RESUMEN
Mycobacteria have a cell envelope that can act as a shield against host defense. This study shows that mycobacteria survive in host macrophages by targeting the innate host receptor dectin-1 through a noncanonical ligand. Compared with wild-type (WT) mice, dectin-1-deficient mice were more resistant to infection to mycobacteria. Dectin-1-deficient mice presented with substantially reduced bacterial burdens, inflammatory cytokines, and infiltrating myeloid cells, such as neutrophils and macrophages. Intracellular survival of these bacteria was reduced in macrophages derived from dectin-1-deficient mice compared with those from WT mice. Cellular characterization of mycobacteria-infected macrophages indicated that the presence of dectin-1 altered phagosomal maturation and association with markers of autophagy. Activity-based purification and nuclear magnetic resonance spectrometry identified branched α-glucan as the dectin-1 mycobacterial ligand. This branched glucan was essential for activating dectin-1. These results show that mycobacterial α-glucan targets dectin-1 to facilitate intracellular bacterial survival.
Asunto(s)
Glucanos , Lectinas Tipo C , Macrófagos , Animales , Lectinas Tipo C/inmunología , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ratones , Macrófagos/inmunología , Macrófagos/microbiología , Ratones Noqueados , Glucanos/inmunología , Glucanos/metabolismo , Ratones Endogámicos C57BL , Viabilidad MicrobianaRESUMEN
Immunocompetent and experimentally accessible alveolar systems to study human respiratory diseases are lacking. Here, we developed a single-donor human induced pluripotent stem cell-derived lung-on-chip (iLoC) containing type II and I alveolar epithelial cells, vascular endothelial cells, and macrophages in a microfluidic device that mimic lung three-dimensional mechanical stretching and air-liquid interface. Imaging and single-cell RNA sequencing analysis revealed that the iLoC recapitulated cellular profiles present in the human distal lung. Infection of the iLoC with the human pathogen Mycobacterium tuberculosis (Mtb) showed that both macrophages and epithelial cells were infected but not permissive to bacterial replication. Stochastically, large macrophage clusters containing necrotic macrophages supporting Mtb replication were observed. A genetically engineered autophagy-deficient iLoC revealed that after Mtb infection, macrophage necrosis was higher upon ATG14 deficiency without bacterial replication. Together, we report an autologous, genetically tractable human alveolar model to study lung diseases and therapies.
Asunto(s)
Células Madre Pluripotentes Inducidas , Dispositivos Laboratorio en un Chip , Mycobacterium tuberculosis , Alveolos Pulmonares , Tuberculosis , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/microbiología , Mycobacterium tuberculosis/fisiología , Mycobacterium tuberculosis/patogenicidad , Alveolos Pulmonares/microbiología , Alveolos Pulmonares/patología , Alveolos Pulmonares/citología , Alveolos Pulmonares/metabolismo , Macrófagos/microbiología , Macrófagos/metabolismo , Macrófagos/patología , Tuberculosis/microbiología , Tuberculosis/patología , Autofagia , Células Epiteliales Alveolares/microbiología , Células Epiteliales Alveolares/metabolismoRESUMEN
Nontuberculous mycobacteria (NTM) refers to all mycobacteria except Mycobacterium tuberculosis (Mtb) and Mycobacterium leprae (M. leprae). In recent years, its incidence and prevalence have been increasing worldwide, which has attracted the attention of medical and scientific research circles. We determined the immune mechanism of Mycobacterium neoaurum (M. neoaurum) and Bacillus Calmette-Guérin (BCG) by studying the association between M. neoaurum and BCG infection and the release of IL-1ß and IL-18, the assembly of inflammatory body, and pyroptosis. Macrophages were infected with M. neoaurum and BCG. The secretion of IL-1ß and IL-18 was detected by ELISA, and the gene expression and protein expression of IL-1ß, IL-18, NLRP3, AIM2, ASC, caspase-1 and GSDMD were detected by RT-PCR and Western Blot. NLRP3 and AIM2 inflammasome assembly was induced by M. neoaurum and BCG infection in mouse macrophages, which activated caspase-1 and induced the maturation and secretion of IL-1ß and IL-18. The secretion of IL-1ß and IL-18 were regulated by ATP-dependent K+ channel. At the same time, M. neoaurum and BCG infection can also induce pyroptosis. This study was limited by the lack of in vivo mouse experiments. The research findings suggest that the activation of NLRP3 and AIM2 inflammasomes may be involved in the host defense against M. neoaurum and BCG, and the process of pyroptosis may also be related to this.
Asunto(s)
Proteínas de Unión al ADN , Inflamasomas , Interleucina-18 , Interleucina-1 , Mycobacterium bovis , Mycobacterium , Proteína con Dominio Pirina 3 de la Familia NLR , Animales , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-18/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Inflamasomas/inmunología , Inflamasomas/metabolismo , Inflamasomas/genética , Ratones , Macrófagos/microbiología , Macrófagos/inmunología , Mycobacterium bovis/fisiología , Mycobacterium bovis/inmunología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/inmunología , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-1/inmunología , Ratones Endogámicos C57BL , Mycobacterium/fisiología , Mycobacterium/inmunología , Interleucina-1beta/genética , Interleucina-1beta/metabolismoRESUMEN
Brucella abortus, an intracellular bacterium, employs intricate mechanisms to manipulate host signaling for persistence. This study investigates the role of STAT3 palmitoylation in B. abortus-infected macrophages. We demonstrate that B. abortus infection induces STAT3 palmitoylation, which is critical for its membrane recruitment. Among zinc finger DHHC-type palmitoyl acyltransferases (ZDHHC) family members, ZDHHC3 specifically mediates STAT3 palmitoylation in infected macrophages. ZDHHC3-induced STAT3 palmitoylation promotes STAT3 phosphorylation at Y705, independently of IL-6. Functionally, ZDHHC3 suppresses pro-inflammatory cytokines (IL-1ß, TNF-α) and nitric oxide (NO) production, while increasing anti-inflammatory IL-10, thereby enhancing intracellular B. abortus survival. In vivo, ZDHHC3 mRNA is upregulated in splenic macrophages during infection, and 2-bromopalmitate (2BP) treatment reduces bacterial burden in mice, associated with elevated TNF-α and IFN-γ. Additionally, ZDHHC3 inhibits macrophage apoptosis (via regulating Bax and Bcl-2), limiting bacterial egress from apoptotic cells. These findings identify ZDHHC3-mediated STAT3 palmitoylation as one of the key regulatory mechanism in B. abortus infection, linking lipid modification to STAT3 activation, inflammation, apoptosis, and bacterial persistence.
Asunto(s)
Aciltransferasas , Brucella abortus , Brucelosis , Factor de Transcripción STAT3 , Animales , Brucella abortus/fisiología , Brucelosis/microbiología , Brucelosis/inmunología , Brucelosis/veterinaria , Aciltransferasas/metabolismo , Aciltransferasas/genética , Ratones , Macrófagos/microbiología , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Lipoilación , Femenino , Ratones Endogámicos C57BLRESUMEN
Leptospira interrogans is the major causative agent of leptospirosis. Humans, canines and agricultural animals are susceptible to Leptospira species and can develop fulminant disease. Rodents serve as reservoir hosts in which the bacteria colonize the renal tubules and are excreted in the urine. The host immune response to Leptospira spp. remains poorly defined. We show that L. interrogans induces a robust type I interferon (IFN) response in human and murine macrophages that is dependent on the cytosolic dsDNA sensor Cyclic GMP-AMP Synthase (cGAS) and the Stimulator of IFN Genes (STING) signaling pathway. Further, we show that mice deficient in the IFNα/ß receptor subunit 1 (IFNAR1) or STING had higher bacterial burdens and increased renal colonization following infection in vivo suggesting that cGAS-STING-driven type I IFN is required for the host defense against L. interrogans. These findings demonstrate the significance of cGAS-STING- dependent type I IFN signaling in mammalian innate immune responses to L. interrogans.
Asunto(s)
Interferón Tipo I , Riñón , Leptospira interrogans , Leptospirosis , Proteínas de la Membrana , Nucleotidiltransferasas , Animales , Nucleotidiltransferasas/inmunología , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Leptospira interrogans/inmunología , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Leptospirosis/inmunología , Leptospirosis/microbiología , Leptospirosis/metabolismo , Ratones , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones Noqueados , Humanos , Riñón/microbiología , Riñón/inmunología , Transducción de Señal/inmunología , Ratones Endogámicos C57BL , Inmunidad Innata , Macrófagos/inmunología , Macrófagos/microbiología , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/inmunologíaRESUMEN
Non-tuberculous mycobacteria (NTM) infections are difficult to cure completely with current treatments, and no specific drugs are available. However, recent reports have indicated that immune checkpoint inhibitors may effectively treat pulmonary NTM infections. In this study, we investigated the expression of immune checkpoint molecules in macrophages, the host cells of NTM, and assessed their impact on the microenvironment of infected lesions. Bulk-RNA sequencing and western blot analyses revealed that macrophages infected with Mycobacterium avium, an NTM species, exhibited a pro-inflammatory phenotype and increased PD-L1 expression. Additionally, immunostaining of an NTM-infected mouse model and human tissues showed that increased PD-L1 expression in macrophages was associated with decreased T cell infiltration and increased T cell exhaustion (upregulated PD-1 expression) within infected lesions. These findings suggest that NTM infections evade cellular immunity by enhancing PD-L1 expression in macrophages. Therefore, PD-L1 inhibition may be a promising therapeutic strategy against NTM infections.
Asunto(s)
Antígeno B7-H1 , Evasión Inmune , Macrófagos , Mycobacterium avium , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Animales , Macrófagos/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Humanos , Ratones , Mycobacterium avium/inmunología , Ratones Endogámicos C57BL , Infecciones por Mycobacterium no Tuberculosas/inmunologíaRESUMEN
The indiscriminate use of antibiotics has led to an increase in multidrug-resistant bacteria, necessitating the search for effective alternative therapies to reduce antimicrobial use. Lactic Acid Bacteria (LAB) have been explored as an alternative to antibiotics due to their multiple beneficial properties. These bacteria secrete membrane vesicles (MVs), key acellular components in combating pathogens. This study aimed to evaluate the effects of Lactiplantibacillus plantarum MVs co-cultured with Escherichia coli (MVsplE) or Salmonella Typhimurium (MVsplS) through inhibition assays using disk diffusion on agar plates, as well as their activation and cytokine expression in the RAW 264.7 macrophage cell line. The results showed that MVsplE and MVsplS were produced in greater quantity and size than non-co-cultured L. plantarum MVs (MVspl). Additionally, MVsplE and MVsplS exhibited a dose-dependent inhibitory effect on the growth of enteropathogenic bacteria. Furthermore, RAW 264.7 cells stimulated with these MVs demonstrated that the expression of IL-1ß, TNF-α and IL-10 depended on the enteropathogenic strain with which L. plantarum was previously co-cultured. Following a challenge with enteropathogenic bacteria, the MVs induced an immunomodulatory response. These findings demonstrate that L. plantarum MVs exert bactericidal and immunomodulatory effects against enteropathogenic bacteria, suggesting their potential use as an alternative treatment to antimicrobials.
Asunto(s)
Antibacterianos , Escherichia coli , Factores Inmunológicos , Lactiplantibacillus plantarum , Salmonella typhimurium , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/inmunología , Salmonella typhimurium/crecimiento & desarrollo , Animales , Ratones , Escherichia coli/efectos de los fármacos , Escherichia coli/inmunología , Escherichia coli/crecimiento & desarrollo , Células RAW 264.7 , Antibacterianos/farmacología , Citocinas/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Técnicas de Cocultivo , Factores Inmunológicos/farmacologíaRESUMEN
Tuberculosis remains the leading cause of death from infectious diseases. While effective therapies are available, their prolonged duration and severe adverse effects compromise patient adherence and promote antimicrobial resistance, highlighting the urgent need for innovative therapeutic approaches. We previously identified polymeric ß-cyclodextrin (pßCD) as a promising drug delivery platform, possessing intrinsic antibacterial activity and serving as a carrier for the second-line drug ethionamide. Here, we investigated the biological properties of pßCD of different molecular weights. Strikingly, only the low-molecular-weight pßCD (LMW-pßCD) displayed antibacterial activity, in contrast to the high-molecular-weight one. Mechanistically, this activity was linked to the inhibition of Mycobacterium tuberculosis entry into macrophages through disruption of lipid rafts. Transcriptomic profiling further revealed that the LMW-pßCD also enhanced pro-inflammatory cytokine secretion, suggesting dual antimicrobial and immunomodulatory functions. Notably, linezolid, an important second-line anti-tuberculosis drug, was efficiently incorporated in pßCD, regardless of its molecular weight. These findings emphasize the critical importance of molecular weight in dictating pßCD bioactivity. Only LMW-pßCD combines antimicrobial and immunomodulatory activities with effective drug delivery, making it a promising candidate for the development of novel anti-tuberculosis therapies.
Asunto(s)
Antituberculosos , Mycobacterium tuberculosis , Tuberculosis , beta-Ciclodextrinas , Mycobacterium tuberculosis/efectos de los fármacos , beta-Ciclodextrinas/química , beta-Ciclodextrinas/farmacología , beta-Ciclodextrinas/administración & dosificación , Peso Molecular , Antituberculosos/farmacología , Antituberculosos/administración & dosificación , Antituberculosos/química , Animales , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Humanos , Ratones , Tuberculosis/tratamiento farmacológico , Citocinas/metabolismo , Linezolid/farmacología , Linezolid/administración & dosificación , Linezolid/química , Portadores de Fármacos/químicaRESUMEN
Nontuberculous mycobacteria (NTM) infections, especially those caused by Mycobacterium abscessus, are extremely difficult to treat due to the organisms' intrinsic resistance to many antimicrobials. Myxin is a naturally occurring phenazine-5,10-dioxide antibiotic from Sorangium sp., notable for its broad-spectrum antimicrobial activity. In this study, we determined the susceptibilities of Myxin against 11 NTM reference strains and 194 clinical M. abscessus isolates using microdilution assays. Additionally, time-kill kinetic experiments were performed to distinguish the clearing efficiency of Myxin in broth. We also evaluated the efficacy of Myxin against intracellular M. abscessus in THP-1 macrophages. Checkerboard assays assessed the interactions between Myxin and eight clinically important anti-NTM antibiotics. Myxin demonstrated potent activity against M. abscessus with low MIC values (MIC50 = 0.125 mg/L and MIC90 = 0.5 mg/L). Treatment with 20 × MIC of Myxin in 7H9 broth resulted in up to 10 log10 CFU/mL reduction after 4 days of incubation. An intracellular concentration-dependent bactericidal effect of Myxin was observed in THP-1 macrophages. Myxin exhibited no antagonism in combination with standard anti-NTM drugs. As such, Myxin represents a promising candidate for the treatment of M. abscessus and other NTM infections.
Asunto(s)
Antibacterianos , Mycobacterium abscessus , Fenazinas , Mycobacterium abscessus/efectos de los fármacos , Mycobacterium abscessus/aislamiento & purificación , Humanos , Antibacterianos/farmacología , Pruebas de Sensibilidad Microbiana , Células THP-1 , Macrófagos/microbiología , Macrófagos/efectos de los fármacos , Fenazinas/farmacología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológicoRESUMEN
Canine brucellosis is a common zoonosis worldwide, but treatment is challenging due to the stealthy and chronic infection caused by the bacterium Brucella (B.) canis. Antimicrobial peptides (AMPs) are promising candidates for combating drug-resistant microbes, and gold nanoparticle aptamer (AuNP-Apt) aids the delivery of peptides to mammalian cells. This study evaluated the therapeutic efficacy of AuNP-AptHis conjugated antimicrobial peptide RW-BP100His (AuNP-AptHis-RW-BP100His) against B. canis in murine macrophage RAW 264.7 cells and BALB/c mice. In vitro, a high concentration of RW-BP100His alone had a bactericidal effect. AuNP-AptHis-RW-BP100His inhibited intracellular bacterial replication and decreased nitric oxide (NO) production in RAW 264.7 cells infected with B. canis. The production of proinflammatory cytokines in RAW 264.7 cells was elevated after AuNP-AptHis-RW-BP100His treatment. In vivo, AuNP-AptHis-RW-BP100His treatment reduced bacterial burden and microgranulomas in the liver and spleen. CD4+, CD8+ T cells, and Th1 cytokines IFN-γ and TNF were elevated in the treated mice. The results indicate that the therapeutic mechanism of AuNP-AptHis-RW-BP100His is immunomodulation of the infected host to upregulate cell-mediated immunity for B. canis clearance. Therefore, it is a promising candidate to treat canine brucellosis as an alternative to conventional antibiotics.
Asunto(s)
Antibacterianos , Péptidos Catiónicos Antimicrobianos , Péptidos Antimicrobianos , Aptámeros de Nucleótidos , Brucella canis , Brucelosis , Oro , Nanopartículas del Metal , Animales , Ratones , Oro/química , Nanopartículas del Metal/química , Células RAW 264.7 , Ratones Endogámicos BALB C , Brucelosis/tratamiento farmacológico , Brucelosis/microbiología , Brucelosis/veterinaria , Citocinas/metabolismo , Brucella canis/efectos de los fármacos , Óxido Nítrico/metabolismo , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/química , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Aptámeros de Nucleótidos/química , Perros , Bazo/microbiología , Bazo/patología , Antibacterianos/farmacología , Modelos Animales de Enfermedad , Hígado/microbiología , Hígado/patología , Péptidos Catiónicos Antimicrobianos/farmacología , FemeninoRESUMEN
OBJECTIVES: Delafloxacin is a fourth-generation fluoroquinolone that is currently approved in Europe and the United States for bacterial infections of the skin and skin structures and for community-acquired pneumonia, including cases where it is caused by atypical pathogens. It has demonstrated superior efficacy against a broad spectrum of bacteria and fewer adverse effects compared with other quinolones, such as levofloxacin, a current first-line treatment against Legionnaires' disease. This study aimed to characterize for the first time the intracellular activity of delafloxacin compared with levofloxacin against various serogroups of Legionella pneumophila and species of Legionella in terms of culturability and viability. METHODS: The infectious capacity of 10 Legionella strains was evaluated individually to ensure a percentage of viable infected macrophage post-infection. Then, the extracellular concentration inhibiting intracellular multiplication (MIEC90) of the antibiotic was determined by gradient antibiotic incubations in the in vitro infection model through culturability assays. Finally, the viability of intracellular Legionella was studied by quantitative polymerase chain reaction after antibiotic treatment at MIEC90 value. RESULTS: Delafloxacin decreased the culturability of Legionella from five to 10 times more than levofloxacin. The MIEC90 obtained was 0.025 mg/l for levofloxacin and 0.005 mg/l for delafloxacin for all strains tested, except L. longbeachae; the same concentration for both antibiotics was required. The bactericidal effect of both antibiotics was confirmed by viability quantitative polymerase chain reaction. CONCLUSIONS: Based on the results obtained, the higher intracellular activity of delafloxacin compared with levofloxacin and its better safety profile should be confirmed in phase IV clinical real-life cases of Legionnaires' disease.
Asunto(s)
Antibacterianos , Fluoroquinolonas , Legionella pneumophila , Legionella , Enfermedad de los Legionarios , Fluoroquinolonas/farmacología , Antibacterianos/farmacología , Humanos , Levofloxacino/farmacología , Enfermedad de los Legionarios/tratamiento farmacológico , Enfermedad de los Legionarios/microbiología , Legionella/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Legionella pneumophila/efectos de los fármacos , Viabilidad Microbiana/efectos de los fármacos , Macrófagos/microbiologíaRESUMEN
Tuberculosis (TB) is an infectious disease caused by infection with Mycobacterium tuberculosis (MTB). The morbidity of TB in the Xinjiang region of China is higher than that in other provinces. Macrophage apoptosis after infection with MTB is considered to serve a key role in killing the bacteria. However, the biological process of apoptosis and the underlying molecular mechanisms triggered by the infection of macrophages with clinical isolates of MTB from Xinjiang (XJMTB) are not clear. The present study aimed to investigate the unique characteristics of XJMTB. Briefly, western blotting and flow cytometry were employed in the present study, and it was demonstrated that macrophages infected with MTB H37Rv or XJMTB underwent G2/M cell cycle arrest and apoptosis. The transcriptome sequencing analysis showed that cyclindependent kinase 1 (CDK1) was a key regulatory gene in regulating the G2/M cell cycle arrest and apoptosis in MTBinfected macrophages, and the p53 gene was most likely involved in the regulation of this. Moreover, the phosphorylation of p53 (Ser315) was elevated with the upregulation of CDK1 activation, leading to a higher proportion of MTBinfected macrophages exhibiting G2/M cell cycle block and apoptosis. The current study also revealed that enhanced activation of CDK1 reversed the attenuation of the G2/M cell cycle block and the reduction in the percentage of apoptosis caused by inhibition of p53 (Ser315) phosphorylation. Furthermore, the coimmunoprecipitation experiment demonstrated an interaction between CDK1 and p53. The present study indicated that, in an in vitro model of macrophage infection with XJMTB, enhanced activation of CDK1 may regulate the phosphorylation of p53 (Ser315), promote the secretion of TNFα, IL6, IL10, IL1ß and IL12, promote G2/M cell cycle arrest and apoptosis of macrophages, and enhance the survival of XJMTB in macrophages. These results provide CDK1 and phosphorylatedp53 as two new potential therapeutic targets for TB in Xinjiang, and lay a foundation for the development of novel TB treatment strategies.
Asunto(s)
Apoptosis , Proteína Quinasa CDC2 , Puntos de Control del Ciclo Celular , Macrófagos , Mycobacterium tuberculosis , Tuberculosis , Proteína p53 Supresora de Tumor , Macrófagos/microbiología , Macrófagos/metabolismo , Macrófagos/patología , Mycobacterium tuberculosis/aislamiento & purificación , Fosforilación , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína Quinasa CDC2/metabolismo , Humanos , Tuberculosis/microbiología , Tuberculosis/metabolismo , Ratones , AnimalesRESUMEN
Autophagy plays a critical role in clearance of Mycobacterium tuberculosis. It has emerged as a promising target for host-directed therapies against drug-resistant tuberculosis (TB). This insight opens up promising therapeutic avenues, suggesting that pharmacological activation of autophagy could effectively combat this highly persistent and harmful bacterium. The current study investigates the anti-mycobacterial properties of the anthracene-dione compound Mitoxantrone (MTX) through the activation of autophagy in differentiated THP-1 cells. The non-cytotoxic dose of MTX reduced the intracellular viability of mycobacteria compared to the control cells, and inhibition of autophagy reversed the effect of MTX on intracellular bacterial burden. Through multiparametric approaches, our investigation established the effect of MTX on mitochondria, the principal source of endogenous reactive oxygen species (ROS), acting as essential signal transducers that promote autophagy. Further, we have demonstrated that MTX decreased ATP production, which caused disruption of mitochondrial membrane proteins and increased mitochondrial ROS generation, resulting in mitochondrial fission and accelerating the initiation of mitophagy, leading to the elimination of intracellular mycobacteria. Our findings collectively demonstrated that MTX-induced mitochondrial dysfunction triggered interplay between two selective autophagic responses, diminishing mycobacterial infection and promoting its clearance. This study highlights MTX as a potential host-directed therapeutic candidate against TB through modulation of mitochondrial signaling pathways and autophagic responses.
Asunto(s)
Macrófagos , Viabilidad Microbiana , Mitocondrias , Mitofagia , Mitoxantrona , Mycobacterium tuberculosis , Especies Reactivas de Oxígeno , Humanos , Especies Reactivas de Oxígeno/metabolismo , Mitoxantrona/farmacología , Mitofagia/efectos de los fármacos , Mitofagia/fisiología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Macrófagos/metabolismo , Células THP-1 , Mycobacterium tuberculosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Autofagia/efectos de los fármacos , Viabilidad Microbiana/efectos de los fármacos , Relación Dosis-Respuesta a DrogaRESUMEN
The difficulty of accurately identifying Candidozyma auris (formerly Candida auris) and the high resistance rates presented have increased the concern in the healthcare setting. Due to this, the aim of this study was to analyse the fungal response to oxidative stress. To achieve this goal, gene and protein expression were examined using qPCR and two-dimensional electrophoresis, respectively, peroxiredoxin Tsa1b being found to be upregulated under oxidative stress. Subsequently, the significance of Tsa1b was next investigated by characterizing the C. auris Δtsa1b and C. auris Δtsa1b::TSA1B strains generated by CRISPR-Cas9. The findings demonstrated that the Δtsa1b strain was more susceptible to oxidative and cell wall stressors than the wild-type strain, which was consistent with an increase in the cell wall ß-glucan amounts when grown in the presence of oxidative stress. Importantly, Tsa1b has also been detected as an antigen by patient sera. Furthermore, the Δtsa1b strain was also more vulnerable to the presence of dendritic cells and bone marrow-derived macrophages. Finally, in vivo infections performed in Galleria mellonella and mice showed a slower progression of the disease in animals infected with the mutant strain. In conclusion, the peroxiredoxin Tsa1b has been identified as a potential candidate to be studied as a virulence factor implicated in the oxidative stress response of C. auris. These findings advance the knowledge of the pathobiology of C. auris and point out the potential of Tsa1b for the development of new diagnostic and therapeutic approaches.
Asunto(s)
Candida auris , Proteínas Fúngicas , Estrés Oxidativo , Peroxirredoxinas , Animales , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Ratones , Virulencia , Candida auris/patogenicidad , Candida auris/genética , Candida auris/enzimología , Candida auris/metabolismo , Humanos , Pared Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Macrófagos/microbiología , Mariposas Nocturnas/microbiología , Regulación Fúngica de la Expresión Génica , Modelos Animales de EnfermedadRESUMEN
The limitations of the current standard therapy for pulmonary diseases caused by Mycobacterium avium complex (MAC) underscore the urgent need for novel therapeutic agents. Among potential candidates, thiopeptide antibiotics have attracted attention due to their antibacterial activity but their poor solubility has limited clinical application. In this study, we developed AJ-099, a thiopeptide derivative with improved solubility and enhanced antibacterial potency. The in vitro antibacterial activity of AJ-099 was confirmed across macrolide-susceptible and- resistant clinical isolates, with minimum inhibitory concentration values consistently falling within the range of ≤0.125-0.5 µg/mL. In addition, AJ-099 exhibited a marked growth inhibition against both macrolide-susceptible and -resistant MAC clinical isolates in macrophages. Importantly, AJ-099 displayed synergistic effects in combination with clarithromycin (CLR), a macrolide drug, which resulted in significantly greater reductions in intracellular MAC burden compared to either agent alone. The synergistic effect was consistently observed in vivo, where the AJ-099 and CLR combination achieved superior bacterial clearance and reduced lung inflammation to the current standard therapy consisting of CLR, ethambutol, and rifampicin. Collectively, these results highlight that AJ-099 in combination with macrolides is a promising candidate for treating MAC pulmonary infections. Moreover, its potent activity against macrolide-resistant MAC strains suggests it may offer an effective therapeutic option for refractory MAC pulmonary infections.
Asunto(s)
Antibacterianos , Macrólidos , Complejo Mycobacterium avium , Infección por Mycobacterium avium-intracellulare , Sinergismo Farmacológico , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/uso terapéutico , Animales , Complejo Mycobacterium avium/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Macrólidos/farmacología , Macrólidos/uso terapéutico , Ratones , Infección por Mycobacterium avium-intracellulare/tratamiento farmacológico , Infección por Mycobacterium avium-intracellulare/microbiología , Humanos , Claritromicina/farmacología , Macrófagos/microbiología , Macrófagos/efectos de los fármacos , Femenino , Farmacorresistencia BacterianaRESUMEN
Coxiella burnetii is an intracellular bacterial pathogen that causes the disease Q fever. Both secretion of effector proteins by a type IVB secretion system and expression of lipopolysaccharide (LPS) are critical for C. burnetii virulence. The C. burnetii Nine Mile strain lineage is routinely used in laboratories, with Nine Mile I RSA493 (fully virulent) expressing phase I LPS, Nine Mile Crazy RSA514 (lightly attenuated) expressing intermediate LPS, and NMII RSA439 clone 4 (NMII, attenuated) expressing phase II LPS. NMII was exempted from the U.S. Centers for Disease Control and Prevention Federal Select Agent Program due to a genetic deletion predicted to prevent phase I LPS synthesis; however, recent findings demonstrated that NMII LPS elongation occurs in vivo and in vitro. These findings raise concerns regarding the suitability of NMII for manipulation at biosafety level-2 (BSL-2) containment. Here, we evaluated a mutant strain lacking the gene responsible for LPS elongation (C. burnetii NMII Δcbu0533) as an alternative strain for BSL-2 containment studies. Growth of wild-type NMII and NMII Δcbu0533 in axenic media and macrophages was evaluated. Neither growth kinetics nor replication vacuole morphology significantly differed between strains. NMII Δcbu0533 also prevented staurosporine-induced cell death, indicating that the mutant strain maintains the capacity to alter cell signaling events similar to wild-type C. burnetii. Cumulatively, these data reveal that C. burnetii NMII Δcbu0533 is a suitable alternative to wild-type NMII in BSL-2 laboratory experiments that can be distributed to the field for use.IMPORTANCEAttenuated C. burnetii NMII RSA439 clone 4 (NMII) expresses truncated lipopolysaccharide (LPS) and was long predicted to be unable to produce elongated LPS due to a large chromosomal deletion. As a result, this strain was exempted from the CDC Federal Select Agent Program's regulation and is commonly used under biosafety level-2 laboratory conditions. Recently, it was shown that C. burnetii NMII LPS can elongate due to a mutation reversion in cbu0533, resulting in increased virulence in guinea pigs. As a result, the previously exempted NMII strain is subject to regulation if the cbu0533 mutation reversion is present, posing concerns regarding laboratory use. Experiments described here provide a suitable alternative strain to address these concerns.
Asunto(s)
Coxiella burnetii , Coxiella burnetii/genética , Coxiella burnetii/patogenicidad , Coxiella burnetii/metabolismo , Coxiella burnetii/crecimiento & desarrollo , Animales , Lipopolisacáridos/metabolismo , Lipopolisacáridos/genética , Fiebre Q/microbiología , Ratones , Virulencia , Macrófagos/microbiología , Contención de Riesgos Biológicos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , HumanosRESUMEN
Aspergillus fumigatus, a major cause of invasive aspergillosis, relies on oxidoreductases for stress adaptation. The role of the oxidoreductase gene fabG in fungal physiology and virulence remains unclear. This study aims to investigate the function of fabG in regulating A. fumigatus growth, virulence, redox homeostasis, and cell wall integrity (CWI). fabG knockout (ΔfabG) and complemented strains were constructed via homologous recombination. Phenotypic assays evaluated hyphal growth, virulence in Galleria mellonella, and antifungal susceptibility. Transcriptomic profiling, cell wall composition analysis (chitin/ß-glucan), biochemical assays (superoxide dismutase [SOD] activity, reactive oxygen species [ROS] levels, and stress tolerance), and macrophage co-culture experiments (phagocytosis/killing) were performed. CWI-related gene expression was assessed under caspofungin/rapamycin treatment. ΔfabG showed inhibited hyphal growth, reduced virulence, and decreased caspofungin sensitivity. Transcriptomics revealed altered oxidoreductase and mitogen-activated protein kinase signaling genes. Cell wall thickening (increased chitin) and enhanced tolerance to cell wall stressors indicated CWI activation. ΔfabG exhibited decreased SOD activity, elevated ROS, reduced stress tolerance, and increased susceptibility to macrophage killing. CWI-related gene expression was unchanged under caspofungin/rapamycin. fabG critically regulates A. fumigatus growth, environmental adaptation, and virulence by modulating CWI and redox homeostasis, highlighting its potential as an antifungal target.IMPORTANCEAspergillus fumigatus is a harmful fungus that can cause severe and often deadly infections in people with weakened immune systems. Understanding how this fungus adapts to stress and causes disease is crucial for developing new treatments. Our study reveals that the gene fabG plays a vital role in regulating the growth, stress response, and virulence of A. fumigatus. When FabG is missing, the fungus becomes less able to grow, resists environmental stresses poorly, and is less harmful to infected hosts. This gene controls the fungus's ability to maintain internal balance during oxidative stress and strengthens its cell wall, a protective structure. Since FabG is critical for the fungus's survival and virulence, it could be a promising target for new antifungal drugs. By blocking fabG, we might be able to develop more effective treatments against this dangerous pathogen.
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Aspergillus fumigatus , Pared Celular , Proteínas Fúngicas , Oxidorreductasas , Pared Celular/metabolismo , Pared Celular/genética , Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidad , Aspergillus fumigatus/crecimiento & desarrollo , Aspergillus fumigatus/enzimología , Aspergillus fumigatus/efectos de los fármacos , Virulencia/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Animales , Antifúngicos/farmacología , Aspergilosis/microbiología , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Regulación Fúngica de la Expresión Génica , Especies Reactivas de Oxígeno/metabolismo , Hifa/crecimiento & desarrollo , Hifa/genética , Mariposas Nocturnas/microbiología , Macrófagos/microbiología , Quitina/metabolismo , Caspofungina/farmacología , Perfilación de la Expresión GénicaRESUMEN
Prostate inflammation and fibrosis are linked to lower urinary tract symptoms (LUTS) in men. Uropathogenic Escherichia coli (E. coli) infection of the mouse prostate triggers a cascade of immune responses that drive inflammation and fibrosis. A recent study found that lysosome 2-positive (LYZ2+) myeloid cells (fibrocytes) are recruited in a C-C motif chemokine receptor 2 (Ccr2)-dependent manner to the E. coli-infected prostate, where they produce collagen. This study aims to identify factors that drive collagen synthesis in LYZ2+ myeloid cells during E. coli infection. We show that lymphocyte antigen 6 family member (Ly6C)hi monocytes and their maturation products, Ly6Chi macrophages, infiltrate the E. coli infected prostate in a Ccr2-dependent manner, that monocytes in the infected prostate produce Tgfb1 RNA, and that E. coli infection activates TGFB signaling and collagen synthesis in LYZ2+ cells. Blockade of macrophage colony-stimulating factor (M-CSF), a factor required for monocyte differentiation into macrophages, reduces macrophage density, TGFB signaling in LYZ2+ cells, and collagen density in the E. coli-infected prostate. These findings highlight a critical role of macrophages in activating collagen synthesis in fibrocytes to drive a fibrotic response to E. coli infection in the mouse prostate.NEW & NOTEWORTHY Prostatic/urethral fibrosis is an emerging cause of urinary voiding dysfunction in aging men and has been linked to prostate inflammation, but cellular mediators and mechanisms of this process are incompletely understood. We provide evidence that Ly6Chi monocytes and LYZ2+ myeloid cells are recruited to the E. coli infected mouse prostate. Macrophages in the infected prostate synthesize TGFB1 and stimulate collagen synthesis in LYZ2+ cells.
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Infecciones por Escherichia coli , Macrófagos , Próstata , Prostatitis , Factor de Crecimiento Transformador beta1 , Escherichia coli Uropatógena , Animales , Masculino , Macrófagos/metabolismo , Macrófagos/microbiología , Macrófagos/inmunología , Fibrosis , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/patología , Infecciones por Escherichia coli/microbiología , Transducción de Señal , Próstata/patología , Próstata/microbiología , Próstata/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Ratones Endogámicos C57BL , Receptores CCR2/metabolismo , Receptores CCR2/genética , Colágeno/metabolismo , Colágeno/biosíntesis , Modelos Animales de Enfermedad , Escherichia coli Uropatógena/patogenicidad , Ratones , Factor Estimulante de Colonias de Macrófagos/metabolismo , Monocitos/metabolismo , Antígenos Ly/metabolismo , Prostatitis/metabolismo , Prostatitis/patología , Prostatitis/microbiologíaRESUMEN
The Gram-negative facultative intracellular pathogen Piscirickettsia salmonis infects salmonid fish species and causes economic losses in the Chilean salmon farming industry. Until now, the virulence factors and the mechanisms involved in establishing the fish infection are scarcely understood. P. salmonis can infect and survive in salmonid macrophages, and reports have shown that the Icm/Dot type-IV secretion system is part of the virulence factors that are essential for bacterial infection and replication inside bacteria-induced vacuoles. Since the Icm/Dot substrate SdhA has been identified as a key protein factor in maintaining the integrity of the Legionella-induced vacuolar membrane, we seek to explore the possibility that this substrate can also play a role in P. salmonis infection in fish. In this study we first confirmed the presence of the orthologous gene encoding SdhA of P. salmonis, demonstrating an evolutionary relationship with the sdhA genes of Coxiella burnetii and Legionella pneumophila. The in-silico analysis of the protein revealed a conserved structure and short linear motifs that suggest several potential functions related to vacuolar stability, autophagy, apoptosis, and vesicular transport processes that probably enable the pathogen to manipulate the defense mechanisms of the host cell. Additionally, for the first time, we demonstrated the early expression of sdhA at both the transcript and protein levels in infected salmon cells, suggesting that SdhA could interfere with the host pathogen eradication mechanisms shortly after infection. These results set up the basis to further studies on the functional role of SdhA in the host-pathogen interaction mechanisms associated with P. salmonis infection of salmonid fish.
Asunto(s)
Proteínas Bacterianas , Enfermedades de los Peces , Piscirickettsia , Infecciones por Piscirickettsiaceae , Salmo salar , Animales , Piscirickettsia/genética , Piscirickettsia/fisiología , Infecciones por Piscirickettsiaceae/veterinaria , Infecciones por Piscirickettsiaceae/inmunología , Infecciones por Piscirickettsiaceae/microbiología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Macrófagos/microbiología , Macrófagos/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Filogenia , Alineación de Secuencia/veterinaria , Secuencia de Aminoácidos , CinéticaRESUMEN
Listeria monocytogenes infection, which has a higher mortality rate than other foodborne pathogens, is a major concern in global food safety. Among various interventions, lactic acid bacteria and their metabolites have gained significant attention. Our previous study showed that the surface layer proteins (SLPs) of Enterococcus faecium WEFA23, isolated from the gastrointestinal tract of newborn infants, exhibited strong inhibitory activity against L. monocytogenes CMCC57007; however, the underlying mechanism remained unclear. In this study, the immunomodulatory effects of E. faecium WEFA23 SLPs against L. monocytogenes infection were investigated in RAW 264.7 macrophage cells, focusing on phagocytic and bactericidal activity, as well as cytokine production. Furthermore, LC-MS/MS analysis indicated that ornithine carbamoyltransferase (OTC) could be a functional component of the SLPs. Consequently, otc gene knockout and heterologous expression strains were constructed. The results showed that deletion of the otc gene eliminated the inhibitory activity, while recombinant OTC maintained a satisfactory inhibitory effect. Mechanistically, both SLPs and purified OTC suppressed the activation of the NF-κB/MAPK signalling pathways, which were likely mediated through TLR2. Overall, our findings provide a scientific basis for the application of SLPs and E. faecium in food systems to prevent pathogenic infections.