Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 142
Filtrar
Más filtros

Base de datos
País/Región como asunto
Tipo del documento
Intervalo de año de publicación
1.
Biology (Basel) ; 13(9)2024 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-39336115

RESUMEN

Zebrafish is a natural host of various Mycobacterium species and a surrogate model organism for tuberculosis research. Mycobacterium marinum is evolutionarily one of the closest non-tuberculous species related to M. tuberculosis and shares the majority of virulence genes. Although zebrafish is not a natural host of the human pathogen, we have previously demonstrated successful robotic infection of zebrafish embryos with M. tuberculosis and performed drug treatment of the infected larvae. In the present study, we examined for how long M. tuberculosis can be propagated in zebrafish larvae and tested a time series of infected larvae to study the transcriptional response via Illumina RNA deep sequencing (RNAseq). Bacterial aggregates carrying fluorescently labeled M. tuberculosis could be detected up to 9 days post-infection. The infected larvae showed a clear and specific transcriptional immune response with a high similarity to the inflammatory response of zebrafish larvae infected with the surrogate species M. marinum. We conclude that M. tuberculosis can be propagated in zebrafish larvae for at least one week after infection and provide further evidence that M. marinum is a good surrogate model for M. tuberculosis. The generated extensive transcriptome data sets will be of great use to add translational value to zebrafish as a model for infection of tuberculosis using the M. marinum infection system. In addition, we identify new marker genes such as dusp8 and CD180 that are induced by M. tuberculosis infection in zebrafish and in human macrophages at later stages of infection that can be further investigated.

2.
Sci Rep ; 14(1): 17225, 2024 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-39060313

RESUMEN

The emergence of antimicrobial resistance has created an urgent need for alternative treatments against bacterial pathogens. Here, we investigated kinase inhibitors as potential host-directed therapies (HDTs) against intracellular bacteria, specifically Salmonella Typhimurium (Stm) and Mycobacterium tuberculosis (Mtb). We screened 827 ATP-competitive kinase inhibitors with known target profiles from two Published Kinase Inhibitor Sets (PKIS1 and PKIS2) using intracellular infection models for Stm and Mtb, based on human cell lines and primary macrophages. Additionally, the in vivo safety and efficacy of the compounds were assessed using zebrafish embryo infection models. Our screen identified 11 hit compounds for Stm and 17 hit compounds for Mtb that were effective against intracellular bacteria and non-toxic for host cells. Further experiments were conducted to prioritize Stm hit compounds that were able to clear the intracellular infection in primary human macrophages. From these, two structurally related Stm hit compounds, GSK1379738A and GSK1379760A, exhibited significant activity against Stm in infected zebrafish embryos. In addition, we identified compounds that were active against intracellular Mtb, including morpholino-imidazo/triazolo-pyrimidinones that target PIK3CB, as well as 2-aminobenzimidazoles targeting ABL1. Overall, this study provided insights into kinase targets acting at the host-pathogen interface and identified several kinase inhibitors as potential HDTs.


Asunto(s)
Macrófagos , Mycobacterium tuberculosis , Inhibidores de Proteínas Quinasas , Salmonella typhimurium , Pez Cebra , Animales , Humanos , Salmonella typhimurium/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Macrófagos/microbiología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Infecciones por Salmonella/tratamiento farmacológico , Infecciones por Salmonella/microbiología , Antibacterianos/farmacología , Línea Celular , Embrión no Mamífero/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología
3.
Microbiol Spectr ; 12(8): e0016724, 2024 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-38916320

RESUMEN

Mycobacterium tuberculosis (Mtb) as well as nontuberculous mycobacteria are intracellular pathogens whose treatment is extensive and increasingly impaired due to the rise of mycobacterial drug resistance. The loss of antibiotic efficacy has raised interest in the identification of host-directed therapeutics (HDT) to develop novel treatment strategies for mycobacterial infections. In this study, we identified amiodarone as a potential HDT candidate that inhibited both intracellular Mtb and Mycobacterium avium in primary human macrophages without directly impairing bacterial growth, thereby confirming that amiodarone acts in a host-mediated manner. Moreover, amiodarone induced the formation of (auto)phagosomes and enhanced autophagic targeting of mycobacteria in macrophages. The induction of autophagy by amiodarone is likely due to enhanced transcriptional regulation, as the nuclear intensity of the transcription factor EB, the master regulator of autophagy and lysosomal biogenesis, was strongly increased. Furthermore, blocking lysosomal degradation with bafilomycin impaired the host-beneficial effect of amiodarone. Finally, amiodarone induced autophagy and reduced bacterial burden in a zebrafish embryo model of tuberculosis, thereby confirming the HDT activity of amiodarone in vivo. In conclusion, we have identified amiodarone as an autophagy-inducing antimycobacterial HDT that improves host control of mycobacterial infections. IMPORTANCE: Due to the global rise in antibiotic resistance, there is a strong need for alternative treatment strategies against intracellular bacterial infections, including Mycobacterium tuberculosis (Mtb) and non-tuberculous mycobacteria. Stimulating host defense mechanisms by host-directed therapy (HDT) is a promising approach for treating mycobacterial infections. This study identified amiodarone, an antiarrhythmic agent, as a potential HDT candidate that inhibits the survival of Mtb and Mycobacterium avium in primary human macrophages. The antimycobacterial effect of amiodarone was confirmed in an in vivo tuberculosis model based on Mycobacterium marinum infection of zebrafish embryos. Furthermore, amiodarone induced autophagy and inhibition of the autophagic flux effectively impaired the host-protective effect of amiodarone, supporting that activation of the host (auto)phagolysosomal pathway is essential for the mechanism of action of amiodarone. In conclusion, we have identified amiodarone as an autophagy-inducing HDT that improves host control of a wide range of mycobacteria.


Asunto(s)
Amiodarona , Autofagia , Macrófagos , Mycobacterium tuberculosis , Tuberculosis , Pez Cebra , Amiodarona/farmacología , Autofagia/efectos de los fármacos , Animales , Pez Cebra/microbiología , Humanos , Macrófagos/microbiología , Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Modelos Animales de Enfermedad , Mycobacterium avium/efectos de los fármacos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/microbiología
4.
Front Cell Infect Microbiol ; 14: 1367938, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38590439

RESUMEN

The increasing prevalence of antimicrobial-resistant Staphylococcus aureus strains, especially methicillin-resistant S. aureus (MRSA), poses a threat to successful antibiotic treatment. Unsuccessful attempts to develop a vaccine and rising resistance to last-resort antibiotics urge the need for alternative treatments. Host-directed therapy (HDT) targeting critical intracellular stages of S. aureus emerges as a promising alternative, potentially acting synergistically with antibiotics and reducing the risk of de novo drug resistance. We assessed 201 ATP-competitive kinase inhibitors from Published Kinase Inhibitor Sets (PKIS1 and PKIS2) against intracellular MRSA. Seventeen hit compounds were identified, of which the two most effective and well-tolerated hit compounds (i.e., GW633459A and GW296115X) were selected for further analysis. The compounds did not affect planktonic bacterial cultures, while they were active in a range of human cell lines of cervical, skin, lung, breast and monocyte origin, confirming their host-directed mechanisms. GW633459A, structurally related to lapatinib, exhibited an HDT effect on intracellular MRSA independently of its known human epidermal growth factor receptor (EGFR)/(HER) kinase family targets. GW296115X activated adenosine monophosphate-activated protein kinase (AMPK), thereby enhancing bacterial degradation via autophagy. Finally, GW296115X not only reduced MRSA growth in human cells but also improved the survival rates of MRSA-infected zebrafish embryos, highlighting its potential as HDT.


Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Humanos , Animales , Staphylococcus aureus , Pez Cebra , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Infecciones Estafilocócicas/microbiología , Pruebas de Sensibilidad Microbiana
5.
Sci Rep ; 13(1): 15406, 2023 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-37717068

RESUMEN

The ß-lactamase of Mycobacterium tuberculosis, BlaC, hydrolyzes ß-lactam antibiotics, hindering the use of these antibiotics for the treatment of tuberculosis. Inhibitors, such as avibactam, can reversibly inhibit the enzyme, allowing for the development of combination therapies using both antibiotic and inhibitor. However, laboratory evolution studies using Escherichia coli resulted in the discovery of single amino acid variants of BlaC that reduce the sensitivity for inhibitors or show higher catalytic efficiency against antibiotics. Here, we tested these BlaC variants under more physiological conditions using the M. marinum infection model of zebrafish, which recapitulates hallmark features of tuberculosis, including the intracellular persistence of mycobacteria in macrophages and the induction of granuloma formation. To this end, the M. tuberculosis blaC gene was integrated into the chromosome of a blaC frameshift mutant of M. marinum. Subsequently, the resulting strains were used to infect zebrafish embryos in order to test the combinatorial effect of ampicillin and avibactam. The results show that embryos infected with an M. marinum strain producing BlaC show lower infection levels after treatment than untreated embryos. Additionally, BlaC K234R showed higher infection levels after treatment than those infected with bacteria producing the wild-type enzyme, demonstrating that the zebrafish host is less sensitive to the combinatorial therapy of ß-lactam antibiotic and inhibitor. These findings are of interest for future development of combination therapies to treat tuberculosis.


Asunto(s)
Mycobacterium marinum , Mycobacterium tuberculosis , Tuberculosis , Animales , Mycobacterium tuberculosis/genética , Pez Cebra , Mycobacterium marinum/genética , beta-Lactamasas/genética , Tuberculosis/tratamiento farmacológico , Ampicilina , Antibacterianos , Escherichia coli/genética
6.
Biology (Basel) ; 12(6)2023 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-37372102

RESUMEN

Existing drug treatment against tuberculosis is no match against the increasing number of multi-drug resistant strains of its causative agent, Mycobacterium tuberculosis (Mtb). A better understanding of how mycobacteria subvert the host immune defenses is crucial for developing novel therapeutic strategies. A potential approach is enhancing the activity of the autophagy machinery, which can direct bacteria to autophagolysosomal degradation. However, the interplay specifics between mycobacteria and the autophagy machinery must be better understood. Here, we analyzed live imaging data from the zebrafish model of tuberculosis to characterize mycobacteria-autophagy interactions during the early stages of infection in vivo. For high-resolution imaging, we microinjected fluorescent Mycobacterium marinum (Mm) into the tail fin tissue of zebrafish larvae carrying the GFP-LC3 autophagy reporter. We detected phagocytosed Mm clusters and LC3-positive Mm-containing vesicles within the first hour of infection. LC3 associations with these vesicles were transient and heterogeneous, ranging from simple vesicles to complex compound structures, dynamically changing shape by fusions between Mm-containing and empty vesicles. LC3-Mm-vesicles could adopt elongated shapes during cell migration or alternate between spacious and compact morphologies. LC3-Mm-vesicles were also observed in cells reverse migrating from the infection site, indicating that the autophagy machinery fails to control infection before tissue dissemination.

7.
Dis Model Mech ; 16(6)2023 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-37078586

RESUMEN

Innate immune responses to inflammation and infection are complex and represent major challenges for developing much needed new treatments for chronic inflammatory diseases and drug-resistant infections. To be ultimately successful, the immune response must be balanced to allow pathogen clearance without excess tissue damage, processes controlled by pro- and anti-inflammatory signals. The roles of anti-inflammatory signalling in raising an appropriate immune response are underappreciated, representing overlooked potential drug targets. This is especially true in neutrophils, a difficult cell type to study ex vivo owing to a short lifespan, dogmatically seen as being highly pro-inflammatory. Here, we have generated and describe the first zebrafish transgenic line [TgBAC(arg2:eGFP)sh571] that labels expression of the anti-inflammatory gene arginase 2 (arg2) and show that a subpopulation of neutrophils upregulate arginase soon after immune challenge with injury and infection. At wound-healing stages, arg2:GFP is expressed in subsets of neutrophils and macrophages, potentially representing anti-inflammatory, polarised immune cell populations. Our findings identify nuanced responses to immune challenge in vivo, responses that represent new opportunities for therapeutic interventions during inflammation and infection.


Asunto(s)
Arginasa , Pez Cebra , Animales , Pez Cebra/metabolismo , Arginasa/genética , Arginasa/metabolismo , Animales Modificados Genéticamente , Neutrófilos , Inflamación , Antiinflamatorios/metabolismo
8.
Cells ; 12(6)2023 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-36980169

RESUMEN

Damage-Regulated Autophagy Modulator 1 (DRAM1) is an infection-inducible membrane protein, whose function in the immune response is incompletely understood. Based on previous results in a zebrafish infection model, we have proposed that DRAM1 is a host resistance factor against intracellular mycobacterial infection. To gain insight into the cellular processes underlying DRAM1-mediated host defence, here we studied the interaction of DRAM1 with Mycobacterium marinum in murine RAW264.7 macrophages. We found that, shortly after phagocytosis, DRAM1 localised in a punctate pattern to mycobacteria, which gradually progressed to full DRAM1 envelopment of the bacteria. Within the same time frame, DRAM1-positive mycobacteria colocalised with the LC3 marker for autophagosomes and LysoTracker and LAMP1 markers for (endo)lysosomes. Knockdown analysis revealed that DRAM1 is required for the recruitment of LC3 and for the acidification of mycobacteria-containing vesicles. A reduction in the presence of LAMP1 further suggested reduced fusion of lysosomes with mycobacteria-containing vesicles. Finally, we show that DRAM1 knockdown impairs the ability of macrophages to defend against mycobacterial infection. Together, these results support that DRAM1 promotes the trafficking of mycobacteria through the degradative (auto)phagolysosomal pathway. Considering its prominent effect on host resistance to intracellular infection, DRAM1 is a promising target for therapeutic modulation of the microbicidal capacity of macrophages.


Asunto(s)
Proteínas de la Membrana , Infecciones por Mycobacterium , Mycobacterium marinum , Animales , Ratones , Autofagia , Lisosomas/metabolismo , Macrófagos/metabolismo , Proteínas de la Membrana/metabolismo
9.
mBio ; 14(1): e0302422, 2023 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-36475748

RESUMEN

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.


Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Animales , Humanos , Pez Cebra , Tamoxifeno/farmacología , Tamoxifeno/uso terapéutico , Reposicionamiento de Medicamentos , Tuberculosis/microbiología , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Mycobacterium tuberculosis/genética
10.
Front Cell Infect Microbiol ; 13: 1331818, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38264729

RESUMEN

Anti-bacterial autophagy, also known as xenophagy, is a crucial innate immune process that helps maintain cellular homeostasis by targeting invading microbes. This defense pathway is widely studied in the context of infections with mycobacteria, the causative agents of human tuberculosis and tuberculosis-like disease in animal models. Our previous work in a zebrafish tuberculosis model showed that host defense against Mycobacterium marinum (Mm) is impaired by deficiencies in xenophagy receptors, optineurin (Optn) or sequestome 1 (p62), and Damage-regulated autophagy modulator 1 (Dram1). However, the interdependency of these receptors and their interaction with Dram1 remained unknown. In the present study, we used single and double knockout zebrafish lines in combination with overexpression experiments. We show that Optn and p62 can compensate for the loss of each other's function, as their overexpression restores the infection susceptibility of the mutant phenotypes. Similarly, Dram1 can compensate for deficiencies in Optn and p62, and, vice versa, Optn and p62 compensate for the loss of Dram1, indicating that these xenophagy receptors and Dram1 do not rely on each other for host defense against Mm. In agreement, Dram1 overexpression in optn/p62 double mutants restored the interaction of autophagosome marker Lc3 with Mm. Finally, optn/p62 double mutants displayed more severe infection susceptibility than the single mutants. Taken together, these results suggest that Optn and p62 do not function downstream of each other in the anti-mycobacterial xenophagy pathway, and that the Dram1-mediated defense against Mm infection does not rely on specific xenophagy receptors.


Asunto(s)
Mycobacterium marinum , Tuberculosis , Proteínas de Pez Cebra , Animales , Autofagia , Macroautofagia , Pez Cebra , Proteínas de Pez Cebra/genética
11.
J Lipid Res ; 63(5): 100199, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35315333

RESUMEN

In Gaucher disease (GD), the deficiency of glucocerebrosidase causes lysosomal accumulation of glucosylceramide (GlcCer), which is partly converted by acid ceramidase to glucosylsphingosine (GlcSph) in the lysosome. Chronically elevated blood and tissue GlcSph is thought to contribute to symptoms in GD patients as well as to increased risk for Parkinson's disease. On the other hand, formation of GlcSph may be beneficial since the water soluble sphingoid base is excreted via urine and bile. To study the role of excessive GlcSph formation during glucocerebrosidase deficiency, we studied zebrafish that have two orthologs of acid ceramidase, Asah1a and Asah1b. Only the latter is involved in the formation of GlcSph in glucocerebrosidase-deficient zebrafish as revealed by knockouts of Asah1a or Asah1b with glucocerebrosidase deficiency (either pharmacologically induced or genetic). Comparison of zebrafish with excessive GlcSph (gba1-/- fish) and without GlcSph (gba1-/-:asah1b-/- fish) allowed us to study the consequences of chronic high levels of GlcSph. Prevention of excessive GlcSph in gba1-/-:asah1b-/- fish did not restrict storage cells, GlcCer accumulation, or neuroinflammation. However, GD fish lacking excessive GlcSph show an ameliorated course of disease reflected by significantly increased lifespan, delayed locomotor abnormality, and delayed development of an abnormal curved back posture. The loss of tyrosine hydroxylase 1 (th1) mRNA, a marker of dopaminergic neurons, is slowed down in brain of GD fish lacking excessive GlcSph. In conclusion, in the zebrafish GD model, excess GlcSph has little impact on (neuro)inflammation or the presence of GlcCer-laden macrophages but rather seems harmful to th1-positive dopaminergic neurons.


Asunto(s)
Enfermedad de Gaucher , Glucosilceramidasa/metabolismo , Proteínas de Pez Cebra/metabolismo , Ceramidasa Ácida , Animales , Enfermedad de Gaucher/genética , Glucosilceramidasa/genética , Glucosilceramidas , Humanos , Psicosina/análogos & derivados , Pez Cebra/genética
12.
Mol Microbiol ; 117(3): 661-669, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-34714579

RESUMEN

The zebrafish has earned its place among animal models to study tuberculosis and other infections caused by pathogenic mycobacteria. This model host is especially useful to study the role of granulomas, the inflammatory lesions characteristic of mycobacterial disease. The optically transparent zebrafish larvae provide a window on the initial stages of granuloma development in the context of innate immunity. Application of fluorescent dyes and transgenic markers enabled real-time visualization of how innate immune mechanisms, such as autophagy and inflammasomes, are activated in infected macrophages and how propagating calcium signals drive communication between macrophages during granuloma formation. A combination of imaging, genetic, and chemical approaches has revealed that the interplay between macrophages and mycobacteria is the main driver of tissue dissemination and granuloma development, while neutrophils have a protective function in early granulomas. Different chemokine signaling axes, conserved between humans and zebrafish, have been shown to recruit macrophages permissive to mycobacterial growth, control their microbicidal capacity, drive their spreading and aggregation, and mediate granuloma vascularization. Finally, zebrafish larvae are now exploited to explore cell death processes, emerging as crucial factors in granuloma expansion. In this review, we discuss recent advances in the understanding of mycobacterial pathogenesis contributed by zebrafish models.


Asunto(s)
Mycobacterium , Tuberculosis , Animales , Granuloma/microbiología , Granuloma/patología , Larva , Tuberculosis/microbiología , Virulencia , Pez Cebra/microbiología
13.
Nucleic Acids Res ; 50(2): e10, 2022 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-34734265

RESUMEN

The interplay between three-dimensional chromosome organisation and genomic processes such as replication and transcription necessitates in vivo studies of chromosome dynamics. Fluorescent organic dyes are often used for chromosome labelling in vivo. The mode of binding of these dyes to DNA cause its distortion, elongation, and partial unwinding. The structural changes induce DNA damage and interfere with the binding dynamics of chromatin-associated proteins, consequently perturbing gene expression, genome replication, and cell cycle progression. We have developed a minimally-perturbing, genetically encoded fluorescent DNA label consisting of a (photo-switchable) fluorescent protein fused to the DNA-binding domain of H-NS - a bacterial nucleoid-associated protein. We show that this DNA label, abbreviated as HI-NESS (H-NS-based indicator for nucleic acid stainings), is minimally-perturbing to genomic processes and labels chromosomes in eukaryotic cells in culture, and in zebrafish embryos with preferential binding to AT-rich chromatin.


Asunto(s)
Proteínas Bacterianas/metabolismo , Bioensayo/métodos , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Coloración y Etiquetado/métodos , Animales , Proteínas Bacterianas/genética , Línea Celular , Clonación Molecular , Replicación del ADN , ADN Bacteriano/química , Proteínas de Unión al ADN/genética , Colorantes Fluorescentes , Expresión Génica , Vectores Genéticos , Microscopía Fluorescente
14.
Front Immunol ; 12: 618569, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34046029

RESUMEN

Glucocorticoids are effective drugs for treating immune-related diseases, but prolonged therapy is associated with an increased risk of various infectious diseases, including tuberculosis. In this study, we have used a larval zebrafish model for tuberculosis, based on Mycobacterium marinum (Mm) infection, to study the effect of glucocorticoids. Our results show that the synthetic glucocorticoid beclomethasone increases the bacterial burden and the dissemination of a systemic Mm infection. The exacerbated Mm infection was associated with a decreased phagocytic activity of macrophages, higher percentages of extracellular bacteria, and a reduced rate of infected cell death, whereas the bactericidal capacity of the macrophages was not affected. The inhibited phagocytic capacity of macrophages was associated with suppression of the transcription of genes involved in phagocytosis in these cells. The decreased bacterial phagocytosis by macrophages was not specific for Mm, since it was also observed upon infection with Salmonella Typhimurium. In conclusion, our results show that glucocorticoids inhibit the phagocytic activity of macrophages, which may increase the severity of bacterial infections like tuberculosis.


Asunto(s)
Glucocorticoides/efectos adversos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Infecciones por Mycobacterium no Tuberculosas/inmunología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium marinum/efectos de los fármacos , Mycobacterium marinum/inmunología , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Animales , Carga Bacteriana , Beclometasona/metabolismo , Inmunofenotipificación , Inmunosupresores/efectos adversos , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/genética , Activación de Macrófagos/inmunología , Macrófagos/metabolismo , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Glucocorticoides/metabolismo , Pez Cebra
15.
Cell Rep ; 35(2): 109000, 2021 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-33852860

RESUMEN

Chemotaxis and lysosomal function are closely intertwined processes essential for the inflammatory response and clearance of intracellular bacteria. We used the zebrafish model to examine the link between chemotactic signaling and lysosome physiology in macrophages during mycobacterial infection and wound-induced inflammation in vivo. Macrophages from zebrafish larvae carrying a mutation in a chemokine receptor of the Cxcr3 family display upregulated expression of vesicle trafficking and lysosomal genes and possess enlarged lysosomes that enhance intracellular bacterial clearance. This increased microbicidal capacity is phenocopied by inhibiting the lysosomal transcription factor EC, while its overexpression counteracts the protective effect of chemokine receptor mutation. Tracking macrophage migration in zebrafish revealed that lysosomes of chemokine receptor mutants accumulate in the front half of cells, preventing macrophage polarization during chemotaxis and reaching sites of inflammation. Our work shows that chemotactic signaling affects the bactericidal properties and localization during chemotaxis, key aspects of the inflammatory response.


Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Lisosomas/inmunología , Macrófagos/inmunología , Infecciones por Mycobacterium/genética , Receptores CXCR3/genética , Transducción de Señal/inmunología , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/inmunología , Rastreo Celular , Quimiotaxis/genética , Quimiotaxis/inmunología , Embrión no Mamífero , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genes Reporteros , Larva/inmunología , Larva/microbiología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/inmunología , Lisosomas/metabolismo , Lisosomas/microbiología , Lisosomas/ultraestructura , Activación de Macrófagos , Macrófagos/microbiología , Macrófagos/ultraestructura , Mutación , Infecciones por Mycobacterium/inmunología , Infecciones por Mycobacterium/microbiología , Mycobacterium marinum/inmunología , Mycobacterium marinum/patogenicidad , Receptores CXCR3/inmunología , Análisis de Secuencia de ARN , Transducción de Señal/genética , Pez Cebra/inmunología , Pez Cebra/microbiología , Proteínas de Pez Cebra/inmunología , Proteína Fluorescente Roja
16.
Virchows Arch ; 479(2): 265-275, 2021 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-33559740

RESUMEN

Tuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm) infection in zebrafish larvae as an animal model for this disease to study the role of the myeloid differentiation factor 88 (Myd88), the key adapter protein of Toll-like receptors. Previously, Myd88 has been shown to enhance innate immune responses against bacterial infections, and in the present study, we have investigated the effect of Myd88 deficiency on the granuloma morphology and the intracellular distribution of bacteria during Mm infection. Our results show that granulomas formed in the tail fin from myd88 mutant larvae have a more compact structure and contain a reduced number of leukocytes compared to the granulomas observed in wild-type larvae. These morphological differences were associated with an increased bacterial burden in the myd88 mutant. Electron microscopy analysis showed that the majority of Mm in the myd88 mutant are located extracellularly, whereas in the wild type, most bacteria were intracellular. In the myd88 mutant, intracellular bacteria were mainly present in compartments that were not electron-dense, suggesting that these compartments had not undergone fusion with a lysosome. In contrast, approximately half of the intracellular bacteria in wild-type larvae were found in electron-dense compartments. These observations in a zebrafish model for tuberculosis suggest a role for Myd88-dependent signalling in two important phenomena that limit mycobacterial growth in the infected tissue. It reduces the number of leukocytes at the site of infection and the acidification of bacteria-containing compartments inside these cells.


Asunto(s)
Granuloma/microbiología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium marinum/crecimiento & desarrollo , Factor 88 de Diferenciación Mieloide/metabolismo , Tuberculosis/microbiología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/microbiología , Animales , Animales Modificados Genéticamente , Carga Bacteriana , Modelos Animales de Enfermedad , Granuloma/genética , Granuloma/metabolismo , Granuloma/patología , Concentración de Iones de Hidrógeno , Leucocitos/metabolismo , Leucocitos/microbiología , Leucocitos/ultraestructura , Lisosomas/metabolismo , Lisosomas/microbiología , Lisosomas/ultraestructura , Microscopía Electrónica de Transmisión , Infecciones por Mycobacterium no Tuberculosas/genética , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Infecciones por Mycobacterium no Tuberculosas/patología , Mycobacterium marinum/ultraestructura , Factor 88 de Diferenciación Mieloide/genética , Transducción de Señal , Tuberculosis/genética , Tuberculosis/metabolismo , Tuberculosis/patología , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética
17.
Toxicol Lett ; 342: 20-25, 2021 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-33581288

RESUMEN

Many bony features of the face develop from endochondral ossification of preexisting collagen-rich cartilage structures. The proper development of these cartilage structures is essential to the morphological formation of the face. The developmental programs governing the formation of the pre-bone facial cartilages are sensitive to chemical compounds that disturb histone acetylation patterns and chromatin structure. We have taken advantage of this fact to develop a quantitative morphological assay of craniofacial developmental toxicity based on the distortion and deterioration of facial cartilage structures in zebrafish larvae upon exposure to increasing concentrations of several well-described histone deacetylase inhibitors. In this assay, we measure the angle formed by the developing ceratohyal bone as a precise, sensitive and quantitative proxy for the overall developmental status of facial cartilages. Using the well-established developmental toxicant and histone deacetylase-inhibiting compound valproic acid along with 12 structurally related compounds, we demonstrate the applicability of the ceratohyal angle assay to investigate structure-activity relationships.


Asunto(s)
Butiratos/toxicidad , Colágeno Tipo II/metabolismo , Anomalías Craneofaciales/inducido químicamente , Histona Desacetilasas/metabolismo , Ácidos Hidroxámicos/toxicidad , Péptidos/toxicidad , Animales , Animales Modificados Genéticamente , Antibióticos Antineoplásicos/toxicidad , Anticonvulsivantes/toxicidad , Antifúngicos/toxicidad , Colágeno Tipo II/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ácido Valproico/toxicidad , Pez Cebra , Proteína Fluorescente Roja
18.
Front Cell Infect Microbiol ; 11: 809121, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-35047422

RESUMEN

Cells of the innate immune system continuously patrol the extracellular environment for potential microbial threats that are to be neutralized by phagocytosis and delivery to lysosomes. In addition, phagocytes employ autophagy as an innate immune mechanism against pathogens that succeed to escape the phagolysosomal pathway and invade the cytosol. In recent years, LC3-associated phagocytosis (LAP) has emerged as an intermediate between phagocytosis and autophagy. During LAP, phagocytes target extracellular microbes while using parts of the autophagic machinery to label the cargo-containing phagosomes for lysosomal degradation. LAP contributes greatly to host immunity against a multitude of bacterial pathogens. In the pursuit of survival, bacteria have developed elaborate strategies to disarm or circumvent the LAP process. In this review, we will outline the nature of the LAP mechanism and discuss recent insights into its interplay with bacterial pathogens.


Asunto(s)
Proteínas Asociadas a Microtúbulos , Fagocitosis , Autofagia , Bacterias , Fagosomas
19.
Autophagy ; 17(4): 888-902, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-32174246

RESUMEN

Staphylococcus aureus is a major human pathogen causing multiple pathologies, from cutaneous lesions to life-threatening sepsis. Although neutrophils contribute to immunity against S. aureus, multiple lines of evidence suggest that these phagocytes can provide an intracellular niche for staphylococcal dissemination. However, the mechanism of neutrophil subversion by intracellular S. aureus remains unknown. Targeting of intracellular pathogens by macroautophagy/autophagy is recognized as an important component of host innate immunity, but whether autophagy is beneficial or detrimental to S. aureus-infected hosts remains controversial. Here, using larval zebrafish, we showed that the autophagy marker Lc3 rapidly decorates S. aureus following engulfment by macrophages and neutrophils. Upon phagocytosis by neutrophils, Lc3-positive, non-acidified spacious phagosomes are formed. This response is dependent on phagocyte NADPH oxidase as both cyba/p22phox knockdown and diphenyleneiodonium (DPI) treatment inhibited Lc3 decoration of phagosomes. Importantly, NADPH oxidase inhibition diverted neutrophil S. aureus processing into tight acidified vesicles, which resulted in increased host resistance to the infection. Some intracellular bacteria within neutrophils were also tagged by Sqstm1/p62-GFP fusion protein and loss of Sqstm1 impaired host defense. Together, we have shown that intracellular handling of S. aureus by neutrophils is best explained by Lc3-associated phagocytosis (LAP), which appears to provide an intracellular niche for bacterial pathogenesis, while the selective autophagy receptor Sqstm1 is host-protective. The antagonistic roles of LAP and Sqstm1-mediated pathways in S. aureus-infected neutrophils may explain the conflicting reports relating to anti-staphylococcal autophagy and provide new insights for therapeutic strategies against antimicrobial-resistant Staphylococci.Abbreviations: ATG: autophagy related; CFU: colony-forming units; CMV: cytomegalovirus; Cyba/P22phox: cytochrome b-245, alpha polypeptide; DMSO: dimethyl sulfoxide; DPI: diphenyleneiodonium; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein; hpf: hours post-fertilization; hpi: hours post-infection; Irf8: interferon regulatory factor 8; LAP: LC3-associated phagocytosis; lyz: lysozyme; LWT: london wild type; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NADPH oxidase: nicotinamide adenine dinucleotide phosphate oxidase; RFP: red fluorescent protein; ROS: reactive oxygen species; RT-PCR: reverse transcriptase polymerase chain reaction; Sqstm1/p62: sequestosome 1; Tg: transgenic; TSA: tyramide signal amplification.


Asunto(s)
Autofagia , Espacio Intracelular/microbiología , Neutrófilos/microbiología , Staphylococcus aureus/fisiología , Animales , Animales Modificados Genéticamente , Cinética , Macrófagos/metabolismo , Macrófagos/microbiología , Proteínas Asociadas a Microtúbulos/metabolismo , NADPH Oxidasas/metabolismo , Neutrófilos/metabolismo , Fagocitosis , Fagosomas/metabolismo , Agregado de Proteínas , Proteína Sequestosoma-1/metabolismo , Pez Cebra/embriología , Pez Cebra/microbiología , Proteínas de Pez Cebra/metabolismo
20.
Dev Comp Immunol ; 116: 103932, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33238180

RESUMEN

The chemokine signaling axes CCR2-CCL2 and CXCR3-CXCL11 participate in the inflammatory response by recruiting leukocytes to damaged tissue or sites of infection and are, therefore, potential pharmacological targets to treat inflammatory disorders. Although multiple CCR2 orthosteric and allosteric inhibitors have been developed, none of these compounds has been approved for clinical use, highlighting the need for a fast, simple and robust preclinical test system to determine the in vivo efficacy of CCR2 inhibitors. Herein we show that human CCL2 and CXCL11 drive macrophage recruitment in zebrafish larvae and that CCR2 inhibitors designed for humans also limit macrophage recruitment in this model organism due to the high conservation of the chemokine system. We demonstrated anti-inflammatory activities of three orthosteric and two allosteric CCR2 inhibitors using macrophage recruitment to injury as a functional read-out of their efficiency, while simultaneously evaluating toxicity. These results provide proof-of-principle for screening CCR2 inhibitors in the zebrafish model.


Asunto(s)
Antiinflamatorios/farmacología , Movimiento Celular/efectos de los fármacos , Macrófagos/efectos de los fármacos , Receptores CCR2/antagonistas & inhibidores , Animales , Movimiento Celular/inmunología , Quimiocina CCL2/metabolismo , Quimiocina CXCL11/metabolismo , Evaluación Preclínica de Medicamentos , Humanos , Inflamación/metabolismo , Macrófagos/inmunología , Modelos Animales , Receptores CCR2/metabolismo , Receptores CXCR3/metabolismo , Transducción de Señal , Pez Cebra
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA