RESUMEN
Mycobacterium tuberculosis (Mtb) infection of macrophages reprograms cellular metabolism to promote lipid retention. While it is clearly known that intracellular Mtb utilize host derived fatty acids and cholesterol to fuel the majority of its metabolic demands, the role of macrophage lipid catabolism on the bacteria's ability to access the intracellular lipid pool remains undefined. We utilized a CRISPR genetic knockdown approach to assess the impact of sequential steps in fatty acid metabolism on the growth of intracellular Mtb. Our analyzes demonstrate that knockdown of lipid import, sequestration and metabolism genes collectively impair the intracellular growth of Mtb in macrophages. We further demonstrate that modulating fatty acid homeostasis in macrophages impairs Mtb replication through diverse pathways like enhancing production of pro- inflammatory cytokines, autophagy, restricting the bacteria access to nutrients and increasing oxidative stress. We also show that impaired macrophage lipid droplet biogenesis is restrictive to intracellular Mtb replication, but increased induction of the same by blockade of downstream fatty acid oxidation fails to rescue Mtb growth. Our work expands our understanding of how host fatty acid homeostasis impacts Mtb growth in the macrophage.
RESUMEN
The eukaryotic GID/CTLH complex is a highly conserved E3 ubiquitin ligase involved in a broad range of biological processes. However, a role of this complex in host antimicrobial defenses has not been described. We exploited Mycobacterium tuberculosis ( Mtb ) induced cytotoxicity in macrophages in a FACS based CRISPR genetic screen to identify host determinants of intracellular Mtb growth restriction. Our screen identified 5 ( GID8 , YPEL5 , WDR26 , UBE2H , MAEA ) of the 10 predicted members of the GID/CTLH complex as determinants of intracellular growth of both Mtb and Salmonella serovar Typhimurium. We show that the antimicrobial properties of the GID/CTLH complex knockdown macrophages are mediated by enhanced GABAergic signaling, activated AMPK, increased autophagic flux and resistance to cell death. Meanwhile, Mtb isolated from GID/CTLH knockdown macrophages are nutritionally starved and oxidatively stressed. Our study identifies the GID/CTLH complex activity as broadly suppressive of host antimicrobial responses against intracellular bacterial infections.