The global burden of tuberculosis (TB) is aggravated by the continuously increasing emergence of drug resistance, highlighting the need for innovative therapeutic options. The concept of host... Show moreThe global burden of tuberculosis (TB) is aggravated by the continuously increasing emergence of drug resistance, highlighting the need for innovative therapeutic options. The concept of host-directed therapy (HDT) as adjunctive to classical antibacterial therapy with antibiotics represents a novel and promising approach for treating TB. Here, we have focused on repurposing the clinically used anticancer drug tamoxifen, which was identified as a molecule with strong host-directed activity against intracellular Mycobacterium tuberculosis (Mtb). Using a primary human macrophage Mtb infection model, we demonstrate the potential of tamoxifen against drug-sensitive as well as drug-resistant Mtb bacteria. The therapeutic effect of tamoxifen was confirmed in an in vivo TB model based on Mycobacterium marinum infection of zebrafish larvae. Tamoxifen had no direct antimicrobial effects at the concentrations used, confirming that tamoxifen acted as an HDT drug. Furthermore, we demonstrate that the antimycobacterial effect of tamoxifen is independent of its well-known target the estrogen receptor (ER) pathway, but instead acts by modulating autophagy, in particular the lysosomal pathway. Through RNA sequencing and microscopic colocalization studies, we show that tamoxifen stimulates lysosomal activation and increases the localization of mycobacteria in lysosomes both in vitro and in vivo, while inhibition of lysosomal activity during tamoxifen treatment partly restores mycobacterial survival. Thus, our work highlights the HDT potential of tamoxifen and proposes it as a repurposed molecule for the treatment of TB. Show less
Tuberculosis (TB) is the world's most lethal infectious disease caused by a bacterial pathogen, Mycobacterium tuberculosis. This pathogen evades the immune defenses of its host and grows... Show moreTuberculosis (TB) is the world's most lethal infectious disease caused by a bacterial pathogen, Mycobacterium tuberculosis. This pathogen evades the immune defenses of its host and grows intracellularly in immune cells, particularly inside macrophages.The global burden of tuberculosis (TB) is aggravated by the continuously increasing emergence of drug resistance, highlighting the need for innovative therapeutic options. The concept of host-directed therapy (HDT) as adjunctive to classical antibacterial therapy with antibiotics represents a novel and promising approach for treating TB. Here, we have focused on repurposing the clinically used anticancer drug tamoxifen, which was identified as a molecule with strong host-directed activity against intracellular Mycobacterium tuberculosis (Mtb). Using a primary human macrophage Mtb infection model, we demonstrate the potential of tamoxifen against drug-sensitive as well as drug-resistant Mtb bacteria. The therapeutic effect of tamoxifen was confirmed in an in vivo TB model based on Mycobacterium marinum infection of zebrafish larvae. Tamoxifen had no direct antimicrobial effects at the concentrations used, confirming that tamoxifen acted as an HDT drug. Furthermore, we demonstrate that the antimycobacterial effect of tamoxifen is independent of its well-known target the estrogen receptor (ER) pathway, but instead acts by modulating autophagy, in particular the lysosomal pathway. Through RNA sequencing and microscopic colocalization studies, we show that tamoxifen stimulates lysosomal activation and increases the localization of mycobacteria in lysosomes both in vitro and in vivo, while inhibition of lysosomal activity during tamoxifen treatment partly restores mycobacterial survival. Thus, our work highlights the HDT potential of tamoxifen and proposes it as a repurposed molecule for the treatment of TB.IMPORTANCE Tuberculosis (TB) is the world's most lethal infectious disease caused by a bacterial pathogen, Mycobacterium tuberculosis. This pathogen evades the immune defenses of its host and grows intracellularly in immune cells, particularly inside macrophages. There is an urgent need for novel therapeutic strategies because treatment of TB patients is increasingly complicated by rising antibiotic resistance. In this study, we explored a breast cancer drug, tamoxifen, as a potential anti-TB drug. We show that tamoxifen acts as a so-called host-directed therapeutic, which means that it does not act directly on the bacteria but helps the host macrophages combat the infection more effectively. We confirmed the antimycobacterial effect of tamoxifen in a zebrafish model for TB and showed that it functions by promoting the delivery of mycobacteria to digestive organelles, the lysosomes. These results support the high potential of tamoxifen to be repurposed to fight antibiotic-resistant TB infections by host-directed therapy. Show less
The persistent increase of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) infections negatively impacts Tuberculosis treatment outcomes. Host-directed therapies (HDT) pose an... Show moreThe persistent increase of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) infections negatively impacts Tuberculosis treatment outcomes. Host-directed therapies (HDT) pose an complementing strategy, particularly since Mtb is highly successful in evading host-defense by manipulating host-signaling pathways. Here, we screened a library containing autophagy-modulating compounds for their ability to inhibit intracellular Mtb-bacteria. Several active compounds were identified, including two drugs of the diphenylbutylpiperidine-class, Fluspirilene and Pimozide, commonly used as antipsychotics. Both molecules inhibited intracellular Mtb in pro- as well as anti-inflammatory primary human macrophages in a host-directed manner and synergized with conventional anti-bacterials. Importantly, these inhibitory effects extended to MDR-Mtb strains and the unrelated intracellular pathogen, Salmonella enterica serovar Typhimurium (Stm). Mechanistically Fluspirilene and Pimozide were shown to regulate autophagy and alter the lysosomal response, partly correlating with increased bacterial localization to autophago(lyso)somes. Pimozide's and Fluspirilene's efficacy was inhibited by antioxidants, suggesting involvement of the oxidative-stress response in Mtb growth control. Furthermore, Fluspirilene and especially Pimozide counteracted Mtb-induced STAT5 phosphorylation, thereby reducing Mtb phagosome-localized CISH that promotes phagosomal acidification. In conclusion, two approved antipsychotic drugs, Pimozide and Fluspirilene, constitute highly promising and rapidly translatable candidates for HDT against Mtb and Stm and act by modulating the autophagic/lysosomal response by multiple mechanisms. Show less
Global increases in the prevalence of antimicrobial resistance highlight the urgent need for novel strategies to combat infectious diseases. Recent studies suggest that host metabolic pathways play... Show moreGlobal increases in the prevalence of antimicrobial resistance highlight the urgent need for novel strategies to combat infectious diseases. Recent studies suggest that host metabolic pathways play a key role in host control of intracellular bacterial pathogens. In this study we explored the potential of targeting host metabolic pathways for innovative host-directed therapy (HDT) against intracellular bacterial infections. Through gene expression profiling in human macrophages, pyruvate metabolism was identified as potential key pathway involved in Salmonella enterica serovar Typhimurium (Stm) infections. Next, the effect of targeting pyruvate dehydrogenase kinases (PDKs) - which are regulators of the metabolic checkpoint pyruvate dehydrogenase complex (PDC) - on macrophage function and bacterial control was studied. Chemical inhibition of PDKs by dichloroacetate (DCA) induced PDC activation and was accompanied with metabolic rewiring in classically activated macrophages (M1) but not in alternatively activated macrophages (M2), suggesting cell-type specific effects of dichloroacetate on host metabolism. Furthermore, DCA treatment had minor impact on cytokine and chemokine secretion on top of infection, but induced significant ROS production by M1 and M2. DCA markedly and rapidly reduced intracellular survival of Stm, but interestingly not Mycobacterium tuberculosis, in human macrophages in a host-directed manner. In conclusion, DCA represents a promising novel HDT compound targeting pyruvate metabolism for the treatment of Stm infections. Show less
Bakkum, T.; Heemskerk, M.T.; Bos, E.; Groenewold, M.; Oikonomeas-Koppasis, N.; Walburg, K.V.; ... ; Kasteren, S.I. van 2020
Bioorthogonal correlative light-electron microscopy (BCLEM) can give a detailed overview of multicomponent biological systems. It can provide information on the ultrastructural context of... Show moreBioorthogonal correlative light-electron microscopy (BCLEM) can give a detailed overview of multicomponent biological systems. It can provide information on the ultrastructural context of bioorthogonal handles and other fluorescent signals, as well as information about subcellular organization. We have here applied B-CLEM to the study of the intracellular pathogen Mycobacterium tuberculosis (Mtb) by generating a triply labeled Mtb through combined metabolic labeling of the cell wall and the proteome of a DsRed-expressing Mtb strain. Study of this pathogen in a B-CLEM setting was used to provide information about the intracellular distribution of the pathogen, as well as its in situ response to various clinical antibiotics, supported by flow cytometric analysis of the bacteria, after recovery from the host cell (ex cellula). The RNA polymerase-targeting drug rifampicin displayed the most prominent effect on subcellular distribution, suggesting the most direct effect on pathogenicity and/or viability, while the cell wall synthesis-targeting drugs isoniazid and ethambutol effectively rescued bacterial division-induced loss of metabolic labels. The three drugs combined did not give a more pronounced effect but rather an intermediate response, whereas gentamicin displayed a surprisingly strong additive effect on subcellular distribution. Show less
Bioorthogonal correlative light-electron microscopy (B-CLEM) can give a detailed overview of multicomponent biological systems. It can provide information on the ultrastructural context of... Show moreBioorthogonal correlative light-electron microscopy (B-CLEM) can give a detailed overview of multicomponent biological systems. It can provide information on the ultrastructural context of bioorthogonal handles and other fluorescent signals, as well as information about subcellular organization. We have here applied B-CLEM to the study of the intracellular pathogen Mycobacterium tuberculosis (Mtb) by generating a triply labeled Mtb through combined metabolic labeling of the cell wall and the proteome of a DsRed-expressing Mtb strain. Study of this pathogen in a B-CLEM setting was used to provide information about the intracellular distribution of the pathogen, as well as its in situ response to various clinical antibiotics, supported by flow cytometric analysis of the bacteria, after recovery from the host cell (ex cellula). The RNA polymerase-targeting drug rifampicin displayed the most prominent effect on subcellular distribution, suggesting the most direct effect on pathogenicity and/or viability, while the cell wall synthesis-targeting drugs isoniazid and ethambutol effectively rescued bacterial division-induced loss of metabolic labels. The three drugs combined did not give a more pronounced effect but rather an intermediate response, whereas gentamicin displayed a surprisingly strong additive effect on subcellular distribution. Show less
The rapid and persistent increase of drug-resistant Mycobacterium tuberculosis (Mtb) infections poses increasing global problems in combatting tuberculosis (TB), prompting for the development of... Show moreThe rapid and persistent increase of drug-resistant Mycobacterium tuberculosis (Mtb) infections poses increasing global problems in combatting tuberculosis (TB), prompting for the development of alternative strategies including host-directed therapy (HDT). Since Mtb is an intracellular pathogen with a remarkable ability to manipulate host intracellular signaling pathways to escape from host defense, pharmacological reprogramming of the immune system represents a novel, potentially powerful therapeutic strategy that should be effective also against drug-resistant Mtb. Here, we found that host-pathogen interactions in Mtb-infected primary human macrophages affected host epigenetic features by modifying histone deacetylase (HDAC) transcriptomic levels. In addition, broad spectrum inhibition of HDACs enhanced the antimicrobial response of both pro-inflammatory macrophages (M phi 1) and anti-inflammatory macrophages (M phi 2), while selective inhibition of class IIa HDACs mainly decreased bacterial outgrowth in M phi 2. Moreover, chemical inhibition of HDAC activity during differentiation polarized macrophages into a more bactericidal phenotype with a concomitant decrease in the secretion levels of inflammatory cytokines. Importantly, in vivo chemical inhibition of HDAC activity in Mycobacterium marinum-infected zebrafish embryos, a well-characterized animal model for tuberculosis, significantly reduced mycobacterial burden, validating our in vitro findings in primary human macrophages. Collectively, these data identify HDACs as druggable host targets for HDT against intracellular Mtb. Show less
