RESUMEN
Mycobacterium abscessus (Mab) is an opportunistic pathogen afflicting individuals with underlying lung disease such as Cystic Fibrosis (CF) or immunodeficiencies. Current treatment strategies for Mab infections are limited by its inherent antibiotic resistance and limited drug access to Mab in its in vivo niches resulting in poor cure rates of 30-50%. Mab's ability to survive within macrophages, granulomas and the mucus laden airways of the CF lung requires adaptation via transcriptional remodeling to counteract stresses like hypoxia, increased levels of nitrate, nitrite, and reactive nitrogen intermediates. Mycobacterium tuberculosis (Mtb) is known to coordinate hypoxic adaptation via induction of respiratory nitrate assimilation through the nitrate reductase narGHJI. Mab, on the other hand, does not encode a respiratory nitrate reductase. In addition, our recent study of the transcriptional responses of Mab to hypoxia revealed marked down-regulation of a locus containing putative nitrate assimilation genes, including the orphan response regulator nnaR (nitrate/nitrite assimilation regulator). These putative nitrate assimilation genes, narK3 (nitrate/nitrite transporter), nirBD (nitrite reductase), nnaR, and sirB (ferrochelatase) are arranged contiguously while nasN (assimilatory nitrate reductase identified in this work) is encoded in a different locus. Absence of a respiratory nitrate reductase in Mab and down-regulation of nitrogen metabolism genes in hypoxia suggest interplay between hypoxia adaptation and nitrate assimilation are distinct from what was previously documented in Mtb. The mechanisms used by Mab to fine-tune the transcriptional regulation of nitrogen metabolism in the context of stresses e.g. hypoxia, particularly the role of NnaR, remain poorly understood. To evaluate the role of NnaR in nitrate metabolism we constructed a Mab nnaR knockout strain (MabΔnnaR ) and complement (MabΔnnaR+C ) to investigate transcriptional regulation and phenotypes. qRT-PCR revealed NnaR is necessary for regulating nitrate and nitrite reductases along with a putative nitrate transporter. Loss of NnaR compromised the ability of Mab to assimilate nitrate or nitrite as sole nitrogen sources highlighting its necessity. This work provides the first insights into the role of Mab NnaR setting a foundation for future work investigating NnaR's contribution to pathogenesis.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Mycobacterium abscessus , Nitratos , Nitritos , Mycobacterium abscessus/metabolismo , Mycobacterium abscessus/genética , Nitratos/metabolismo , Nitritos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Humanos , Infecciones por Mycobacterium no Tuberculosas/microbiología , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Nitrito Reductasas/metabolismo , Nitrito Reductasas/genética , Nitrato-Reductasa/metabolismo , Nitrato-Reductasa/genéticaRESUMEN
Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), still ranks among the leading causes of annual human death by infectious disease. Mtb has developed several strategies to survive for years at a time within the host despite the presence of a robust immune response, including manipulating the progression of the inflammatory response and forming granulomatous lesions. Here we demonstrate that IQGAP1, a highly conserved scaffolding protein, compartmentalizes and coordinates multiple signaling pathways in macrophages infected with Mycobacterium marinum (Mm or M.marinum), the closest relative of Mtb. Upregulated IQGAP1 ultimately suppresses TNF-α production by repressing the MKK3 signal and reducing NF-κBp65 translocation, deactivating the p38MAPK pathway. Accordingly, IQGAP1 silencing and overexpression significantly alter p38MAPK activity by modulating the production of phosphorylated MKK3 during mycobacterial infection. Pharmacological inhibition of IQGAP1-associated microtubule assembly not only alleviates tissue damage caused by M.marinum infection but also significantly decreases the production of VEGF-a critical player for granuloma-associated angiogenesis during pathogenic mycobacterial infection. Similarly, IQGAP1 silencing in Mm-infected macrophages diminishes VEGF production, while IQGAP1 overexpression upregulates VEGF. Our data indicate that mycobacteria induce IQGAP1 to hijack NF-κBp65 activation, preventing the expression of proinflammatory cytokines as well as promoting VEGF production during infection and granuloma formation. Thus, therapies targeting host IQGAP1 may be a promising strategy for treating tuberculosis, particularly in drug-resistant diseases.
Asunto(s)
Macrófagos , FN-kappa B , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular , Proteínas Activadoras de ras GTPasa , Animales , Ratones , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa 3/genética , Ratones Endogámicos C57BL , Infecciones por Mycobacterium no Tuberculosas/inmunología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Infecciones por Mycobacterium no Tuberculosas/metabolismo , FN-kappa B/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Células RAW 264.7 , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Ohmyungsamycin A (OMS) is a newly identified cyclic peptide that exerts antimicrobial effects against Mycobacterium tuberculosis. However, its role in nontuberculous mycobacteria (NTMs) infections has not been clarified. Mycobacteroides abscessus (Mabc) is a rapidly growing NTM that has emerged as a human pathogen in both immunocompetent and immunosuppressed individuals. In this study, we demonstrated that OMS had significant antimicrobial effects against Mabc infection in both immunocompetent and immunodeficient mice, and in macrophages. OMS treatment amplified Mabc-induced expression of M1-related proinflammatory cytokines and inducible nitric oxide synthase, and significantly downregulated arginase-1 expression in murine macrophages. In addition, OMS augmented Mabc-mediated production of mitochondrial reactive oxygen species (mtROS), which promoted M1-like proinflammatory responses in Mabc-infected macrophages. OMS-induced production of mtROS and nitric oxide was critical for OMS-mediated antimicrobial responses during Mabc infections. Notably, the combination of OMS and rifabutin had a synergistic effect on the antimicrobial responses against Mabc infections in vitro, in murine macrophages, and in zebrafish models in vivo. Collectively, these data strongly suggest that OMS may be an effective M1-like adjunctive therapeutic against Mabc infections, either alone or in combination with antibiotics.
Asunto(s)
Infecciones por Mycobacterium no Tuberculosas , Mycobacterium abscessus , Humanos , Ratones , Animales , Pez Cebra , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antibacterianos/metabolismo , Macrófagos/microbiologíaRESUMEN
Human macrophages are a natural host of many mycobacterium species, including Mycobacterium abscessus (M. abscessus), an emerging pathogen affecting immunocompromised and cystic fibrosis patients with few available treatments. The search for an effective treatment is hindered by the lack of a tractable in vitro intracellular infection model. Here, we established a reliable model for M. abscessus infection using human pluripotent stem cell-derived macrophages (hPSC-macrophages). hPSC differentiation permitted reproducible generation of functional macrophages that were highly susceptible to M. abscessus infection. Electron microscopy demonstrated that M. abscessus was present in the hPSC-macrophage vacuoles. RNA sequencing analysis revealed a time-dependent host cell response, with differing gene and protein expression patterns post-infection. Engineered tdTOMATO-expressing hPSC-macrophages with GFP-expressing mycobacteria enabled rapid image-based high-throughput analysis of intracellular infection and quantitative assessment of antibiotic efficacy. Our study describes the first to our knowledge hPSC-based model for M. abscessus infection, representing a novel and accessible system for studying pathogen-host interaction and drug discovery.
Asunto(s)
Infecciones por Mycobacterium no Tuberculosas , Mycobacterium abscessus , Mycobacterium , Células Madre Pluripotentes , Humanos , Macrófagos/metabolismo , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Infecciones por Mycobacterium no Tuberculosas/microbiologíaRESUMEN
Mycobacterium abscessus (MAB) is one of the rapidly growing, multidrug-resistant non-tuberculous mycobacteria (NTM) causing various diseases including pulmonary disorder. Although it has been known that type I interferons (IFNs) contribute to host defense against bacterial infections, the role of type I IFNs against MAB infection is still unclear. In the present study, we show that rIFN-ß treatment reduced the intracellular growth of MAB in macrophages. Deficiency of IFN-α/ß receptor (IFNAR) led to the reduction of nitric oxide (NO) production in MAB-infected macrophages. Consistently, rIFN-ß treatment enhanced the expression of iNOS gene and protein, and NO production in response to MAB. We also found that NO is essential for the intracellular growth control of MAB within macrophages in an inhibitor assay using iNOS-deficient cells. In addition, pretreatment of rIFN-ß before MAB infection in mice increased production of NO in the lungs at day 1 after infection and promoted the bacterial clearance at day 5. However, when alveolar macrophages were depleted by treatment of clodronate liposome, rIFN-ß did not promote the bacterial clearance in the lungs. Moreover, we found that a cytosolic receptor nucleotide-binding oligomerization domain 2 (NOD2) is required for MAB-induced TANK binding kinase 1 (TBK1) phosphorylation and IFN-ß gene expression in macrophages. Finally, increase in the bacterial loads caused by reduction of NO levels was reversed by rIFN-ß treatment in the lungs of NOD2-deficient mice. Collectively, our findings suggest that type I IFNs act as an intermediator of NOD2-induced NO production in macrophages and thus contribute to host defense against MAB infection.
Asunto(s)
Interferón Tipo I/metabolismo , Pulmón/microbiología , Macrófagos Alveolares/microbiología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium abscessus/crecimiento & desarrollo , Óxido Nítrico/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Interacciones Huésped-Patógeno , Pulmón/inmunología , Pulmón/metabolismo , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Mycobacterium no Tuberculosas/inmunología , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Mycobacterium abscessus/inmunología , Mycobacterium abscessus/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteína Adaptadora de Señalización NOD2/genética , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Transducción de SeñalRESUMEN
Phagosome-lysosome fusion in innate immune cells like macrophages and neutrophils marshal an essential role in eliminating intracellular microorganisms. In microbe-challenged macrophages, phagosome-lysosome fusion occurs 4 to 6 h after the phagocytic uptake of the microbe. However, live pathogenic mycobacteria hinder the transfer of phagosomes to lysosomes, up to 20 h post-phagocytic uptake. This period is required to evade pro-inflammatory response and upregulate the acid-stress tolerant proteins. The exact sequence of events through which mycobacteria retards phagolysosome formation remains an enigma. The macrophage coat protein Coronin1(Cor1) is recruited and retained by mycobacteria on the phagosome membrane to retard its maturation by hindering the access of phagosome maturation factors. Mycobacteria-infected macrophages exhibit an increased cAMP level, and based on receptor stimulus, Cor1 expressing cells show a higher level of cAMP than non-Cor1 expressing cells. Here we have shown that infection of bone marrow-derived macrophages with H37Rv causes a Cor1 dependent rise of intracellular cAMP levels at the vicinity of the phagosomes. This increased cAMP fuels cytoskeletal protein Cofilin1 to depolymerize F-actin around the mycobacteria-containing phagosome. Owing to reduced F-actin levels, the movement of the phagosome toward the lysosomes is hindered, thus contributing to the retarded phagosome maturation process. Additionally, Cor1 mediated upregulation of Cofilin1 also contributes to the prevention of phagosomal acidification, which further aids in the retardation of phagosome maturation. Overall, our study provides first-hand information on Cor1 mediated retardation of phagosome maturation, which can be utilized in developing novel peptidomimetics as part of host-directed therapeutics against tuberculosis.
Asunto(s)
Cofilina 1/metabolismo , AMP Cíclico/metabolismo , Macrófagos/microbiología , Proteínas de Microfilamentos/metabolismo , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium bovis/patogenicidad , Mycobacterium smegmatis/patogenicidad , Mycobacterium tuberculosis/patogenicidad , Fagosomas/microbiología , Tuberculosis/microbiología , Animales , Línea Celular , Interacciones Huésped-Patógeno , Concentración de Iones de Hidrógeno , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Proteínas de Microfilamentos/genética , Infecciones por Mycobacterium no Tuberculosas/inmunología , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Mycobacterium bovis/inmunología , Mycobacterium smegmatis/inmunología , Mycobacterium tuberculosis/inmunología , Fagosomas/inmunología , Fagosomas/metabolismo , Sistemas de Mensajero Secundario , Tuberculosis/inmunología , Tuberculosis/metabolismoRESUMEN
Autophagy is critically involved in host defense pathways through targeting and elimination of numerous pathogens via autophagic machinery. Nontuberculous mycobacteria (NTMs) are ubiquitous microbes, have become increasingly prevalent, and are emerging as clinically important strains due to drug-resistant issues. Compared to Mycobacterium tuberculosis (Mtb), the causal pathogen for human tuberculosis, the roles of autophagy remain largely uncharacterized in the context of a variety of NTM infections. Compelling evidence suggests that host autophagy activation plays an essential role in the enhancement of antimicrobial immune responses and controlling pathological inflammation against various NTM infections. As similar to Mtb, it is believed that NTM bacteria evolve multiple strategies to manipulate and hijack host autophagy pathways. Despite this, we are just beginning to understand the molecular mechanisms underlying the crosstalk between pathogen and the host autophagy system in a battle with NTM bacteria. In this review, we will explore the function of autophagy, which is involved in shaping host-pathogen interaction and disease outcomes during NTM infections. These efforts will lead to the development of autophagy-based host-directed therapeutics against NTM infection.
Asunto(s)
Autofagia , Inmunidad Innata , Infecciones por Mycobacterium no Tuberculosas/microbiología , Micobacterias no Tuberculosas/patogenicidad , Animales , Antibacterianos/uso terapéutico , Autofagia/efectos de los fármacos , Evolución Biológica , Farmacorresistencia Bacteriana , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata/efectos de los fármacos , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/inmunología , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Micobacterias no Tuberculosas/efectos de los fármacos , Micobacterias no Tuberculosas/inmunologíaRESUMEN
Galanin is a peptide that is conserved among different species and plays various roles in an organism, although its entire role is not completely understood. For many years, galanin has been linked mainly with the neurotransmission in the nervous system; however, recent reports underline its role in immunity. Zebrafish (Danio rerio) is an intensively developing animal model to study infectious diseases. In this study, we used larval zebrafish to determine the role of galanin in bacterial infection. We showed that knockout of galanin in zebrafish leads to a higher bacterial burden and mortality during Mycobacterium marinum and Staphylococcus aureus infection, whereas administration of a galanin analogue, NAX 5055, improves the ability of fish to control the infection caused by both pathogens. Moreover, the transcriptomics data revealed that a lower number of genes were regulated in response to mycobacterial infection in gal-/- mutants compared with their gal+/+ wild-type counterparts. We also found that galanin deficiency led to significant changes in immune-related pathways, mostly connected with cytokine and chemokine functions. The results show that galanin acts not only as a neurotransmitter but is also involved in immune response to bacterial infections, demonstrating the complexity of the neuroendocrine system and its possible connection with immunity.
Asunto(s)
Galanina/metabolismo , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium marinum/patogenicidad , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/patogenicidad , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Galanina/genética , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Interacciones Huésped-Patógeno , Mediadores de Inflamación/metabolismo , Infecciones por Mycobacterium no Tuberculosas/genética , Infecciones por Mycobacterium no Tuberculosas/inmunología , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Mycobacterium marinum/inmunología , Transducción de Señal , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus/inmunología , Transcriptoma , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
To describe the clinical features and the risk factors for nontuberculous mycobacteria (NTM) and Talaromyces marneffei (TM) co-infections in HIV-negative patients. A multicenter retrospective study in 13 hospitals, and a systematic literature review were performed of original articles published in English related to TM/NTM co-infections. HIV-negative patients with TM and NTM co-infections comprised Group 1; TM-only infection Group 2; NTM-only infection Group 3; and healthy volunteers Group 4. Univariate logistic analysis was used to estimate the potential risk factors of TM/NTM co-infections. A total of 22 cases of TM and NTM co-infections were enrolled. Of these, 17 patients (77.3%) had a missed diagnosis of one of the TM or NTM pathogens. The anti-IFN-γ autoantibodies (AIGAs) titer, white blood cell (WBC), neutrophil counts (N), erythrocyte sedimentation rate (ESR), C reactive protein (CRP), globulin, and immunoglobulin G (IgG) levels of Group 1 were higher than those of the other groups, whereas the levels of CD4+T cells was lower than those of other groups. There was a significant negative correlation between the AIGA titers and the number of CD4+T cells (P < 0.05). Factors including the ratio of the actual values to the cut-off values of AIGAs, WBC, N, HGB, CD4+T cells, IgG, IgM, IgA, serum globulin, ESR, and CRP were taken as potential risk factors for TM and NTM co-infection. Most patients with TM and NTM co-infection had a missed diagnosis of one of the TM or NTM pathogens. The levels of AIGAs, WBC, N, ESR, and CRP in TM and NTM co-infections were remarkably higher than in mono-infection. High-titer AIGAs may be a potential risk factor and susceptibility factor for co-infection of TM and NTM in HIV-negative hosts.
Asunto(s)
Coinfección/epidemiología , Citocinas/metabolismo , Infecciones por VIH , Infecciones por Mycobacterium no Tuberculosas/epidemiología , Micobacterias no Tuberculosas/aislamiento & purificación , Talaromyces/aislamiento & purificación , Adulto , Anciano , Estudios de Casos y Controles , China/epidemiología , Coinfección/diagnóstico , Coinfección/metabolismo , Coinfección/microbiología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Infecciones por Mycobacterium no Tuberculosas/microbiología , Pronóstico , Estudios Retrospectivos , Factores de RiesgoRESUMEN
Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, kills 1.5 to 1.7 million people every year. Macrophages are Mtb's main host cells and their inflammatory response is an essential component of the host defense against Mtb. However, Mtb is able to circumvent the macrophages' defenses by triggering an inappropriate inflammatory response. The ability of Mtb to hinder phagolysosome maturation and acidification, and to escape the phagosome into the cytosol, is closely linked to its virulence. The modulation of the host inflammatory response relies on Mtb virulence factors, but remains poorly studied. Understanding macrophage interactions with Mtb is crucial to develop strategies to control tuberculosis. The present study aims to determine the inflammatory response transcriptome and miRNome of human macrophages infected with the virulent H37Rv Mtb strain, to identify macrophage genetic networks specifically modulated by Mtb virulence. Using human macrophages infected with two different live strains of mycobacteria (live or heat-inactivated Mtb H37Rv and M. marinum), we quantified and analyzed 184 inflammatory mRNAs and 765 micro(mi)RNAs. Transcripts and miRNAs differently modulated by H37Rv in comparison with the two other conditions were analyzed using in silico approaches. We identified 30 host inflammatory response genes and 37 miRNAs specific for H37Rv virulence, and highlight evidence suggesting that Mtb intracellular-linked virulence depends on the inhibition of IL-1ß-dependent pro-inflammatory response, the repression of apoptosis and the delay of the recruitment and activation of adaptive immune cells. Our findings provide new potential targets for the development of macrophage-based therapeutic strategies against TB.
Asunto(s)
Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Pulmón/microbiología , Macrófagos/microbiología , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/microbiología , Inmunidad Adaptativa , Apoptosis , Citocinas/genética , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Pulmón/inmunología , Pulmón/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Infecciones por Mycobacterium no Tuberculosas/inmunología , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium marinum/inmunología , Mycobacterium marinum/patogenicidad , Mycobacterium tuberculosis/inmunología , Transducción de Señal , Células THP-1 , Transcriptoma , Tuberculosis/genética , Tuberculosis/inmunología , Tuberculosis/metabolismo , VirulenciaRESUMEN
Tuberculosis caused by Mycobacterium tuberculosis remains a serious global public health threat. Macrophage polarization is crucial for the innate immunity against M. tuberculosis. However, how M. tuberculosis interferes with macrophage polarization is elusive. We demonstrated here that M. tuberculosis PPE36 (Rv2108) blocked macrophage M1 polarization, preventing the cytokine storm, and alleviating inflammatory damage to mouse immune organs. PPE36 inhibited the polarization of THP-1 cell differentiation to M1 macrophages, reduced mitochondrial dehydrogenase activity, inhibited the expression of CD16, and repressed the expression of pro-inflammatory cytokines IL-6 and TNF-α, as well as chemokines CXCL9, CXCL10, CCL3, and CCL5. Intriguingly, in the mouse infection model, PPE36 significantly alleviated the inflammatory damage of immune organs caused by a cytokine storm. Furthermore, we found that PPE36 inhibited the polarization of macrophages into mature M1 macrophages by suppressing the ERK signaling. The study provided novel insights into the function and mechanism of action of M. tuberculosis effector PPE36 both at the cellular and animal level.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Síndrome de Liberación de Citoquinas/prevención & control , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Macrófagos/microbiología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium smegmatis/metabolismo , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Síndrome de Liberación de Citoquinas/inmunología , Síndrome de Liberación de Citoquinas/metabolismo , Síndrome de Liberación de Citoquinas/microbiología , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Interacciones Huésped-Patógeno , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Infecciones por Mycobacterium no Tuberculosas/inmunología , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/inmunología , Fenotipo , Transducción de Señal , Células THP-1RESUMEN
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 CebraRESUMEN
Host-derived fatty acids are an important carbon source for pathogenic mycobacteria during infection. How mycobacterial cells regulate the catabolism of fatty acids to serve the pathogenicity, however, remains unknown. Here, we identified a TetR-family transcriptional factor, FdmR, as the key regulator of fatty acid catabolism in the pathogen Mycobacterium marinum by combining use of transcriptomics, chromatin immunoprecipitation followed by sequencing, dynamic 13C-based flux analysis, metabolomics, and lipidomics. An M. marinum mutant deficient in FdmR was severely attenuated in zebrafish larvae and adult zebrafish. The mutant showed defective growth but high substrate consumption on fatty acids. FdmR was identified as a long-chain acyl-coenzyme A (acyl-CoA)-responsive repressor of genes involved in fatty acid degradation and modification. We demonstrated that FdmR functions as a valve to direct the flux of exogenously derived fatty acids away from ß-oxidation toward lipid biosynthesis, thereby avoiding the overactive catabolism and accumulation of biologically toxic intermediates. Moreover, we found that FdmR suppresses degradation of long-chain acyl-CoAs endogenously synthesized through the type I fatty acid synthase. By modulating the supply of long-chain acyl-CoAs for lipogenesis, FdmR controls the abundance and chain length of virulence-associated lipids and mycolates and plays an important role in the impermeability of the cell envelope. These results reveal that despite the fact that host-derived fatty acids are used as an important carbon source, overactive catabolism of fatty acids is detrimental to mycobacterial cell growth and pathogenicity. This study thus presents FdmR as a potentially attractive target for chemotherapy.
Asunto(s)
Ácidos Grasos/metabolismo , Lipogénesis/fisiología , Mycobacterium marinum/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Lipólisis , Metabolismo/fisiología , Modelos Animales , Mycobacterium/metabolismo , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Infecciones por Mycobacterium no Tuberculosas/fisiopatología , Oxidación-Reducción , Factores de Transcripción/metabolismo , Virulencia/fisiología , Pez Cebra/metabolismo , Pez Cebra/microbiologíaRESUMEN
Tuberculosis (TB) is the leading cause of death among HIV-1-infected individuals and Mycobacterium tuberculosis (Mtb) co-infection is an early precipitate to AIDS. We aimed to determine whether Mtb strains differentially modulate cellular susceptibility to HIV-1 infection (cis- and trans-infection), via surface receptor interaction by their cell envelope lipids. Total lipids from pathogenic (lineage 4 Mtb H37Rv, CDC1551 and lineage 2 Mtb HN878, EU127) and non-pathogenic (Mycobacterium bovis BCG and Mycobacterium smegmatis) Mycobacterium strains were integrated into liposomes mimicking the lipid distribution and antigen accessibility of the mycobacterial cell wall. The resulting liposomes were tested for modulating in vitro HIV-1 cis- and trans-infection of TZM-bl cells using single-cycle infectious virus particles. Mtb glycolipids did not affect HIV-1 direct infection however, trans-infection of both R5 and X4 tropic HIV-1 strains were impaired in the presence of glycolipids from M. bovis, Mtb H37Rv and Mtb EU127 strains when using Raji-DC-SIGN cells or immature and mature dendritic cells (DCs) to capture virus. SL1, PDIM and TDM lipids were identified to be involved in DC-SIGN recognition and impairment of HIV-1 trans-infection. These findings indicate that variant strains of Mtb have differential effect on HIV-1 trans-infection with the potential to influence HIV-1 disease course in co-infected individuals.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/metabolismo , Coinfección/metabolismo , Glucolípidos/metabolismo , VIH-1/fisiología , Liposomas/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculosis/metabolismo , Infecciones Oportunistas Relacionadas con el SIDA/virología , Moléculas de Adhesión Celular/metabolismo , Pared Celular/metabolismo , Células HEK293 , Humanos , Lectinas Tipo C/metabolismo , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium bovis/metabolismo , Mycobacterium smegmatis/metabolismo , Receptores de Superficie Celular/metabolismo , Tuberculosis/microbiología , Internalización del VirusRESUMEN
There is no validated clinical biomarker for disease severity or treatment response for nontuberculous mycobacterial pulmonary disease (NTM-PD). We investigated the correlation between elevated serum carbohydrate antigen (CA) 19-9 levels and NTM-PD disease activity, defined using an imaging severity score based on chest computed tomography (CT). We retrospectively examined 79 patients with NTM-PD who underwent serum CA19-9 level assessments and chest CT less than 1 month apart. NTM-PD severity was rated using a CT-based scoring system. The correlation between the CT score and serum CA19-9 levels was evaluated. Chest CT revealed nodular bronchiectasis without cavitation in most patients (78.5%). Serum CA19-9 levels were elevated in 19 (24%) patients. Serum CA19-9 levels were positively correlated with the total CT score and bronchiectasis, bronchiolitis, cavity, and consolidation subscores. Partial correlation analysis revealed a significant positive correlation between serum CA19-9 levels and CT scores for total score and bronchiectasis, bronchiolitis, cavitation, and consolidation subscores after controlling for age, sex, and BMI. Serum CA19-9 levels were positively correlated with the CT severity score for NTM-PD. Serum CA19-9 may be useful in evaluating disease activity or therapeutic response in patients with NTM-PD.
Asunto(s)
Antígeno CA-19-9/sangre , Pulmón/diagnóstico por imagen , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Anciano , Biomarcadores/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Índice de Severidad de la EnfermedadRESUMEN
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/genéticaRESUMEN
Infection with rapidly growing nontuberculous mycobacteria is emerging as a global health issue; however, key host factors remain elusive. Here, we investigated the characteristic immune profiles of peripheral blood mononuclear cells (PBMCs) from patients infected with Mycobacteroides abscessus subsp. abscessus (Mabc) and M. abscessus subsp. massiliense (Mmass). Using an integrated analysis of global mRNA and microRNA expression profiles, we found that several inflammatory cytokines/chemokines [interleukin (IL)-1ß, IL-6, C-X-C motif chemokine ligand 2, and C-C motif chemokine ligand 2] and miR-144-3p were significantly upregulated in PBMCs from patients compared with those from healthy controls (HCs). Notably, there was a strong correlation between the expression levels of miR-144-3p and proinflammatory cytokines/chemokines. Similarly, upregulated expression of miR-144-3p and proinflammatory cytokines/chemokines was found in macrophages and lungs from mice after infection with Mabc and Mmass. We showed that the expression of negative regulators of inflammation (SARM1 and TNIP3) was significantly downregulated in PBMCs from the patients, although they were not putative targets of miR-144-3p. Furthermore, overexpression of miR-144-3p led to a marked increase in proinflammatory cytokines/chemokines and promoted bacterial growth in macrophages. Together, our results highlight the importance of miR-144-3p linking to pathological inflammation during M. abscessus infection.
Asunto(s)
MicroARNs , Infecciones por Mycobacterium no Tuberculosas , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Proteínas del Dominio Armadillo/genética , Proteínas del Dominio Armadillo/metabolismo , Células Cultivadas , Quimiocina CXCL2/genética , Quimiocina CXCL2/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Mycobacterium abscessus/patogenicidad , Infecciones por Mycobacterium no Tuberculosas/genética , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Infecciones por Mycobacterium no Tuberculosas/microbiología , Infecciones por Mycobacterium no Tuberculosas/patologíaRESUMEN
Several virulence lipids populate the outer cell wall of pathogenic mycobacteria. Phthiocerol dimycocerosate (PDIM), one of the most abundant outer membrane lipids, plays important roles in both defending against host antimicrobial programs and in evading these programs altogether. Immediately following infection, mycobacteria rely on PDIM to evade Myd88-dependent recruitment of microbicidal monocytes which can clear infection. To circumvent the limitations in using genetics to understand virulence lipids, we developed a chemical approach to track PDIM during Mycobacterium marinum infection of zebrafish. We found that PDIM's methyl-branched lipid tails enabled it to spread into host epithelial membranes to prevent immune activation. Additionally, PDIM's affinity for cholesterol promoted this phenotype; treatment of zebrafish with statins, cholesterol synthesis inhibitors, decreased spreading and provided protection from infection. This work establishes that interactions between host and pathogen lipids influence mycobacterial infectivity and suggests the use of statins as tuberculosis preventive therapy by inhibiting PDIM spread.
Asunto(s)
Membrana Celular/microbiología , Células Epiteliales/microbiología , Lípidos , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium marinum/patogenicidad , Factores de Virulencia/metabolismo , Células A549 , Animales , Animales Modificados Genéticamente , Antibacterianos/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Interacciones Huésped-Patógeno , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Lípidos/química , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/microbiología , Estructura Molecular , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Infecciones por Mycobacterium no Tuberculosas/prevención & control , Mycobacterium marinum/efectos de los fármacos , Mycobacterium marinum/genética , Mycobacterium marinum/metabolismo , Relación Estructura-Actividad , Células THP-1 , Virulencia , Factores de Virulencia/química , Pez CebraRESUMEN
Outside of Mycobacterium tuberculosis and Mycobacterium leprae, nontuberculous mycobacteria (NTM) are environmental mycobacteria (>190 species) and are classified as slow- or rapid-growing mycobacteria. Infections caused by NTM show an increased incidence in immunocompromised patients and patients with underlying structural lung disease. The true global prevalence of NTM infections remains unknown because many countries do not require mandatory reporting of the infection. This is coupled with a challenging diagnosis and identification of the species. Current therapies for treatment of NTM infections require multidrug regimens for a minimum of 18 months and are associated with serious adverse reactions, infection relapse, and high reinfection rates, necessitating discovery of novel antimycobacterial agents. Robust drug discovery processes have discovered inhibitors targeting mycobacterial membrane protein large 3 (MmpL3), a protein responsible for translocating mycolic acids from the inner membrane to periplasm in the biosynthesis of the mycobacterial cell membrane. This review focuses on promising new chemical scaffolds that inhibit MmpL3 function and represent interesting and promising putative drug candidates for the treatment of NTM infections. Additionally, agents (FS-1, SMARt-420, C10) that promote reversion of drug resistance are also reviewed.
Asunto(s)
Antibacterianos/farmacología , Proteínas de Transporte de Membrana/metabolismo , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Micobacterias no Tuberculosas/metabolismo , Antibacterianos/uso terapéutico , Proteínas Bacterianas/metabolismo , Transporte Biológico/efectos de los fármacos , Descubrimiento de Drogas , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Humanos , Yodóforos/farmacología , Yodóforos/uso terapéutico , Isoxazoles/farmacología , Isoxazoles/uso terapéutico , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Ácidos Micólicos/metabolismo , Micobacterias no Tuberculosas/efectos de los fármacos , Polisacáridos/farmacología , Polisacáridos/uso terapéutico , Compuestos de Espiro/farmacología , Compuestos de Espiro/uso terapéuticoRESUMEN
Lung inflammation due to sarcoidosis is characterized by a complex cascade of immunopathologic events, including leukocyte recruitment and granuloma formation. α-melanocyte stimulating hormone (α-MSH) is a melanocortin signaling peptide with anti-inflammatory properties. We aimed to evaluate the effects of α-MSH in a novel in vitro sarcoidosis model. An in vitro sarcoidosis-like granuloma model was developed by challenging peripheral blood mononuclear cells (PBMCs) derived from patients with confirmed treatment-naïve sarcoidosis with microparticles generated from Mycobacterium abscessus cell walls. Unchallenged PBMCsand developed granulomas were treated daily with 10 µM α-MSH or saline as control. Cytokine concentrations in supernatants of culture and in cell extracts were measured using Illumina multiplex Elisa and western blot, respectively. Gene expression was analyzed using RNA-Seq and RT-PCR. Protein secretion and gene expression of IL-7, IL-7R, IFN-γ, MC1R, NF-κB, phosphorylated NF-κB (p-NF-κB), MARCO, and p-CREB were measured with western blot and RNAseq. A significant increase in IL-7, IL-7R, and IFN-γ protein expression was found in developed granulomas comparing to microparticle unchallenged PBMCs. IL-7, IL-7R, and IFN-γ protein expression was significantly reduced in developed granulomas after exposure to α-MSH compared with saline treated granulomas. Compared with microparticle unchallenged PBMCs, total NF-κB and p-NF-κB were significantly increased in developed granulomas, while expression of p-CREB was not changed. Treatment with α-MSH promoted a significantly higher concentration of p-CREB in granulomas. The anti-inflammatory effects of α-MSH were blocked by specific p-CREB inhibition. α-MSH has anti-inflammatory properties in this in vitro granuloma model, which is an effect mediated by induction of phosphorylation of CREB.