Projects & Collaborations 24 foundShow per page10 10 20 50 TOXIN-MEDIATED DEPLETION OF NICOTINAMIDE DINUCLEOTIDES DRIVES PERSISTER FORMATION IN A HUMAN PATHOGEN Research Project | 1 Project MembersImported from Grants Tool 4701438 Efficient Characterization of Biomolecular Interactions by Automated Isothermal Titration Calorimetry Research Project | 5 Project MembersInhalt und Ziel des Forschungsprojekts Im Gegensatz zu anderen Methoden ist die isothermale Titrationskalorimetrie (ITC) in einzigartiger Weise geeignet, die Wechselwirkung von unmodifizierten Bindungspartnern anhand der aufgenommenen oder abgegebenen Bindungswärme zu messen. Messungen mittels ITC erlauben dabei eine thermodynamische Charakterisierung der Wechselwirkungen zwischen Bindungspartnern. Diese erlaubt, zusammen mit Strukturinformationen, die Mechanismen der Bindung im Detail zu verstehen und zu erklären, warum verschiedene Bindungspartner unterschiedlich stark interagieren. In diesem Projekt wird ein automatisiertes System für ITC Messungen aufgebaut, das hilft, wesentliche Nachteile von manuellen Messungen hinsichtlich des Durchsatzes und der Datenqualität zu überkommen. Das neue ITC System wird in der Biophysik Forschungsserviceeinheit (BF) des Biozentrums der Universität Basel installiert und betrieben, und steht dort auch externen Nutzern zur Verfügung. Erste Anwendungen des neuen Geräts sind in den Bereichen Antibiotikaentwicklung und Antibiotikaresistenzen, sowie der molekularen und zellulären Krebsforschung vorgesehen. Wissenschaftlicher und gesellschaftlicher Kontext Wir erwarten, dass dieses Projekt zu einem besseren Verständnis fundamentaler biologischer Prozesse beitragen wird und dass die entsprechenden Erkenntnisse einen wertvollen Beitrag zu der Entwicklung von Molekülen leisten, die als Werkzeuge in der Biologie oder als Vorstufen zukünftiger Medikamente dienen. Unmasking the spatial code - How global and local c-di-GMP signaling modules regulate bacterial behavior and virulence Research Project | 1 Project MembersThe concentration of bacteria on surfaces (including animals and plants) is orders of magnitudes higher than in the surrounding environment, offering bacteria ample opportunity for mutualistic, symbiotic, and pathogenic interactions. To efficiently populate surfaces, bacteria have evolved mechanisms to sense mechanical or chemical cues upon contact with solid substrata. This is of particular importance for pathogens that interact with host tissue surfaces as they need to rapidly adapt to this environment to optimize adherence, tissue dissemination and virulence. Recent work has revealed that small signaling molecules like c-di-GMP and cAMP play key roles in this process, but the mechanisms by which these molecules instruct bacteria on surfaces have remained largely unchallenged. Moreover, experimental systems that allow scrutinizing mechanisms and processes involved in tissue colonization by important pathogens under realistic, human-like conditions are missing. Here, we propose to dissect the role of c-di-GMP in surface colonization of Pseudomonas aeruginosa , an opportunistic human pathogen that is able to invade the human host by effectively colonizing mucosal surfaces. Our studies will probe the initial stages of this process including surface attachment, virulence induction and surface motility to better understand the powerful invasion and dissemination strategies of this pathogen. We will investigate these processes on abiotic surfaces and on human lung organoids to be able to challenge the relevance of in vitro studies. We will explore how c-di-GMP signaling is coordinated with cAMP and how these signaling molecules regulate P. aeruginosa surface colonization with high precision and specificity. Our studies will focus on two c-di-GMP binding proteins, FimW and FimX, which regulate type IV pili, a prime virulence factor, to drive distinct and antagonistic processes, surface adherence and twitching motility. To dissect the dynamic regulation of these processes, we will develop powerful biosensors that allow monitoring changes of c-di-GMP and cAMP in real time and with high temporal and spatial resolution. Together with state-of-the-art imaging and powerful genetic analyses of the FimW and FimX pathways, this will uncover distinct second messenger signaling modes operating either on the global level or in local, spatially confined modules, thereby providing unprecedented insight into how P. aeruginosa colonizes tissue surfaces. Studies with P. aeruginosa will be complemented and instructed by investigations of c-di-GMP signaling in two powerful model organisms, Caulobacter crescentus and Escherichia coli . We propose to investigate mechanisms responsible for local c-di-GMP-dependent cell polarity and rapid surface attachment of C. crescentus . As processes involved in Caulobacter surface colonization are strikingly similar to those in P. aeruginosa , these studies will serve as a blueprint for our work with the human pathogen. Studies in E. coli are geared towards a detailed understanding of a newly discovered surface glycan, NGR, which serves as receptor for several bacteriophages. NGR biogenesis and secretion was proposed to be regulated by a local pool of c-di-GMP in a highly specific manner. These studies will provide a detailed understanding of how c-di-GMP controls bacterial processes within confined 'microcompartments' and thus will greatly influence our work with P. aeruginosa both on the conceptual and the experimental level. SNiB-cdG-P - Global mapping of second messenger c-di-GMP signaling networks in bacteria using proteomics Research Project | 2 Project MembersTo survive in diverse niches, bacteria must adapt to changes in their local environment by sensing and responding to environmental cues. External cues are transduced through complex signaling networks throughout a cell and drive diverse changes in cellular behavior. In bacteria, cyclic di-guanosine-monophosphate (c-di-GMP) is a nucleotidederived second messenger that mediates signal transduction of important biological processes for bacterial growth and survival e.g. motility, biofilm formation and metabolism. These biological processes are also crucial in clinical settings as they underlay antibiotic resistance in important pathogenic bacteria. Recent advances in MS-based proteomics have provided different tools to investigate the proteome of an organism in a systematic and global manner. Especially, thermal proteome profiling (TPP) and limited-proteolysis MS (LiP-MS) are pioneering methods to study change of protein states proteome-wide. I aim to employ these proteomics based approaches to achieve the global map of the c-di-GMP signaling network in two different bacteria, Escherichia coli and Caulobacter crescentus, both are model organisms of Gram-negative bacteria. Furthermore, I will apply these methods to investigate signaling network of another important second messenger, (p)ppGpp, and explore how the networks of these two messenger molecules interact in bacteria to dictate cellular physiology. Lung tissue-like culture system for bacterial pathogens to support antibiotic drug discovery (Efficacy and PK) Research Project | 2 Project MembersThe focus of the Roche antibiotics discovery group is to identify broad-spectrum antibiotics with novel Mode of Action (MOA). A key indication is the treatment of Gram-negative, Difficult-to-treat (DTR), hospital-acquired infections;specifically, Hospital Acquired Pneumonia (HAP) / Ventilator Acquired Pneumonia (VAP), that are among the mostchallenging and life-threatening bacterial infections today and therefore, deserve a close attention (Petite andNguyen, 2018; Weiss. et al., 2019). In order to validate novel molecular entities identified in high throughput screens,Roche aim to establish an advanced cellular system that allows simulation of human lung infections, especiallyHAP/VAP. Epithelix, a Geneva based company, has engineered an in vivo-like 3D cell culture model, MucilAir, mimicking lung(bronchial) airway system. Infection of the MucilAir system with P. aeruginosa led to a loss of the "epithelial barrier"function of the lung epithelium as observed during in vivo infections (Bertinetti C. et al, poster from Epithelix). In this project, Roche propose to adapt the MucilAir system and establish infection conditions for other Gram-negative bacteria to be able to: assess potency and PK properties of prioritized hit series, enable selection of clinical candidates improve the translation of in vitro data to human infections, thereby reducing the use of animal models (3R principle). Similar interest for developing novel tools to support antibacterial discovery and development is a major goal of Biozentrum University of Basel and ETH Zurich within the frame of National Center of Competence in Research (NCCR) AntiResist. Specifically, Prof. Christoph Dehio together with two other Biozentrum professors, Dirk Bumann and Urs Jenal have been awarded research funding to better understand pathogen physiology and heterogeneity in infected patients and development of in vitro systems that mimic relevant aspects of patient tissues. This project will be funded by Roche Center of Excellence (CoE, Roche Pharma Sciences) as part of an umbrella initiative of lung models for infection diseases, immunology and safety. Such environment is an opportunity of crossfertilization for technical knowledge, as the above-mentioned 3D cell system could also be adapted to study viral lung infections, inflammatory processes of the lung and to address the safety aspects of the developmental compounds. Direct collaborative connection between Roche and the research groups at Biozentrum and ETH Zurich will help to learn first-hand what type of progress is made in the field, with the possibility to gain access to patient sample analysis data and to the top advanced technologies available within the AntiResist initiative. 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. The Role of Toxin-Antitoxin Modules in Pseudomonas aeruginosa Phenotypic Heterogeneity and Antibiotic Tolerance Research Project | 1 Project MembersStochastic binary decisions generate phenotypic heterogeneity in bacterial populations, thereby contributing to bet-hedging processes and to division-of-labor. While specific bimodal programs were implicated in bacterial virulence 1 and antibiotic tolerance 2, the mechanisms leading to bi-nary cellular responses and the resulting consequences for population fitness and resilience are often unclear. Here, we aim at dissecting the role of two novel toxin-antitoxin (TA) modules in Pseudomonas aeruginosa, one of three human pathogens recently listed as critical-priority by the WHO. One of the hallmarks of bacterial TA systems is the binary expression of toxins or toxin-like factors, leading to the generation of functional heterogeneity in bacterial populations. We hypoth-esize that the TA modules PA1029-PA1030 and PA2780-PA2781 contribute to behavioral heteroge-neity and antibiotic tolerance of cultures of P. aeruginosa and by that contribute to the establish-ment of successful infections. To strengthen this idea, we propose to embark on a thorough func-tional examination of both modules.In the first part of the project, we plan to dissect the regulation and function of the toxin PA1030 and its antitoxin PA1029. Activating mutations in the PA1030 gene were originally identified in isolates of chronically infected CF patients and were shown to confer strongly increased tolerance against different classes of antibiotics. Our preliminary data suggest that stochastic expression of PA1030 generates persisters by modulating the cellular NAD pool. We propose a combination of biochemical, structural and cell biology experiments to determine the mechanisms of toxin action on the cells' metabolism and its stochastic control. These experiments will test the central hypoth-esis that the PA1029-PA1030 TA module generates persisters upon sensing the depletion of a cen-tral metabolite. We will investigate the role of this TA module in chronic infections by characterizing TA variants from clinical isolates and by quantifying the expression of the TA components in pa-tient samples. In the second part of the project, we will dissect the TA-like module PA2780-PA2781, which through its stochastic expression generates behavioral diversity in P. aeruginosa cells colonizing surfaces to form biofilms. Biofilms are multicellular communities that strongly promote chronic infections by protecting pathogens from phagocytic clearance and safeguarding bacteria from an-tibiotic killing. Based on preliminary results, we hypothesize that PA2780-PA2781 converts regu-latory input from the global Gac/Rsm cascade into a binary cellular response, generating pheno-typic heterogeneity through the control of c-di-GMP-dependent processes like virulence, biofilm formation and persistence. We postulate that this system provides P. aeruginosa with a bet-hedging strategy to functionally diversify during infection and colonization of host tissues. Our studies will uncover the mechanisms of stochastic expression and downstream processes of this TA-like mod-ule and will investigate its role in P. aeruginosa virulence and persistence. Speed and robustness: c-di-GMP signaling in bacterial surface colonization and virulence Research Project | 1 Project MembersWhen pathogenic or non-pathogenic bacteria colonize surfaces or host tissues they rapidly change their behavior and develop surface-based motility, express virulence traits and eventually mature into multi-cellular communities, called biofilms. Biofilms are difficult to eradicate in the host as they feature in-creased drug resistance and persister rates and provide tolerance against phagocytic clearance. In the past years, c-di-GMP (cdG) was identified as a key regulator of bacterial surface colonization and biofilm for-mation. This provides an opportunity for pharmacological intervention with clinically problematic forms of bacterial growth. Here we analyze the role of cdG signaling in the initial steps of surface colonization and biofilm formation in several bacterial model organisms. We address how bacteria perceive mechani-cal stimuli and how such cues are transmitted into rapid and robust changes in cell behavior including surface-based motility, attachment and expression of virulence factors. Basilea Research Agreement 102834 Research Project | 1 Project MembersNo abstract Tolerance as a potential reservoir for the development of antibiotic resistance (NPR72) Research Project | 2 Project MembersThe widespread use of antibiotics promotes the spread of existing resistance mechanisms and the evolution of novel resistance traits. While resistant microorganisms are able to grow in the presence of the antibiotic, drug tolerance and drug persistence allow survival during transient antibiotic treatment windows. The mechanisms responsible for drug tolerance and persistence are currently unknown, representing a major obstacle for the development of anti-persistence drugs and other intervention strategies to cure persistent bacterial infection and interfere with resistance development. The often rare and transient nature of the multi-drug tolerant phenotype represents a particular challenge for the experimental and clinical exploration of persisting bacteria calling for a concerted and multi-pronged research approach to uncover their specific properties, clinical significance, and possible eradication strategies. While drug persistence mechanisms have been pioneered primarily in E. coli, it remains unclear how universal these features are. Importantly, it is also unclear if tolerance or persistence relates to the emergence of resistance traits and what their relevance is in the human patient. To uncover the different mechanisms of antibiotic survival and their implications for resistance development, we use Pseudomonas aeruginosa, an important human pathogen causing both acute and chronic infections. 123 123
TOXIN-MEDIATED DEPLETION OF NICOTINAMIDE DINUCLEOTIDES DRIVES PERSISTER FORMATION IN A HUMAN PATHOGEN Research Project | 1 Project MembersImported from Grants Tool 4701438
Efficient Characterization of Biomolecular Interactions by Automated Isothermal Titration Calorimetry Research Project | 5 Project MembersInhalt und Ziel des Forschungsprojekts Im Gegensatz zu anderen Methoden ist die isothermale Titrationskalorimetrie (ITC) in einzigartiger Weise geeignet, die Wechselwirkung von unmodifizierten Bindungspartnern anhand der aufgenommenen oder abgegebenen Bindungswärme zu messen. Messungen mittels ITC erlauben dabei eine thermodynamische Charakterisierung der Wechselwirkungen zwischen Bindungspartnern. Diese erlaubt, zusammen mit Strukturinformationen, die Mechanismen der Bindung im Detail zu verstehen und zu erklären, warum verschiedene Bindungspartner unterschiedlich stark interagieren. In diesem Projekt wird ein automatisiertes System für ITC Messungen aufgebaut, das hilft, wesentliche Nachteile von manuellen Messungen hinsichtlich des Durchsatzes und der Datenqualität zu überkommen. Das neue ITC System wird in der Biophysik Forschungsserviceeinheit (BF) des Biozentrums der Universität Basel installiert und betrieben, und steht dort auch externen Nutzern zur Verfügung. Erste Anwendungen des neuen Geräts sind in den Bereichen Antibiotikaentwicklung und Antibiotikaresistenzen, sowie der molekularen und zellulären Krebsforschung vorgesehen. Wissenschaftlicher und gesellschaftlicher Kontext Wir erwarten, dass dieses Projekt zu einem besseren Verständnis fundamentaler biologischer Prozesse beitragen wird und dass die entsprechenden Erkenntnisse einen wertvollen Beitrag zu der Entwicklung von Molekülen leisten, die als Werkzeuge in der Biologie oder als Vorstufen zukünftiger Medikamente dienen.
Unmasking the spatial code - How global and local c-di-GMP signaling modules regulate bacterial behavior and virulence Research Project | 1 Project MembersThe concentration of bacteria on surfaces (including animals and plants) is orders of magnitudes higher than in the surrounding environment, offering bacteria ample opportunity for mutualistic, symbiotic, and pathogenic interactions. To efficiently populate surfaces, bacteria have evolved mechanisms to sense mechanical or chemical cues upon contact with solid substrata. This is of particular importance for pathogens that interact with host tissue surfaces as they need to rapidly adapt to this environment to optimize adherence, tissue dissemination and virulence. Recent work has revealed that small signaling molecules like c-di-GMP and cAMP play key roles in this process, but the mechanisms by which these molecules instruct bacteria on surfaces have remained largely unchallenged. Moreover, experimental systems that allow scrutinizing mechanisms and processes involved in tissue colonization by important pathogens under realistic, human-like conditions are missing. Here, we propose to dissect the role of c-di-GMP in surface colonization of Pseudomonas aeruginosa , an opportunistic human pathogen that is able to invade the human host by effectively colonizing mucosal surfaces. Our studies will probe the initial stages of this process including surface attachment, virulence induction and surface motility to better understand the powerful invasion and dissemination strategies of this pathogen. We will investigate these processes on abiotic surfaces and on human lung organoids to be able to challenge the relevance of in vitro studies. We will explore how c-di-GMP signaling is coordinated with cAMP and how these signaling molecules regulate P. aeruginosa surface colonization with high precision and specificity. Our studies will focus on two c-di-GMP binding proteins, FimW and FimX, which regulate type IV pili, a prime virulence factor, to drive distinct and antagonistic processes, surface adherence and twitching motility. To dissect the dynamic regulation of these processes, we will develop powerful biosensors that allow monitoring changes of c-di-GMP and cAMP in real time and with high temporal and spatial resolution. Together with state-of-the-art imaging and powerful genetic analyses of the FimW and FimX pathways, this will uncover distinct second messenger signaling modes operating either on the global level or in local, spatially confined modules, thereby providing unprecedented insight into how P. aeruginosa colonizes tissue surfaces. Studies with P. aeruginosa will be complemented and instructed by investigations of c-di-GMP signaling in two powerful model organisms, Caulobacter crescentus and Escherichia coli . We propose to investigate mechanisms responsible for local c-di-GMP-dependent cell polarity and rapid surface attachment of C. crescentus . As processes involved in Caulobacter surface colonization are strikingly similar to those in P. aeruginosa , these studies will serve as a blueprint for our work with the human pathogen. Studies in E. coli are geared towards a detailed understanding of a newly discovered surface glycan, NGR, which serves as receptor for several bacteriophages. NGR biogenesis and secretion was proposed to be regulated by a local pool of c-di-GMP in a highly specific manner. These studies will provide a detailed understanding of how c-di-GMP controls bacterial processes within confined 'microcompartments' and thus will greatly influence our work with P. aeruginosa both on the conceptual and the experimental level.
SNiB-cdG-P - Global mapping of second messenger c-di-GMP signaling networks in bacteria using proteomics Research Project | 2 Project MembersTo survive in diverse niches, bacteria must adapt to changes in their local environment by sensing and responding to environmental cues. External cues are transduced through complex signaling networks throughout a cell and drive diverse changes in cellular behavior. In bacteria, cyclic di-guanosine-monophosphate (c-di-GMP) is a nucleotidederived second messenger that mediates signal transduction of important biological processes for bacterial growth and survival e.g. motility, biofilm formation and metabolism. These biological processes are also crucial in clinical settings as they underlay antibiotic resistance in important pathogenic bacteria. Recent advances in MS-based proteomics have provided different tools to investigate the proteome of an organism in a systematic and global manner. Especially, thermal proteome profiling (TPP) and limited-proteolysis MS (LiP-MS) are pioneering methods to study change of protein states proteome-wide. I aim to employ these proteomics based approaches to achieve the global map of the c-di-GMP signaling network in two different bacteria, Escherichia coli and Caulobacter crescentus, both are model organisms of Gram-negative bacteria. Furthermore, I will apply these methods to investigate signaling network of another important second messenger, (p)ppGpp, and explore how the networks of these two messenger molecules interact in bacteria to dictate cellular physiology.
Lung tissue-like culture system for bacterial pathogens to support antibiotic drug discovery (Efficacy and PK) Research Project | 2 Project MembersThe focus of the Roche antibiotics discovery group is to identify broad-spectrum antibiotics with novel Mode of Action (MOA). A key indication is the treatment of Gram-negative, Difficult-to-treat (DTR), hospital-acquired infections;specifically, Hospital Acquired Pneumonia (HAP) / Ventilator Acquired Pneumonia (VAP), that are among the mostchallenging and life-threatening bacterial infections today and therefore, deserve a close attention (Petite andNguyen, 2018; Weiss. et al., 2019). In order to validate novel molecular entities identified in high throughput screens,Roche aim to establish an advanced cellular system that allows simulation of human lung infections, especiallyHAP/VAP. Epithelix, a Geneva based company, has engineered an in vivo-like 3D cell culture model, MucilAir, mimicking lung(bronchial) airway system. Infection of the MucilAir system with P. aeruginosa led to a loss of the "epithelial barrier"function of the lung epithelium as observed during in vivo infections (Bertinetti C. et al, poster from Epithelix). In this project, Roche propose to adapt the MucilAir system and establish infection conditions for other Gram-negative bacteria to be able to: assess potency and PK properties of prioritized hit series, enable selection of clinical candidates improve the translation of in vitro data to human infections, thereby reducing the use of animal models (3R principle). Similar interest for developing novel tools to support antibacterial discovery and development is a major goal of Biozentrum University of Basel and ETH Zurich within the frame of National Center of Competence in Research (NCCR) AntiResist. Specifically, Prof. Christoph Dehio together with two other Biozentrum professors, Dirk Bumann and Urs Jenal have been awarded research funding to better understand pathogen physiology and heterogeneity in infected patients and development of in vitro systems that mimic relevant aspects of patient tissues. This project will be funded by Roche Center of Excellence (CoE, Roche Pharma Sciences) as part of an umbrella initiative of lung models for infection diseases, immunology and safety. Such environment is an opportunity of crossfertilization for technical knowledge, as the above-mentioned 3D cell system could also be adapted to study viral lung infections, inflammatory processes of the lung and to address the safety aspects of the developmental compounds. Direct collaborative connection between Roche and the research groups at Biozentrum and ETH Zurich will help to learn first-hand what type of progress is made in the field, with the possibility to gain access to patient sample analysis data and to the top advanced technologies available within the AntiResist initiative.
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.
The Role of Toxin-Antitoxin Modules in Pseudomonas aeruginosa Phenotypic Heterogeneity and Antibiotic Tolerance Research Project | 1 Project MembersStochastic binary decisions generate phenotypic heterogeneity in bacterial populations, thereby contributing to bet-hedging processes and to division-of-labor. While specific bimodal programs were implicated in bacterial virulence 1 and antibiotic tolerance 2, the mechanisms leading to bi-nary cellular responses and the resulting consequences for population fitness and resilience are often unclear. Here, we aim at dissecting the role of two novel toxin-antitoxin (TA) modules in Pseudomonas aeruginosa, one of three human pathogens recently listed as critical-priority by the WHO. One of the hallmarks of bacterial TA systems is the binary expression of toxins or toxin-like factors, leading to the generation of functional heterogeneity in bacterial populations. We hypoth-esize that the TA modules PA1029-PA1030 and PA2780-PA2781 contribute to behavioral heteroge-neity and antibiotic tolerance of cultures of P. aeruginosa and by that contribute to the establish-ment of successful infections. To strengthen this idea, we propose to embark on a thorough func-tional examination of both modules.In the first part of the project, we plan to dissect the regulation and function of the toxin PA1030 and its antitoxin PA1029. Activating mutations in the PA1030 gene were originally identified in isolates of chronically infected CF patients and were shown to confer strongly increased tolerance against different classes of antibiotics. Our preliminary data suggest that stochastic expression of PA1030 generates persisters by modulating the cellular NAD pool. We propose a combination of biochemical, structural and cell biology experiments to determine the mechanisms of toxin action on the cells' metabolism and its stochastic control. These experiments will test the central hypoth-esis that the PA1029-PA1030 TA module generates persisters upon sensing the depletion of a cen-tral metabolite. We will investigate the role of this TA module in chronic infections by characterizing TA variants from clinical isolates and by quantifying the expression of the TA components in pa-tient samples. In the second part of the project, we will dissect the TA-like module PA2780-PA2781, which through its stochastic expression generates behavioral diversity in P. aeruginosa cells colonizing surfaces to form biofilms. Biofilms are multicellular communities that strongly promote chronic infections by protecting pathogens from phagocytic clearance and safeguarding bacteria from an-tibiotic killing. Based on preliminary results, we hypothesize that PA2780-PA2781 converts regu-latory input from the global Gac/Rsm cascade into a binary cellular response, generating pheno-typic heterogeneity through the control of c-di-GMP-dependent processes like virulence, biofilm formation and persistence. We postulate that this system provides P. aeruginosa with a bet-hedging strategy to functionally diversify during infection and colonization of host tissues. Our studies will uncover the mechanisms of stochastic expression and downstream processes of this TA-like mod-ule and will investigate its role in P. aeruginosa virulence and persistence.
Speed and robustness: c-di-GMP signaling in bacterial surface colonization and virulence Research Project | 1 Project MembersWhen pathogenic or non-pathogenic bacteria colonize surfaces or host tissues they rapidly change their behavior and develop surface-based motility, express virulence traits and eventually mature into multi-cellular communities, called biofilms. Biofilms are difficult to eradicate in the host as they feature in-creased drug resistance and persister rates and provide tolerance against phagocytic clearance. In the past years, c-di-GMP (cdG) was identified as a key regulator of bacterial surface colonization and biofilm for-mation. This provides an opportunity for pharmacological intervention with clinically problematic forms of bacterial growth. Here we analyze the role of cdG signaling in the initial steps of surface colonization and biofilm formation in several bacterial model organisms. We address how bacteria perceive mechani-cal stimuli and how such cues are transmitted into rapid and robust changes in cell behavior including surface-based motility, attachment and expression of virulence factors.
Tolerance as a potential reservoir for the development of antibiotic resistance (NPR72) Research Project | 2 Project MembersThe widespread use of antibiotics promotes the spread of existing resistance mechanisms and the evolution of novel resistance traits. While resistant microorganisms are able to grow in the presence of the antibiotic, drug tolerance and drug persistence allow survival during transient antibiotic treatment windows. The mechanisms responsible for drug tolerance and persistence are currently unknown, representing a major obstacle for the development of anti-persistence drugs and other intervention strategies to cure persistent bacterial infection and interfere with resistance development. The often rare and transient nature of the multi-drug tolerant phenotype represents a particular challenge for the experimental and clinical exploration of persisting bacteria calling for a concerted and multi-pronged research approach to uncover their specific properties, clinical significance, and possible eradication strategies. While drug persistence mechanisms have been pioneered primarily in E. coli, it remains unclear how universal these features are. Importantly, it is also unclear if tolerance or persistence relates to the emergence of resistance traits and what their relevance is in the human patient. To uncover the different mechanisms of antibiotic survival and their implications for resistance development, we use Pseudomonas aeruginosa, an important human pathogen causing both acute and chronic infections.
