Molecular Microbiology (Bumann)Head of Research Unit Prof. Dr.Dirk BumannOverviewMembersPublicationsProjects & CollaborationsProjects & Collaborations OverviewMembersPublicationsProjects & Collaborations Projects & Collaborations 18 foundShow per page10 10 20 50 Impact of Magnesium on Salmonella Infection and Therapy Research Project | 1 Project MembersImported from Grants Tool 4697853 Phage activation in Staphylococcus aureus during deep-seated human infections Research Project | 2 Project MembersThe Gram-positive bacterium Staphylococcus aureus causes a wide variety of infectious diseases that are associated with substantial morbidity and mortality. Clinical isolates of S. aureus display vast genetic diversity, which is mainly determined by the presence of mobile genetic elements, comprising phages, and genomic islands. Phages mediate most genetic exchange among S. aureus strains, including the dissemination of resistance genes and virulence factors. Previous research has suggested that phage activation in S. aureus occurs regularly in infected host tissues. On the other hand, phage therapy has regained interest as a non-conventional strategy for urgently needed novel approaches to control infection. Endogenous phage activation might interfere with such approaches. This proposal aims to quantify the extent of phage activation in Staphylococcus aureus during deep-seated human infections and to determine its impact on therapy. I will first quantify phage activation in freshly frozen surgical samples from patients with deep-seated S. aureus infections. I will focus on samples containing S. aureus strains that are representative of major lineages as determined by whole-genome sequencing. I will then establish patient-mimicking in-vitro conditions to recapitulate in-vivo phage activation for identifying underlying molecular mechanisms and potential heterogeneity at the single-cell level. Finally, I will determine the impact of in vivo -like phage activation on S. aureus susceptibility to antimicrobials and phage therapy. Overall, this project will determine the relevance and impact of an understudied, but likely critical aspect of the major human pathogen S. aureus during human infections. Characterization of the ELF for better PK/PD models of human lung infection Research Project | 2 Project MembersThe objective of this collaboration is knowledge generation of the composition of Epithelial Lining Fluid (ELF) of human (healthy volunteers and patients with bacterial pneumonia) and pre-clinical species. This knowledge will be used for constructing simulated ELF matrixes mimicking the most important components of the physiological fluids. An in vitro assay is consequently set up to assess the free fraction of drugs in these fluids. Finally, the data generated will be used to establish a machine learning tool to predict free fraction in ELF based on chemical descriptors and will be further used to develop advanced Physiologically Based PharmacoKinetic (PBPK) model including ELF as additional compartment and its validation for human dose prediction on marketed drugs. The mechanisms of action of macrolide antibiotics RUS_ST2019-124 Research Project | 1 Project MembersCurrently, increasing antibiotic resistance levels has been identified as a major health threat World wide. The World Health Organization has identified several pathogens with critical resistance levels. One of the most worrisome group of bacteria is Enterobacteriacea, which are becoming resistant against cephalosporins and carbapenems. Macrolides is a widely used group of antibiotics. Based on the in vitro activity tests these antibiotics are not used against Enterobacteriacea. Surprisingly, recent clinical data indicate that macrolides can be used against Salmonella infections. Here we aim to elucidate the molecular mechanisms of this unexplained activity. In addition to antibacterial activity, we hypothesize that macrolides can act on the human mitochondrial ribosomes leading potentially to immunomodulation and treatment side effects. We will use novel bioreporters for measuring the effects of macrolides on bacterial protein synthesis in Salmonella and uropathogenic Escherichia coli infection models. We also measure the effects of macrolide treatment on mitochondrial protein synthesis and the levels of mitochondrial proteins. The results will provide information for potentially using macrolides against wider range of infections caused by Enterobacteriacea. The test systems developed will become important tools for the development of new antibiotics. 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. Living Microbial Diagnostics to Enable Individualized Child Health Interventions Research Project | 1 Project MembersWe are in the midst of a global health tragedy where over 3 million children in the developing world die annually before the age of 5 years(GBD 2013, Lancet, 2014) predominantly through infectious diseases as well as malnutrition and related disorders. A further 200 million children annually do not reach their developmental potential(Grantham, Lancet, 2007). A major challenge in diagnosing and treating these children in the developing world lies in the paucity of options for objectively measuring the nutritional, infection, and inflammation status of the gastrointestinal tract. To overcome this challenge, we recently engineered a strain of bacteria that is capable of sensing, remembering, and reporting on the environment within the gastrointestinal tract of animals. These engineered bacteria can safely traverse the gastrointestinal tract, analogously to probiotics, and then be retrieved from feces, analyzed in the laboratory, and enable predictive therapeutic interventions. Our proposal focuses on further developing these engineered bacteria and comprehensively testing their safety and capacity to reveal clinically-meaningful and therapeutically-actionable information. In the future, our technology could be deployed throughout the world, in a sustainable and scalable fashion, and aid in improving child health and wellbeing in both the developed and developing worlds. Horizon 2020 Framework Project MIBEst Research Project | 1 Project MembersInfectious diseases have been the leading cause of death for many centuries. The development of vaccination and antibiotic treatments combined with improved hygiene has decreased the number of deaths, but the mortality and morbidity associated with infections remain considerable, requiring constant societal awareness and scientific research. An increasing concern are the latent and chronic infections that are often refractory to treatments. As the frequency of latent infections increases with age, it is a major concern for aging societies. The great diversity of the infectious agents, and the multidisciplinary nature of the infectious biology research demand a convergence of various competencies: microbiology, cell biology, animal infection models, immunology etc, emphasizing the need for collaboration between research centres. Especially important are joint activities for smaller countries, e.g. Estonia, where establishment of full-scale stand-alone programs is not economically feasible. Despite the strong positions in basic molecular biology, virology and microbiology, Estonia often fails to capitalize on the excellence in basic research by transitioning to the development of therapeutics targeting medically relevant processes. The main objective of the MIBEst project is to strengthen the research capacity on latent and chronic infections of Institute of Technology at University of Tartu by creating long-lasting links with internationally-leading research institutions: Molecular Infection Medicine Sweden at Umeå University, Sweden, and Basel Biozentrum, University of Basel, Switzerland. As an outcome of MIBEst, Estonian scientists will have new knowledge in infection biology with particular focus on advancement in models for latent infections and high throughput screening for promising candidates for antiinfective compounds. Altogether, it enables development of new anti-infection strategies that will have major impact at the national, European and global scale. Tracking and manipulating Salmonella subsets in infected tissues over time Research Project | 1 Project MembersThis project intends to build a basis for entirely novel strategies in infection control , by broadening successful host antimicrobial attacks , and closing permissive niches in which pathogens can thrive. Systemic Salmonella enterica infections are a major cause of mortality worldwide 1-5 , and become increasingly untreatable 6,7 . Our single-cell data show in a mouse model that the host immune system actually eradicates many Salmonella cells , while other Salmonella thrive at the same time in the same tissue, causing lethal disease progression 8,9 . Emerging evidence suggests that similar heterogeneous host-pathogen encounters might be a key feature of many infectious diseases 10-20 . This heterogeneity offers fascinating opportunities for research and application . If we understand the mechanisms that determine the disparate local outcomes, we might be able to tip the balance in favor of the host, by closing permissive niches in which pathogens can thrive . Current research mostly relies on snapshots that cannot capture the crucial temporal dynamics . We know that Salmonella cells differentially access host nutrients, experience oxidative or nitrosative stress, etc.; but what is the consequence of these encounters and what impact do they have for overall disease outcome ? We detect many dead Salmonella cells in tissues; but which events led to their killing , and why did this not happen to the thriving Salmonella subset? Our goals are, therefore, to track Salmonella cells that have experienced a specific host encounter, to manipulate their fate, and to elucidate underlying mechanisms in vivo. To achieve these goals, we will follow four specific aims, in which we test eight specific hypotheses: To unravel encounter consequences over the first few divisions (6-36 h). We will exploit a fluorescent TIMER reporter variant with very slow color maturation as a novel tool. To unravel encounter consequences over several days . We will link Salmonella subset-specific promoters with genetic switches and reporters useful for lineage tracing. To determine the impact of specific encounters for overall disease outcome . We will deplete Salmonella in defined encounters using suicide lysis and/or DNA damage. To determine the underlying molecular mechanisms . We will correlate local abundance of host factors with key encounter types and validate causal relationships using perturbations. Combination of these innovative approaches with our recently developed 3D imaging technique for whole spleen will enable us to validate, improve, and extend our spatio-temporal model for Salmonella infection dynamics . This will be a key step for translating the increasingly detailed knowledge about Salmonella and host heterogeneity during infection, into new strategies for controlling life-threatening systemic Salmonella infections . We hope that our methods and concepts may inspire similar studies in other major diseases with a decisive role of heterogeneous host-pathogen encounters, such as tuberculosis 21 . ESBL-MS: Early diagnosis of ESBL Enteriobacteriaceae in patient samples Research Project | 3 Project MembersEnterobacteriaceae are a major cause of life-threatening infections such as sepsis. Treatment of Enterobacteriaceae infections relies mostly on β-lactams and β-lactam-β-lactamase inhibitor combinations, but the efficacy of these drugs is endangered by the accelerating spread of extended spectrum β-lactamases (ESBL). ESBLs can be detected using biochemical test, isothermal amplification, PCR and microarrays, but these data cannot predictive quantitative susceptibility as needed for targeted treatment decisions. In this project, we use ultrasensitive mass spectrometry to detect ESBLs and other resistance proteins directly in patient samples. In parallel, we determine how these proteins affect capabilities of clinical strains to rapidly evolve increased resistance in vitro, e.g. by upregulating ESBL expression. We also engineer mutants of clinical strains with precisely altered expression of ESBL and other genes, to quantify their role in resistance. Based on all these data, we will build a classifier algorithm for accurate prediction of resistance within 5 h after patient sample acquisition. Finally, we translate sensitive detection methods for relevant proteins to widely available MALDI-TOF mass instruments for simple and rapid implementation. If successful, the results of our project will enable rapid diagnostics for early targeted treatment of serious infections, while avoiding unnecessary usage of last-resort antibiotics. This strategy has large potential to enhance antibiotic stewardship in face of rising resistance. Cyst wall formation: a persistent challenge in Toxoplasmosis Research Project | 2 Project MembersThe acute phase of Toxoplasma gondii infection initiates with the rapid proliferation and dissemination of the fast-replicating form of the parasite (tachyzoite) throughout the vertebrate host. At the onset of the immune response, the tachyzoites are efficiently neutralized and the infection enters in a chronic phase with conversion to a slow-replicating developmental stage (bradyzoite) that forms tissue cysts predominantly in the central nervous system and in striated and heart muscle. This process of encystation is vital to the parasite's life cycle because i) it ensures survival and life-long persistence in intermediate hosts, and ii) it allows peroral transmission to the feline definitive host, initiating the sexual cycle. Tissue cysts not only prevent eradication of the parasite, but also pose a significant threat of reactivation in the context of host immunosuppression and can lead to encephalitis and other severe clinical manifestations. Despite the central importance of cyst formation for pathogenesis and transmission, our insight into how T. gondii defies the innate and adaptive immune responses to take up permanent residence in the immunocompetent hosts is rudimentary. Very little is known about the cyst wall composition and the molecular processes governing its formation. We have a very fragmented view of the temporal and spatial dissemination of cysts in the host and especially in the brain. Progress in biology is driven both by medical necessity and scientific curiosity and this project, which proposes to investigate the process of encystation, lies at the intersection of these two forces. It is the most propitious time to address the challenging question of cyst wall formation in light of the most recent breakthroughs in the sensitivity of -omics approaches, the power of the CRISPR/Cas9 system in genome editing, the revolution in high-throughput microscopy, and ex-vivo tissue examination at the highest level of resolution. This ambitious and highly synergistic project tackles key biological questions on tissue cyst formation and capitalizes heavily on cutting-edge technologies to address three specific objectives: 1. A comprehensive definition of the cyst wall composition. We will generate an unprecedentedly accurate transcriptome of the bradyzoite stage and a differential proteome of bradyzoites and the surrounding cyst wall. The data will be curated via a powerful comparative genomics approach spanning cyst-forming and cyst-lacking Apicomplexa species. A complementary mutagenesis and selection strategy is designed to identify specific defects in cyst formation or maturation by large-scale genetic screens using quantitative high-throughput microscopy. 2. An uncovering of molecular mechanisms governing the cyst wall formation. This aim will be accomplished by taking a targeted as well as a global functional approach. Starting from recent findings on parasitophorous vacuole formation of tachyzoites we will implement a semi-targeted gene knockout- or conditional knockout approach. Moreover, the creation of a comprehensive gene disruption collection in subproject 1 will allow identification of all non-essential tachyzoites genes that display trafficking defects during cyst formation. Key mutants will be mechanistically dissected in vitro with a subset to be investigated further in vivo. 3. A spatiotemporal cartography of cyst formation in the brain. We will harness cutting-edge imaging methods to investigate the in vivo dynamics of dissemination and cyst formation in the whole body and foremost in the brain of a mouse infection model. These findings will inform development of spatiotemporal models describing parasite dissemination and differentiation, to be correlated with data from post-mortem biopsies. By combining unbiased and targeted experimental approaches, we are poised to achieve major conceptual advances in deciphering the molecular events leading to cyst wall formation. In the long term, the data and technology created in this project will lay the foundation to decoding the molecular information exchange between host and pathogen during establishment of life-long latent infections. These studies fill a significant knowledge gap and will provide i) highly valuable web-accessible integrated gene expression data, ii) fundamental discoveries about the regulatory and trafficking circuits that govern formation of the cyst wall as a biological barrier during encystation iii) invaluable paradigms of how the parasite initiates and sustains molecular programs required for disease progression and persistence. 12 12 OverviewMembersPublicationsProjects & Collaborations
Projects & Collaborations 18 foundShow per page10 10 20 50 Impact of Magnesium on Salmonella Infection and Therapy Research Project | 1 Project MembersImported from Grants Tool 4697853 Phage activation in Staphylococcus aureus during deep-seated human infections Research Project | 2 Project MembersThe Gram-positive bacterium Staphylococcus aureus causes a wide variety of infectious diseases that are associated with substantial morbidity and mortality. Clinical isolates of S. aureus display vast genetic diversity, which is mainly determined by the presence of mobile genetic elements, comprising phages, and genomic islands. Phages mediate most genetic exchange among S. aureus strains, including the dissemination of resistance genes and virulence factors. Previous research has suggested that phage activation in S. aureus occurs regularly in infected host tissues. On the other hand, phage therapy has regained interest as a non-conventional strategy for urgently needed novel approaches to control infection. Endogenous phage activation might interfere with such approaches. This proposal aims to quantify the extent of phage activation in Staphylococcus aureus during deep-seated human infections and to determine its impact on therapy. I will first quantify phage activation in freshly frozen surgical samples from patients with deep-seated S. aureus infections. I will focus on samples containing S. aureus strains that are representative of major lineages as determined by whole-genome sequencing. I will then establish patient-mimicking in-vitro conditions to recapitulate in-vivo phage activation for identifying underlying molecular mechanisms and potential heterogeneity at the single-cell level. Finally, I will determine the impact of in vivo -like phage activation on S. aureus susceptibility to antimicrobials and phage therapy. Overall, this project will determine the relevance and impact of an understudied, but likely critical aspect of the major human pathogen S. aureus during human infections. Characterization of the ELF for better PK/PD models of human lung infection Research Project | 2 Project MembersThe objective of this collaboration is knowledge generation of the composition of Epithelial Lining Fluid (ELF) of human (healthy volunteers and patients with bacterial pneumonia) and pre-clinical species. This knowledge will be used for constructing simulated ELF matrixes mimicking the most important components of the physiological fluids. An in vitro assay is consequently set up to assess the free fraction of drugs in these fluids. Finally, the data generated will be used to establish a machine learning tool to predict free fraction in ELF based on chemical descriptors and will be further used to develop advanced Physiologically Based PharmacoKinetic (PBPK) model including ELF as additional compartment and its validation for human dose prediction on marketed drugs. The mechanisms of action of macrolide antibiotics RUS_ST2019-124 Research Project | 1 Project MembersCurrently, increasing antibiotic resistance levels has been identified as a major health threat World wide. The World Health Organization has identified several pathogens with critical resistance levels. One of the most worrisome group of bacteria is Enterobacteriacea, which are becoming resistant against cephalosporins and carbapenems. Macrolides is a widely used group of antibiotics. Based on the in vitro activity tests these antibiotics are not used against Enterobacteriacea. Surprisingly, recent clinical data indicate that macrolides can be used against Salmonella infections. Here we aim to elucidate the molecular mechanisms of this unexplained activity. In addition to antibacterial activity, we hypothesize that macrolides can act on the human mitochondrial ribosomes leading potentially to immunomodulation and treatment side effects. We will use novel bioreporters for measuring the effects of macrolides on bacterial protein synthesis in Salmonella and uropathogenic Escherichia coli infection models. We also measure the effects of macrolide treatment on mitochondrial protein synthesis and the levels of mitochondrial proteins. The results will provide information for potentially using macrolides against wider range of infections caused by Enterobacteriacea. The test systems developed will become important tools for the development of new antibiotics. 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. Living Microbial Diagnostics to Enable Individualized Child Health Interventions Research Project | 1 Project MembersWe are in the midst of a global health tragedy where over 3 million children in the developing world die annually before the age of 5 years(GBD 2013, Lancet, 2014) predominantly through infectious diseases as well as malnutrition and related disorders. A further 200 million children annually do not reach their developmental potential(Grantham, Lancet, 2007). A major challenge in diagnosing and treating these children in the developing world lies in the paucity of options for objectively measuring the nutritional, infection, and inflammation status of the gastrointestinal tract. To overcome this challenge, we recently engineered a strain of bacteria that is capable of sensing, remembering, and reporting on the environment within the gastrointestinal tract of animals. These engineered bacteria can safely traverse the gastrointestinal tract, analogously to probiotics, and then be retrieved from feces, analyzed in the laboratory, and enable predictive therapeutic interventions. Our proposal focuses on further developing these engineered bacteria and comprehensively testing their safety and capacity to reveal clinically-meaningful and therapeutically-actionable information. In the future, our technology could be deployed throughout the world, in a sustainable and scalable fashion, and aid in improving child health and wellbeing in both the developed and developing worlds. Horizon 2020 Framework Project MIBEst Research Project | 1 Project MembersInfectious diseases have been the leading cause of death for many centuries. The development of vaccination and antibiotic treatments combined with improved hygiene has decreased the number of deaths, but the mortality and morbidity associated with infections remain considerable, requiring constant societal awareness and scientific research. An increasing concern are the latent and chronic infections that are often refractory to treatments. As the frequency of latent infections increases with age, it is a major concern for aging societies. The great diversity of the infectious agents, and the multidisciplinary nature of the infectious biology research demand a convergence of various competencies: microbiology, cell biology, animal infection models, immunology etc, emphasizing the need for collaboration between research centres. Especially important are joint activities for smaller countries, e.g. Estonia, where establishment of full-scale stand-alone programs is not economically feasible. Despite the strong positions in basic molecular biology, virology and microbiology, Estonia often fails to capitalize on the excellence in basic research by transitioning to the development of therapeutics targeting medically relevant processes. The main objective of the MIBEst project is to strengthen the research capacity on latent and chronic infections of Institute of Technology at University of Tartu by creating long-lasting links with internationally-leading research institutions: Molecular Infection Medicine Sweden at Umeå University, Sweden, and Basel Biozentrum, University of Basel, Switzerland. As an outcome of MIBEst, Estonian scientists will have new knowledge in infection biology with particular focus on advancement in models for latent infections and high throughput screening for promising candidates for antiinfective compounds. Altogether, it enables development of new anti-infection strategies that will have major impact at the national, European and global scale. Tracking and manipulating Salmonella subsets in infected tissues over time Research Project | 1 Project MembersThis project intends to build a basis for entirely novel strategies in infection control , by broadening successful host antimicrobial attacks , and closing permissive niches in which pathogens can thrive. Systemic Salmonella enterica infections are a major cause of mortality worldwide 1-5 , and become increasingly untreatable 6,7 . Our single-cell data show in a mouse model that the host immune system actually eradicates many Salmonella cells , while other Salmonella thrive at the same time in the same tissue, causing lethal disease progression 8,9 . Emerging evidence suggests that similar heterogeneous host-pathogen encounters might be a key feature of many infectious diseases 10-20 . This heterogeneity offers fascinating opportunities for research and application . If we understand the mechanisms that determine the disparate local outcomes, we might be able to tip the balance in favor of the host, by closing permissive niches in which pathogens can thrive . Current research mostly relies on snapshots that cannot capture the crucial temporal dynamics . We know that Salmonella cells differentially access host nutrients, experience oxidative or nitrosative stress, etc.; but what is the consequence of these encounters and what impact do they have for overall disease outcome ? We detect many dead Salmonella cells in tissues; but which events led to their killing , and why did this not happen to the thriving Salmonella subset? Our goals are, therefore, to track Salmonella cells that have experienced a specific host encounter, to manipulate their fate, and to elucidate underlying mechanisms in vivo. To achieve these goals, we will follow four specific aims, in which we test eight specific hypotheses: To unravel encounter consequences over the first few divisions (6-36 h). We will exploit a fluorescent TIMER reporter variant with very slow color maturation as a novel tool. To unravel encounter consequences over several days . We will link Salmonella subset-specific promoters with genetic switches and reporters useful for lineage tracing. To determine the impact of specific encounters for overall disease outcome . We will deplete Salmonella in defined encounters using suicide lysis and/or DNA damage. To determine the underlying molecular mechanisms . We will correlate local abundance of host factors with key encounter types and validate causal relationships using perturbations. Combination of these innovative approaches with our recently developed 3D imaging technique for whole spleen will enable us to validate, improve, and extend our spatio-temporal model for Salmonella infection dynamics . This will be a key step for translating the increasingly detailed knowledge about Salmonella and host heterogeneity during infection, into new strategies for controlling life-threatening systemic Salmonella infections . We hope that our methods and concepts may inspire similar studies in other major diseases with a decisive role of heterogeneous host-pathogen encounters, such as tuberculosis 21 . ESBL-MS: Early diagnosis of ESBL Enteriobacteriaceae in patient samples Research Project | 3 Project MembersEnterobacteriaceae are a major cause of life-threatening infections such as sepsis. Treatment of Enterobacteriaceae infections relies mostly on β-lactams and β-lactam-β-lactamase inhibitor combinations, but the efficacy of these drugs is endangered by the accelerating spread of extended spectrum β-lactamases (ESBL). ESBLs can be detected using biochemical test, isothermal amplification, PCR and microarrays, but these data cannot predictive quantitative susceptibility as needed for targeted treatment decisions. In this project, we use ultrasensitive mass spectrometry to detect ESBLs and other resistance proteins directly in patient samples. In parallel, we determine how these proteins affect capabilities of clinical strains to rapidly evolve increased resistance in vitro, e.g. by upregulating ESBL expression. We also engineer mutants of clinical strains with precisely altered expression of ESBL and other genes, to quantify their role in resistance. Based on all these data, we will build a classifier algorithm for accurate prediction of resistance within 5 h after patient sample acquisition. Finally, we translate sensitive detection methods for relevant proteins to widely available MALDI-TOF mass instruments for simple and rapid implementation. If successful, the results of our project will enable rapid diagnostics for early targeted treatment of serious infections, while avoiding unnecessary usage of last-resort antibiotics. This strategy has large potential to enhance antibiotic stewardship in face of rising resistance. Cyst wall formation: a persistent challenge in Toxoplasmosis Research Project | 2 Project MembersThe acute phase of Toxoplasma gondii infection initiates with the rapid proliferation and dissemination of the fast-replicating form of the parasite (tachyzoite) throughout the vertebrate host. At the onset of the immune response, the tachyzoites are efficiently neutralized and the infection enters in a chronic phase with conversion to a slow-replicating developmental stage (bradyzoite) that forms tissue cysts predominantly in the central nervous system and in striated and heart muscle. This process of encystation is vital to the parasite's life cycle because i) it ensures survival and life-long persistence in intermediate hosts, and ii) it allows peroral transmission to the feline definitive host, initiating the sexual cycle. Tissue cysts not only prevent eradication of the parasite, but also pose a significant threat of reactivation in the context of host immunosuppression and can lead to encephalitis and other severe clinical manifestations. Despite the central importance of cyst formation for pathogenesis and transmission, our insight into how T. gondii defies the innate and adaptive immune responses to take up permanent residence in the immunocompetent hosts is rudimentary. Very little is known about the cyst wall composition and the molecular processes governing its formation. We have a very fragmented view of the temporal and spatial dissemination of cysts in the host and especially in the brain. Progress in biology is driven both by medical necessity and scientific curiosity and this project, which proposes to investigate the process of encystation, lies at the intersection of these two forces. It is the most propitious time to address the challenging question of cyst wall formation in light of the most recent breakthroughs in the sensitivity of -omics approaches, the power of the CRISPR/Cas9 system in genome editing, the revolution in high-throughput microscopy, and ex-vivo tissue examination at the highest level of resolution. This ambitious and highly synergistic project tackles key biological questions on tissue cyst formation and capitalizes heavily on cutting-edge technologies to address three specific objectives: 1. A comprehensive definition of the cyst wall composition. We will generate an unprecedentedly accurate transcriptome of the bradyzoite stage and a differential proteome of bradyzoites and the surrounding cyst wall. The data will be curated via a powerful comparative genomics approach spanning cyst-forming and cyst-lacking Apicomplexa species. A complementary mutagenesis and selection strategy is designed to identify specific defects in cyst formation or maturation by large-scale genetic screens using quantitative high-throughput microscopy. 2. An uncovering of molecular mechanisms governing the cyst wall formation. This aim will be accomplished by taking a targeted as well as a global functional approach. Starting from recent findings on parasitophorous vacuole formation of tachyzoites we will implement a semi-targeted gene knockout- or conditional knockout approach. Moreover, the creation of a comprehensive gene disruption collection in subproject 1 will allow identification of all non-essential tachyzoites genes that display trafficking defects during cyst formation. Key mutants will be mechanistically dissected in vitro with a subset to be investigated further in vivo. 3. A spatiotemporal cartography of cyst formation in the brain. We will harness cutting-edge imaging methods to investigate the in vivo dynamics of dissemination and cyst formation in the whole body and foremost in the brain of a mouse infection model. These findings will inform development of spatiotemporal models describing parasite dissemination and differentiation, to be correlated with data from post-mortem biopsies. By combining unbiased and targeted experimental approaches, we are poised to achieve major conceptual advances in deciphering the molecular events leading to cyst wall formation. In the long term, the data and technology created in this project will lay the foundation to decoding the molecular information exchange between host and pathogen during establishment of life-long latent infections. These studies fill a significant knowledge gap and will provide i) highly valuable web-accessible integrated gene expression data, ii) fundamental discoveries about the regulatory and trafficking circuits that govern formation of the cyst wall as a biological barrier during encystation iii) invaluable paradigms of how the parasite initiates and sustains molecular programs required for disease progression and persistence. 12 12
Impact of Magnesium on Salmonella Infection and Therapy Research Project | 1 Project MembersImported from Grants Tool 4697853
Phage activation in Staphylococcus aureus during deep-seated human infections Research Project | 2 Project MembersThe Gram-positive bacterium Staphylococcus aureus causes a wide variety of infectious diseases that are associated with substantial morbidity and mortality. Clinical isolates of S. aureus display vast genetic diversity, which is mainly determined by the presence of mobile genetic elements, comprising phages, and genomic islands. Phages mediate most genetic exchange among S. aureus strains, including the dissemination of resistance genes and virulence factors. Previous research has suggested that phage activation in S. aureus occurs regularly in infected host tissues. On the other hand, phage therapy has regained interest as a non-conventional strategy for urgently needed novel approaches to control infection. Endogenous phage activation might interfere with such approaches. This proposal aims to quantify the extent of phage activation in Staphylococcus aureus during deep-seated human infections and to determine its impact on therapy. I will first quantify phage activation in freshly frozen surgical samples from patients with deep-seated S. aureus infections. I will focus on samples containing S. aureus strains that are representative of major lineages as determined by whole-genome sequencing. I will then establish patient-mimicking in-vitro conditions to recapitulate in-vivo phage activation for identifying underlying molecular mechanisms and potential heterogeneity at the single-cell level. Finally, I will determine the impact of in vivo -like phage activation on S. aureus susceptibility to antimicrobials and phage therapy. Overall, this project will determine the relevance and impact of an understudied, but likely critical aspect of the major human pathogen S. aureus during human infections.
Characterization of the ELF for better PK/PD models of human lung infection Research Project | 2 Project MembersThe objective of this collaboration is knowledge generation of the composition of Epithelial Lining Fluid (ELF) of human (healthy volunteers and patients with bacterial pneumonia) and pre-clinical species. This knowledge will be used for constructing simulated ELF matrixes mimicking the most important components of the physiological fluids. An in vitro assay is consequently set up to assess the free fraction of drugs in these fluids. Finally, the data generated will be used to establish a machine learning tool to predict free fraction in ELF based on chemical descriptors and will be further used to develop advanced Physiologically Based PharmacoKinetic (PBPK) model including ELF as additional compartment and its validation for human dose prediction on marketed drugs.
The mechanisms of action of macrolide antibiotics RUS_ST2019-124 Research Project | 1 Project MembersCurrently, increasing antibiotic resistance levels has been identified as a major health threat World wide. The World Health Organization has identified several pathogens with critical resistance levels. One of the most worrisome group of bacteria is Enterobacteriacea, which are becoming resistant against cephalosporins and carbapenems. Macrolides is a widely used group of antibiotics. Based on the in vitro activity tests these antibiotics are not used against Enterobacteriacea. Surprisingly, recent clinical data indicate that macrolides can be used against Salmonella infections. Here we aim to elucidate the molecular mechanisms of this unexplained activity. In addition to antibacterial activity, we hypothesize that macrolides can act on the human mitochondrial ribosomes leading potentially to immunomodulation and treatment side effects. We will use novel bioreporters for measuring the effects of macrolides on bacterial protein synthesis in Salmonella and uropathogenic Escherichia coli infection models. We also measure the effects of macrolide treatment on mitochondrial protein synthesis and the levels of mitochondrial proteins. The results will provide information for potentially using macrolides against wider range of infections caused by Enterobacteriacea. The test systems developed will become important tools for the development of new antibiotics.
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.
Living Microbial Diagnostics to Enable Individualized Child Health Interventions Research Project | 1 Project MembersWe are in the midst of a global health tragedy where over 3 million children in the developing world die annually before the age of 5 years(GBD 2013, Lancet, 2014) predominantly through infectious diseases as well as malnutrition and related disorders. A further 200 million children annually do not reach their developmental potential(Grantham, Lancet, 2007). A major challenge in diagnosing and treating these children in the developing world lies in the paucity of options for objectively measuring the nutritional, infection, and inflammation status of the gastrointestinal tract. To overcome this challenge, we recently engineered a strain of bacteria that is capable of sensing, remembering, and reporting on the environment within the gastrointestinal tract of animals. These engineered bacteria can safely traverse the gastrointestinal tract, analogously to probiotics, and then be retrieved from feces, analyzed in the laboratory, and enable predictive therapeutic interventions. Our proposal focuses on further developing these engineered bacteria and comprehensively testing their safety and capacity to reveal clinically-meaningful and therapeutically-actionable information. In the future, our technology could be deployed throughout the world, in a sustainable and scalable fashion, and aid in improving child health and wellbeing in both the developed and developing worlds.
Horizon 2020 Framework Project MIBEst Research Project | 1 Project MembersInfectious diseases have been the leading cause of death for many centuries. The development of vaccination and antibiotic treatments combined with improved hygiene has decreased the number of deaths, but the mortality and morbidity associated with infections remain considerable, requiring constant societal awareness and scientific research. An increasing concern are the latent and chronic infections that are often refractory to treatments. As the frequency of latent infections increases with age, it is a major concern for aging societies. The great diversity of the infectious agents, and the multidisciplinary nature of the infectious biology research demand a convergence of various competencies: microbiology, cell biology, animal infection models, immunology etc, emphasizing the need for collaboration between research centres. Especially important are joint activities for smaller countries, e.g. Estonia, where establishment of full-scale stand-alone programs is not economically feasible. Despite the strong positions in basic molecular biology, virology and microbiology, Estonia often fails to capitalize on the excellence in basic research by transitioning to the development of therapeutics targeting medically relevant processes. The main objective of the MIBEst project is to strengthen the research capacity on latent and chronic infections of Institute of Technology at University of Tartu by creating long-lasting links with internationally-leading research institutions: Molecular Infection Medicine Sweden at Umeå University, Sweden, and Basel Biozentrum, University of Basel, Switzerland. As an outcome of MIBEst, Estonian scientists will have new knowledge in infection biology with particular focus on advancement in models for latent infections and high throughput screening for promising candidates for antiinfective compounds. Altogether, it enables development of new anti-infection strategies that will have major impact at the national, European and global scale.
Tracking and manipulating Salmonella subsets in infected tissues over time Research Project | 1 Project MembersThis project intends to build a basis for entirely novel strategies in infection control , by broadening successful host antimicrobial attacks , and closing permissive niches in which pathogens can thrive. Systemic Salmonella enterica infections are a major cause of mortality worldwide 1-5 , and become increasingly untreatable 6,7 . Our single-cell data show in a mouse model that the host immune system actually eradicates many Salmonella cells , while other Salmonella thrive at the same time in the same tissue, causing lethal disease progression 8,9 . Emerging evidence suggests that similar heterogeneous host-pathogen encounters might be a key feature of many infectious diseases 10-20 . This heterogeneity offers fascinating opportunities for research and application . If we understand the mechanisms that determine the disparate local outcomes, we might be able to tip the balance in favor of the host, by closing permissive niches in which pathogens can thrive . Current research mostly relies on snapshots that cannot capture the crucial temporal dynamics . We know that Salmonella cells differentially access host nutrients, experience oxidative or nitrosative stress, etc.; but what is the consequence of these encounters and what impact do they have for overall disease outcome ? We detect many dead Salmonella cells in tissues; but which events led to their killing , and why did this not happen to the thriving Salmonella subset? Our goals are, therefore, to track Salmonella cells that have experienced a specific host encounter, to manipulate their fate, and to elucidate underlying mechanisms in vivo. To achieve these goals, we will follow four specific aims, in which we test eight specific hypotheses: To unravel encounter consequences over the first few divisions (6-36 h). We will exploit a fluorescent TIMER reporter variant with very slow color maturation as a novel tool. To unravel encounter consequences over several days . We will link Salmonella subset-specific promoters with genetic switches and reporters useful for lineage tracing. To determine the impact of specific encounters for overall disease outcome . We will deplete Salmonella in defined encounters using suicide lysis and/or DNA damage. To determine the underlying molecular mechanisms . We will correlate local abundance of host factors with key encounter types and validate causal relationships using perturbations. Combination of these innovative approaches with our recently developed 3D imaging technique for whole spleen will enable us to validate, improve, and extend our spatio-temporal model for Salmonella infection dynamics . This will be a key step for translating the increasingly detailed knowledge about Salmonella and host heterogeneity during infection, into new strategies for controlling life-threatening systemic Salmonella infections . We hope that our methods and concepts may inspire similar studies in other major diseases with a decisive role of heterogeneous host-pathogen encounters, such as tuberculosis 21 .
ESBL-MS: Early diagnosis of ESBL Enteriobacteriaceae in patient samples Research Project | 3 Project MembersEnterobacteriaceae are a major cause of life-threatening infections such as sepsis. Treatment of Enterobacteriaceae infections relies mostly on β-lactams and β-lactam-β-lactamase inhibitor combinations, but the efficacy of these drugs is endangered by the accelerating spread of extended spectrum β-lactamases (ESBL). ESBLs can be detected using biochemical test, isothermal amplification, PCR and microarrays, but these data cannot predictive quantitative susceptibility as needed for targeted treatment decisions. In this project, we use ultrasensitive mass spectrometry to detect ESBLs and other resistance proteins directly in patient samples. In parallel, we determine how these proteins affect capabilities of clinical strains to rapidly evolve increased resistance in vitro, e.g. by upregulating ESBL expression. We also engineer mutants of clinical strains with precisely altered expression of ESBL and other genes, to quantify their role in resistance. Based on all these data, we will build a classifier algorithm for accurate prediction of resistance within 5 h after patient sample acquisition. Finally, we translate sensitive detection methods for relevant proteins to widely available MALDI-TOF mass instruments for simple and rapid implementation. If successful, the results of our project will enable rapid diagnostics for early targeted treatment of serious infections, while avoiding unnecessary usage of last-resort antibiotics. This strategy has large potential to enhance antibiotic stewardship in face of rising resistance.
Cyst wall formation: a persistent challenge in Toxoplasmosis Research Project | 2 Project MembersThe acute phase of Toxoplasma gondii infection initiates with the rapid proliferation and dissemination of the fast-replicating form of the parasite (tachyzoite) throughout the vertebrate host. At the onset of the immune response, the tachyzoites are efficiently neutralized and the infection enters in a chronic phase with conversion to a slow-replicating developmental stage (bradyzoite) that forms tissue cysts predominantly in the central nervous system and in striated and heart muscle. This process of encystation is vital to the parasite's life cycle because i) it ensures survival and life-long persistence in intermediate hosts, and ii) it allows peroral transmission to the feline definitive host, initiating the sexual cycle. Tissue cysts not only prevent eradication of the parasite, but also pose a significant threat of reactivation in the context of host immunosuppression and can lead to encephalitis and other severe clinical manifestations. Despite the central importance of cyst formation for pathogenesis and transmission, our insight into how T. gondii defies the innate and adaptive immune responses to take up permanent residence in the immunocompetent hosts is rudimentary. Very little is known about the cyst wall composition and the molecular processes governing its formation. We have a very fragmented view of the temporal and spatial dissemination of cysts in the host and especially in the brain. Progress in biology is driven both by medical necessity and scientific curiosity and this project, which proposes to investigate the process of encystation, lies at the intersection of these two forces. It is the most propitious time to address the challenging question of cyst wall formation in light of the most recent breakthroughs in the sensitivity of -omics approaches, the power of the CRISPR/Cas9 system in genome editing, the revolution in high-throughput microscopy, and ex-vivo tissue examination at the highest level of resolution. This ambitious and highly synergistic project tackles key biological questions on tissue cyst formation and capitalizes heavily on cutting-edge technologies to address three specific objectives: 1. A comprehensive definition of the cyst wall composition. We will generate an unprecedentedly accurate transcriptome of the bradyzoite stage and a differential proteome of bradyzoites and the surrounding cyst wall. The data will be curated via a powerful comparative genomics approach spanning cyst-forming and cyst-lacking Apicomplexa species. A complementary mutagenesis and selection strategy is designed to identify specific defects in cyst formation or maturation by large-scale genetic screens using quantitative high-throughput microscopy. 2. An uncovering of molecular mechanisms governing the cyst wall formation. This aim will be accomplished by taking a targeted as well as a global functional approach. Starting from recent findings on parasitophorous vacuole formation of tachyzoites we will implement a semi-targeted gene knockout- or conditional knockout approach. Moreover, the creation of a comprehensive gene disruption collection in subproject 1 will allow identification of all non-essential tachyzoites genes that display trafficking defects during cyst formation. Key mutants will be mechanistically dissected in vitro with a subset to be investigated further in vivo. 3. A spatiotemporal cartography of cyst formation in the brain. We will harness cutting-edge imaging methods to investigate the in vivo dynamics of dissemination and cyst formation in the whole body and foremost in the brain of a mouse infection model. These findings will inform development of spatiotemporal models describing parasite dissemination and differentiation, to be correlated with data from post-mortem biopsies. By combining unbiased and targeted experimental approaches, we are poised to achieve major conceptual advances in deciphering the molecular events leading to cyst wall formation. In the long term, the data and technology created in this project will lay the foundation to decoding the molecular information exchange between host and pathogen during establishment of life-long latent infections. These studies fill a significant knowledge gap and will provide i) highly valuable web-accessible integrated gene expression data, ii) fundamental discoveries about the regulatory and trafficking circuits that govern formation of the cyst wall as a biological barrier during encystation iii) invaluable paradigms of how the parasite initiates and sustains molecular programs required for disease progression and persistence.
