A hallmark of tuberculosis is the ability of the causative agent,Mycobacterium tuberculosis, to persist for decades despite a vigorous host immune response. Previously, we identified a mycobacterial gene cluster,mce4, that was specifically required for bacterial survival during this prolonged infection. We now show thatmce4encodes a cholesterol import system that enablesM. tuberculosisto derive both carbon and energy from this ubiquitous component of host membranes. Cholesterol import is not required for establishing infection in mice or for growth in resting macrophages. However, this function is essential for persistence in the lungs of chronically infected animals and for growth within the IFN-γ-activated macrophages that predominate at this stage of infection. This finding indicates that a major effect of IFN-γ stimulation may be to sequester potential pathogens in a compartment devoid of more commonly used nutrients. The unusual capacity to catabolize sterols allowsM. tuberculosisto circumvent this defense and thereby sustain a persistent infection.

The pathways that comprise cellular metabolism are highly interconnected, and alterations in individual enzymes can have far-reaching effects. As a result, global profiling methods that measure gene expression are of limited value in predicting how the loss of an individual function will affect the cell. In this work, we employed a new method of global phenotypic profiling to directly define the genes required for the growth of Mycobacterium tuberculosis. A combination of high-density mutagenesis and deep-sequencing was used to characterize the composition of complex mutant libraries exposed to different conditions. This allowed the unambiguous identification of the genes that are essential for Mtb to grow in vitro, and proved to be a significant improvement over previous approaches. To further explore functions that are required for persistence in the host, we defined the pathways necessary for the utilization of cholesterol, a critical carbon source during infection. Few of the genes we identified had previously been implicated in this adaptation by transcriptional profiling, and only a fraction were encoded in the chromosomal region known to encode sterol catabolic functions. These genes comprise an unexpectedly large percentage of those previously shown to be required for bacterial growth in mouse tissue. Thus, this single nutritional change accounts for a significant fraction of the adaption to the host. This work provides the most comprehensive genetic characterization of a sterol catabolic pathway to date, suggests putative roles for uncharacterized virulence genes, and precisely maps genes encoding potential drug targets. The adaptation of a bacterial pathogen to the environments encountered during infection requires the wholesale remodeling of cellular physiology. One of the major changes encountered upon infection is nutritional, as the bacterium is forced to utilize compounds scavenged from the host. Mycobacterium tuberculosis has the unusual ability to use host cholesterol as a source of carbon and energy, and this capacity is required for persistence in animal models. However, our ignorance of the biochemical pathways involved in sterol degradation has limited our ability to assess the importance of this carbon shift in shaping the metabolic state of the bacterium. In this work, we developed a new method to quantitatively profile the fitness of thousands of mutants simultaneously. This allowed the identification of each bacterial gene that is required for the bacterium to grow using cholesterol as a carbon source. Reconstruction of the pathways comprised by these essential gene products revealed that adaptation to cholesterol required widespread metabolic changes. These genes account for a significant fraction of the bacterial functions important for growth in animal tissues, suggesting that the physiology of this intracellular pathogen is shaped by carbon sources available in this environment.

Chronic infections can result in harmful production of the proinflammatory cytokine IL-1 generated via the NLRP3 inflammasome. Sassetti et al. demonstrate that IL-1 activated by Mycobacteria results in nitrosylation and consequent regulation of the NLRP3 inflammasome. Interleukin 1 (IL-1) is an important mediator of innate immunity but can also promote inflammatory tissue damage. During chronic infections such as tuberculosis, the beneficial antimicrobial role of IL-1 must be balanced with the need to prevent immunopathology. By exogenously controlling the replication of Mycobacterium tuberculosis in vivo, we obviated the requirement for antimicrobial immunity and discovered that both IL-1 production and infection-induced immunopathology were suppressed by lymphocyte-derived interferon-γ (IFN-γ). This effect was mediated by nitric oxide (NO), which we found specifically inhibited assembly of the NLRP3 inflammasome via thiol nitrosylation. Our data indicate that the NO produced as a result of adaptive immunity is indispensable in modulating the destructive innate inflammatory responses elicited during persistent infections.

A greater understanding of hematopoietic stem cell (HSC) regulation is required for dissecting protective versus detrimental immunity to pathogens that cause chronic infections such as Mycobacterium tuberculosis (Mtb). We have shown that systemic administration of Bacille Calmette-Guérin (BCG) or β-glucan reprograms HSCs in the bone marrow (BM) via a type II interferon (IFN-II) or interleukin-1 (IL1) response, respectively, which confers protective trained immunity against Mtb. Here, we demonstrate that, unlike BCG or β-glucan, Mtb reprograms HSCs via an IFN-I response that suppresses myelopoiesis and impairs development of protective trained immunity to Mtb. Mechanistically, IFN-I signaling dysregulates iron metabolism, depolarizes mitochondrial membrane potential, and induces cell death specifically in myeloid progenitors. Additionally, activation of the IFN-I/iron axis in HSCs impairs trained immunity to Mtb infection. These results identify an unanticipated immune evasion strategy of Mtb in the BM that controls the magnitude and intrinsic anti-microbial capacity of innate immunity to infection.