Systems analysis reveals alternate metabolic states adopted by <i>Mycobacterium tuberculosis</i> across species

ABSTRACT Mycobacterium tuberculosis (Mtb) survives within macrophages by adapting its metabolism to host-imposed constraints, yet how host species shape bacterial metabolic state remains poorly defined. Here, we directly compared the intracellular transcriptional and metabolic states of Mtb during infection of human and mouse macrophages. Cross-species transcriptomics revealed distinct host-imposed microenvironments that drive Mtb into separable metabolic programs, with mouse macrophages inducing stress-associated pathways and human macrophages promoting fatty acid import and catabolism. Strikingly, these differences manifested in a species-specific phenotype: Mtb formed intracellular lipid inclusions (ILIs) in murine macrophages but not in human macrophages, despite equivalent lipid availability. This divergence was independent of macrophage ontogeny, culture conditions, nitric oxide, or itaconate. Instead, bacterial lipid storage in ILIs was inversely correlated with host triacylglycerol synthesis. Pharmacologic inhibition of diacylglycerol acyltransferase 1 in human macrophages partially restored Mtb ILI formation, identifying host lipid sequestration as a metabolic gate that restricts bacterial lipid storage. Together, these findings establish host lipid metabolism as a key determinant of intracellular Mtb metabolic state and reveal a species-specific barrier that limits bacterial access to host lipid stores in human macrophages. IMPORTANCE Tuberculosis remains a leading cause of infectious death worldwide, yet much of what we know about how Mycobacterium tuberculosis survives inside immune cells comes from studies in animal models. This work shows that human and mouse macrophages impose fundamentally different metabolic constraints on the bacterium, leading to distinct survival strategies. In mouse cells, the pathogen adopts a stress-associated state and stores lipids, whereas in human cells, it does not. We identify host lipid metabolism as a key factor limiting bacterial access to these resources in human macrophages. These findings highlight an important species-specific difference that may influence how well animal models predict human infection and suggest that targeting host lipid pathways could offer new strategies to control tuberculosis.