Glutamine metabolism inhibition has dual immunomodulatory and antibacterial activities against Mycobacterium tuberculosis.

As one of the most successful human pathogens, Mycobacterium tuberculosis (Mtb) has evolved a diverse array of determinants to subvert host immunity and alter host metabolic patterns. However, the mechanisms of pathogen interference with host metabolism remain poorly understood. Here we show that a glutamine metabolism antagonist, JHU083, inhibits Mtb proliferation in vitro and in vivo. JHU083-treated mice exhibit weight gain, improved survival, a 2.5 log lower lung bacillary burden at 35 days post-infection, and reduced lung pathology. JHU083 treatment also initiates earlier T-cell recruitment, increased proinflammatory myeloid cell infiltration, and a reduced frequency of immunosuppressive myeloid cells when compared to uninfected and rifampin-treated controls. Metabolomic analysis of lungs from JHU083-treated Mtb-infected mice reveals citrulline accumulation, suggesting elevated nitric oxide (NO) synthesis, and lowered levels of quinolinic acid which is derived from the immunosuppressive metabolite kynurenine. JHU083-treated macrophages also produce more NO potentiating their antibacterial activity. When tested in an immunocompromised mouse model of Mtb infection, JHU083 loses its therapeutic efficacy suggesting the drug's host-directed effects are likely to be predominant. Collectively, these data reveal that JHU083-mediated glutamine metabolism inhibition results in dual antibacterial and host-directed activity against tuberculosis.

Glutamine metabolism inhibition has dual immunomodulatory and antibacterial activities against Mycobacterium tuberculosis.

As one of the most successful human pathogens, Mycobacterium tuberculosis (Mtb) has evolved a diverse array of determinants to subvert host immunity and alter host metabolic patterns. However, the mechanisms of pathogen interference with host metabolism remain poorly understood. Here we show that a novel glutamine metabolism antagonist, JHU083, inhibits Mtb proliferation in vitro and in vivo. JHU083-treated mice exhibit weight gain, improved survival, a 2.5 log lower lung bacillary burden at 35 days post-infection, and reduced lung pathology. JHU083 treatment also initiates earlier T-cell recruitment, increased proinflammatory myeloid cell infiltration, and a reduced frequency of immunosuppressive myeloid cells when compared to uninfected and rifampin-treated controls. Metabolomics analysis of lungs from JHU083-treated Mtb-infected mice revealed reduced glutamine levels, citrulline accumulation suggesting elevated NOS activity, and lowered levels of quinolinic acid which is derived from the immunosuppressive metabolite kynurenine. When tested in an immunocompromised mouse model of Mtb infection, JHU083 lost its therapeutic efficacy suggesting the drug's host-directed effects are likely to be predominant. Collectively, these data reveal that JHU083-mediated glutamine metabolism inhibition results in dual antibacterial and host-directed activity against tuberculosis.

In vivo interferon-gamma induced changes in gene expression dramatically alter neutrophil phenotype.

The cytokine Interferon-γ (IFN-γ) exerts powerful immunoregulatory effects on the adaptive immune system and also enhances functions of the neutrophil (PMN). The clinical use of IFN-γ has been driven by the finding that its administration to patients with chronic granulomatous disease (CGD) results in decreased incidence and severity of infections. However, IFN-γ has no effect on the characteristic defect of CGD, the inability to convert oxygen to microbicidal metabolites including superoxide anion (O2-) during the phagocytosis associated oxidative burst. We administered varying doses of IFN-γ to adult volunteers and studied the effects on plasma drug levels and response molecules and PMNs isolated from blood drawn at intervals over a 96- hour period. Plasma concentrations of IFN-γ, IP-10 and neopterin, and stimulated release of O2- from PMNs exhibited dose- and time-dependent increases after IFN-γ administration. Gene expression in PMNs was altered for 2775 genes; changes occurred rapidly after administration and returned to baseline in 24-36 hours. Several genes involved with neutrophil host defense were upregulated including those for components of the O2- generating NADPH oxidase; innate-immune and Fc receptors; proteins involved in MHCI and II; a regulator of circulating PMN number; guanylate binding proteins; and a key enzyme in synthesis of an essential NOS cofactor. Coordinate changes were detected in protein levels of representative products from several of these genes. Lysates from isolated neutrophils also demonstrated a spike in NO following IFN-γ administration. IFN-γ appears to increase non-oxygen dependent microbicidal functions of PMNs which could provide strategies to compensate for deficiencies, explain its clinical benefit for CGD patients and expand therapeutic applications of IFN-γ to other disorders. Trial registration: Protocol registered in ClinicalTrials.gov, NCT02609932, Effect of IFN-γ on Innate Immune Cells.

Effective Host-Directed Therapy for Tuberculosis by Depletion of Myeloid-Derived Suppressor Cells and Related Cells Using a Diphtheria Toxin Fusion Protein.

Myeloid-derived suppressor cells (MDSCs) are present in elevated numbers in tuberculosis patients and have been found to be permissive for Mycobacterium tuberculosis proliferation. To determine whether depletion of MDSCs may improve host control of tuberculosis, we used a novel diphtheria toxin-based fusion protein DABIL-4 that targets and depletes interleukin 4 (IL-4) receptor-positive cells. We show that DABIL-4 depletes both polymorphonuclear MDSCs and monocytic MDSCs, increases interferon-γ + T cells, and reduces the lung bacillary burden in a mouse tuberculosis model. These results indicate that MDSC-depleting therapies targeting the IL-4 receptor are beneficial in tuberculosis and offer an avenue towards host-directed tuberculosis therapy.

Therapeutic targeting with DABIL-4 depletes myeloid suppressor cells in 4T1 triple-negative breast cancer model.

In many solid tumors including triple-negative breast cancer (TNBC), upregulation of the interleukin-4 receptor (IL-4R) has been shown to promote cancer cell proliferation, apoptotic resistance, metastatic potential, and a Th2 response in the tumor microenvironment (TME). Since immunosuppressive cells in the TME and spleen including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) also express the IL-4R, we hypothesized that selective depletion of IL-4R-bearing cells in TNBC would result in the direct killing of tumor cells and the depletion of immunosuppressive cells and lead to an enhanced antitumor response. To selectively target IL-4R+ cells, we employed DABIL-4, a fusion protein toxin consisting of the catalytic and translocation domains of diphtheria toxin fused to murine IL-4. As anticipated, DABIL-4 has potent cytotoxic activity against TNBC cells both in vitro and in vivo. We demonstrate in the murine 4T1 TNBC model that DABIL-4 significantly reduces tumor growth, splenomegaly, and lung metastases. Importantly, we also show that the administration of DABIL-4 results in the selective depletion of MDSCs, TAMs, and regulatory T cells in treated mice, with a concomitant increase in IFN-γ+ CD8 effector T cells in the TME. Since the 4T1 antitumor activity of DABIL-4 was largely diminished in IL-4R knockout mice, we postulate that DABIL-4 functions primarily as an immunotherapeutic by the depletion of MDSCs, TAMs, and regulatory T cells. NanoString analysis of control and treated tumors confirmed and extended these observations by showing a marked decline of mRNA transcripts that are associated with tumorigenesis and metastasis. In conclusion, we demonstrate that DABIL-4 targeting of both tumor and immunosuppressive host cells likely represents a novel and effective treatment strategy for 4T1 TNBC and warrants further study.

Recent advances with Treg depleting fusion protein toxins for cancer immunotherapy.

T regulatory cells (Tregs) are an important T cell population for immune tolerance, prevention of autoimmune diseases and inhibition of antitumor immunity. The tumor-promoting role played by Tregs in cancer has prompted numerous approaches to develop immunotherapeutics targeting Tregs. One approach to depletion of Treg cells is retargeting the highly potent cytotoxic activity of bacterial toxins. These agents capitalize on the well-characterized bacterial toxins, diphtheria toxin and Pseudomonas aeruginosa exotoxin A-both of which harbor membrane translocation domains and enzymatic domains that catalytically halt protein synthesis within intoxicated eukaryotic cells and act at picomolar or subpicomolar concentrations. In this review, we summarize the preclinical and clinical development of several Treg-depleting cancer immunotherapies based on these two bacterial toxins.

Corynebacterium diphtheriae: Diphtheria Toxin, the tox Operon, and Its Regulation by Fe2+ Activation of apo-DtxR.

Diphtheria is one of the most well studied of all the bacterial infectious diseases. These milestone studies of toxigenic Corynebacterium diphtheriae along with its primary virulence determinant, diphtheria toxin, have established the paradigm for the study of other related bacterial protein toxins. This review highlights those studies that have contributed to our current understanding of the structure-function relationships of diphtheria toxin, the molecular mechanism of its entry into the eukaryotic cell cytosol, the regulation of diphtheria tox expression by holo-DtxR, and the molecular basis of transition metal ion activation of apo-DtxR itself. These seminal studies have laid the foundation for the protein engineering of diphtheria toxin and the development of highly potent eukaryotic cell-surface receptor-targeted fusion protein toxins for the treatment of human diseases that range from T cell malignancies to steroid-resistant graft-versus-host disease to metastatic melanoma. This deeper scientific understanding of diphtheria toxin and the regulation of its expression have metamorphosed the third-most-potent bacterial toxin known into a life-saving targeted protein therapeutic, thereby at least partially fulfilling Paul Erlich's concept of a magic bullet-"a chemical that binds to and specifically kills microbes or tumor cells."

Second-generation IL-2 receptor-targeted diphtheria fusion toxin exhibits antitumor activity and synergy with anti-PD-1 in melanoma.

Denileukin diftitox (DAB-IL-2, Ontak) is a diphtheria-toxin-based fusion protein that depletes CD25-positive cells including regulatory T cells and has been approved for the treatment of persistent or recurrent cutaneous T cell lymphoma. However, the clinical use of denileukin diftitox was limited by vascular leak toxicity and production issues related to drug aggregation and purity. We found that a single amino acid substitution (V6A) in a motif associated with vascular leak induction yields a fully active, second-generation biologic, s-DAB-IL-2(V6A), which elicits 50-fold less human umbilical vein endothelial cell monolayer permeation and is 3.7-fold less lethal to mice by LD50 analysis than s-DAB-IL-2. Additionally, to overcome aggregation problems, we developed a production method for the fusion toxin using Corynebacterium diphtheriae that secretes fully folded, biologically active, monomeric s-DAB-IL-2 into the culture medium. Using the poorly immunogenic mouse B16F10 melanoma model, we initiated treatment 7 days after tumor challenge and observed that, while both s-DAB-IL-2(V6A) and s-DAB-IL-2 are inhibitors of tumor growth, the capacity to treat with higher doses of s-DAB-IL-2(V6A) could provide a superior activity window. In a sequential dual-therapy study in tumors that have progressed for 10 days, both s-DAB-IL-2(V6A) and s-DAB-IL-2 given before checkpoint inhibition with anti-programmed cell death-1 (anti-PD-1) antibodies inhibited tumor growth, while either drug given as monotherapy had less effect. s-DAB-IL-2(V6A), a fully monomeric protein with reduced vascular leak, is a second-generation diphtheria-toxin-based fusion protein with promise as a cancer immunotherapeutic both alone and in conjunction with PD-1 blockade.

Suppressor Cell-Depleting Immunotherapy With Denileukin Diftitox is an Effective Host-Directed Therapy for Tuberculosis.

Host-directed therapies that augment host immune effector mechanisms may serve as important adjunctive therapies for tuberculosis treatment. We evaluated the activity of denileukin diftitox in an acute mouse model of tuberculosis (TB) infection and analyzed the cellular composition and bacterial burden in lungs and spleens. These in vivo studies show that denileukin diftitox potentiates standard TB treatment in the mouse model, an effect which may be due to depletion of T-regulatory and myeloid-derived suppressor cells during TB infection. Our results indicate that denileukin diftitox and other suppressor cell-depleting therapies may be useful adjunctive, host-directed therapies for TB.

Design and Synthesis of a Pan-Janus Kinase Inhibitor Clinical Candidate (PF-06263276) Suitable for Inhaled and Topical Delivery for the Treatment of Inflammatory Diseases of the Lungs and Skin.

By use of a structure-based computational method for identification of structurally novel Janus kinase (JAK) inhibitors predicted to bind beyond the ATP binding site, a potent series of indazoles was identified as selective pan-JAK inhibitors with a type 1.5 binding mode. Optimization of the series for potency and increased duration of action commensurate with inhaled or topical delivery resulted in potent pan-JAK inhibitor 2 (PF-06263276), which was advanced into clinical studies.

Therapeutic targeting of regulatory T cells enhances tumor-specific CD8+ T cell responses in Epstein-Barr virus associated nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) is associated with multiple malignancies including nasopharyngeal carcinoma (NPC). In nasopharynx cancer, CD8+ T cells specific for EBV Nuclear Antigen-1 (EBNA-1) and Latent Membrane Protein 2 (LMP2) are important components of anti-tumor immunity since both are consistently expressed in NPC. We have previously shown that EBNA-1-specific CD8+ T cell responses were suppressed in NPC patients compared to healthy controls. We now find that CD8+ T cell responses specific for LMP2 are also abnormal in NPC patients, and both EBNA-1- and LMP2-specific responses are suppressed by regulatory T cells (Treg). EBNA-1 and LMP2-specific CD8+ T cell responses, as well as immune control of EBV-infected cells in vitro, could be restored by the depletion of Tregs and by use of a clinically approved drug targeting Tregs. Thus, in vivo modulation of Tregs may be an effective means of enhancing these anti-tumor immune responses in NPC patients.

Mechanism of diphtheria toxin catalytic domain delivery to the eukaryotic cell cytosol and the cellular factors that directly participate in the process.

Research on diphtheria and anthrax toxins over the past three decades has culminated in a detailed understanding of their structure function relationships (e.g., catalytic (C), transmembrane (T), and receptor binding (R) domains), as well as the identification of their eukaryotic cell surface receptor, an understanding of the molecular events leading to the receptor-mediated internalization of the toxin into an endosomal compartment, and the pH triggered conformational changes required for pore formation in the vesicle membrane. Recently, a major research effort has been focused on the development of a detailed understanding of the molecular interactions between each of these toxins and eukaryotic cell factors that play an essential role in the efficient translocation of their respective catalytic domains through the trans-endosomal vesicle membrane pore and delivery into the cell cytosol. In this review, I shall focus on recent findings that have led to a more detailed understanding of the mechanism by which the diphtheria toxin catalytic domain is delivered to the eukaryotic cell cytosol. While much work remains, it is becoming increasingly clear that the entry process is facilitated by specific interactions with a number of cellular factors in an ordered sequential fashion. In addition, since diphtheria, anthrax lethal factor and anthrax edema factor all carry multiple coatomer I complex binding motifs and COPI complex has been shown to play an essential role in entry process, it is likely that the initial steps in catalytic domain entry of these divergent toxins follow a common mechanism.

GRP78(BiP) facilitates the cytosolic delivery of anthrax lethal factor (LF) in vivo and functions as an unfoldase in vitro.

Anthrax toxin is an A/B bacterial protein toxin which is composed of the enzymatically active Lethal Factor (LF) and/or Oedema Factor (EF) bound to Protective Antigen 63 (PA63) which functions as both the receptor binding and transmembrane domains. Once the toxin binds to its cell surface receptors it is internalized into the cell and traffics through Rab5- and Rab7-associated endosomal vesicles. Following acidification of the vesicle lumen, PA63 undergoes a dynamic change forming a beta-barrel that inserts into and forms a pore through the endosomal membrane. It is widely recognized that LF, and the related fusion protein LFnDTA, must be completely denatured in order to transit through the PA63 formed pore and enter the eukaryotic cell cytosol. We demonstrate by protease protection assays that the molecular chaperone GRP78 mediates the unfolding of LFnDTA and LF at neutral pH and thereby converts these proteins from a trypsin resistant to sensitive conformation. We have used immunoelectron microscopy and gold-labelled antibodies to demonstrate that both GRP78 and GRP94 chaperones are present in the lumen of endosomal vesicles. Finally, we have used siRNA to demonstrate that knock-down of GRP78 results in the emergence of resistance to anthrax lethal toxin and oedema toxin action.

Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry.

The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre-loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 plays an essential, but as yet undefined role in the entry process. We have used both site-directed mutagenesis and a COPI complex precipitation assay to demonstrate that interaction(s) between at least three lysine residues in transmembrane helix 1 are essential for both COPI complex binding and the delivery of the catalytic domain into the target cell cytosol. Finally, a COPI binding domain swap was used to demonstrate that substitution of the lysine-rich transmembrane helix 1 with the COPI binding portion of the p23 adaptor cytoplasmic tail results in a mutant that displays full wild-type activity. Thus, irrespective of sequence, the ability of transmembrane helix 1 to bind to COPI complex appears to be the essential feature for catalytic domain delivery to the cytosol.

Effective delivery of antisense peptide nucleic acid oligomers into cells by anthrax protective antigen.

Peptide nucleic acid (PNA) is highly stable and binds to complementary RNA and DNA with high affinity, but it resists cellular uptake, thereby limiting its bioavailability. We investigated whether protectiveantigen (PA, a non-toxic component of anthrax toxin) could transport antisense PNA oligomers into reporter cells that contain luciferase transgenes with mutant beta-globin IVS2 intronic inserts, which permit aberrant pre-mRNA splicing and impair luciferase expression. PNA oligomers antisense to mutant splice sites in these IVS2 inserts induced luciferase expression when effectively delivered into the cells. PNA 18-mers with C-terminal poly-lysine tails [PNA(Lys)(8)] demonstrated modest sequence-specific antisense activity by themselves at micromolar concentrations in luc-IVS2 reporter cell cultures. However, this activity was greatly amplified by PA. Antisense PNA(Lys)(8) with but not without PA also corrected the IVS2-654 beta-globin splice defect in cultured erythroid precursor cells from a patient with beta-thalassemia [genotype, IVS2-654(beta(0)/beta(E))], providing further evidence that anthrax PA can effectively transport antisense PNA oligomers into cells.

Neisseria meningitidis PorB, a Toll-like receptor 2 ligand, improves the capacity of Francisella tularensis lipopolysaccharide to protect mice against experimental tularemia.

Francisella tularensis causes severe pneumonia that can be fatal if it is left untreated. Due to its potential use as a biological weapon, research is being conducted to develop an effective vaccine and to select and study adjuvant molecules able to generate a better and long-lasting protective effect. PorB, a porin from Neisseria meningitidis, is a well-established Toll-like receptor 2 ligand and has been shown to be a promising vaccine adjuvant candidate due to its ability to enhance the T-cell costimulatory activity of antigen-presenting cells both in vitro and in vivo. BALB/c mice were immunized with lipopolysaccharide (LPS) isolated from the F. tularensis subsp. holarctica live vaccine strain (LVS), with or without PorB from N. meningitidis, and the antibody levels induced during the vaccination regimen and the level of protection against intranasal challenge with LVS were determined. Antigen administered alone induced a specific F. tularensis LPS immunoglobulin M (IgM) response that was not maintained over the weeks and that conferred protection to only 25% of the mice. In contrast, F. tularensis LPS given in combination with neisserial PorB induced consistent levels of specific IgM throughout the immunization and increased the proportion of surviving mice to 70%. Postchallenge cytokine analysis showed that interleukin-6 (IL-6), monocyte chemoattractant protein 1, and gamma interferon were markers of mortality and that IL-1beta was a correlate of survival, independent of the presence of PorB as an adjuvant. These data indicate that neisserial PorB might be an optimal candidate adjuvant for improving the protective effect of F. tularensis LPS and other subunit vaccines against tularemia, but there is still a need to test its efficacy against virulent type A and type B F. tularensis strains.

COPI coatomer complex proteins facilitate the translocation of anthrax lethal factor across vesicular membranes in vitro.

The delivery of the diphtheria toxin catalytic domain (DTA) from acidified endosomes into the cytoplasm of eukaryotic cells requires protein-protein interactions between the toxin and a cytosolic translocation factor (CTF) complex. A conserved peptide motif, T1, within the DT transmembrane helix 1 mediates these interactions. Because the T1 motif is also present in the N-terminal segments of lethal factor (LF) and edema factor (EF) in anthrax toxin, we asked whether LF entry into the cell might also be facilitated by target cell cytosolic proteins. In this study, we have used LFnDTA and its associated ADP-ribosyltransferase activity (DTA) to determine the requirements for LF translocation from the lumen of endosomal vesicles to the external medium in vitro. Although low-level release of LFnDTA from enriched endosomal vesicles occurs in the absence of added factors, translocation was enhanced by the addition of cytosolic proteins and ATP to the reaction mixture. We show by GST-LFn pull-down assays that LFn specifically interacts with at least zeta-COP and beta-COP of the COPI coatomer complex. Immunodepletion of COPI coatomer complex and associated proteins from cytosolic extracts blocks in vitro LFnDTA translocation. Translocation may be reconstituted by the addition of partially purified bovine COPI to the translocation assay mixture. Taken together, these data suggest that the delivery of LF to the cytosol requires either COPI coatomer complex or a COPI subcomplex for translocation from the endosomal lumen. This facilitated delivery appears to use a mechanism that is analogous to that of DT entry.

Identification of immunologic and pathologic parameters of death versus survival in respiratory tularemia.

Francisella tularensis can cause severe disseminated disease after respiratory infection. The identification of factors involved in mortality or recovery following induction of tularemia in the mouse will improve our understanding of the natural history of this disease and facilitate future evaluation of vaccine candidate preparations. BALB/c mice were infected intranasally with the live vaccine strain (LVS) of F. tularensis subsp. holarctica and euthanized at different stages of disease to analyze the induction of immune molecules, gross anatomical features of organs, bacterial burdens, and progression of the histopathological changes in lung and spleen. Tissue-specific interleukin-6 (IL-6), macrophage inflammatory protein 2, and monocyte chemotactic protein 1 were immune markers of mortality, while anti-LVS immunoglobulin M and IL-1beta were associated with survival. Moribund mice had enlarged spleens and lungs, while surviving mice had even more prominent splenomegaly and normal-appearing lungs. Histopathology of the spleens of severely ill mice was characterized by disrupted lymphoid follicles and fragmented nuclei, while the spleens of survivors appeared healthy but with increased numbers of megakaryocytes and erythrocytes. Histopathology of the lungs of severely ill mice indicated severe pneumonia. Lungs of survivors at early time points showed increased inflammation, while at late times they appeared healthy with peribronchial lymphoid aggregates. Our results suggest that host immune factors are able to affect bacterial dissemination after respiratory tularemia, provide new insights regarding the pathological characteristics of pulmonary tularemia leading to systemic disease, and potentially identify immune markers associated with recovery from the disease.

A conserved motif in transmembrane helix 1 of diphtheria toxin mediates catalytic domain delivery to the cytosol.

A 10-aa motif in transmembrane helix 1 of diphtheria toxin that is conserved in anthrax edema factor, anthrax lethal factor, and botulinum neurotoxin serotypes A, C, and D was identified by blast, clustal w, and meme computational analysis. Using the diphtheria toxin-related fusion protein toxin DAB(389)IL-2, we demonstrate that introduction of the L221E mutation into a highly conserved residue within this motif results in a nontoxic catalytic domain translocation deficient phenotype. To further probe the function of this motif in the process by which the catalytic domain is delivered from the lumen of early endosomes to the cytosol, we constructed a gene encoding a portion of diphtheria toxin transmembrane helix 1, T1, which carries the motif and is expressed from a CMV promoter. We then isolated stable transfectants of Hut102/6TG cells that express the T1 peptide, Hut102/6TG-T1. In contrast to the parental cell line, Hut102/6TG-T1 cells are ca. 10(4)-fold more resistant to the fusion protein toxin. This resistance is completely reversed by coexpression of small interfering RNA directed against the gene encoding the T1 peptide in Hut102/6TG-T1 cells. We further demonstrate by GST-DT140-271 pull-down experiments in the presence and absence of synthetic T1 peptides the specific binding of coatomer protein complex subunit beta to this region of the diphtheria toxin transmembrane domain.

Both Corynebacterium diphtheriae DtxR(E175K) and Mycobacterium tuberculosis IdeR(D177K) are dominant positive repressors of IdeR-regulated genes in M. tuberculosis.

The diphtheria toxin repressor (DtxR) is an important iron-dependent transcriptional regulator of known virulence genes in Corynebacterium diphtheriae. The mycobacterial iron-dependent repressor (IdeR) is phylogenetically closely related to DtxR, with high amino acid similarity in the DNA binding and metal ion binding site domains. We have previously shown that an iron-insensitive, dominant-positive dtxR(E175K) mutant allele from Corynebacterium diphtheriae can be expressed in Mycobacterium tuberculosis and results in an attenuated phenotype in mice. In this paper, we report the M. tuberculosis IdeR(D177K) strain that has the cognate point mutation. We tested four known and predicted IdeR-regulated gene promoters (mbtI, Rv2123, Rv3402c, and Rv1519) using a promoterless green fluorescent protein (GFP) construct. GFP expression from these promoters was abrogated under low-iron conditions in the presence of both IdeR(D177K) and DtxR(E175K), a result confirmed by reverse transcription-PCR. The IdeR regulon can be constitutively repressed in the presence of an integrated copy of ideR containing this point mutation. These data also suggest that mutant IdeR(D177K) has a mechanism similar to that of DtxR(E175K); iron insensitivity occurs as a result of SH3-like domain binding interactions that stabilize the intermediate form of the repressor after ancillary metal ion binding. This construct can be used to elucidate further the IdeR regulon and its virulence genes and to differentiate these from genes regulated by SirR, which does not have this domain.