Host-directed therapy for intracellular bacterial Infections

Antibiotic resistance is an increasing problem in the battle against (bacterial) infectious diseases. The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) threatens to render tuberculosis (TB) untreatable. Efforts to develop novel antibiotics have so far been unsuccessful, calling for additional approaches for treatment of bacterial infections. Intracellular pathogens like Mtb and Salmonella can survive in the host by manipulating host cell signaling. This provides opportunities for novel therapeutic strategies by targeting the host, rather than the bacterium (host-directed therapy). In this thesis we report the development and application of novel (in vitro and in vivo) methods for identifying host genes and proteins involved in host control of intracellular bacteria, as well as chemical compounds that target host molecules as a basis for drug development for host-directed therapies. As a result, we report the identification of RTK inhibitors, the novel kinase inhibitor 97i, the human kinase family PCTAIRE and the host protein DRAM1 as promising leads for further drug development for host-directed therapeutic strategies for intracellular bacterial infections.LUMC / Geneeskund

The DNA Damage-Regulated Autophagy Modulator DRAM1 Links Mycobacterial Recognition via TLR-MYD88 to Autophagic Defense

Animal science

Combined chemical genetics and data-driven bioinformatics approach identifies receptor tyrosine kinase inhibitors as host-directed antimicrobials

Immunogenetics and cellular immunology of bacterial infectious disease

Repurposing diphenylbutylpiperidine-class antipsychotic drugs for host-directed therapy of Mycobacterium tuberculosis and Salmonella enterica infections

The 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.Immunogenetics and cellular immunology of bacterial infectious disease

Host-directed therapy for intracellular bacterial Infections

Antibiotic resistance is an increasing problem in the battle against (bacterial) infectious diseases. The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) threatens to render tuberculosis (TB) untreatable. Efforts to develop novel antibiotics have so far been unsuccessful, calling for additional approaches for treatment of bacterial infections. Intracellular pathogens like Mtb and Salmonella can survive in the host by manipulating host cell signaling. This provides opportunities for novel therapeutic strategies by targeting the host, rather than the bacterium (host-directed therapy). In this thesis we report the development and application of novel (in vitro and in vivo) methods for identifying host genes and proteins involved in host control of intracellular bacteria, as well as chemical compounds that target host molecules as a basis for drug development for host-directed therapies. As a result, we report the identification of RTK inhibitors, the novel kinase inhibitor 97i, the human kinase family PCTAIRE and the host protein DRAM1 as promising leads for further drug development for host-directed therapeutic strategies for intracellular bacterial infections.</p

Host-directed therapy for intracellular bacterial Infections

Antibiotic resistance is an increasing problem in the battle against (bacterial) infectious diseases. The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) threatens to render tuberculosis (TB) untreatable. Efforts to develop novel antibiotics have so far been unsuccessful, calling for additional approaches for treatment of bacterial infections. Intracellular pathogens like Mtb and Salmonella can survive in the host by manipulating host cell signaling. This provides opportunities for novel therapeutic strategies by targeting the host, rather than the bacterium (host-directed therapy). In this thesis we report the development and application of novel (in vitro and in vivo) methods for identifying host genes and proteins involved in host control of intracellular bacteria, as well as chemical compounds that target host molecules as a basis for drug development for host-directed therapies. As a result, we report the identification of RTK inhibitors, the novel kinase inhibitor 97i, the human kinase family PCTAIRE and the host protein DRAM1 as promising leads for further drug development for host-directed therapeutic strategies for intracellular bacterial infections.</p

Mycobacterial Secretion Systems ESX-1 and ESX-5 Play Distinct Roles in Host Cell Death and Inflammasome Activation

During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5 - two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators - during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1b activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread. Copyright © 2011 by The American Association of Immunologists, Inc

Repurposing tamoxifen as potential host-directed therapeutic for tuberculosis

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.</p

Repurposing tamoxifen as potential host-directed therapeutic for tuberculosis

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.</p

Repurposing Tamoxifen as Potential Host-Directed Therapeutic for Tuberculosis

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.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.Immunogenetics and cellular immunology of bacterial infectious disease