Immunoevasion mechanisms by Mycobacterium tuberculosis

Tuberculosis affects 2 billion people worldwide. The bacteria causing TB, Mycobacterium tuberculosis can lie dormant within an infected host for decades in a chronic state. In order to survive within the host, TB evolved means to evade the immune response of the human host. Immune responses are initiated by specialised cells, known as antigen presenting cells (APC). The aim of this project will be to identify and characterise the TB-derived factors that help TB evade detection by the arm of the immune response that employs T lymphocytes. A TB strain will be genetically modified to express a protein, ovalbumin. There are many useful immunological tools that can be used to precisely quantify immune responses against ovalbumin, as a surrogate for immunity to the TB itself. A library of mutant strains of these bacteria, with all non-essential genes knocked out, will be generated. Two screens will be employed to identify which genes are responsible for down-regulation of T lymphocyte responses against TB: using APC in cell culture, and also using live mice. The candidate genes identified with the screens will have their functions characterised using biochemical methods. Through better understanding how TB subverts immunity, better vaccines and therapeutic targets can be developed.

Tuberculosis is a major global health crisis. Despite widespread use of the bacillus Calmette-Guerin (BCG) vaccine and availability of effective drug therapy, Mycobacterium tuberculosis (Mtb) remains the most common causes of infectious disease morbidity worldwide. Like other pathogens that establish a state of chronic, controlled infection, M. tuberculosis has evolved mechanisms to evade the host immune response. These immune responses are initiated by professional antigen presenting cells (APC), such as dendritic cells (DC). The objectives of this project are to determine: (i) What mycobacterial genetic factors are necessary for access of M. tuberculosis to the antigen loading machinery of APC, and (ii) how mycobacteria evade the adaptive immune response. I will make a transgenic strain of M. tuberculosis expressing ovalbumin and a high density random insertion mutant library of this strain will be constructed. The mutant library will be used in an in vivo screen using wild-type and ova-immunised mice. Decreased survival of mycobacteria in the immunised mice will imply those strains also have defects in immunoevasion. These mutants will be identified using a novel screen - transposon site hybridisation (TraSH). A quantitative in vitro antigen presentation assay will also be set-up using primary murine DC and macrophages; and OVA-peptide specific hybridoma cell-lines and the clonotypic antibody 25.D1.16. The mutant TB library will be tested using this assay to identify strains that exhibit enhanced antigen presentation, implying a defect in immunoevasion. The functions of the identified candidate gene products will be characterised by biochemical and cell-biological means.