DictyMyc: Using Dictyostelium to study the genetic basis of Mycobacterium bovis intracellular infection.
Lead Research Organisation:
University of Surrey
Department Name: Microbial & Cellular Sciences
Abstract
Bovine tuberculosis (TB) caused by Mycobacterium bovis is one of the most important veterinary health problems in the UK. In the absence of improved control, the projected economic burden to the UK over the next decade is predicted to be £1 billion. Control is likely to require an integrated approach with vaccination of cattle representing a key component. Presently there is no licensed vaccine against bovine TB. However, the live M.bovis BCG vaccine, presently used against human TB, represents an encouraging vaccination option, yet studies suggest that it only provides protection to ~70% of animals.
To form a rational platform from which to design an improved vaccine that stimulates protective immunity in all individuals, we propose it is necessary to better understand the basic mechanisms of the host-pathogen interaction during mycobacterial infection. Evidence suggests that manipulating the mycobacterial interaction with phagocytic cells can increase immunogenicity.
The central feature of mycobacterial infection is an ability to replicate inside mammalian immune cells called phagocytes. We and others have previously studied the bacterial genes involved in these processes. However, analysis of the HOST genes involved in the interactions has been difficult because it is hard to manipulate the genetics of mammalian cells. One solution is to use phagocytes from genetically modified- (GM-) mice which are becoming more widely used in research.
THE AIM OF THIS PROJECT IS TO DEVELOP AND CHARACTERISE NON-VERTEBRATE MODELS OF MYCOBACTERIAL INFECTION AS A REPLACEMENT FOR USING PHAGOCYTES FROM GM-MICE.
In this project we propose to use the free living amoebae Dictyostelium discoideum as a model phagocyte because it is easy to manipulate its genetics, and mycobacteria are able to survive in dictyostelium in a similar way to how they survive in phagocytes. Thus experiments in which dictyostelium is infected with mycobacteria provide a way to easily study both the host and pathogen genes during mycobacterial infection whilst avoiding the use of sentient animals. To characterise the Dictyostelium model systems and validate them against bovine infection, we will compare the bacterial genes used in infection in bovine macrophages with those required for infection in dictyostelium. We will also use a newly developed technology that will allow the simultaneous analysis of a library of 40,000 dictyostelium (host) mutants during mycobacterial infection. Our experiments will comprehensively identify which host and pathogen genes are involved in the M.bovis survival in dictyostelium informing how the mycobacterium controls its host cell and providing information to help design a better vaccine against bovine and human TB.
This project will provide the technology and evidence to justify other researchers working on tuberculosis and other infectious diseases to utilise dictyostelium as a model organism.
To form a rational platform from which to design an improved vaccine that stimulates protective immunity in all individuals, we propose it is necessary to better understand the basic mechanisms of the host-pathogen interaction during mycobacterial infection. Evidence suggests that manipulating the mycobacterial interaction with phagocytic cells can increase immunogenicity.
The central feature of mycobacterial infection is an ability to replicate inside mammalian immune cells called phagocytes. We and others have previously studied the bacterial genes involved in these processes. However, analysis of the HOST genes involved in the interactions has been difficult because it is hard to manipulate the genetics of mammalian cells. One solution is to use phagocytes from genetically modified- (GM-) mice which are becoming more widely used in research.
THE AIM OF THIS PROJECT IS TO DEVELOP AND CHARACTERISE NON-VERTEBRATE MODELS OF MYCOBACTERIAL INFECTION AS A REPLACEMENT FOR USING PHAGOCYTES FROM GM-MICE.
In this project we propose to use the free living amoebae Dictyostelium discoideum as a model phagocyte because it is easy to manipulate its genetics, and mycobacteria are able to survive in dictyostelium in a similar way to how they survive in phagocytes. Thus experiments in which dictyostelium is infected with mycobacteria provide a way to easily study both the host and pathogen genes during mycobacterial infection whilst avoiding the use of sentient animals. To characterise the Dictyostelium model systems and validate them against bovine infection, we will compare the bacterial genes used in infection in bovine macrophages with those required for infection in dictyostelium. We will also use a newly developed technology that will allow the simultaneous analysis of a library of 40,000 dictyostelium (host) mutants during mycobacterial infection. Our experiments will comprehensively identify which host and pathogen genes are involved in the M.bovis survival in dictyostelium informing how the mycobacterium controls its host cell and providing information to help design a better vaccine against bovine and human TB.
This project will provide the technology and evidence to justify other researchers working on tuberculosis and other infectious diseases to utilise dictyostelium as a model organism.
Technical Summary
Bovine TB caused by Mycobacterium bovis is the most important veterinary health problem in the UK. Development of an efficacious cattle vaccine is a priority and gaining a better understanding of how mycobacteria manipulate phagocytic cells will help rational design of an improved live vaccine.
We and others have used parallel genetic screens like TnSeq to study the bacterial genes important for survival in mammalian phagocytes. However, it has been difficult to study the host genes involved in the interaction because the genetics of mammalian cells are less easily manipulated. Many studies have used phagocytes from gene-KO mice. The aim of this project is to develop non-sentient models for mycobacterial infection using the genetically tractable Dictyostelium discoideum as a model phagocyte. We will characterise the Dictyostelium infection models using TnSeq to compare the M.bovis genes needed for survival in bovine macrophages with those required by M.bovis and M.marinum to survive in Dictyostelium. This will tell us which M.bovis virulence mechanisms in macrophages also occur and can be studied in the Dictyostelium models.
The unique advantage of Dictyostelium as a model host cell is that libraries of KO mutants are available. Thus we will be able to study the host genes involved in the mycobacterium/phagocyte interaction without using gene-KO mice. We will use a new parallel genetics technique, REMI-seq, to comprehensively identify the host genes involved in susceptibility or resistance of Dictyostelium to M.marinum (requires flow-cytometry at CL2) and subsequently test identified host genes for roles in M.bovis infection using individual Dictyostelium mutants (CL3).
The data will be validated by assessment of individual bacterial and dictyostelium mutants during infection using microscopy. Finally, using bioinformatic and modelling techniques we will compile the molecular pathways involved in the mycobacterium phagocyte interaction.
We and others have used parallel genetic screens like TnSeq to study the bacterial genes important for survival in mammalian phagocytes. However, it has been difficult to study the host genes involved in the interaction because the genetics of mammalian cells are less easily manipulated. Many studies have used phagocytes from gene-KO mice. The aim of this project is to develop non-sentient models for mycobacterial infection using the genetically tractable Dictyostelium discoideum as a model phagocyte. We will characterise the Dictyostelium infection models using TnSeq to compare the M.bovis genes needed for survival in bovine macrophages with those required by M.bovis and M.marinum to survive in Dictyostelium. This will tell us which M.bovis virulence mechanisms in macrophages also occur and can be studied in the Dictyostelium models.
The unique advantage of Dictyostelium as a model host cell is that libraries of KO mutants are available. Thus we will be able to study the host genes involved in the mycobacterium/phagocyte interaction without using gene-KO mice. We will use a new parallel genetics technique, REMI-seq, to comprehensively identify the host genes involved in susceptibility or resistance of Dictyostelium to M.marinum (requires flow-cytometry at CL2) and subsequently test identified host genes for roles in M.bovis infection using individual Dictyostelium mutants (CL3).
The data will be validated by assessment of individual bacterial and dictyostelium mutants during infection using microscopy. Finally, using bioinformatic and modelling techniques we will compile the molecular pathways involved in the mycobacterium phagocyte interaction.
Planned Impact
Impact on the 3Rs
Our experiments represent an unbiased, comprehensive screen of the host genes involved in mycobacterium intracellular survival. Such a broad screen is unlikely to be performed using phagocytes from genetically manipulated (GM)-mice but theoretically would require the use of more than 190,000 animals. A more realistic estimate is that the Dictyostelium experiments in our project will replace 120 targeted experiments using phagocytes from GM-mice. These experiments would use 6720 GM-mice.
Most importantly, we will show that the Dictyostelium model not only has technical/scientific advantages over vertebrate-based models but we will delineate the extent of its relevance such that other researchers will be able to make an evidence based decision to use these models when they are appropriate. We anticipate that many mycobacterial research laboratories will adopt these models.
The application of REMI-SEQ screening of Dictyostelium mutants in an intracellular infection model is a step change in our ability to study the pathogenesis of intracellular infection. We anticipate that this technology will be utilised by researchers working on a variety of intracellular pathogens including salmonella, legionella, vibrio and burkholderia. Each REMI-Seq study has the potential to replace thousands of GM-mice.
Our project will contribute to the the rational design of more effective vaccines against M.bovis (and potentially M.tuberculosis). Better design of new vaccines will reduce unneccesary animal testing.
This project will train two PDRAs in the use of Dictyostelium as a genetically tractable model organism for studying human and animal diseases. The collaborators, applicants, technicians and core staff involved in the project will also learn about the principles and potential benefits of well designed 3Rs research. The PI and CoIs will integrate this 3Rs research and this project in particular into the FHEQ level 6 and 7 teaching modules on the Pathogenesis of Infectious Diseases.
Impact on M.bovis vaccine development
This project is basic research on the interaction between M.bovis (and M.marinum) and its host phagocytic cell. However, we anticipate that the knowledge gained will be useful in the rational development of novel live vaccine candidates against M.bovis. Published studies suggest that recombinant mycobacterial strains with altered properties associated with manipulation of the phagosome or host cell redox state have enhanced immunogenicity in animal models. This project will identify candidate molecular mechanisms that can be targeted in new recombinant vaccines.
Further academic impact
This project will generate a comprehensive genome-wide list of the Mycobacterium bovis genes that are important for survival in primary bovine macrophages. This will be a valuable reference to many research programmes studying the molecular basis of pathogenesis in Mycobacterium tuberculosis complex organisms. It will be particularly useful to those studying the pathogenomic differences between M.bovis and M.tuberculosis because this will be the first such dataset for virulent M.bovis.
We will produce an experimentally validated genome scale dataset showing the importance of all mutable Dictyostelium genes during infection with M.marinum. THIS WILL BE THE FIRST SUCH DATASET FOR ANY MYCOBACTERIUM HOST CELL. M.marinum is closely related to M.bovis/tuberculosis and research on M.marinum infection in zebrafish and Dictyostelium has led to significant discoveries of mycobacterial pathogenesis that hold true in M.tuberculosis complex bacteria. This dataset will be an invaluable resource for researchers examining the host-pathogen interaction in any mycobacterial infection.
The project will also generate understanding/hypotheses of how M.bovis is able to survive in the environment. This is important to understanding how M.bovis is transmitted.
Our experiments represent an unbiased, comprehensive screen of the host genes involved in mycobacterium intracellular survival. Such a broad screen is unlikely to be performed using phagocytes from genetically manipulated (GM)-mice but theoretically would require the use of more than 190,000 animals. A more realistic estimate is that the Dictyostelium experiments in our project will replace 120 targeted experiments using phagocytes from GM-mice. These experiments would use 6720 GM-mice.
Most importantly, we will show that the Dictyostelium model not only has technical/scientific advantages over vertebrate-based models but we will delineate the extent of its relevance such that other researchers will be able to make an evidence based decision to use these models when they are appropriate. We anticipate that many mycobacterial research laboratories will adopt these models.
The application of REMI-SEQ screening of Dictyostelium mutants in an intracellular infection model is a step change in our ability to study the pathogenesis of intracellular infection. We anticipate that this technology will be utilised by researchers working on a variety of intracellular pathogens including salmonella, legionella, vibrio and burkholderia. Each REMI-Seq study has the potential to replace thousands of GM-mice.
Our project will contribute to the the rational design of more effective vaccines against M.bovis (and potentially M.tuberculosis). Better design of new vaccines will reduce unneccesary animal testing.
This project will train two PDRAs in the use of Dictyostelium as a genetically tractable model organism for studying human and animal diseases. The collaborators, applicants, technicians and core staff involved in the project will also learn about the principles and potential benefits of well designed 3Rs research. The PI and CoIs will integrate this 3Rs research and this project in particular into the FHEQ level 6 and 7 teaching modules on the Pathogenesis of Infectious Diseases.
Impact on M.bovis vaccine development
This project is basic research on the interaction between M.bovis (and M.marinum) and its host phagocytic cell. However, we anticipate that the knowledge gained will be useful in the rational development of novel live vaccine candidates against M.bovis. Published studies suggest that recombinant mycobacterial strains with altered properties associated with manipulation of the phagosome or host cell redox state have enhanced immunogenicity in animal models. This project will identify candidate molecular mechanisms that can be targeted in new recombinant vaccines.
Further academic impact
This project will generate a comprehensive genome-wide list of the Mycobacterium bovis genes that are important for survival in primary bovine macrophages. This will be a valuable reference to many research programmes studying the molecular basis of pathogenesis in Mycobacterium tuberculosis complex organisms. It will be particularly useful to those studying the pathogenomic differences between M.bovis and M.tuberculosis because this will be the first such dataset for virulent M.bovis.
We will produce an experimentally validated genome scale dataset showing the importance of all mutable Dictyostelium genes during infection with M.marinum. THIS WILL BE THE FIRST SUCH DATASET FOR ANY MYCOBACTERIUM HOST CELL. M.marinum is closely related to M.bovis/tuberculosis and research on M.marinum infection in zebrafish and Dictyostelium has led to significant discoveries of mycobacterial pathogenesis that hold true in M.tuberculosis complex bacteria. This dataset will be an invaluable resource for researchers examining the host-pathogen interaction in any mycobacterial infection.
The project will also generate understanding/hypotheses of how M.bovis is able to survive in the environment. This is important to understanding how M.bovis is transmitted.