Molecular Microbiology (Dehio)Head of Research Unit Prof. Dr.Christoph DehioOverviewMembersPublicationsProjects & CollaborationsProjects & Collaborations OverviewMembersPublicationsProjects & Collaborations Projects & Collaborations 15 foundShow per page10 10 20 50 Providing immunocompetence to human microtissue bladder models Research Project | 1 Project MembersImported from Grants Tool 4705864 A human bladder model to decipher host-uropathogen interactions from tissue to single-cells Research Project | 2 Project MembersImported from Grants Tool 4705120 Bacterial Type IV Secretion (T4S): Cellular, Molecular and Evolutionary Basis of the Subversion of Host Cell Functions by Translocated Effector Proteins Research Project | 2 Project MembersIn the ongoing funding period of SNSF grant 31003A-173119 we are studying the cellular, molecular and evolutionary basis of subversion of host cell functions by type IV secretion (T4S) effector proteins during chronic bacterial infection by the related zoonotic pathogens Bartonella and Brucella. The three-year pro rata temporis extension in the frame of the excellence grant will allow us to extend our studies towards a better understanding (i) of the evolutionary origin and function of these host cell-targeted effector proteins and (ii) of the nature of their signal for T4S-dependent translocation. In Subproject A we will continue the functional characterization of Bartonella effector proteins (Beps) translocated by the VirB T4S system and the structure/function analysis of the BID domain as principle constituent of their T4S signal. Furthermore, we will extend our multidisciplinary studies to an unrelated class of newly identified T4S effectors in Bartonella with homology to a family of widespread type III secretion (T3S) effectors. Furthermore, we will study the role of the Bartonella Gene Transfer Agent (BaGTA) in facilitating adaptive evolution of Beps and associated virulence factors in processes like host adaptation. Subproject B will focus on the functional analysis of effector proteins translocated by the distinct VirB T4S system of Brucella with particular emphasis on delineating the unknown T4S signal. Together, these studies will contribute fundamentally to our molecular understanding of the widespread T4S mechanism in the context of chronic bacterial infections. NCCR AntiResist: New approaches to combat antibiotic-resistant bacteria Umbrella Project | 32 Project MembersAntibiotics are powerful and indispensable drugs to treat life threatening bacterial infections such as sepsis or pneumonia. Antibiotics also play a central role in many other areas of modern medicine, in particular to protect patients with compromised immunity during cancer therapies, transplantations or surgical interventions. These achievements are now at risk, with the fraction of bacterial pathogens that are resistant to one or more antibiotics steadily increasing. In addition, development of novel antimicrobials lags behind, suffering from inherently high attrition rates in particular for drug candidates against the most problematic Gram-negative bacteria. Together, these factors increasingly limit the options clinicians have for treating bacterial infections. The overarching goal of NCCR AntiResist is to elucidate the physiological properties of bacterial pathogens in infected human patients in order to find new ways of combatting superbugs. Among the many societal, economic, and scientific factors that impact on the development of alternative strategies for antibiotic discovery, our limited understanding of the physiology and heterogeneity of bacterial pathogens in patients ranks highly. Bacteria growing in tissues of patients experience environments very different from standard laboratory conditions, resulting in radically different microbial physiology and population heterogeneity compared to conditions generally used for antibacterial discovery. There is currently no systematic strategy to overcome this fundamental problem. This has resulted in: (i) suboptimal screens that identify new antibiotics, which do not target the special properties of bacteria growing within the patient; (ii) an inability to properly evaluate the efficacy of non-conventional antibacterial strategies; (iii) missed opportunities for entirely new treatment strategies. This NCCR utilizes patient samples from ongoing clinical studies and establishes a unique multidisciplinary network of clinicians, biologists, engineers, chemists, computational scientists and drug developers that will overcome this problem. We are excited to merge these disciplines in order to determine the properties of pathogens infecting patients, establish conditions in the lab that reproduce these properties and utilize these in-vitro models for antimicrobial discovery and development. In addition, clinical-trial networks and the pharmaceutical industry have major footprints in antimicrobial R&D. Exploiting synergies between these players has great potential for making transformative progress in this critical field of human health. This NCCR maintains active collaborations with Biotech SMEs and large pharmaceutical companies with the goal to: accelerate antibiotic discovery by providing relevant read-outs for early prioritization of compounds; enable innovative screens for non-canonical strategies such as anti-virulence inhibitors and immunomodulators; identify new antibacterial strategies that effectively combat bacteria either by targeting refractory subpopulations or by synergizing with bacterial stresses imposed by the patients' own immune system. This NCCR proposes a paradigm shift in antibiotic discovery by investigating the physiology of bacterial pathogens in human patients. This knowledge will be used to develop assays for molecular analyses and drug screening under relevant conditions and to accelerate antibacterial discovery, improve treatment regimens, and uncover novel targets for eradicating pathogens. Through this concerted effort, this NCCR will make a crucial and unique contribution to winning the race against superbugs. Bacterial Type IV Secretion (T4S): Cellular, Molecular and Evolutionary Basis of the Subversion of Host Cell Functions by Translocated Effector Proteins Research Project | 4 Project MembersThe type IV secretion (T4S) systems of Gram-negative bacteria are versatile nanomachines ancestrally related to bacterial conjugation systems. Numerous bacterial pathogens targeting eukaryotic host cells have adopted these supramolecular protein assemblies for the intracellular delivery of bacterial effector proteins from the bacterial cytoplasm directly into the host cell cytoplasm. We are using zoonotic pathogens belonging to the closely related genus Bartonella (causing bartonellosis) and Brucella (causing brucellosis) to address fundamental questions related to the roles of T4S systems and their effector proteins in the establishment of chronic bacterial infection.Over the past 16 years we have - with support from the SNSF (grants 61777, 109925, 132979, and 149886) - established Bartonella as a powerful model for studying the cellular, molecular and evolutionary basis of T4S in bacterial pathogenesis. In early studies we have shown that the VirB T4S system represents an essential virulence device that translocates a cocktail of Bartonella effector proteins (Beps) into mammalian host cells, which subverts multiple cellular functions that facilitate chronic infection. We have then functionally characterized the bipartite secretion signal of Beps composed of a C-terminal BID domain and a charged tail. In recent years, we have assigned physiological functions to several Beps, identified some of their host cellular targets and performed corresponding structure-function analysis. We have also shown that all Beps are derived from a single ancestral effector that resulted from the fusion of a FIC domain derived from a bacterial toxin-antitoxin system that mediates AMPylation of target proteins and a BID domain derived from the secreted substrate (relaxase) of a conjugation system. We have further shown that independent Bep arsenals evolved in parallel in three Bartonella sublineages by gene duplication and diversification events, eventually resulting in Bep arsenals that facilitated adaptation of the host-restricted bartonellae to novel mammalian hosts. In the frame of the proposed project (subproject A), we want to deepen our understanding of the molecular functions of representatives of the growing repertoire of Beps by identifying their host targets and performing molecular and structure-function analysis. A major goal will be to understand the functional versatility of the limited set of Bep effector domains - FIC, BID and phosphorylated tyrosine arrays - to subvert a wide spectrum of host functions. Moreover, we want to characterize the physiological functions of representative Beps during infection using cell culture and animal infection models, with a focus of understanding how they facilitate evasion of innate immune responses by the pathogen and support bacterial spreading from the dermal infection site towards the replicative niches in deep tissues and blood.The Brucella project - funded by SNSF grants 132979 and 149886 - was initiated six years ago as a new research line. We are studying the role of the T4S system and its effectors in trafficking of the Brucella containing vacuole (BCV) and the establishment of an intracellular replication niche in the endoplasmic reticulum (ER). We have shown that the T4S system-dependent escape of the early BCV from the degradative endocytic network and its trafficking towards the ER depends on retrograde endosome-to-Golgi trafficking pathways. Moreover, using yeast as surrogate model we have been able to map the wiring of T4S effectors to conserved eukaryotic signaling and trafficking pathways and we identified candidates of some of their mammalian target proteins by yeast two-hybrid screens. In the frame of the proposed project (subproject B) we intend to (i) refine our understanding of the T4S-dependent intracellular trafficking route of the BCV towards the replicative niche in the ER and (ii) study the molecular functions of individual T4S effectors in this process. Due to the small size of the biosafety 3 (BSL3) laboratory presently used by us at the nearby Swiss TPH Institute this project will remain a rather small activity and limited to cell culture infection models until 2018 when we will move into the new Biozentrum building with its state-of-the-art BSL3 facility that will allow us to significantly expand our activities in this project, including animal experimentation. Bacterial Type IV Secretion (T4S): Cellular, Molecular, and Evolutionary Basis of the Subversion of Host Cell Functions by Translocated Effector Proteins Research Project | 9 Project MembersThe type IV secretion (T4S) systems of Gram-negative bacteria are versatile nanomachines ancestrally related to bacterial conjugation systems. Numerous bacterial pathogens targeting eukaryotic host cells have adopted these supramolecular protein assemblies for the intracellular delivery of bacterial effector proteins from the bacterial cytoplasm directly into the host cell cytoplasm. We are using zoonotic pathogens belonging to the closely related genus Bartonella (causing bartonellosis) and Brucella (causing brucellosis) to address fundamental questions related to the roles of T4S systems and their effector proteins in the establishment of chronic bacterial infection. Over the past 16 years we have established Bartonella as a powerful model for studying the cellular, molecular and evolutionary basis of T4S in bacterial pathogenesis. In early studies we have shown that the VirB T4S system represents an essential virulence device that translocates a cocktail of Bartonella effector proteins (Beps) into mammalian host cells, which subverts multiple cellular functions that facilitate chronic infection. We have then functionally characterized the bipartite secretion signal of Beps composed of a C-terminal BID domain and a charged tail. In recent years, we have assigned physiological functions to several Beps, identified some of their host cellular targets and performed corresponding structure-function analysis. We have also shown that all Beps are derived from a single ancestral effector that resulted from the fusion of a FIC domain derived from a bacterial toxin-antitoxin system that mediates AMPylation of target proteins and a BID domain derived from the secreted substrate (relaxase) of a conjugation system. We have further shown that independent Bep arsenals evolved in parallel in three Bartonella sublineages by gene duplication and diversification events, eventually resulting in Bep arsenals that facilitated adaptation of the host-restricted bartonellae to novel mammalian hosts. In the frame of the proposed project (subproject A), we want to deepen our understanding of the molecular functions of representatives of the growing repertoire of Beps by identifying their host targets and performing molecular and structure-function analysis. A major goal will be to understand the functional versatility of the limited set of Bep effector domains - FIC, BID and phosphorylated tyrosine arrays - to subvert a wide spectrum of host functions. Moreover, we want to characterize the physiological functions of representative Beps during infection using cell culture and animal infection models, with a focus of understanding how they facilitate evasion of innate immune responses by the pathogen and support bacterial spreading from the dermal infection site towards the replicative niches in deep tissues and blood. FIC-Mediated Posttranslational Modifications at the Pathogen-Host Interface: From Studying FIC Structure, Function and Role in Pathogen-Host Interaction to the Engineering of Synthetic Activities (Akronym: FICModFun) Research Project | 6 Project MembersThe ubiquitous FIC domain catalyzes post-translational modifications (PTMs) of target proteins; i.e. adenylylation (=AMPylation) and, more rarely, uridylylation and phosphocholination. Fic proteins are thought to play critical roles in intrinsic signaling processes of prokaryotes and eukaryotes; however, a subset encoded by bacterial pathogens is translocated via dedicated secretion systems into the cytoplasm of mammalian host cells. Some of these host-targeted Fic proteins modify small GTPases leading to collapse of the actin cytoskeleton and other drastic cellular changes. Recently, we described a large set of functionally diverse homologues in pathogens of the genus Bartonella that are required for their "stealth attack" strategy and persistent course of infection [1, 2]. Our preliminary functional analysis of some of these host-targeted Fic proteins of Bartonella demonstrated adenylylation activity towards novel host targets (e.g. tubulin and vimentin). Moreover, in addition to the canonical adenylylation activity they may also display a competing kinase activity resulting from altered ATP binding to the FIC active site. Finally, we described a conserved mechanism of FIC active site auto- inhibition that is relieved by a single amino acid exchange [1], thus facilitating functional analysis of any Fic protein of interest. Despite this recent progress only a few Fic proteins have been functionally characterized to date; our understanding of the functional plasticity of the FIC domain in mediating diverse target PTMs and their specific roles in infection thus remains limited. In this project, we aim to study the vast repertoire of host-targeted Fic proteins of Bartonella to: 1) identify novel target proteins and types of PTMs; 2) study their physiological consequences and molecular mechanisms of action; and 3) analyze structure-function relationships critical for FIC-mediated PTMs and infer from these data determinants of target specificity, type of PTM and mode of regulation. At the forefront of infection biology research, this project is ground-breaking as (i) we will identify a plethora of novel host target PTMs that are critical for a "stealth attack" infection strategy and thus will open new avenues for investigating fundamental mechanisms of persistent infection; and (ii), we will unveil the molecular basis of the remarkable functional versatility of the structurally conserved FIC domain. TargetInfectX - Multi-Pronged Perturbation of Pathogen Infection in Human Cells Research Project | 21 Project MembersTo uncover the human protein network underlying infection and to identify targets for novel host-directed anti‐infectives, InfectX (2009‐2013) employed RNA interference (RNAi) screens. In that project, libraries of small inhibitory RNAs (siRNAs) that systematically target a large fraction of human genes were screened, and the effects of individual siRNA perturbations on the cellular infection processes by various human pathogens were measured. The image‐based high‐content RNAi datasets that were generated through a standardized experimental and computational approach are of unprecedented quality and breadth and should thus enable systems‐level analyses of general gene‐phenotype relationships. SiRNAs are designed to be perfectly complementary to 19‐ 23 nucleotide regions in the intended mRNA targets. As siRNA targets undergo degradation upon siRNA transfection, it is generally assumed that the observed phenotypes are due to the direct effect of the siRNA on these targets ('on‐target effect'). However, our systematic exploration of the impact of siRNA‐based RNAi on two phenotypic readouts (cell number and infection index) in InfectX has highlighted the prevalence and magnitude of 'off‐target effects' that are mediated by the siRNA 'seed sequences' (nucleotides 2‐8 from the 5' end of the siRNA) through a microRNA (miRNA)‐type mechanism. A major contribution that InfectX made to the RNAi screening field was the development of modeling approaches to comprehensively quantify off‐target effects, partly deconvolute combinatorial effects, and finally provide improved methods for establishing individual gene‐phenotype relationships. The present proposal, TargetInfectX (2014‐2017), will build on the image‐based high‐content RNAi datasets established within InfectX , but will have more far‐reaching goals. From the image data generated in InfectX we will extract a rich set of phenotypic features on the single‐cell level. Modeling miRNAtarget mRNA interactions, their effect on gene expression, and the gene‐phenotype relationships emerging from the imaging data, we will attempt to generally reconstruct the genotypic basis of elementary cell behaviors such as those involved in the response to pathogen intrusion. On the translational side, we will explore the potential of siRNAs as novel combination therapy approach to interfere with the course of infections. Moreover, beyond the public release of our data via publications and publically accessible databases, we will encourage knowledge and technology transfer of unpublished work in progress with partners outside of TargetInfectX via a collaboration suite that should foster long‐term collaborations and ensure the impact of our work beyond SystemsX.ch . Bacterial Type IV Secretion (T4S): Cellular, Molecular, and Evolutionary Basis of the Subversion of Host Cell Functions by Translocated Effector Proteins Research Project | 4 Project MembersIn the initial funding period of the SNSF grant 31003A-132979 we used Bartonella and Brucella - two related zoonotic pathogens engaging the widespread type IV secretion (T4S) mechanism for establishing chronic bacterial infection - as models to study the cellular, molecular and evolutionary basis of the subversion of host cell functions by T4S-translocated effector proteins. For the three year prolongation period the established multidisciplinary experimental approach will be extended by adopting yeast as surrogate model for studying conserved eukaryotic processes targeted by T4S effectors. Subproject A will focus on the structure/function analysis of Bartonella effector proteins (Beps) translocated by the VirB T4S system of Bartonella and their physiological consequences on the host, with particular emphasis on studying the subversion of immune signaling processes. Subproject B will focus on the functional analysis of effector proteins translocated by the distinct VirB T4S system of Brucella and their physiological consequences on host cell interaction, with particular emphasis on studying the intracellular trafficking events from a late endosomal compartment, where the VirB system is activated in response to acidification, to the endoplasmic reticulum (ER)-related replicative niche of Brucella. Together, these studies will contribute to establishing a molecular paradigm for the role of T4S effectors in triggering chronic bacterial infection. Bacterial Type IV Secretion: Cellular, Molecular, and Evolutionary Basis of the Subversion of Host Cell Functions by Translocated Effector Proteins. Research Project | 1 Project MembersThe type IV secretion (T4S) systems of gram-negative bacteria are versatile nano-machines involved in processes relevant to bacterial infection, such as horizontal transfer of virulence and antibiotic resistance genes between bacteria, and the translocation of bacterial effector proteins into eukaryotic target cells. Effector-translocating T4S systems are widely distributed among human pathogenic bacteria that are mostly causing chronic infections, including Helicobacter pylori (causing gastritis and gastric cancer), Legionella pneumophila (causing Legionnaires disease), Bartonella spp. (causing bartonellosis) and Brucella spp. (causing brucellosis). In recent years we have - with support from the SNF (grants 3100-061777 and 31003A-109925) - established Bartonella as a powerful model for studying the cellular, molecular and evolutionary basis of T4S in bacterial pathogenesis. In the frame of the proposed project we will continue to explore this paradigmatic model of T4S. Moreover, we will expand the successful approach for studying T4S as established for Bartonella to the closely related pathogen Brucella - the etiological agent of the most important bacterial zoonosis worldwide that causes chronic infections in various mammals. We will apply a multidisciplinary experimental approach including bacterial genetics, cell biology, molecular biology, biochemistry, structural biology, genomics, and animal experimentation. In subproject A targeting T4S in Bartonella, we will perform a structure/function analysis of the T4S system-translocated effector proteins and their cellular targets, and study molecular evolutionary mechanisms of host adaptation mediated by these effectors. In subproject B targeting T4S in Brucella , we will focus on the characterization of the T4S-dependent intracellular trafficking route of Brucella resulting in the establishment of an endoplasmic reticulum-associated replication niche, the systematic identification of the involved host factors and the structure/function analysis of the T4S effectors interacting with the identified host factors. By studying the molecular mechanism of T4S in the closely related pathogens Bartonella and Brucella , we anticipate to provide fundamental contributions to our understanding how this wide-spread virulence mechanism facilitates chronic bacterial infection, which may also help to design novel strategies to combat bacterial virulence. Based on the specific subversion of multiple cellular functions by the T4S effectors of these pathogens we also expect to contribute new basic knowledge and novel tools to different fields of cell biology. 12 12 OverviewMembersPublicationsProjects & Collaborations
Projects & Collaborations 15 foundShow per page10 10 20 50 Providing immunocompetence to human microtissue bladder models Research Project | 1 Project MembersImported from Grants Tool 4705864 A human bladder model to decipher host-uropathogen interactions from tissue to single-cells Research Project | 2 Project MembersImported from Grants Tool 4705120 Bacterial Type IV Secretion (T4S): Cellular, Molecular and Evolutionary Basis of the Subversion of Host Cell Functions by Translocated Effector Proteins Research Project | 2 Project MembersIn the ongoing funding period of SNSF grant 31003A-173119 we are studying the cellular, molecular and evolutionary basis of subversion of host cell functions by type IV secretion (T4S) effector proteins during chronic bacterial infection by the related zoonotic pathogens Bartonella and Brucella. The three-year pro rata temporis extension in the frame of the excellence grant will allow us to extend our studies towards a better understanding (i) of the evolutionary origin and function of these host cell-targeted effector proteins and (ii) of the nature of their signal for T4S-dependent translocation. In Subproject A we will continue the functional characterization of Bartonella effector proteins (Beps) translocated by the VirB T4S system and the structure/function analysis of the BID domain as principle constituent of their T4S signal. Furthermore, we will extend our multidisciplinary studies to an unrelated class of newly identified T4S effectors in Bartonella with homology to a family of widespread type III secretion (T3S) effectors. Furthermore, we will study the role of the Bartonella Gene Transfer Agent (BaGTA) in facilitating adaptive evolution of Beps and associated virulence factors in processes like host adaptation. Subproject B will focus on the functional analysis of effector proteins translocated by the distinct VirB T4S system of Brucella with particular emphasis on delineating the unknown T4S signal. Together, these studies will contribute fundamentally to our molecular understanding of the widespread T4S mechanism in the context of chronic bacterial infections. NCCR AntiResist: New approaches to combat antibiotic-resistant bacteria Umbrella Project | 32 Project MembersAntibiotics are powerful and indispensable drugs to treat life threatening bacterial infections such as sepsis or pneumonia. Antibiotics also play a central role in many other areas of modern medicine, in particular to protect patients with compromised immunity during cancer therapies, transplantations or surgical interventions. These achievements are now at risk, with the fraction of bacterial pathogens that are resistant to one or more antibiotics steadily increasing. In addition, development of novel antimicrobials lags behind, suffering from inherently high attrition rates in particular for drug candidates against the most problematic Gram-negative bacteria. Together, these factors increasingly limit the options clinicians have for treating bacterial infections. The overarching goal of NCCR AntiResist is to elucidate the physiological properties of bacterial pathogens in infected human patients in order to find new ways of combatting superbugs. Among the many societal, economic, and scientific factors that impact on the development of alternative strategies for antibiotic discovery, our limited understanding of the physiology and heterogeneity of bacterial pathogens in patients ranks highly. Bacteria growing in tissues of patients experience environments very different from standard laboratory conditions, resulting in radically different microbial physiology and population heterogeneity compared to conditions generally used for antibacterial discovery. There is currently no systematic strategy to overcome this fundamental problem. This has resulted in: (i) suboptimal screens that identify new antibiotics, which do not target the special properties of bacteria growing within the patient; (ii) an inability to properly evaluate the efficacy of non-conventional antibacterial strategies; (iii) missed opportunities for entirely new treatment strategies. This NCCR utilizes patient samples from ongoing clinical studies and establishes a unique multidisciplinary network of clinicians, biologists, engineers, chemists, computational scientists and drug developers that will overcome this problem. We are excited to merge these disciplines in order to determine the properties of pathogens infecting patients, establish conditions in the lab that reproduce these properties and utilize these in-vitro models for antimicrobial discovery and development. In addition, clinical-trial networks and the pharmaceutical industry have major footprints in antimicrobial R&D. Exploiting synergies between these players has great potential for making transformative progress in this critical field of human health. This NCCR maintains active collaborations with Biotech SMEs and large pharmaceutical companies with the goal to: accelerate antibiotic discovery by providing relevant read-outs for early prioritization of compounds; enable innovative screens for non-canonical strategies such as anti-virulence inhibitors and immunomodulators; identify new antibacterial strategies that effectively combat bacteria either by targeting refractory subpopulations or by synergizing with bacterial stresses imposed by the patients' own immune system. This NCCR proposes a paradigm shift in antibiotic discovery by investigating the physiology of bacterial pathogens in human patients. This knowledge will be used to develop assays for molecular analyses and drug screening under relevant conditions and to accelerate antibacterial discovery, improve treatment regimens, and uncover novel targets for eradicating pathogens. Through this concerted effort, this NCCR will make a crucial and unique contribution to winning the race against superbugs. Bacterial Type IV Secretion (T4S): Cellular, Molecular and Evolutionary Basis of the Subversion of Host Cell Functions by Translocated Effector Proteins Research Project | 4 Project MembersThe type IV secretion (T4S) systems of Gram-negative bacteria are versatile nanomachines ancestrally related to bacterial conjugation systems. Numerous bacterial pathogens targeting eukaryotic host cells have adopted these supramolecular protein assemblies for the intracellular delivery of bacterial effector proteins from the bacterial cytoplasm directly into the host cell cytoplasm. We are using zoonotic pathogens belonging to the closely related genus Bartonella (causing bartonellosis) and Brucella (causing brucellosis) to address fundamental questions related to the roles of T4S systems and their effector proteins in the establishment of chronic bacterial infection.Over the past 16 years we have - with support from the SNSF (grants 61777, 109925, 132979, and 149886) - established Bartonella as a powerful model for studying the cellular, molecular and evolutionary basis of T4S in bacterial pathogenesis. In early studies we have shown that the VirB T4S system represents an essential virulence device that translocates a cocktail of Bartonella effector proteins (Beps) into mammalian host cells, which subverts multiple cellular functions that facilitate chronic infection. We have then functionally characterized the bipartite secretion signal of Beps composed of a C-terminal BID domain and a charged tail. In recent years, we have assigned physiological functions to several Beps, identified some of their host cellular targets and performed corresponding structure-function analysis. We have also shown that all Beps are derived from a single ancestral effector that resulted from the fusion of a FIC domain derived from a bacterial toxin-antitoxin system that mediates AMPylation of target proteins and a BID domain derived from the secreted substrate (relaxase) of a conjugation system. We have further shown that independent Bep arsenals evolved in parallel in three Bartonella sublineages by gene duplication and diversification events, eventually resulting in Bep arsenals that facilitated adaptation of the host-restricted bartonellae to novel mammalian hosts. In the frame of the proposed project (subproject A), we want to deepen our understanding of the molecular functions of representatives of the growing repertoire of Beps by identifying their host targets and performing molecular and structure-function analysis. A major goal will be to understand the functional versatility of the limited set of Bep effector domains - FIC, BID and phosphorylated tyrosine arrays - to subvert a wide spectrum of host functions. Moreover, we want to characterize the physiological functions of representative Beps during infection using cell culture and animal infection models, with a focus of understanding how they facilitate evasion of innate immune responses by the pathogen and support bacterial spreading from the dermal infection site towards the replicative niches in deep tissues and blood.The Brucella project - funded by SNSF grants 132979 and 149886 - was initiated six years ago as a new research line. We are studying the role of the T4S system and its effectors in trafficking of the Brucella containing vacuole (BCV) and the establishment of an intracellular replication niche in the endoplasmic reticulum (ER). We have shown that the T4S system-dependent escape of the early BCV from the degradative endocytic network and its trafficking towards the ER depends on retrograde endosome-to-Golgi trafficking pathways. Moreover, using yeast as surrogate model we have been able to map the wiring of T4S effectors to conserved eukaryotic signaling and trafficking pathways and we identified candidates of some of their mammalian target proteins by yeast two-hybrid screens. In the frame of the proposed project (subproject B) we intend to (i) refine our understanding of the T4S-dependent intracellular trafficking route of the BCV towards the replicative niche in the ER and (ii) study the molecular functions of individual T4S effectors in this process. Due to the small size of the biosafety 3 (BSL3) laboratory presently used by us at the nearby Swiss TPH Institute this project will remain a rather small activity and limited to cell culture infection models until 2018 when we will move into the new Biozentrum building with its state-of-the-art BSL3 facility that will allow us to significantly expand our activities in this project, including animal experimentation. Bacterial Type IV Secretion (T4S): Cellular, Molecular, and Evolutionary Basis of the Subversion of Host Cell Functions by Translocated Effector Proteins Research Project | 9 Project MembersThe type IV secretion (T4S) systems of Gram-negative bacteria are versatile nanomachines ancestrally related to bacterial conjugation systems. Numerous bacterial pathogens targeting eukaryotic host cells have adopted these supramolecular protein assemblies for the intracellular delivery of bacterial effector proteins from the bacterial cytoplasm directly into the host cell cytoplasm. We are using zoonotic pathogens belonging to the closely related genus Bartonella (causing bartonellosis) and Brucella (causing brucellosis) to address fundamental questions related to the roles of T4S systems and their effector proteins in the establishment of chronic bacterial infection. Over the past 16 years we have established Bartonella as a powerful model for studying the cellular, molecular and evolutionary basis of T4S in bacterial pathogenesis. In early studies we have shown that the VirB T4S system represents an essential virulence device that translocates a cocktail of Bartonella effector proteins (Beps) into mammalian host cells, which subverts multiple cellular functions that facilitate chronic infection. We have then functionally characterized the bipartite secretion signal of Beps composed of a C-terminal BID domain and a charged tail. In recent years, we have assigned physiological functions to several Beps, identified some of their host cellular targets and performed corresponding structure-function analysis. We have also shown that all Beps are derived from a single ancestral effector that resulted from the fusion of a FIC domain derived from a bacterial toxin-antitoxin system that mediates AMPylation of target proteins and a BID domain derived from the secreted substrate (relaxase) of a conjugation system. We have further shown that independent Bep arsenals evolved in parallel in three Bartonella sublineages by gene duplication and diversification events, eventually resulting in Bep arsenals that facilitated adaptation of the host-restricted bartonellae to novel mammalian hosts. In the frame of the proposed project (subproject A), we want to deepen our understanding of the molecular functions of representatives of the growing repertoire of Beps by identifying their host targets and performing molecular and structure-function analysis. A major goal will be to understand the functional versatility of the limited set of Bep effector domains - FIC, BID and phosphorylated tyrosine arrays - to subvert a wide spectrum of host functions. Moreover, we want to characterize the physiological functions of representative Beps during infection using cell culture and animal infection models, with a focus of understanding how they facilitate evasion of innate immune responses by the pathogen and support bacterial spreading from the dermal infection site towards the replicative niches in deep tissues and blood. FIC-Mediated Posttranslational Modifications at the Pathogen-Host Interface: From Studying FIC Structure, Function and Role in Pathogen-Host Interaction to the Engineering of Synthetic Activities (Akronym: FICModFun) Research Project | 6 Project MembersThe ubiquitous FIC domain catalyzes post-translational modifications (PTMs) of target proteins; i.e. adenylylation (=AMPylation) and, more rarely, uridylylation and phosphocholination. Fic proteins are thought to play critical roles in intrinsic signaling processes of prokaryotes and eukaryotes; however, a subset encoded by bacterial pathogens is translocated via dedicated secretion systems into the cytoplasm of mammalian host cells. Some of these host-targeted Fic proteins modify small GTPases leading to collapse of the actin cytoskeleton and other drastic cellular changes. Recently, we described a large set of functionally diverse homologues in pathogens of the genus Bartonella that are required for their "stealth attack" strategy and persistent course of infection [1, 2]. Our preliminary functional analysis of some of these host-targeted Fic proteins of Bartonella demonstrated adenylylation activity towards novel host targets (e.g. tubulin and vimentin). Moreover, in addition to the canonical adenylylation activity they may also display a competing kinase activity resulting from altered ATP binding to the FIC active site. Finally, we described a conserved mechanism of FIC active site auto- inhibition that is relieved by a single amino acid exchange [1], thus facilitating functional analysis of any Fic protein of interest. Despite this recent progress only a few Fic proteins have been functionally characterized to date; our understanding of the functional plasticity of the FIC domain in mediating diverse target PTMs and their specific roles in infection thus remains limited. In this project, we aim to study the vast repertoire of host-targeted Fic proteins of Bartonella to: 1) identify novel target proteins and types of PTMs; 2) study their physiological consequences and molecular mechanisms of action; and 3) analyze structure-function relationships critical for FIC-mediated PTMs and infer from these data determinants of target specificity, type of PTM and mode of regulation. At the forefront of infection biology research, this project is ground-breaking as (i) we will identify a plethora of novel host target PTMs that are critical for a "stealth attack" infection strategy and thus will open new avenues for investigating fundamental mechanisms of persistent infection; and (ii), we will unveil the molecular basis of the remarkable functional versatility of the structurally conserved FIC domain. TargetInfectX - Multi-Pronged Perturbation of Pathogen Infection in Human Cells Research Project | 21 Project MembersTo uncover the human protein network underlying infection and to identify targets for novel host-directed anti‐infectives, InfectX (2009‐2013) employed RNA interference (RNAi) screens. In that project, libraries of small inhibitory RNAs (siRNAs) that systematically target a large fraction of human genes were screened, and the effects of individual siRNA perturbations on the cellular infection processes by various human pathogens were measured. The image‐based high‐content RNAi datasets that were generated through a standardized experimental and computational approach are of unprecedented quality and breadth and should thus enable systems‐level analyses of general gene‐phenotype relationships. SiRNAs are designed to be perfectly complementary to 19‐ 23 nucleotide regions in the intended mRNA targets. As siRNA targets undergo degradation upon siRNA transfection, it is generally assumed that the observed phenotypes are due to the direct effect of the siRNA on these targets ('on‐target effect'). However, our systematic exploration of the impact of siRNA‐based RNAi on two phenotypic readouts (cell number and infection index) in InfectX has highlighted the prevalence and magnitude of 'off‐target effects' that are mediated by the siRNA 'seed sequences' (nucleotides 2‐8 from the 5' end of the siRNA) through a microRNA (miRNA)‐type mechanism. A major contribution that InfectX made to the RNAi screening field was the development of modeling approaches to comprehensively quantify off‐target effects, partly deconvolute combinatorial effects, and finally provide improved methods for establishing individual gene‐phenotype relationships. The present proposal, TargetInfectX (2014‐2017), will build on the image‐based high‐content RNAi datasets established within InfectX , but will have more far‐reaching goals. From the image data generated in InfectX we will extract a rich set of phenotypic features on the single‐cell level. Modeling miRNAtarget mRNA interactions, their effect on gene expression, and the gene‐phenotype relationships emerging from the imaging data, we will attempt to generally reconstruct the genotypic basis of elementary cell behaviors such as those involved in the response to pathogen intrusion. On the translational side, we will explore the potential of siRNAs as novel combination therapy approach to interfere with the course of infections. Moreover, beyond the public release of our data via publications and publically accessible databases, we will encourage knowledge and technology transfer of unpublished work in progress with partners outside of TargetInfectX via a collaboration suite that should foster long‐term collaborations and ensure the impact of our work beyond SystemsX.ch . Bacterial Type IV Secretion (T4S): Cellular, Molecular, and Evolutionary Basis of the Subversion of Host Cell Functions by Translocated Effector Proteins Research Project | 4 Project MembersIn the initial funding period of the SNSF grant 31003A-132979 we used Bartonella and Brucella - two related zoonotic pathogens engaging the widespread type IV secretion (T4S) mechanism for establishing chronic bacterial infection - as models to study the cellular, molecular and evolutionary basis of the subversion of host cell functions by T4S-translocated effector proteins. For the three year prolongation period the established multidisciplinary experimental approach will be extended by adopting yeast as surrogate model for studying conserved eukaryotic processes targeted by T4S effectors. Subproject A will focus on the structure/function analysis of Bartonella effector proteins (Beps) translocated by the VirB T4S system of Bartonella and their physiological consequences on the host, with particular emphasis on studying the subversion of immune signaling processes. Subproject B will focus on the functional analysis of effector proteins translocated by the distinct VirB T4S system of Brucella and their physiological consequences on host cell interaction, with particular emphasis on studying the intracellular trafficking events from a late endosomal compartment, where the VirB system is activated in response to acidification, to the endoplasmic reticulum (ER)-related replicative niche of Brucella. Together, these studies will contribute to establishing a molecular paradigm for the role of T4S effectors in triggering chronic bacterial infection. Bacterial Type IV Secretion: Cellular, Molecular, and Evolutionary Basis of the Subversion of Host Cell Functions by Translocated Effector Proteins. Research Project | 1 Project MembersThe type IV secretion (T4S) systems of gram-negative bacteria are versatile nano-machines involved in processes relevant to bacterial infection, such as horizontal transfer of virulence and antibiotic resistance genes between bacteria, and the translocation of bacterial effector proteins into eukaryotic target cells. Effector-translocating T4S systems are widely distributed among human pathogenic bacteria that are mostly causing chronic infections, including Helicobacter pylori (causing gastritis and gastric cancer), Legionella pneumophila (causing Legionnaires disease), Bartonella spp. (causing bartonellosis) and Brucella spp. (causing brucellosis). In recent years we have - with support from the SNF (grants 3100-061777 and 31003A-109925) - established Bartonella as a powerful model for studying the cellular, molecular and evolutionary basis of T4S in bacterial pathogenesis. In the frame of the proposed project we will continue to explore this paradigmatic model of T4S. Moreover, we will expand the successful approach for studying T4S as established for Bartonella to the closely related pathogen Brucella - the etiological agent of the most important bacterial zoonosis worldwide that causes chronic infections in various mammals. We will apply a multidisciplinary experimental approach including bacterial genetics, cell biology, molecular biology, biochemistry, structural biology, genomics, and animal experimentation. In subproject A targeting T4S in Bartonella, we will perform a structure/function analysis of the T4S system-translocated effector proteins and their cellular targets, and study molecular evolutionary mechanisms of host adaptation mediated by these effectors. In subproject B targeting T4S in Brucella , we will focus on the characterization of the T4S-dependent intracellular trafficking route of Brucella resulting in the establishment of an endoplasmic reticulum-associated replication niche, the systematic identification of the involved host factors and the structure/function analysis of the T4S effectors interacting with the identified host factors. By studying the molecular mechanism of T4S in the closely related pathogens Bartonella and Brucella , we anticipate to provide fundamental contributions to our understanding how this wide-spread virulence mechanism facilitates chronic bacterial infection, which may also help to design novel strategies to combat bacterial virulence. Based on the specific subversion of multiple cellular functions by the T4S effectors of these pathogens we also expect to contribute new basic knowledge and novel tools to different fields of cell biology. 12 12
Providing immunocompetence to human microtissue bladder models Research Project | 1 Project MembersImported from Grants Tool 4705864
A human bladder model to decipher host-uropathogen interactions from tissue to single-cells Research Project | 2 Project MembersImported from Grants Tool 4705120
Bacterial Type IV Secretion (T4S): Cellular, Molecular and Evolutionary Basis of the Subversion of Host Cell Functions by Translocated Effector Proteins Research Project | 2 Project MembersIn the ongoing funding period of SNSF grant 31003A-173119 we are studying the cellular, molecular and evolutionary basis of subversion of host cell functions by type IV secretion (T4S) effector proteins during chronic bacterial infection by the related zoonotic pathogens Bartonella and Brucella. The three-year pro rata temporis extension in the frame of the excellence grant will allow us to extend our studies towards a better understanding (i) of the evolutionary origin and function of these host cell-targeted effector proteins and (ii) of the nature of their signal for T4S-dependent translocation. In Subproject A we will continue the functional characterization of Bartonella effector proteins (Beps) translocated by the VirB T4S system and the structure/function analysis of the BID domain as principle constituent of their T4S signal. Furthermore, we will extend our multidisciplinary studies to an unrelated class of newly identified T4S effectors in Bartonella with homology to a family of widespread type III secretion (T3S) effectors. Furthermore, we will study the role of the Bartonella Gene Transfer Agent (BaGTA) in facilitating adaptive evolution of Beps and associated virulence factors in processes like host adaptation. Subproject B will focus on the functional analysis of effector proteins translocated by the distinct VirB T4S system of Brucella with particular emphasis on delineating the unknown T4S signal. Together, these studies will contribute fundamentally to our molecular understanding of the widespread T4S mechanism in the context of chronic bacterial infections.
NCCR AntiResist: New approaches to combat antibiotic-resistant bacteria Umbrella Project | 32 Project MembersAntibiotics are powerful and indispensable drugs to treat life threatening bacterial infections such as sepsis or pneumonia. Antibiotics also play a central role in many other areas of modern medicine, in particular to protect patients with compromised immunity during cancer therapies, transplantations or surgical interventions. These achievements are now at risk, with the fraction of bacterial pathogens that are resistant to one or more antibiotics steadily increasing. In addition, development of novel antimicrobials lags behind, suffering from inherently high attrition rates in particular for drug candidates against the most problematic Gram-negative bacteria. Together, these factors increasingly limit the options clinicians have for treating bacterial infections. The overarching goal of NCCR AntiResist is to elucidate the physiological properties of bacterial pathogens in infected human patients in order to find new ways of combatting superbugs. Among the many societal, economic, and scientific factors that impact on the development of alternative strategies for antibiotic discovery, our limited understanding of the physiology and heterogeneity of bacterial pathogens in patients ranks highly. Bacteria growing in tissues of patients experience environments very different from standard laboratory conditions, resulting in radically different microbial physiology and population heterogeneity compared to conditions generally used for antibacterial discovery. There is currently no systematic strategy to overcome this fundamental problem. This has resulted in: (i) suboptimal screens that identify new antibiotics, which do not target the special properties of bacteria growing within the patient; (ii) an inability to properly evaluate the efficacy of non-conventional antibacterial strategies; (iii) missed opportunities for entirely new treatment strategies. This NCCR utilizes patient samples from ongoing clinical studies and establishes a unique multidisciplinary network of clinicians, biologists, engineers, chemists, computational scientists and drug developers that will overcome this problem. We are excited to merge these disciplines in order to determine the properties of pathogens infecting patients, establish conditions in the lab that reproduce these properties and utilize these in-vitro models for antimicrobial discovery and development. In addition, clinical-trial networks and the pharmaceutical industry have major footprints in antimicrobial R&D. Exploiting synergies between these players has great potential for making transformative progress in this critical field of human health. This NCCR maintains active collaborations with Biotech SMEs and large pharmaceutical companies with the goal to: accelerate antibiotic discovery by providing relevant read-outs for early prioritization of compounds; enable innovative screens for non-canonical strategies such as anti-virulence inhibitors and immunomodulators; identify new antibacterial strategies that effectively combat bacteria either by targeting refractory subpopulations or by synergizing with bacterial stresses imposed by the patients' own immune system. This NCCR proposes a paradigm shift in antibiotic discovery by investigating the physiology of bacterial pathogens in human patients. This knowledge will be used to develop assays for molecular analyses and drug screening under relevant conditions and to accelerate antibacterial discovery, improve treatment regimens, and uncover novel targets for eradicating pathogens. Through this concerted effort, this NCCR will make a crucial and unique contribution to winning the race against superbugs.
Bacterial Type IV Secretion (T4S): Cellular, Molecular and Evolutionary Basis of the Subversion of Host Cell Functions by Translocated Effector Proteins Research Project | 4 Project MembersThe type IV secretion (T4S) systems of Gram-negative bacteria are versatile nanomachines ancestrally related to bacterial conjugation systems. Numerous bacterial pathogens targeting eukaryotic host cells have adopted these supramolecular protein assemblies for the intracellular delivery of bacterial effector proteins from the bacterial cytoplasm directly into the host cell cytoplasm. We are using zoonotic pathogens belonging to the closely related genus Bartonella (causing bartonellosis) and Brucella (causing brucellosis) to address fundamental questions related to the roles of T4S systems and their effector proteins in the establishment of chronic bacterial infection.Over the past 16 years we have - with support from the SNSF (grants 61777, 109925, 132979, and 149886) - established Bartonella as a powerful model for studying the cellular, molecular and evolutionary basis of T4S in bacterial pathogenesis. In early studies we have shown that the VirB T4S system represents an essential virulence device that translocates a cocktail of Bartonella effector proteins (Beps) into mammalian host cells, which subverts multiple cellular functions that facilitate chronic infection. We have then functionally characterized the bipartite secretion signal of Beps composed of a C-terminal BID domain and a charged tail. In recent years, we have assigned physiological functions to several Beps, identified some of their host cellular targets and performed corresponding structure-function analysis. We have also shown that all Beps are derived from a single ancestral effector that resulted from the fusion of a FIC domain derived from a bacterial toxin-antitoxin system that mediates AMPylation of target proteins and a BID domain derived from the secreted substrate (relaxase) of a conjugation system. We have further shown that independent Bep arsenals evolved in parallel in three Bartonella sublineages by gene duplication and diversification events, eventually resulting in Bep arsenals that facilitated adaptation of the host-restricted bartonellae to novel mammalian hosts. In the frame of the proposed project (subproject A), we want to deepen our understanding of the molecular functions of representatives of the growing repertoire of Beps by identifying their host targets and performing molecular and structure-function analysis. A major goal will be to understand the functional versatility of the limited set of Bep effector domains - FIC, BID and phosphorylated tyrosine arrays - to subvert a wide spectrum of host functions. Moreover, we want to characterize the physiological functions of representative Beps during infection using cell culture and animal infection models, with a focus of understanding how they facilitate evasion of innate immune responses by the pathogen and support bacterial spreading from the dermal infection site towards the replicative niches in deep tissues and blood.The Brucella project - funded by SNSF grants 132979 and 149886 - was initiated six years ago as a new research line. We are studying the role of the T4S system and its effectors in trafficking of the Brucella containing vacuole (BCV) and the establishment of an intracellular replication niche in the endoplasmic reticulum (ER). We have shown that the T4S system-dependent escape of the early BCV from the degradative endocytic network and its trafficking towards the ER depends on retrograde endosome-to-Golgi trafficking pathways. Moreover, using yeast as surrogate model we have been able to map the wiring of T4S effectors to conserved eukaryotic signaling and trafficking pathways and we identified candidates of some of their mammalian target proteins by yeast two-hybrid screens. In the frame of the proposed project (subproject B) we intend to (i) refine our understanding of the T4S-dependent intracellular trafficking route of the BCV towards the replicative niche in the ER and (ii) study the molecular functions of individual T4S effectors in this process. Due to the small size of the biosafety 3 (BSL3) laboratory presently used by us at the nearby Swiss TPH Institute this project will remain a rather small activity and limited to cell culture infection models until 2018 when we will move into the new Biozentrum building with its state-of-the-art BSL3 facility that will allow us to significantly expand our activities in this project, including animal experimentation.
Bacterial Type IV Secretion (T4S): Cellular, Molecular, and Evolutionary Basis of the Subversion of Host Cell Functions by Translocated Effector Proteins Research Project | 9 Project MembersThe type IV secretion (T4S) systems of Gram-negative bacteria are versatile nanomachines ancestrally related to bacterial conjugation systems. Numerous bacterial pathogens targeting eukaryotic host cells have adopted these supramolecular protein assemblies for the intracellular delivery of bacterial effector proteins from the bacterial cytoplasm directly into the host cell cytoplasm. We are using zoonotic pathogens belonging to the closely related genus Bartonella (causing bartonellosis) and Brucella (causing brucellosis) to address fundamental questions related to the roles of T4S systems and their effector proteins in the establishment of chronic bacterial infection. Over the past 16 years we have established Bartonella as a powerful model for studying the cellular, molecular and evolutionary basis of T4S in bacterial pathogenesis. In early studies we have shown that the VirB T4S system represents an essential virulence device that translocates a cocktail of Bartonella effector proteins (Beps) into mammalian host cells, which subverts multiple cellular functions that facilitate chronic infection. We have then functionally characterized the bipartite secretion signal of Beps composed of a C-terminal BID domain and a charged tail. In recent years, we have assigned physiological functions to several Beps, identified some of their host cellular targets and performed corresponding structure-function analysis. We have also shown that all Beps are derived from a single ancestral effector that resulted from the fusion of a FIC domain derived from a bacterial toxin-antitoxin system that mediates AMPylation of target proteins and a BID domain derived from the secreted substrate (relaxase) of a conjugation system. We have further shown that independent Bep arsenals evolved in parallel in three Bartonella sublineages by gene duplication and diversification events, eventually resulting in Bep arsenals that facilitated adaptation of the host-restricted bartonellae to novel mammalian hosts. In the frame of the proposed project (subproject A), we want to deepen our understanding of the molecular functions of representatives of the growing repertoire of Beps by identifying their host targets and performing molecular and structure-function analysis. A major goal will be to understand the functional versatility of the limited set of Bep effector domains - FIC, BID and phosphorylated tyrosine arrays - to subvert a wide spectrum of host functions. Moreover, we want to characterize the physiological functions of representative Beps during infection using cell culture and animal infection models, with a focus of understanding how they facilitate evasion of innate immune responses by the pathogen and support bacterial spreading from the dermal infection site towards the replicative niches in deep tissues and blood.
FIC-Mediated Posttranslational Modifications at the Pathogen-Host Interface: From Studying FIC Structure, Function and Role in Pathogen-Host Interaction to the Engineering of Synthetic Activities (Akronym: FICModFun) Research Project | 6 Project MembersThe ubiquitous FIC domain catalyzes post-translational modifications (PTMs) of target proteins; i.e. adenylylation (=AMPylation) and, more rarely, uridylylation and phosphocholination. Fic proteins are thought to play critical roles in intrinsic signaling processes of prokaryotes and eukaryotes; however, a subset encoded by bacterial pathogens is translocated via dedicated secretion systems into the cytoplasm of mammalian host cells. Some of these host-targeted Fic proteins modify small GTPases leading to collapse of the actin cytoskeleton and other drastic cellular changes. Recently, we described a large set of functionally diverse homologues in pathogens of the genus Bartonella that are required for their "stealth attack" strategy and persistent course of infection [1, 2]. Our preliminary functional analysis of some of these host-targeted Fic proteins of Bartonella demonstrated adenylylation activity towards novel host targets (e.g. tubulin and vimentin). Moreover, in addition to the canonical adenylylation activity they may also display a competing kinase activity resulting from altered ATP binding to the FIC active site. Finally, we described a conserved mechanism of FIC active site auto- inhibition that is relieved by a single amino acid exchange [1], thus facilitating functional analysis of any Fic protein of interest. Despite this recent progress only a few Fic proteins have been functionally characterized to date; our understanding of the functional plasticity of the FIC domain in mediating diverse target PTMs and their specific roles in infection thus remains limited. In this project, we aim to study the vast repertoire of host-targeted Fic proteins of Bartonella to: 1) identify novel target proteins and types of PTMs; 2) study their physiological consequences and molecular mechanisms of action; and 3) analyze structure-function relationships critical for FIC-mediated PTMs and infer from these data determinants of target specificity, type of PTM and mode of regulation. At the forefront of infection biology research, this project is ground-breaking as (i) we will identify a plethora of novel host target PTMs that are critical for a "stealth attack" infection strategy and thus will open new avenues for investigating fundamental mechanisms of persistent infection; and (ii), we will unveil the molecular basis of the remarkable functional versatility of the structurally conserved FIC domain.
TargetInfectX - Multi-Pronged Perturbation of Pathogen Infection in Human Cells Research Project | 21 Project MembersTo uncover the human protein network underlying infection and to identify targets for novel host-directed anti‐infectives, InfectX (2009‐2013) employed RNA interference (RNAi) screens. In that project, libraries of small inhibitory RNAs (siRNAs) that systematically target a large fraction of human genes were screened, and the effects of individual siRNA perturbations on the cellular infection processes by various human pathogens were measured. The image‐based high‐content RNAi datasets that were generated through a standardized experimental and computational approach are of unprecedented quality and breadth and should thus enable systems‐level analyses of general gene‐phenotype relationships. SiRNAs are designed to be perfectly complementary to 19‐ 23 nucleotide regions in the intended mRNA targets. As siRNA targets undergo degradation upon siRNA transfection, it is generally assumed that the observed phenotypes are due to the direct effect of the siRNA on these targets ('on‐target effect'). However, our systematic exploration of the impact of siRNA‐based RNAi on two phenotypic readouts (cell number and infection index) in InfectX has highlighted the prevalence and magnitude of 'off‐target effects' that are mediated by the siRNA 'seed sequences' (nucleotides 2‐8 from the 5' end of the siRNA) through a microRNA (miRNA)‐type mechanism. A major contribution that InfectX made to the RNAi screening field was the development of modeling approaches to comprehensively quantify off‐target effects, partly deconvolute combinatorial effects, and finally provide improved methods for establishing individual gene‐phenotype relationships. The present proposal, TargetInfectX (2014‐2017), will build on the image‐based high‐content RNAi datasets established within InfectX , but will have more far‐reaching goals. From the image data generated in InfectX we will extract a rich set of phenotypic features on the single‐cell level. Modeling miRNAtarget mRNA interactions, their effect on gene expression, and the gene‐phenotype relationships emerging from the imaging data, we will attempt to generally reconstruct the genotypic basis of elementary cell behaviors such as those involved in the response to pathogen intrusion. On the translational side, we will explore the potential of siRNAs as novel combination therapy approach to interfere with the course of infections. Moreover, beyond the public release of our data via publications and publically accessible databases, we will encourage knowledge and technology transfer of unpublished work in progress with partners outside of TargetInfectX via a collaboration suite that should foster long‐term collaborations and ensure the impact of our work beyond SystemsX.ch .
Bacterial Type IV Secretion (T4S): Cellular, Molecular, and Evolutionary Basis of the Subversion of Host Cell Functions by Translocated Effector Proteins Research Project | 4 Project MembersIn the initial funding period of the SNSF grant 31003A-132979 we used Bartonella and Brucella - two related zoonotic pathogens engaging the widespread type IV secretion (T4S) mechanism for establishing chronic bacterial infection - as models to study the cellular, molecular and evolutionary basis of the subversion of host cell functions by T4S-translocated effector proteins. For the three year prolongation period the established multidisciplinary experimental approach will be extended by adopting yeast as surrogate model for studying conserved eukaryotic processes targeted by T4S effectors. Subproject A will focus on the structure/function analysis of Bartonella effector proteins (Beps) translocated by the VirB T4S system of Bartonella and their physiological consequences on the host, with particular emphasis on studying the subversion of immune signaling processes. Subproject B will focus on the functional analysis of effector proteins translocated by the distinct VirB T4S system of Brucella and their physiological consequences on host cell interaction, with particular emphasis on studying the intracellular trafficking events from a late endosomal compartment, where the VirB system is activated in response to acidification, to the endoplasmic reticulum (ER)-related replicative niche of Brucella. Together, these studies will contribute to establishing a molecular paradigm for the role of T4S effectors in triggering chronic bacterial infection.
Bacterial Type IV Secretion: Cellular, Molecular, and Evolutionary Basis of the Subversion of Host Cell Functions by Translocated Effector Proteins. Research Project | 1 Project MembersThe type IV secretion (T4S) systems of gram-negative bacteria are versatile nano-machines involved in processes relevant to bacterial infection, such as horizontal transfer of virulence and antibiotic resistance genes between bacteria, and the translocation of bacterial effector proteins into eukaryotic target cells. Effector-translocating T4S systems are widely distributed among human pathogenic bacteria that are mostly causing chronic infections, including Helicobacter pylori (causing gastritis and gastric cancer), Legionella pneumophila (causing Legionnaires disease), Bartonella spp. (causing bartonellosis) and Brucella spp. (causing brucellosis). In recent years we have - with support from the SNF (grants 3100-061777 and 31003A-109925) - established Bartonella as a powerful model for studying the cellular, molecular and evolutionary basis of T4S in bacterial pathogenesis. In the frame of the proposed project we will continue to explore this paradigmatic model of T4S. Moreover, we will expand the successful approach for studying T4S as established for Bartonella to the closely related pathogen Brucella - the etiological agent of the most important bacterial zoonosis worldwide that causes chronic infections in various mammals. We will apply a multidisciplinary experimental approach including bacterial genetics, cell biology, molecular biology, biochemistry, structural biology, genomics, and animal experimentation. In subproject A targeting T4S in Bartonella, we will perform a structure/function analysis of the T4S system-translocated effector proteins and their cellular targets, and study molecular evolutionary mechanisms of host adaptation mediated by these effectors. In subproject B targeting T4S in Brucella , we will focus on the characterization of the T4S-dependent intracellular trafficking route of Brucella resulting in the establishment of an endoplasmic reticulum-associated replication niche, the systematic identification of the involved host factors and the structure/function analysis of the T4S effectors interacting with the identified host factors. By studying the molecular mechanism of T4S in the closely related pathogens Bartonella and Brucella , we anticipate to provide fundamental contributions to our understanding how this wide-spread virulence mechanism facilitates chronic bacterial infection, which may also help to design novel strategies to combat bacterial virulence. Based on the specific subversion of multiple cellular functions by the T4S effectors of these pathogens we also expect to contribute new basic knowledge and novel tools to different fields of cell biology.
