bacterial phlyogenetic and metabolic analysis of stool samples collected from a large cohort of children living in a helminth endemic region of Ecuador. We further propose to test our hypothesis that helminth-bacterial interactions contribute to disease modulation using experimental models of infection and disease. We plan to develop and utilize mouse models to elucidate the mechanisms through which bacterial dysbiosis and helminth infection influence the development of chronic inflammatory diseases. These models will be utilized for germ-free and recolonization experiments, investigating the relative contribution of bacteria versus helminthes to host immunity, co-metabolism and disease modulation.
Taking a trans-disciplinary approach, this research will break new ground in our understanding of the crosstalk and pressures between intestinal helminth infection and commensal bacterial communities, and the implications this has for human health."
Summary
"Throughout evolution both intestinal helminths and commensal bacteria have inhabited our intestines. This ""ménage à trois"" situation is likely to have exerted a strong selective pressure on the development of our metabolic and immune systems. Such pressures remain in developing countries, whilst the eradication of helminths in industrialized countries has shifted this evolutionary balance—possibly underlying the increased development of chronic inflammatory diseases. We hypothesize that helminth-bacterial interactions are a key determinant of healthy homeostasis.
Preliminary findings from our laboratory indicate that helminth infection of mice alters the abundance and diversity of intestinal bacteria and impacts on the availability of immuno-modulatory metabolites; this altered environment correlates with a direct health advantage, protecting against inflammatory diseases such as asthma and rheumatoid arthritis. We intend to validate and extend these data in humans by performing bacterial phlyogenetic and metabolic analysis of stool samples collected from a large cohort of children living in a helminth endemic region of Ecuador. We further propose to test our hypothesis that helminth-bacterial interactions contribute to disease modulation using experimental models of infection and disease. We plan to develop and utilize mouse models to elucidate the mechanisms through which bacterial dysbiosis and helminth infection influence the development of chronic inflammatory diseases. These models will be utilized for germ-free and recolonization experiments, investigating the relative contribution of bacteria versus helminthes to host immunity, co-metabolism and disease modulation.
Taking a trans-disciplinary approach, this research will break new ground in our understanding of the crosstalk and pressures between intestinal helminth infection and commensal bacterial communities, and the implications this has for human health."
Max ERC Funding
1 480 612 €
Duration
Start date: 2013-04-01, End date: 2018-03-31
Project acronym CytoBacLysis
Project Deciphering cytosolic antibacterial immunity: from triggering bacteriolysis to Aim2 inflammasome activation
Researcher (PI) Thomas Henry
Host Institution (HI) INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE
Call Details Starting Grant (StG), LS6, ERC-2012-StG_20111109
Summary Bacteria replicating within host cells either multiply in membrane-bound compartment or escape into the host cytosol. The host cytosol has long been considered as a safe haven for bacteria. However, the host cytosol is armed with an array of innate immune receptors detecting cytosolic invasion. Furthermore, the macrophage cytosol displays a bacteriolytic activity, which is inducible by IFN. Surprisingly, the molecular mechanisms of this innate immune effector response are still largely uncharacterized. A ubiquitously expressed antimicrobial peptide, ubiquicidin has been described in the macrophage cytosol. Its relevance, its connection with macrophage-specific bacteriolytic activity and with IFN, remain to be deciphered. While cytosol-adapted bacteria are largely resistant to the bactericidal activity of the macrophage, lysis of a single bacterium triggers activation of the Aim2 inflammasome. Cytosolic bacteriolysis is thus key to orchestrate inflammasome-mediated innate immune responses. We propose here to characterize the bacteriolytic effector mechanisms, the regulation of this response and of the Aim2 inflammasome by IFN in infected macrophages. We will use two complementary bacterial models: F. tularensis, a cytosol-adapted bacterium and S. typhimurium sifA mutant, a bacterium lysed in the macrophage cytosol. We will develop three synergistic approaches:
i) the generation of novel tools to monitor cytosolic bacteriolysis
ii) hypothesis-driven investigations on the antimicrobial activity of the macrophage cytosol focusing on ubiquicidin to uncover the mechanisms of processing and targeting of this antimicrobial peptide
iii) screening of IFN-inducible genes to identify novel players involved in the cytosolic bacteriolytic activity and in inflammasome regulation.
We believe this project should reveal the innate immune effector mechanisms of the macrophage cytosol i.e. how the macrophage kills cytosolic bacteria and orchestrates further immune responses.
Summary
Bacteria replicating within host cells either multiply in membrane-bound compartment or escape into the host cytosol. The host cytosol has long been considered as a safe haven for bacteria. However, the host cytosol is armed with an array of innate immune receptors detecting cytosolic invasion. Furthermore, the macrophage cytosol displays a bacteriolytic activity, which is inducible by IFN. Surprisingly, the molecular mechanisms of this innate immune effector response are still largely uncharacterized. A ubiquitously expressed antimicrobial peptide, ubiquicidin has been described in the macrophage cytosol. Its relevance, its connection with macrophage-specific bacteriolytic activity and with IFN, remain to be deciphered. While cytosol-adapted bacteria are largely resistant to the bactericidal activity of the macrophage, lysis of a single bacterium triggers activation of the Aim2 inflammasome. Cytosolic bacteriolysis is thus key to orchestrate inflammasome-mediated innate immune responses. We propose here to characterize the bacteriolytic effector mechanisms, the regulation of this response and of the Aim2 inflammasome by IFN in infected macrophages. We will use two complementary bacterial models: F. tularensis, a cytosol-adapted bacterium and S. typhimurium sifA mutant, a bacterium lysed in the macrophage cytosol. We will develop three synergistic approaches:
i) the generation of novel tools to monitor cytosolic bacteriolysis
ii) hypothesis-driven investigations on the antimicrobial activity of the macrophage cytosol focusing on ubiquicidin to uncover the mechanisms of processing and targeting of this antimicrobial peptide
iii) screening of IFN-inducible genes to identify novel players involved in the cytosolic bacteriolytic activity and in inflammasome regulation.
We believe this project should reveal the innate immune effector mechanisms of the macrophage cytosol i.e. how the macrophage kills cytosolic bacteria and orchestrates further immune responses.
Max ERC Funding
1 404 688 €
Duration
Start date: 2012-11-01, End date: 2018-10-31
Project acronym Defensinactivity
Project The influence of environmental factors on antimicrobial activity of human intestinal defensins
Researcher (PI) Jan Wehkamp
Host Institution (HI) EBERHARD KARLS UNIVERSITAET TUEBINGEN
Call Details Starting Grant (StG), LS6, ERC-2012-StG_20111109
Summary Human epithelia are permanently challenged by microorganisms. In the gut, the fraction of strict anaerobic bacteria increases from proximal to distal, reaching 99% of bacterial species in the colon. Moreover, microbial metabolism causes a reduction of the environment to a low redox potential of only –200 mV to –300 mV. Defensins, characterised by three intramolecular disulfide-bridges, are key effector molecules of innate immunity that protect the host from infectious microbes. Human β-defensin 1 (hBD-1) is one of the most prominent peptides of its class but comparison with other defensins suggested only minor antibiotic killing activity. We could recently show that hBD-1 becomes a potent antimicrobial peptide against C. albicans and anaerobic, Gram-positive commensals of the human normal flora in a reducing environment (Nature 2011). The effect was attributable to the linear, reduced hBD-1 peptide.
Here we aim to investigate the antimicrobial activity of reduced hBD-1 in more detail. We will study the mechanism of its reduction by cell-culture experiments and in vitro reduction assays. The molecular details of its antibiotic action will be investigated by using bacterial mutants and further in vitro assays. Additionally we aim to characterise the antibiotic spectrum of reduced hBD-1 by using different antimicrobial assays. Also, we plan to systematically test human defensins under reducing conditions and different pH values that occur in the gut
Besides we will screen extracts of human intestinal tissue and stool samples for antimicrobial substances by using the conditions described above. Extracts will be purified by HPLC and antimicrobially active fractions will be examined by MALDI-TOF peptide mass fingerprint technique. We hope to identify novel peptides which have been overlooked due to standardized testing methods. Resembling the natural conditions as close as possible will help to better understand antibiotic mucosal host defense in the intestinal tract.
Summary
Human epithelia are permanently challenged by microorganisms. In the gut, the fraction of strict anaerobic bacteria increases from proximal to distal, reaching 99% of bacterial species in the colon. Moreover, microbial metabolism causes a reduction of the environment to a low redox potential of only –200 mV to –300 mV. Defensins, characterised by three intramolecular disulfide-bridges, are key effector molecules of innate immunity that protect the host from infectious microbes. Human β-defensin 1 (hBD-1) is one of the most prominent peptides of its class but comparison with other defensins suggested only minor antibiotic killing activity. We could recently show that hBD-1 becomes a potent antimicrobial peptide against C. albicans and anaerobic, Gram-positive commensals of the human normal flora in a reducing environment (Nature 2011). The effect was attributable to the linear, reduced hBD-1 peptide.
Here we aim to investigate the antimicrobial activity of reduced hBD-1 in more detail. We will study the mechanism of its reduction by cell-culture experiments and in vitro reduction assays. The molecular details of its antibiotic action will be investigated by using bacterial mutants and further in vitro assays. Additionally we aim to characterise the antibiotic spectrum of reduced hBD-1 by using different antimicrobial assays. Also, we plan to systematically test human defensins under reducing conditions and different pH values that occur in the gut
Besides we will screen extracts of human intestinal tissue and stool samples for antimicrobial substances by using the conditions described above. Extracts will be purified by HPLC and antimicrobially active fractions will be examined by MALDI-TOF peptide mass fingerprint technique. We hope to identify novel peptides which have been overlooked due to standardized testing methods. Resembling the natural conditions as close as possible will help to better understand antibiotic mucosal host defense in the intestinal tract.
Max ERC Funding
1 500 000 €
Duration
Start date: 2013-02-01, End date: 2018-01-31
Project acronym Droso-parasite
Project Drosophila as a model host to study infections by kinetoplastid parasites
Researcher (PI) Petros Ligoxygakis
Host Institution (HI) THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD
Call Details Starting Grant (StG), LS6, ERC-2012-StG_20111109
Summary For the vast majority of vector borne parasites the ability to overcome the insect midgut defences is central to transmission. However, for many such diseases we know virtually nothing about the molecular mechanisms involved. For vectors such as tsetse flies and sand flies the prospects for rapidly improving our understanding of key interactions occurring in the midgut when challenged by parasites is bleak. This is because the ‘tool box’ required untangling the interactions is very unlikely to be rapidly developed. For example, there is no realistic prospect of producing transgenic technology for tsetse flies because eggs are inaccessible due to intrauterine development of larvae; maintenance of multiple lines of either sand or tsetse flies permitting genetic studies is impossible because of the cost and complexity of culturing colonies; bioinformatic resources are still in their infancy. In this application we suggest that under these circumstances a comparative approach, in which kinetoplastid interactions in Drosophila melanogaster are studied in the first instance, will permit us to make significant progress in understanding the more important cases of insect-parasite interactions (Trypanosome brucei spp in tsetse and Leishmania in sandflies). Herpetomonas ampelophilae is a natural kinetoplastid parasite of Drosophila melanogaster, which establishes infection in the midgut of the fruit fly and can go on to invade the salivary glands. We now have this protozoan in culture and intend, through a combination of genomics, cell biology and RNAi experiments to identify the gut-specific host genomic contingent involved in parasite challenge. In addition, we will study the interaction between the indigenous flora and the parasite and the role of the former in protecting the host from parasite infection. These studies will outline the major immune pathways and interactions by which insects and their gut microflora respond to kinetoplastid challenge in the midgut.
Summary
For the vast majority of vector borne parasites the ability to overcome the insect midgut defences is central to transmission. However, for many such diseases we know virtually nothing about the molecular mechanisms involved. For vectors such as tsetse flies and sand flies the prospects for rapidly improving our understanding of key interactions occurring in the midgut when challenged by parasites is bleak. This is because the ‘tool box’ required untangling the interactions is very unlikely to be rapidly developed. For example, there is no realistic prospect of producing transgenic technology for tsetse flies because eggs are inaccessible due to intrauterine development of larvae; maintenance of multiple lines of either sand or tsetse flies permitting genetic studies is impossible because of the cost and complexity of culturing colonies; bioinformatic resources are still in their infancy. In this application we suggest that under these circumstances a comparative approach, in which kinetoplastid interactions in Drosophila melanogaster are studied in the first instance, will permit us to make significant progress in understanding the more important cases of insect-parasite interactions (Trypanosome brucei spp in tsetse and Leishmania in sandflies). Herpetomonas ampelophilae is a natural kinetoplastid parasite of Drosophila melanogaster, which establishes infection in the midgut of the fruit fly and can go on to invade the salivary glands. We now have this protozoan in culture and intend, through a combination of genomics, cell biology and RNAi experiments to identify the gut-specific host genomic contingent involved in parasite challenge. In addition, we will study the interaction between the indigenous flora and the parasite and the role of the former in protecting the host from parasite infection. These studies will outline the major immune pathways and interactions by which insects and their gut microflora respond to kinetoplastid challenge in the midgut.
Max ERC Funding
1 110 126 €
Duration
Start date: 2013-03-01, End date: 2018-02-28
Project acronym ENDOTOX
Project Endosomal dependent transport to the unique secretory organelles of apicomplexan parasites
Researcher (PI) Markus Meissner
Host Institution (HI) UNIVERSITY OF GLASGOW
Call Details Starting Grant (StG), LS6, ERC-2012-StG_20111109
Summary Apicomplexan parasites invade the host cell in an active process that involves gliding motility, formation and movement through the moving junction (MJ) and establishment of a prasitophorous vacuole (PV) around the parasite. In order to invade the host cell they employ an arsenal of virulence factors derived from the specialised secretory organelles; the micronemes, rhoptries and dense granules that are sequentially secreted during the invasion process.
While the content of the secretory organelles has been well described and individual virulence factors have been the focus of research, our knowledge on the evolution, biogenesis, maintenance and regulation of these unique organelles is incomplete. Our recent research has established endosomal-like compartments as a key organelle for the specific sorting to micronemes and rhoptries.
The goals of this research project are to i) Analyse biogenesis and segregation of endosomal-like compartments during parasite development within the host cell, ii) Analyse the content of endosomal-like compartments, iii) Systematically dissect the pathways involved in the organisation of the endosomal-like compartments and therefore in the biogenesis, maintenance and regulation of micronemes and rhoptries and iv) Compare the function of identified key factors in other apicomplexans, such as Plasmodium falciparum, the causative agent of malaria.
The evolution of these specialised secretory organelles as an adaptation to an intracellular life style is not only a fascinating, unique feature of apicomplexan parasites that deserves detailed characterisation, but will lead to the identification of novel pathways and interference with these will also represent a new treatment option against these pathogens.
Summary
Apicomplexan parasites invade the host cell in an active process that involves gliding motility, formation and movement through the moving junction (MJ) and establishment of a prasitophorous vacuole (PV) around the parasite. In order to invade the host cell they employ an arsenal of virulence factors derived from the specialised secretory organelles; the micronemes, rhoptries and dense granules that are sequentially secreted during the invasion process.
While the content of the secretory organelles has been well described and individual virulence factors have been the focus of research, our knowledge on the evolution, biogenesis, maintenance and regulation of these unique organelles is incomplete. Our recent research has established endosomal-like compartments as a key organelle for the specific sorting to micronemes and rhoptries.
The goals of this research project are to i) Analyse biogenesis and segregation of endosomal-like compartments during parasite development within the host cell, ii) Analyse the content of endosomal-like compartments, iii) Systematically dissect the pathways involved in the organisation of the endosomal-like compartments and therefore in the biogenesis, maintenance and regulation of micronemes and rhoptries and iv) Compare the function of identified key factors in other apicomplexans, such as Plasmodium falciparum, the causative agent of malaria.
The evolution of these specialised secretory organelles as an adaptation to an intracellular life style is not only a fascinating, unique feature of apicomplexan parasites that deserves detailed characterisation, but will lead to the identification of novel pathways and interference with these will also represent a new treatment option against these pathogens.
Max ERC Funding
1 497 083 €
Duration
Start date: 2013-02-01, End date: 2019-01-31
