RESUMEN
BACKGROUND: Efficient high-throughput drug screening assays are necessary to enable the discovery of new anti-mycobacterial drugs. The purpose of our work was to develop and validate an assay based on live-cell imaging which can monitor the growth of two distinct phenotypes of Mycobacterium tuberculosis and to test their susceptibility to commonly used TB drugs. RESULTS: Both planktonic and cording phenotypes were successfully monitored as fluorescent objects using the live-cell imaging system IncuCyte S3, allowing collection of data describing distinct characteristics of aggregate size and growth. The quantification of changes in total area of aggregates was used to define IC50 and MIC values of selected TB drugs which revealed that the cording phenotype grew more rapidly and displayed a higher susceptibility to rifampicin. In checkerboard approach, testing pair-wise combinations of sub-inhibitory concentrations of drugs, rifampicin, linezolid and pretomanid demonstrated superior growth inhibition of cording phenotype. CONCLUSIONS: Our results emphasize the efficiency of using automated live-cell imaging and its potential in high-throughput whole-cell screening to evaluate existing and search for novel antimycobacterial drugs.
Asunto(s)
Antituberculosos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Pruebas de Sensibilidad Microbiana/métodos , Microscopía/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/microbiología , Automatización , Humanos , Isoniazida/farmacología , Linezolid/farmacología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Fenotipo , Rifampin/farmacologíaRESUMEN
Drug resistance in Mycobacterium tuberculosis is an emerging threat that makes the discovery of new candidate drugs a priority. In particular, drugs with high sterilizing activity within host cells are needed to improve efficacy and reduce treatment duration. We aimed to develope and validate a High Content Screening assay based on Mycobacterium tuberculosis-infected primary human monocyte-derived macrophages as its natural reservoir. Infected primary human monocyte-derived macrophages were exposed to control antibiotics or tested compounds on 384 well plates. Intracellular bacterial growth and macrophage numbers were evaluated using an ImageXpress High Content Screening system and Z'-factor was calculated to assess the reproducibility. The combination of isoniazid and rifampicin as a positive control rendered a Z'-factor above 0.4, demonstrating suitability of the assay for screening and compound profiling purposes. In a validation experiment, isoniazid, rifampicin, moxifloxacin and levofloxacin all effectively inhibited intracellular growth as expected. Finally, a pilot screening campaign including 5700 compounds from diverse libraries resulted in the identification of three compounds with confirmed antimycobacterial activity in the low micromolar range and low host cell toxicity. The assay represents an attractive screening platform for both academic research on host-pathogen mechanisms in tuberculosis and for the identification and characterization of novel antimycobacterial compounds.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis Ganglionar , Antituberculosos/farmacología , Humanos , Isoniazida/farmacología , Macrófagos/microbiología , Reproducibilidad de los Resultados , Rifampin/farmacologíaRESUMEN
Sprouting angiogenesis is the formation of new capillaries from existing vessels in response to tissue hypoxia due to growth/development, repair/healing, and also chronic inflammation. In this study, we aimed to elucidate the effect of IL-6, a pleiotropic cytokine with both pro-inflammatory and anti-inflammatory functions, in regulating the sprouting angiogenic response of endothelial cells (ECs). We found that activation of IL-6 trans-signaling inhibited the migration, proliferation, and tube formation ability of ECs. In addition, inhibition of the autocrine IL-6 classic-signaling by depleting endogenous IL-6 from ECs impaired their tube formation ability. At the molecular level, we found that IL-6 trans-signaling in ECs upregulated established endogenous anti-angiogenic factors such as CXCL10 and SERPINF1 while at the same time downregulated known endogenous pro-angiogenic factors such as cKIT and CXCL8. Furthermore, prior activation of ECs by IL-6 trans-signaling alters their response to vascular endothelial growth factor-A (VEGF-A), causing an increased p38, but decreased Erk1/2 phosphorylation. Collectively, our data demonstrated the dual facets of IL-6 in regulating the sprouting angiogenic function of ECs. In addition, we shed light on molecular mechanisms behind the IL-6 trans-signaling mediated impairment of endothelial sprouting angiogenic response.
Asunto(s)
Movimiento Celular , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Interleucina-6/metabolismo , Neovascularización Fisiológica , Transducción de Señal , Movimiento Celular/genética , Proliferación Celular/genética , Regulación de la Expresión Génica , Humanos , Neovascularización Fisiológica/genéticaRESUMEN
Our aim was to develop a Mycobacterium tuberculosis (Mtb) growth inhibition assay (MGIA) as a summary estimate of host immune control of virulent Mtb. Mycobacterial growth inhibition (MGI) using previously frozen human PBMCs infected with H37Rv was assessed by live-cell imaging (Incucyte©) complemented by imaging flow cytometry analysis of phagocytosis. MGI measured as relative fluorescence units (RFU) was calibrated to time to positive culture (TTP) in BACTEC 960 MGIT. At a MOI (multiplicity of infection) of 5, there was a wide range of MGI of blood donors (1.1*106-2.7*106 RFU, n = 14). Intra- and inter-assay variability were at most 17.5 and 20.7 CV%. Cell viability at day 5 was 57 and 62% monitored by the LDH and Draq7 assays respectively. There was a strong correlation between a readout for Mtb growth using CFU counts or TTP compared to RFU (r2≥0.96). Our MGIA enabling live-cell imaging and monitoring of cell viability was able to detect a wide range of Mtb growth inhibition by PBMCs and was calibrated to several readout options for bacterial growth. This MGIA may be valuable as a surrogate marker of host immunity in a personalized medicine approach.
Asunto(s)
Leucocitos Mononucleares/microbiología , Microscopía Fluorescente , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis/diagnóstico , Supervivencia Celular , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Interacciones Huésped-Patógeno , Humanos , Leucocitos Mononucleares/patología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/metabolismo , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Factores de Tiempo , Tuberculosis/inmunología , Tuberculosis/microbiología , VirulenciaRESUMEN
BACKGROUND: Drugs such as isoniazid (INH) and pretomanid (PRT), used against Mycobacterium tuberculosis are active partly through generation of reactive nitrogen species (RNS). The aim of this study was to explore variability in intracellular susceptibility to nitric oxide (NO) in clinical strains of M. tuberculosis. METHOD: Luciferase-expressing clinical M. tuberculosis strains with or without INH resistance were exposed to RNS donors (DETA/NO and SIN-1) in broth cultures and bacterial survival was analysed by luminometry. NO-dependent intracellular killing in a selection of strains was assessed in interferon gamma/lipopolysaccharide-activated murine macrophages using the NO inhibitor L-NMMA. RESULTS: When M. tuberculosis H37Rv was compared to six clinical isolates and CDC1551, three isolates with inhA mediated INH resistance showed significantly reduced NO-susceptibility in broth culture. All strains showed a variable but dose-dependent susceptibility to RNS donors. Two clinical isolates with increased susceptibility to NO exposure in broth compared to H37Rv were significantly inhibited by activated macrophages whereas there was no effect on growth inhibition when activated macrophages were infected by clinical strains with higher survival to NO exposure in broth. Furthermore, the most NO-tolerant clinical isolate showed increased resistance to PRT both in broth culture and the macrophage model compared to H37Rv in the absence of mutational resistance in genes associated to reduced susceptibility against PRT or NO. CONCLUSION: In a limited number of clinical M. tuberculosis isolates we found a significant difference in susceptibility to NO between clinical isolates, both in broth cultures and in macrophages. Our results indicate that mycobacterial susceptibility to cellular host defence mechanisms such as NO need to be taken into consideration when designing new therapeutic strategies.
Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana/fisiología , Macrófagos/inmunología , Viabilidad Microbiana/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Especies de Nitrógeno Reactivo/farmacología , Animales , Células Cultivadas , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Ratones , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/crecimiento & desarrollo , Óxido Nítrico/farmacología , Organismos Modificados Genéticamente , Ácido Peroxinitroso/farmacologíaRESUMEN
To survive and replicate in macrophages Mycobacterium tuberculosis (Mtb) has developed strategies to subvert host defence mechanisms, including autophagy. Autophagy induction has the potential to clear Mtb, but little is known about its effect during controlled tuberculosis and HIV co-infection. Mammalian target of rapamycin complex1 (mTORC1) inhibitors were used to induce autophagy in human macrophages pre-infected with HIV-1BaL and infected with a low dose of Mtb (co-infected), or single Mtb infected (single infected). The controlled Mtb infection was disrupted upon mTOR inhibition resulting in increased Mtb replication in a dose-dependent manner which was more pronounced during co-infection. The increased Mtb replication could be explained by the marked reduction in phagosome acidification upon mTOR inhibition. Autophagy stimulation targeting mTORC1 clearly induced a basal autophagy with flux that was unlinked to the subcellular environment of the Mtb vacuoles, which showed a concurrent suppression in acidification and maturation/flux. Overall our findings indicate that mTOR inhibition during Mtb or HIV/Mtb co-infection interferes with phagosomal maturation, thereby supporting mycobacterial growth during low-dose and controlled infection. Therefore pharmacological induction of autophagy through targeting of the canonical mTORC1-pathway should be handled with caution during controlled tuberculosis, since this could have serious consequences for patients with HIV/Mtb co-infection.
Asunto(s)
Autofagia/fisiología , Infecciones por VIH/microbiología , Macrófagos/microbiología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Mycobacterium tuberculosis/patogenicidad , Autofagia/efectos de los fármacos , Coinfección , Regulación de la Expresión Génica , Infecciones por VIH/genética , Interacciones Huésped-Patógeno/fisiología , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/virología , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/metabolismo , Naftiridinas/farmacología , Fagosomas/microbiología , Fagosomas/virología , Fosforilación , Proteína Sequestosoma-1/metabolismo , Sirolimus/farmacología , Tuberculosis/metabolismo , Tuberculosis/virologíaRESUMEN
Macrophages in the lung are the primary cells being infected by Mycobacterium tuberculosis (Mtb) during the initial manifestation of tuberculosis. Since the adaptive immune response to Mtb is delayed, innate immune cells such as macrophages and neutrophils mount the early immune protection against this intracellular pathogen. Neutrophils are short-lived cells and removal of apoptotic cells by resident macrophages is a key event in the resolution of inflammation and tissue repair. Since anti-inflammatory activity is not compatible with effective immunity to intracellular pathogens, we therefore investigated how uptake of apoptotic neutrophils modulates the function of Mtb-activated human macrophages. We show that Mtb infection exerts a potent proinflammatory activation of human macrophages with enhanced gene activation and release of proinflammatory cytokines and that this response was augmented by apoptotic neutrophils. The enhanced macrophage response is linked to apoptotic neutrophil-driven activation of the NLRP3 inflammasome and subsequent IL-1ß signalling. We also demonstrate that apoptotic neutrophils not only modulate the inflammatory response, but also enhance the capacity of infected macrophages to control intracellular growth of virulent Mtb. Taken together, these results suggest a novel role for apoptotic neutrophils in the modulation of the macrophage-dependent inflammatory response contributing to the early control of Mtb infection.
Asunto(s)
Apoptosis , Macrófagos/microbiología , Mycobacterium tuberculosis/fisiología , Neutrófilos/citología , Caspasa 1/metabolismo , Activación Enzimática , Humanos , Inflamación/inmunología , Interleucina-1beta/biosíntesis , Interleucina-1beta/metabolismo , Espacio Intracelular/microbiología , Activación de Macrófagos , Macrófagos/citología , Macrófagos/inmunología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Francisella tularensis is a highly virulent intracellular bacterium capable of rapid multiplication in phagocytic cells. Previous studies have revealed that activation of F. tularensis-infected macrophages leads to control of infection and reactive nitrogen and oxygen species make important contributions to the bacterial killing. We investigated the effects of adding S-nitroso-acetyl-penicillamine (SNAP), which generates nitric oxide, or 3-morpholinosydnonimine hydrochloride, which indirectly leads to formation of peroxynitrite, to J774 murine macrophage-like cell cultures infected with F. tularensis LVS. Addition of SNAP led to significantly increased colocalization between LAMP-1 and bacteria, indicating containment of F. tularensis in the phagosome within 2 h, although no killing occurred within 4 h. A specific inhibitory effect on bacterial transcription was observed since the gene encoding the global regulator MglA was inhibited 50-100-fold. F. tularensis-infected J774 cells were incapable of secreting TNF-α in response to Escherichia coli LPS but addition of SNAP almost completely reversed the suppression. Similarly, infection with an MglA mutant did not inhibit LPS-induced TNF-α secretion of J774 cells. Strong staining of nitrotyrosine was observed in SNAP-treated bacteria, and MS identified nitration of two ribosomal 50S proteins, a CBS domain pair protein and bacterioferritin. The results demonstrated that addition of SNAP initially did not affect the viability of intracellular F. tularensis LVS but led to containment of the bacteria in the phagosome. Moreover, the treatment resulted in modification by nitration of several F. tularensis proteins.