RESUMO
Antibiotic resistance poses rapidly increasing global problems in combatting multidrug-resistant (MDR) infectious diseases like MDR tuberculosis, prompting for novel approaches including host-directed therapies (HDT). Intracellular pathogens like Salmonellae and Mycobacterium tuberculosis (Mtb) exploit host pathways to survive. Only very few HDT compounds targeting host pathways are currently known. In a library of pharmacologically active compounds (LOPAC)-based drug-repurposing screen, we identify multiple compounds, which target receptor tyrosine kinases (RTKs) and inhibit intracellular Mtb and Salmonellae more potently than currently known HDT compounds. By developing a data-driven in silico model based on confirmed targets from public databases, we successfully predict additional efficacious HDT compounds. These compounds target host RTK signaling and inhibit intracellular (MDR) Mtb. A complementary human kinome siRNA screen independently confirms the role of RTK signaling and kinases (BLK, ABL1, and NTRK1) in host control of Mtb. These approaches validate RTK signaling as a drugable host pathway for HDT against intracellular bacteria.
Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Infecções por Salmonella/enzimologia , Salmonella typhimurium/efeitos dos fármacos , Tuberculose/enzimologia , Linhagem Celular , Biologia Computacional , Farmacorresistência Bacteriana , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Infecções por Salmonella/genética , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/fisiologia , Transdução de Sinais/efeitos dos fármacos , Tuberculose/genética , Tuberculose/microbiologiaRESUMO
One-third of the world population is infected with Mycobacterium tuberculosis and multi-drug resistant strains are rapidly evolving. The noticeable absence of a whole organism high-throughput screening system for studying the progression of tuberculosis is fast becoming the bottleneck in tuberculosis research. We successfully developed such a system using the zebrafish Mycobacterium marinum infection model, which is a well-characterized model for tuberculosis progression with biomedical significance, mimicking hallmarks of human tuberculosis pathology. Importantly, we demonstrate the suitability of our system to directly study M. tuberculosis, showing for the first time that the human pathogen can propagate in this vertebrate model, resulting in similar early disease symptoms to those observed upon M. marinum infection. Our system is capable of screening for disease progression via robotic yolk injection of early embryos and visual flow screening of late-stage larvae. We also show that this system can reliably recapitulate the standard caudal vein injection method with a throughput level of 2,000 embryos per hour. We additionally demonstrate the possibility of studying signal transduction leading to disease progression using reverse genetics at high-throughput levels. Importantly, we use reference compounds to validate our system in the testing of molecules that prevent tuberculosis progression, making it highly suited for investigating novel anti-tuberculosis compounds in vivo.
Assuntos
Biomarcadores/análise , Ensaios de Triagem em Larga Escala/métodos , Tuberculose/diagnóstico , Animais , Antituberculosos/isolamento & purificação , Antituberculosos/uso terapêutico , Modelos Animais de Doenças , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos/métodos , Embrião não Mamífero , Humanos , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium marinum/fisiologia , Prognóstico , Tuberculose/tratamento farmacológico , Tuberculose/patologia , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/fisiologiaRESUMO
Fe-deficiency anaemia is the most common cause of anaemia in developing countries. In these settings, many chronic infections, including tuberculosis (TB), are highly prevalent. Fe is an essential nutrient for both host and mycobacteria that play a pivotal role in host immunity and mycobacterial growth. A case-control study was performed in a TB-endemic region in Jakarta, Indonesia, among 378 pulmonary TB patients and 436 healthy controls from the same neighbourhood with the same socio-economic status. In a number of these subjects the Fe status could be explored. The distribution of three polymorphisms in the natural resistance-associated macrophage protein gene (NRAMP1) including INT4, D543N and 3'UTR was examined for a possible association with susceptibility to TB. Anaemia (corrected for sex) was present in 63.2 % of active TB compared with 6.8 % of controls, with female patients more often affected. Anaemia was more pronounced in advanced TB as diagnosed by chest radiography. Lower Hb concentrations in TB patients were accompanied by lower plasma Fe concentrations, lower Fe-binding capacity and higher plasma ferritin. After successful TB therapy, Fe parameters improved towards control values and Hb levels normalised, even without Fe supplementation. NRAMP1 gene polymorphisms were not associated with TB susceptibility, TB severity or anaemia. In conclusion, most active TB patients had anaemia, which was probably due to inflammation and not to Fe deficiency since TB treatment without Fe supplementation was sufficient to restore Hb concentration.
Assuntos
Anemia Ferropriva/genética , Proteínas de Transporte de Cátions/genética , Deficiências de Ferro , Polimorfismo Genético , Tuberculose Pulmonar/genética , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Humanos , Indonésia , Masculino , Pessoa de Meia-IdadeRESUMO
The TB Vaccine Cluster project funded by the EU Fifth Framework programme aims to provide novel vaccines against tuberculosis that are suitable for evaluation in humans. This paper describes the studies of the protective efficacy of vaccines in a guinea pig aerosol-infection model of primary tuberculosis. The objective was to conduct comparative evaluations of vaccines that had previously demonstrated efficacy in other animal models. Groups of 6 guinea pigs were immunized with vaccines provided by the relevant EU Vaccine Cluster partners. Survival over 17 or 26 weeks was used as the principal measure of vaccine efficacy following aerosol challenge with H37Rv. Counts of mycobacteria in lungs and spleens, and histopathological changes in the lungs, were also used to provide evidence of protection. A total of 24 vaccines were evaluated in 4 experiments each of a different design. A heterologous prime-boost strategy of DNA and MVA, each expressing Ag85A and a fusion protein of ESAT-6 and Ag85B in adjuvant, protected the guinea pigs to the same extent as BCG. Genetically modified BCG vaccines and boosted BCG strategies also protected guinea pigs to the same extent as BCG but not statistically significantly better. A relatively high aerosol-challenge dose and evaluation over a protracted time post-challenge allowed superior protection over BCG to be demonstrated by BCG boosted with MVA and fowl pox vectors expressing Ag85A.