MECHANISMS OF MYCOBACTERIUM INHIBITION OF HOST ANTIGEN PRESENTATION

<b>Project Methods</b><br> Our research group has developed global genetic approaches to probe the function of both host and bacterial genes during infection of macrophages, an important antigen presenting cell (APC) during Mycobacterium infection and the major reservoir for intracellular Mycobacterium. In the proposed research we will use these approaches to interrogate interactions between Mycobacterium infection and MHCII regulation. Effort: To develop global mammalian genetic approaches to dissect immune evasion during Mycobacterium infection. To investigate the function of host genes we developed genome-wide loss-of-function libraries in macrophages using CRISPR-Cas9 mediated approaches. Using these tools, we will understand the regulation of MHCII following infection with M. bovis and M. tuberculosis. We have already used this library to sort cells that were and were not able to express either MHCII following activation, independently of infection.Evaluation: We will assess these efforts with thorough validation and consider the identification of new pathways that control MHCII on the surface of macrophages during infection as a success.Effort: To develop global Mycobacterial genetic approaches to dissect immune evasion during infection. To investigate the role of Mycobacterial genes we developed transposon-sequencing (Tnseq) approaches to dissect bacterial gene functions in macrophages. Tnseq uses a library of transpon mutants and compares the representation of each mutant following differential selective pressures. These approaches will be used to identify bacterial genes that are required to inhibit the surface expression of MHCII during Mycobacterial infection.Evaluation. We will gauge the success for each pathogen individually. Thorough validation of knockout bacteria will be required to assess the robustness of our effort and will be broadly undertaken.Effort: To dissect the role of validated genes on the immune evasion of Mycobacteria infections. Using a series of in vivo assays including TCR transgenic tools, we will examine how targeting immune evasion tactics of Mycobacterium results in alter protection or bacterial clearance.Evaluation. We will consider these efforts a success if we can identify at least one targetable pathway that improves disease progression and can be potentially targeted by immunization or therapeutically.