A Riboswitch-Based Inducible Gene Expression System for Mycobacteria

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

HIF-1α Is an Essential Mediator of IFN-γ–Dependent Immunity to Mycobacterium tuberculosis

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. In this study, we show that the transcription factor hypoxia-inducible factor-1α (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-sequencing demonstrates that HIF-1α regulates nearly one-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 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

HIF-1α Is an Essential Mediator of IFN-γ–Dependent Immunity to Mycobacterium tuberculosis

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

The Tyrosine Kinase Inhibitor Gefitinib Restricts Mycobacterium tuberculosis Growth through Increased Lysosomal Biogenesis and Modulation of Cytokine Signaling

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 phosphoproteomic 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

The Tyrosine Kinase Inhibitor Gefitinib Restricts Mycobacterium tuberculosis Growth through Increased Lysosomal Biogenesis and Modulation of Cytokine Signaling

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 phosphoproteomic 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

Biosynthesis and Regulation of Sulfomenaquinone, a Metabolite Associated with Virulence in Mycobacterium tuberculosis

Sulfomenaquinone (SMK) is a recently identified metabolite that is unique to the Mycobacterium tuberculosis (M. tuberculosis) complex and is shown to modulate its virulence. Here, we report the identification of the SMK biosynthetic operon that, in addition to a previously identified sulfotransferase stf3, includes a putative cytochrome P450 gene (cyp128) and a gene of unknown function, rv2269c. We demonstrate that cyp128 and stf3 are sufficient for the biosynthesis of SMK from menaquinone and rv2269c exhibits promoter activity in M. tuberculosis. Loss of Stf3 expression, but not that of Cyp128, is correlated with elevated levels of menaquinone-9, an essential component in the electron-transport chain in M. tuberculosis. Finally, we showed in a mouse model of infection that the loss of cyp128 exhibits a hypervirulent phenotype similar to that in previous studies of the stf3 mutant. These findings provide a platform for defining the molecular basis of SMK's role in M. tuberculosis pathogenesis

Biosynthesis and Regulation of Sulfomenaquinone, a Metabolite Associated with Virulence in Mycobacterium tuberculosis

Sulfomenaquinone (SMK) is a recently identified metabolite that is unique to the Mycobacterium tuberculosis (M. tuberculosis) complex and is shown to modulate its virulence. Here, we report the identification of the SMK biosynthetic operon that, in addition to a previously identified sulfotransferase stf3, includes a putative cytochrome P450 gene (cyp128) and a gene of unknown function, rv2269c. We demonstrate that cyp128 and stf3 are sufficient for the biosynthesis of SMK from menaquinone and rv2269c exhibits promoter activity in M. tuberculosis. Loss of Stf3 expression, but not that of Cyp128, is correlated with elevated levels of menaquinone-9, an essential component in the electron-transport chain in M. tuberculosis. Finally, we showed in a mouse model of infection that the loss of cyp128 exhibits a hypervirulent phenotype similar to that in previous studies of the stf3 mutant. These findings provide a platform for defining the molecular basis of SMK's role in M. tuberculosis pathogenesis

Mycobacterium tuberculosis Rv3406 is a type II alkyl sulfatase capable of sulfate scavenging.

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

Biosynthesis and Regulation of Sulfomenaquinone, a Metabolite Associated with Virulence in <i>Mycobacterium tuberculosis</i>

Sulfomenaquinone (SMK) is a recently identified metabolite that is unique to the <i>Mycobacterium tuberculosis</i> (<i>M. tuberculosis</i>) complex and is shown to modulate its virulence. Here, we report the identification of the SMK biosynthetic operon that, in addition to a previously identified sulfotransferase <i>stf3</i>, includes a putative cytochrome P450 gene (<i>cyp128</i>) and a gene of unknown function, <i>rv2269c</i>. We demonstrate that <i>cyp128</i> and <i>stf3</i> are sufficient for the biosynthesis of SMK from menaquinone and <i>rv2269c</i> exhibits promoter activity in <i>M. tuberculosis</i>. Loss of Stf3 expression, but not that of Cyp128, is correlated with elevated levels of menaquinone-9, an essential component in the electron-transport chain in <i>M. tuberculosis</i>. Finally, we showed in a mouse model of infection that the loss of <i>cyp128</i> exhibits a hypervirulent phenotype similar to that in previous studies of the <i>stf3</i> mutant. These findings provide a platform for defining the molecular basis of SMK’s role in <i>M. tuberculosis</i> pathogenesis