Imaging Tumor-Targeting Bacteria Using 18F-Fluorodeoxysorbitol Positron Emission Tomography.

BACKGROUND: Microbial-based cancer treatments are an emerging field, with multiple bacterial species evaluated in animal models and some advancing to clinical trials. Noninvasive bacteria-specific imaging approaches can potentially support the development and clinical translation of bacteria-based cancer treatments by assessing the tumor and off-target bacterial colonization. METHODS: 18F-Fluorodeoxysorbitol (18F-FDS) positron emission tomography (PET), a bacteria-specific imaging approach, was used to visualize an attenuated strain of Yersinia enterocolitica, currently in clinical trials as a microbial-based cancer treatment, in murine models of breast cancer. RESULTS: Y. enterocolitica demonstrated excellent 18F-FDS uptake in in vitro assays. Whole-body 18F-FDS PET demonstrated a significantly higher PET signal in tumors with Y. enterocolitica colonization compared to those not colonized, in murine models utilizing direct intratumor or intravenous administration of bacteria, which were confirmed using ex vivo gamma counting. Conversely, 18F-fluorodeoxyglucose (18F-FDG) PET signal was not different in Y. enterocolitica colonized versus uncolonized tumors. CONCLUSIONS: Given that PET is widely used for the management of cancer patients, 18F-FDS PET could be utilized as a complementary approach supporting the development and clinical translation of Y. enterocolitica-based tumor-targeting bacterial therapeutics.

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.

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.

Identification of YfiH (PgeF) as a factor contributing to the maintenance of bacterial peptidoglycan composition.

Peptidoglycan (PG) is an essential, envelope-fortifying macromolecule of eubacterial cell walls. It is a large polymer with multiple glycan strands interconnected by short peptide chains forming a sac-like structure around cytoplasmic membrane. In most bacteria, the composition of the peptide chain is well-conserved and distinctive; in E. coli, the peptide chain length varies from two to five amino acids with a tetrapeptide consisting of L-alanine - D-glutamic acid - meso-diaminopimelic acid - D-alanine. However, it is not known how bacteria conserve the composition and sequence of peptide chains of PG. Here, we find that a conserved open reading frame of unknown function, YfiH (renamed PgeF) contributes to the maintenance of peptide composition in E. coli. Using genetic, biochemical and mass spectrometrical analyses we demonstrate that absence of yfiH results in incorporation of non-canonical amino acids, L-serine or glycine in place of L-alanine in PG sacculi leading to ß-lactam - sensitivity, lethality in mutants defective in PG remodelling or recycling pathways, altered cell morphology and reduced PG synthesis. yfiH orthologs from other Gram-positive genera were able to compensate the absence of yfiH in E. coli indicating a conserved pathway in bacterial kingdom. Our results suggest editing/quality control mechanisms exist to maintain composition and integrity of bacterial peptidoglycan.

Regulated proteolysis of a cross-link-specific peptidoglycan hydrolase contributes to bacterial morphogenesis.

Bacterial growth and morphogenesis are intimately coupled to expansion of peptidoglycan (PG), an extensively cross-linked macromolecule that forms a protective mesh-like sacculus around the cytoplasmic membrane. Growth of the PG sacculus is a dynamic event requiring the concerted action of hydrolases that cleave the cross-links for insertion of new material and synthases that catalyze cross-link formation; however, the factors that regulate PG expansion during bacterial growth are poorly understood. Here, we show that the PG hydrolase MepS (formerly Spr), which is specific to cleavage of cross-links during PG expansion in Escherichia coli, is modulated by proteolysis. Using combined genetic, molecular, and biochemical approaches, we demonstrate that MepS is rapidly degraded by a proteolytic system comprising an outer membrane lipoprotein of unknown function, NlpI, and a periplasmic protease, Prc (or Tsp). In summary, our results indicate that the NlpI-Prc system contributes to growth and enlargement of the PG sacculus by modulating the cellular levels of the cross-link-cleaving hydrolase MepS. Overall, this study signifies the importance of PG cross-link cleavage and its regulation in bacterial cell wall biogenesis.

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.

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.

Host-Directed Therapies: Modulating Inflammation to Treat Tuberculosis.

Following infection with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), most human hosts are able to contain the infection and avoid progression to active TB disease through expression of a balanced, homeostatic immune response. Proinflammatory mechanisms aiming to kill, slow and sequester the pathogen are key to a successful host response. However, an excessive or inappropriate pro-inflammatory response may lead to granuloma enlargement and tissue damage, which may prolong the TB treatment duration and permanently diminish the lung function of TB survivors. The host also expresses certain anti-inflammatory mediators which may play either beneficial or detrimental roles depending on the timing of their deployment. The balance between the timing and expression levels of pro- and anti-inflammatory responses plays an important role in the fate of infection. Interestingly, M. tuberculosis appears to manipulate both sides of the human immune response to remodel the host environment for its own benefit. Consequently, therapies which modulate either end of this spectrum of immune responses at the appropriate time may have the potential to improve the treatment of TB or to reduce the formation of permanent lung damage after microbiological cure. Here, we highlight host-directed TB therapies targeting pro- or anti-inflammatory processes that have been evaluated in pre-clinical models. The repurposing of already available drugs known to modulate these responses may improve the future of TB 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.

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."

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.

Structural and mechanistic insights into EchAMP: A antimicrobial protein from the Echidna milk.

BACKGROUND: Antibiotic resistance is a problem that necessitates the identification of new antimicrobial molecules. Milk is known to have molecules with antimicrobial properties (AMPs). Echidna Antimicrobial Protein (EchAMP) is one such lactation specific AMP exclusively found in the milk of Echidna, an egg-laying mammal geographically restricted to Australia and New Guinea. Previous studies established that EchAMP exhibits substantial bacteriostatic activity against multiple bacterial genera. However, the subsequent structural and functional studies were hindered due to the unavailability of pure protein. RESULTS: In this study, we expressed EchAMP protein using a heterologous expression system and successfully purified it to >95% homogeneity. The purified recombinant protein exhibits bacteriolytic activity against both Gram-positive and Gram-negative bacteria as confirmed by live-dead staining and scanning electron microscopy. Structurally, this AMP belongs to the family of intrinsically disordered proteins (IDPs) as deciphered by the circular-dichroism, tryptophan fluorescence, and NMR spectroscopy. Nonetheless, EchAMP has the propensity to acquire structure with amphipathic molecules, or membrane mimics like SDS, lipopolysaccharides, and liposomes as again observed through multiple spectroscopic techniques. CONCLUSIONS: Recombinant EchAMP exhibits broad-spectrum bacteriolytic activity by compromising the bacterial cell membrane integrity. Hence, we propose that this intrinsically disordered antimicrobial protein interact with the bacterial cell membrane and undergoes conformational changes to form channels in the membrane resulting in cell lysis. GENERAL SIGNIFICANCE: EchAMP, the evolutionarily conserved, lactation specific AMP from an oviparous mammal may find application as a broad-spectrum antimicrobial against pathogens that affect mammary gland or otherwise cause routine infections in humans and livestock.