The cytokine IFN-γ coordinates macrophage activation and is essential for control of pathogens including Mycobacterium tuberculosis. However, the mechanisms by which IFN-γ controls M. tuberculosis infection are only partially understood. Here, we show that the transcription factor HIF-1α is an essential mediator of IFN-γ dependent control of M. tuberculosis infection both in vitro and in vivo. M. tuberculosis infection of IFN-γ activated macrophages results in a synergistic increase in HIF-1α protein levels. This increase in HIF-1α levels is functionally important, as macrophages lacking HIF-1α are defective for IFN-γ dependent control of infection. RNA-seq profiling demonstrates that HIF-1α regulates nearly half of all IFN-γ inducible genes during infection of macrophages. In particular, HIF-1α regulates production of important immune effectors including inflammatory cytokines and chemokines, eicosanoids, and nitric oxide (NO). In addition, we find that during infection HIF-1α coordinates a metabolic shift to aerobic glycolysis in IFN-γ activated macrophages. We find that this enhanced glycolytic flux is crucial for IFN-γ dependent control of infection in macrophages. Furthermore, we identify a positive feedback loop between HIF-1α and aerobic glycolysis that amplifies macrophage activation. Finally, we demonstrate that HIF-1α is crucial for control of infection in vivo as mice lacking HIF-1α in the myeloid lineage are strikingly susceptible to infection, and exhibit defective production of inflammatory cytokines and microbicidal effectors. In conclusion, we have identified HIF-1α as a novel regulator of IFN-γ dependent immunity that coordinates an immunometabolic program essential for control of M. tuberculosis infection in vitro and in vivo.

Mycobacterium tuberculosis remains a significant threat to global health. Macrophages are the host cell for M. tuberculosis infection, and although bacteria are able to replicate intracellularly under certain conditions, it is also clear that macrophages are capable of killing M. tuberculosis if appropriately activated. The outcome of infection is determined at least in part by the host-pathogen interaction within the macrophage; however, we lack a complete understanding of which host pathways are critical for bacterial survival and replication. To add to our understanding of the molecular processes involved in intracellular infection, we performed a chemical screen using a high-content microscopic assay to identify small molecules that restrict mycobacterial growth in macrophages by targeting host functions and pathways. The identified host-targeted inhibitors restrict bacterial growth exclusively in the context of macrophage infection and predominantly fall into five categories: G-protein coupled receptor modulators, ion channel inhibitors, membrane transport proteins, anti-inflammatories, and kinase modulators. We found that fluoxetine, a selective serotonin reuptake inhibitor, enhances secretion of pro-inflammatory cytokine TNF-α and induces autophagy in infected macrophages, and gefitinib, an inhibitor of the Epidermal Growth Factor Receptor (EGFR), also activates autophagy and restricts growth. We demonstrate that during infection signaling through EGFR activates a p38 MAPK signaling pathway that prevents macrophages from effectively responding to infection. Inhibition of this pathway using gefitinib during in vivo infection reduces growth of M. tuberculosis in the lungs of infected mice. Our results support the concept that screening for inhibitors using intracellular models results in the identification of tool compounds for probing pathways during in vivo infection and may also result in the identification of new anti-tuberculosis agents that work by modulating host pathways. Given the existing experience with some of our identified compounds for other therapeutic indications, further clinically-directed study of these compounds is merited.

SUMMARY There are a limited number of adjuvants that elicit effective cell-based immunity required for protection against intracellular bacterial pathogens. Here, we report that STING-activating cyclic dinucleotides (CDNs) formulated in a protein subunit vaccine elicit long-lasting protective immunity to Mycobacterium tuberculosis in the mouse model. Subcutaneous administration of this vaccine provides equivalent protection to that of the live attenuated vaccine strain Bacille Calmette-Guérin (BCG). Protection is STING dependent but type I IFN independent and correlates with an increased frequency of a recently described subset of CXCR3-expressing T cells that localize to the lung parenchyma. Intranasal delivery results in superior protection compared with BCG, significantly boosts BCG-based immunity, and elicits both Th1 and Th17 immune responses, the latter of which correlates with enhanced protection. Thus, a CDN-adjuvanted protein subunit vaccine has the capability of eliciting a multi-faceted immune response that results in protection from infection by an intracellular pathogen.

Mycobacterium tuberculosis, the causative agent of tuberculosis, produces unique sulfated metabolites associated with virulence. One such metabolite from M. tuberculosis lipid extracts, S881, has been shown to negatively regulate the virulence of M. tuberculosis in mouse infection studies, and its cell-surface localization suggests a role in modulating host-pathogen interactions. However, a detailed structural analysis of S881 has remained elusive. Here we use high resolution, high mass accuracy, and tandem mass spectrometry to characterize the structure of S881. Exact mass measurements showed that S881 is highly unsaturated, tandem mass spectrometry indicated a polyisoprene-derived structure, and characterization of synthetic structural analogs confirmed that S881 is a previously-undescribed sulfated derivative of dihydromenaquinone-9, the primary quinol electron carrier in M. tuberculosis. To our knowledge, this is the first example of a sulfated menaquinone produced in any prokaryote. Together with previous studies, these findings suggest that this redox cofactor may play a role in mycobacterial pathogenesis.Tuberculosis (TB) affects approximately one third of the world's population and kills approximately two million people a year (1). In order to be an effective pathogen, Mycobacterium tuberculosis, the causative agent of TB, must not only survive the initial onslaught of the host immune response, but also carefully modulate adaptive immunity to allow for bacterial persistence. Sulfated metabolites have been shown to serve as signaling molecules between both symbiotic and pathogenic bacteria and their hosts (2-4), and the sulfate modification is also key to a number of mammalian extracellular signaling events (5). A number of sulfated metabolites have been isolated from the mycobacterial family (6-9), many of which are found in the cell wall (10,11). While the best-characterized of these molecules is the M. tuberculosis-specific metabolite sulfolipid-1 (SL-1) (9,12), another sulfated metabolite identified in M. tuberculosis lipid extracts has also been localized to the outer envelope of the cell (8,10). This previously-uncharacterized metabolite was termed S881 based on its measured mass. Isotopic labeling of S881 with 34 SO 4 2− indicated that it contains only one sulfate moiety (8,10). Despite the identification of this novel metabolite in M. tuberculosis

Research on the human pathogen Mycobacterium tuberculosis (Mtb) would benefit from novel tools for regulated gene expression. Here we describe the characterization and application of a synthetic riboswitch-based system, which comprises a mycobacterial promoter for transcriptional control and a riboswitch for translational control. The system was used to induce and repress heterologous protein overexpression reversibly, to create a conditional gene knockdown, and to control gene expression in a macrophage infection model. Unlike existing systems for controlling gene expression in Mtb, the riboswitch does not require the co-expression of any accessory proteins: all of the regulatory machinery is encoded by a short DNA segment directly upstream of the target gene. The inducible riboswitch platform has the potential to be a powerful general strategy for creating customized gene regulation systems in Mtb.

In the Rh 2 (OAc) 4 -catalyzed amidoglycosylation of glucal 3-carbamates, anomeric stereoselectivity and the extent of competing C3-H oxidation depend on the 4O and 6O protecting groups. Acyclic protection permits high α-anomer selectivity with further improvement in less polar solvents, while electron-withdrawing protecting groups limit C3-oxidized byproducts. Stereocontrol and bifurcation between alkene insertion and C3-H oxidation reflect an interplay of conformational, stereoelectronic, and inductive factors.2-Amino sugars having a 2,3-cis stereo array include N-acetylmannosamine (ManNAc, 1), which is the biosynthetic precursor of the sialic acids, 1 and 2-allosamine, a constituent of the potent chitinase inhibitor allosamidin (2) 2 and a useful ligand scaffold (3) 3 for asymmetric catalysis. The challenge of stereoselective C2-N bond construction is acute in these systems, and control of anomeric configuration in the preparation of glycoside derivatives is desirable. Synthetic methods based on intermolecular additions to glycals typically place the C2-N group trans to the C3-oxygen substituent. 4 Gin's activated-sulfoxide-mediated acetamidoglycosylation 5 of glucals is an exception, producing ManNAc structures, though with N-acetylglucosamine (GlcNAc) byproducts. 5cAs an alternative, 6 we have used intramolecular nitrogen atom delivery from allal 3-azidoformates, 7 allal 3-carbamates, 8 and glucal 3-carbamates 9 to establish the 2,3-cis relationship. With the 3O-carbamoyl glycals, we extended Du Bois's C-H amidation method 10 to alkene insertion, 11,12 a new reaction of allylic carbamates. 13 Mechanistic studies 14 imply that these conditions produce rhodium nitrenoids having reactivity strikingly analogous to metal carbenoids. 15 With iodosobenzene (PhIO) 16 instead of PhI(OAc) 2 as the oxidant, we achieved in situ glycosylation of alcohols without nucleophilic competition from acetate, an overall amidoglycosylation process. 8,9,17 Allal frameworks (e.g., 4, Scheme 1) provided high 1,2-trans selectivity, offering a concise route to β-linked 2-amido allopyranosides as found in allosamidin. 7,8 However, in the C3-epimeric series, our one-pot amidoglycosylation process applied to glucal 3-carbamates 6a and 6b, having 4O,6O acetonide or di-tert-butylsilylene protection, gave anomeric mixtures only slighly favoring the 1,2-trans products 7-α and also generated dihydropyranone byproducts 8a and 8b via oxidation at the C3-H bond (Table 1, entries 1 and 5). 9 Using 4-penten-1-ol as the acceptor, we were able to stereoconvergently advance either anomer of n-pentenyl glycoside 18 7a, but the lack of amidoglycosylation selectivity stymied direct access to α-linked ManNAc derivatives. 9Herein we report that proper choice of 4O and 6O protecting groups and solvent enables high levels of stereocontrol and chemoselectivity in amidoglycosylation of glucal 3-carbamates. Our studies also illuminate electronic and conformational aspects of both amidoglycosylation and the competing C3-H oxidation.For comparison, we be...

The genome of Mycobacterium tuberculosis (Mtb) encodes nine putative sulfatases, none of which have a known function or substrate. Here, we characterize Mtb’s single putative type II sulfatase, Rv3406, as a non-heme iron (II) and α-ketoglutarate-dependent dioxygenase that catalyzes the oxidation and subsequent cleavage of alkyl sulfate esters. Rv3406 was identified based on its homology to the alkyl sulfatase AtsK from Pseudomonas putida. Using an in vitro biochemical assay, we confirmed that Rv3406 is a sulfatase with a preference for alkyl sulfate substrates similar to those processed by AtsK. We determined the crystal structure of the apo Rv3406 sulfatase at 2.5 Å. The active site residues of Rv3406 and AtsK are essentially superimposable, suggesting that the two sulfatases share the same catalytic mechanism. Finally, we generated an Rv3406 mutant (Δrv3406) in Mtb to study the sulfatase’s role in sulfate scavenging. The Δrv3406 strain did not replicate in minimal media with 2-ethyl hexyl sulfate as the sole sulfur source, in contrast to wild type Mtb or the complemented strain. We conclude that Rv3406 is an iron and α-ketoglutarate-dependent sulfate ester dioxygenase that has unique substrate specificity that is likely distinct from other Mtb sulfatases.

Host-directed therapeutics have the potential to combat the global tuberculosis pandemic. We previously identified gefitinib, an inhibitor of EGFR, as a potential host-targeted therapeutic effective against Mycobacterium tuberculosis infection of macrophages and mice. Here we examine the functional consequences of gefitinib treatment on M. tuberculosis infected macrophages. Using phosphoproteo-mic and transcriptional profiling, we identify two mechanisms by which gefitinib influences macrophage responses to infection to affect cytokine responses and limit replication of M. tuberculosis in macrophages. First, we find that gefitinib treatment of M. tuberculosis infected macrophages inhibits STAT3, a transcription factor known to repress effective immune responses to M. tuberculosis in vivo. Second, we find that gefitinib treatment of M. tuberculosis infected macrophages leads to increased expression of genes involved in lysosomal biogenesis and function and an increase of functional lysosomes in gefitinib treated cells. Furthermore, we show that gefitinib treatment increases the targeting of bacteria to lysosomes, providing an explanation for the cell intrinsic effects of gefitinib treatment on M. tuberculosis infection. Our data provide novel insights into the effects of gefitinib on mammalian cells and into the possible roles for EGFR signaling in macrophages.