RESUMEN
NOD1 is an intracellular pathogen recognition receptor that contributes to anti-bacterial innate immune responses, adaptive immunity and tissue homeostasis. NOD1-induced signaling relies on actin remodeling, however, the details of the connection of NOD1 and the actin cytoskeleton remained elusive. Here, we identified in a druggable-genome wide siRNA screen the cofilin phosphatase SSH1 as a specific and essential component of the NOD1 pathway. We show that depletion of SSH1 impaired pathogen induced NOD1 signaling evident from diminished NF-κB activation and cytokine release. Chemical inhibition of actin polymerization using cytochalasin D rescued the loss of SSH1. We further demonstrate that NOD1 directly interacted with SSH1 at F-actin rich sites. Finally, we show that enhanced cofilin activity is intimately linked to NOD1 signaling. Our data thus provide evidence that NOD1 requires the SSH1/cofilin network for signaling and to detect bacterial induced changes in actin dynamics leading to NF-κB activation and innate immune responses.
Asunto(s)
Actinas/metabolismo , Cofilina 1/metabolismo , Disentería Bacilar/microbiología , Proteína Adaptadora de Señalización NOD1/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Shigella flexneri/fisiología , Actinas/química , Western Blotting , Células Cultivadas , Cofilina 1/genética , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Regulación de la Expresión Génica , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Inflamación , Mediadores de Inflamación/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Proteína Adaptadora de Señalización NOD1/antagonistas & inhibidores , Proteína Adaptadora de Señalización NOD1/genética , Fosfoproteínas Fosfatasas/genética , Fosforilación , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
Chlamydiaceae are obligate intracellular bacterial pathogens that strictly depend on host metabolites, such as nucleotides, lipids, and amino acids. Depletion of amino acids in cell culture media results in abnormal chlamydial development in vitro. Surprisingly, enrichment of certain amino acids also retards chlamydial growth. Our experiments revealed that the antichlamydial effects are largely independent of changes in the host cell transcriptome or proteome and in the major signal transduction pathway modulated by amino acids, the mTOR (mammalian target of rapamycin) pathway. Furthermore, the chlamydial growth inhibition induced by leucine, isoleucine, methionine, or phenylalanine was completely reversed by concomitant addition of valine. In contrast, the growth inhibition induced by serine, glycine, or threonine was not reversed by valine addition. Functional characterization of the only predicted chlamydial transporter for branched-chain amino acids, BrnQ, revealed that it can be blocked by leucine, isoleucine, methionine, or phenylalanine but not by serine, glycine, or threonine. This chlamydial transporter is the only known BrnQ homolog possessing specificity for methionine, suggesting a unique strategy for methionine uptake among gram-negative bacteria. The antichlamydial effects of leucine, isoleucine, methionine, and phenylalanine could be explained as competitive inhibition of the BrnQ transporter and subsequent valine starvation.
Asunto(s)
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Proteínas Bacterianas/metabolismo , Chlamydia/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/fisiología , Aminoácidos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Transporte Biológico/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Chlamydia/genética , Chlamydia/ultraestructura , Cromatografía Líquida de Alta Presión , Cicloheximida/farmacología , Electroforesis en Gel Bidimensional , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Prueba de Complementación Genética , Glicina/metabolismo , Glicina/farmacología , Células HeLa , Humanos , Isoleucina/metabolismo , Isoleucina/farmacología , Leucina/metabolismo , Leucina/farmacología , Metionina/metabolismo , Metionina/farmacología , Microscopía Confocal , Microscopía Electrónica de Transmisión , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenilalanina/metabolismo , Fenilalanina/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina/metabolismo , Serina/farmacología , Sirolimus/farmacología , Treonina/metabolismo , Treonina/farmacologíaRESUMEN
The differential influence of individual amino acids on the growth of Chlamydia trachomatis versus Chlamydia (Chlamydophila) pneumoniae was investigated. Certain essential amino acids added in excess at the middle of the infection course resulted in varying degrees of abnormality in the development of the two species. If amino acids were added as early as 2 h post-infection, these effects were even more pronounced. The most effective amino acids in terms of C. trachomatis growth inhibition were leucine, isoleucine, methionine and phenylalanine. These amino acids elicited similar effects against C. pneumoniae, except methionine, which, surprisingly, showed a lower inhibitory activity. Tryptophan and valine marginally inhibited C. trachomatis growth and, paradoxically, led to a considerable enhancement of C. pneumoniae growth. On the other hand, some non-essential amino acids administered at the middle of or throughout the infection course differentially affected the development of the two species. For example, C. trachomatis growth was efficiently inhibited by glycine and serine, whereas C. pneumoniae was relatively less sensitive to these agents. Another difference was apparent for glutamate, glutamine and aspartate, which stimulated C. pneumoniae growth more than that of C. trachomatis. Overall, several distinctive patterns of susceptibility to excess amino acid levels were revealed for two representative C. trachomatis and C. pneumoniae isolates. Perturbation of amino acid levels, e.g. of leucine and isoleucine, might form a basis for the development of novel treatment or preventive regimens for chlamydial diseases.
Asunto(s)
Aminoácidos/farmacología , Chlamydia trachomatis/crecimiento & desarrollo , Infecciones por Chlamydophila/microbiología , Chlamydophila pneumoniae/crecimiento & desarrollo , Aminoácidos/metabolismo , Línea Celular Tumoral , Chlamydia trachomatis/metabolismo , Chlamydophila pneumoniae/metabolismo , Humanos , Microscopía Confocal , Microscopía Electrónica de TransmisiónRESUMEN
AIMS: Consumption of cigarette smoke (CS) is a well-known risk factor for early atherosclerosis; yet, the underlying mechanisms of smoking-associated atherosclerosis are poorly understood. Based on the previous results indicating that CS-induced endothelial cell death neither shows typical features of apoptosis nor of necrosis, we investigated the role of autophagy in CS extract (CSE)-induced cell death of human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Here, we demonstrate that overexpression of the classical apoptosis inhibitor BCL-XL had no protective effect on CSE-induced cell death, whereas the autophagy inhibitor 3-methyladenin and an shRNAi-mediated knockdown of the autophagy mediator ATG5 significantly delayed cell death. Our results indicate that CSE induces an excess accumulation of misfolded proteins in the endoplasmic reticulum (ER) and consequently the onset of the unfolded protein response. We provide evidence that the ER-resident kinase PERK is a major transducer of ER stress leading to phosphorylation of eIF2α and attenuation of protein synthesis. Finally, we show that prolonged ER stress in cells subjected to CS is followed by activation of an autophagic programme. CSE-induced autophagy is characterized by an increase in LC3 II/I ratio and activation ATG12. The autophagic signalling pathway via energy depletion and consequent activation AMP-activated protein kinase could be excluded. CONCLUSION: Our results confirm and extend previous findings reporting on the induction of autophagy by CSE in the lung. We show that protein damage caused by CSE activates autophagy, ultimately resulting in necrotic death of HUVECs. Via this mechanism, cigarette smoking may contribute to the deterioration of vascular endothelial function and the initiation of atherosclerosis.
Asunto(s)
Autofagia , Estrés del Retículo Endoplásmico , Células Endoteliales/patología , Nicotiana/toxicidad , Humo , Proteínas Quinasas Activadas por AMP/fisiología , Adenosina Trifosfato/análisis , Aterosclerosis/etiología , Células Cultivadas , Factor 2 Eucariótico de Iniciación/fisiología , Humanos , Venas Umbilicales/citologíaRESUMEN
Microtubule-associated protein 1 (MAP1) light chain 3 (LC3) has proven useful as autophagosomal marker in studies on the interaction between pathogens and the host autophagic machinery. However, the function of LC3 is known to extend above and beyond its role in autophagosome formation. We previously reported that intrinsic LC3 is associated with the intracellular Chlamydia trachomatis inclusion in human epithelial cells. Here we show that LC3, most likely the cytoplasmic nonlipidated form, interacts with the C. trachomatis inclusion as a microtubule-associated protein rather than an autophagosome-associated component. In contrast, N-terminally GFP-tagged LC3 exclusively targets autophagosomes rather than chlamydial inclusions. Immunofluorescence analysis revealed an association of LC3 and MAP1 subunits A and B with the inclusion as early as 18 h post infection. Inclusion-bound LC3 was connected with the microtubular network. Depolymerization of the microtubular architecture disrupted the association of LC3/MAP1s with the inclusion. Furthermore, siRNA-mediated silencing of the MAP1 and LC3 proteins revealed their essential function in the intracellular growth of C. trachomatis. Interestingly, defective autophagy remarkably enhanced chlamydial growth, suggesting a suppressive effect of the autophagic machinery on bacterial development. However, depletion of LC3 in autophagy-deficient cells noticeably reduced chlamydial propagation. Thus, our findings demonstrate a new function for LC3, distinct from autophagy, in intracellular bacterial pathogenesis.
Asunto(s)
Autofagia , Chlamydia trachomatis/crecimiento & desarrollo , Espacio Intracelular/microbiología , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Proteína 5 Relacionada con la Autofagia , Chlamydia trachomatis/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/ultraestructura , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Lípidos/química , Ratones , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Fagosomas/metabolismo , Biosíntesis de Proteínas , Subunidades de Proteína/metabolismo , Transporte de Proteínas , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Chlamydial infection of the host cell induces Gamma interferon (IFNgamma), a central immunoprotector for humans and mice. The primary defense against Chlamydia infection in the mouse involves the IFNgamma-inducible family of IRG proteins; however, the precise mechanisms mediating the pathogen's elimination are unknown. In this study, we identify Irga6 as an important resistance factor against C. trachomatis, but not C. muridarum, infection in IFNgamma-stimulated mouse embryonic fibroblasts (MEFs). We show that Irga6, Irgd, Irgm2 and Irgm3 accumulate at bacterial inclusions in MEFs upon stimulation with IFNgamma, whereas Irgb6 colocalized in the presence or absence of the cytokine. This accumulation triggers a rerouting of bacterial inclusions to autophagosomes that subsequently fuse to lysosomes for elimination. Autophagy-deficient Atg5-/- MEFs and lysosomal acidification impaired cells surrender to infection. Irgm2, Irgm3 and Irgd still localize to inclusions in IFNgamma-induced Atg5-/- cells, but Irga6 localization is disrupted indicating its pivotal role in pathogen resistance. Irga6-deficient (Irga6-/-) MEFs, in which chlamydial growth is enhanced, do not respond to IFNgamma even though Irgb6, Irgd, Irgm2 and Irgm3 still localize to inclusions. Taken together, we identify Irga6 as a necessary factor in conferring host resistance by remodelling a classically nonfusogenic intracellular pathogen to stimulate fusion with autophagosomes, thereby rerouting the intruder to the lysosomal compartment for destruction.
Asunto(s)
Autofagia/inmunología , Chlamydia trachomatis/inmunología , GTP Fosfohidrolasas/inmunología , Interferón gamma/farmacología , Animales , Células Cultivadas , Infecciones por Chlamydia/inmunología , Chlamydia muridarum/inmunología , Fibroblastos/microbiología , Proteínas de Unión al GTP/metabolismo , Cuerpos de Inclusión/metabolismo , Lisosomas/metabolismo , RatonesRESUMEN
Supplementation of culture media with leucine, isoleucine, methionine, or phenylalanine was previously found to inhibit Chlamydia trachomatis growth in HEp-2 cells. Here, we investigated the long-term effects of these additives on C. trachomatis infection in the same cell model. Amino acid addition 30h post-infection (pi) effectively suppressed the generation of infectious progeny monitored for 10 days pi. With the exception of phenylalanine, amino acid treatment beginning at 2h pi for up to 15 days led to a complete lack of infectious progeny. Phenylalanine treatment resulted in residual minimal infectivity. In extended supplementation experiments, very small aberrant chlamydial inclusions formed, whose numbers decreased considerably over time, and the production of infectious chlamydiae could not be rescued even upon amino acid withdrawal. Interestingly, a state of chlamydial persistence was induced under these conditions, as 16S rRNA transcripts were detected throughout treatment. However, expression of several key chlamydial genes including omp1, groEL, omcB, and those functioning for chlamydial DNA replication and cytokinesis was generally very low or even undetected, particularly in monolayers treated with Leu, Ile, or Met. These data revealed a capacity of certain amino acids to eliminate infectious chlamydial progeny. Additionally, supplementation of certain amino acids resulted in the formation of a small persistent population. Extrapolating from these findings may help formulate an anti-chlamydial treatment based on nutritional elements.