Improving and understanding macrophage restriction of Mycobacterium tuberculosis infection

Abstract

Tuberculosis, the disease caused by Mycobacterium tuberculosis (Mtb), is a leading cause of death due to infectious disease. New biomedical interventions are necessary to meet World Health Organization goals for tuberculosis control. However, the creation of these interventions is constrained by insufficient knowledge of how the human immune system fights infection with Mtb, which limits our tools for improving immunity. Macrophages are a protective replicative niche for Mtb but can kill the infecting bacterium when appropriately activated. We sought to identify compounds that best activate macrophage restriction, and elucidate the resulting mechanism of Mtb control. A host-side CRISPR screen during mycobacterial infection identified macrophage genes for which genetic and pharmacologic targeting each increased macrophage survival and Mtb restriction. In a complementary approach, we systematically compared bacterial restriction by human macrophages after treatment with 26 activators, including compounds currently in clinical trials for tuberculosis. Both approaches yielded pharmacological inducers of Mtb restriction that were robust across macrophage contexts. To understand mechanisms of macrophage restriction of Mtb infection we studied the compound all-trans-retinoic acid (ATRA), an active metabolite of vitamin A which most effectively increased Mtb control. By comparing ATRA to closely related yet non-restrictive compounds, we found that bacterial clearance was transcriptionally and functionally associated with macrophage cholesterol efflux; this limitation of macrophage cholesterol was necessary for restriction of Mtb. To determine how cholesterol efflux affected bacterial control, we performed the first Mtb CRISPR interference screen in an infection model, identifying Mtb genes specifically required to survive in ATRA-activated macrophages. These data showed that ATRA treatment starves Mtb of cholesterol and the downstream metabolite propionyl-CoA. Supplementation with sources of propionyl-CoA abrogated the restrictive effect of ATRA, and this effect was dependent on pathways that integrate propionate into central carbon metabolism. The data in this dissertation demonstrate that host genetic screening and systematic comparative analysis can each identify effective activators of Mtb restriction by macrophages, enabling future therapeutic development. They also show that targeting the coupled metabolism of Mtb and the macrophage improves control of infection, and that it is possible to genetically map the mode of bacterial death using CRISPR interference.