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.

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.

Publications

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