RESUMEN
Elevated infant fecal concentrations of the bacterial-derived lipid 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME) increase the risk for childhood atopy and asthma. However, the mechanisms by which this lipid contributes to disease development are largely unknown. We hypothesized that macrophages, which are key to both antimicrobial and antigen responses, are functionally and epigenetically modified by 12,13-diHOME leading to short- and long-term dysfunction with consequences for both antimicrobial and antigenic responses. Macrophages exposed to 12,13-diHOME are skewed toward inflammatory IL-1ß highCD206low cells, a phenomenon that is further amplified in the presence of common microbial-, aero-, and food-allergens. These IL-1ß highCD206low macrophages also exhibit reduced bacterial phagocytic capacity. In primary immune cell coculture assays involving peanut allergen stimulation, 12,13-diHOME promotes both IL-1ß and IL-6 production, memory B cell expansion, and increased IgE production. Exposure to 12,13-diHOME also induces macrophage chromatin remodeling, specifically diminishing access to interferon-stimulated response elements resulting in reduced interferon-regulated gene expression upon bacterial lipopolysaccharide stimulation. Thus 12,13-diHOME reprograms macrophage effector function, B-cell interactions and promotes epigenetic modifications that exacerbate inflammatory response to allergens and mutes antimicrobial response along the interferon axis. These observations offer plausible mechanisms by which this lipid promotes early-life pathogenic microbiome development and innate immune dysfunction associated with childhood allergic sensitization.
Asunto(s)
Alérgenos , Epigénesis Genética , Macrófagos , Macrófagos/inmunología , Macrófagos/metabolismo , Alérgenos/inmunología , Humanos , Interleucina-1beta/metabolismo , Células Cultivadas , Linfocitos B/inmunología , Linfocitos B/metabolismo , Inflamación/inmunología , Inmunoglobulina E/inmunología , AnimalesRESUMEN
The fungal pathogen Candida auris represents a severe threat to hospitalized patients. Its resistance to multiple classes of antifungal drugs and ability to spread and resist decontamination in healthcare settings make it especially dangerous. We screened 1,990 clinically approved and late-stage investigational compounds for the potential to be repurposed as antifungal drugs targeting C. auris and narrowed our focus to five Food and Drug Administration (FDA)-approved compounds with inhibitory concentrations under 10 µM for C. auris and significantly lower toxicity to three human cell lines. These compounds, some of which had been previously identified in independent screens, include three dihalogenated 8-hydroxyquinolines: broxyquinoline, chloroxine, and clioquinol. A subsequent structure-activity study of 32 quinoline derivatives found that 8-hydroxyquinolines, especially those dihalogenated at the C5 and C7 positions, were the most effective inhibitors of C. auris. To pursue these compounds further, we exposed C. auris to clioquinol in an extended experimental evolution study and found that C. auris developed only twofold to fivefold resistance to the compound. DNA sequencing of resistant strains and subsequent verification by directed mutation in naive strains revealed that resistance was due to mutations in the transcriptional regulator CAP1 (causing upregulation of the drug transporter MDR1) and in the drug transporter CDR1. These mutations had only modest effects on resistance to traditional antifungal agents, and the CDR1 mutation rendered C. auris more susceptible to posaconazole. This observation raises the possibility that a combination treatment involving an 8-hydroxyquinoline and posaconazole might prevent C. auris from developing resistance to this established antifungal agent. IMPORTANCE The rapidly emerging fungal pathogen Candida auris represents a growing threat to hospitalized patients, in part due to frequent resistance to multiple classes of antifungal drugs. We identify a class of compounds, the dihalogenated 8-hydroxyquinolines, with broad fungistatic ability against a diverse collection of 13 strains of C. auris. Although this compound has been identified in previous screens, we extended the analysis by showing that C. auris developed only modest twofold to fivefold increases in resistance to this class of compounds despite long-term exposure; a noticeable difference from the 30- to 500-fold increases in resistance reported for similar studies with commonly used antifungal drugs. We also identify the mutations underlying the resistance. These results suggest that the dihalogenated 8-hydroxyquinolines are working inside the fungal cell and should be developed further to combat C. auris and other fungal pathogens. Lohse and colleagues characterize a class of compounds that inhibit the fungal pathogen C. auris. Unlike many other antifungal drugs, C. auris does not readily develop resistance to this class of compounds.
Asunto(s)
Antifúngicos , Clioquinol , Humanos , Antifúngicos/metabolismo , Candida auris , Candida , Clioquinol/farmacología , Clioquinol/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Farmacorresistencia Fúngica/genéticaRESUMEN
The fungal pathogen Candida auris represents a severe threat to hospitalized patients. Its resistance to multiple classes of antifungal drugs and ability to spread and resist decontamination in health-care settings make it especially dangerous. We screened 1,990 clinically approved and late-stage investigational compounds for the potential to be repurposed as antifungal drugs targeting C. auris and narrowed our focus to five FDA-approved compounds with inhibitory concentrations under 10 µM for C. auris and significantly lower toxicity to three human cell lines. These compounds, some of which had been previously identified in independent screens, include three dihalogenated 8-hydroxyquinolines: broxyquinoline, chloroxine, and clioquinol. A subsequent structure-activity study of 32 quinoline derivatives found that 8-hydroxyquinolines, especially those dihalogenated at the C5 and C7 positions, were the most effective inhibitors of C. auris . To pursue these compounds further, we exposed C. auris to clioquinol in an extended experimental evolution study and found that C. auris developed only 2- to 5-fold resistance to the compound. DNA sequencing of resistant strains and subsequent verification by directed mutation in naive strains revealed that resistance was due to mutations in the transcriptional regulator CAP1 (causing upregulation of the drug transporter MDR1 ) and in the drug transporter CDR1 . These mutations had only modest effects on resistance to traditional antifungal agents, and the CDR1 mutation rendered C. auris more sensitive to posaconazole. This observation raises the possibility that a combination treatment involving an 8-hydroxyquinoline and posaconazole might prevent C. auris from developing resistance to this established antifungal agent. Abstract Importance: The rapidly emerging fungal pathogen Candida auris represents a growing threat to hospitalized patients, in part due to frequent resistance to multiple classes of antifungal drugs. We identify a class of compounds, the dihalogenated hydroxyquinolines, with broad fungistatic ability against a diverse collection of 13 strains of C. auris . Although this compound has been identified in previous screens, we extended the analysis by showing that C. auris developed only modest 2- to 5-fold increases in resistance to this class of compounds despite long-term exposure; a noticeable difference from the 30- to 500- fold increases in resistance reported for similar studies with commonly used antifungal drugs. We also identify the mutations underlying the resistance. These results suggest that the dihalogenated hydroxyquinolines are working inside the fungal cell and should be developed further to combat C. auris and other fungal pathogens. Tweet: Lohse and colleagues characterize a class of compounds that inhibit the fungal pathogen C. auris . Unlike many other antifungal drugs, C. auris does not readily develop resistance to this class of compounds.
RESUMEN
Secondary bacterial infections, including ventilator-associated pneumonia (VAP), lead to worse clinical outcomes and increased mortality following viral respiratory infections including in patients with coronavirus disease 2019 (COVID-19). Using a combination of tracheal aspirate bulk and single-cell RNA sequencing (scRNA-seq) we assessed lower respiratory tract immune responses and microbiome dynamics in 28 COVID-19 patients, 15 of whom developed VAP, and eight critically ill uninfected controls. Two days before VAP onset we observed a transcriptional signature of bacterial infection. Two weeks prior to VAP onset, following intubation, we observed a striking impairment in immune signaling in COVID-19 patients who developed VAP. Longitudinal metatranscriptomic analysis revealed disruption of lung microbiome community composition in patients with VAP, providing a connection between dysregulated immune signaling and outgrowth of opportunistic pathogens. These findings suggest that COVID-19 patients who develop VAP have impaired antibacterial immune defense detectable weeks before secondary infection onset.
RESUMEN
Secondary bacterial infections, including ventilator-associated pneumonia (VAP), lead to worse clinical outcomes and increased mortality following viral respiratory infections including in patients with coronavirus disease 2019 (COVID-19). Using a combination of tracheal aspirate bulk and single-cell RNA sequencing we assessed lower respiratory tract immune responses and microbiome dynamics in 23 COVID-19 patients, 10 of whom developed VAP, and eight critically ill uninfected controls. At a median of three days (range: 2-4 days) before VAP onset we observed a transcriptional signature of bacterial infection. At a median of 15 days prior to VAP onset (range: 8-38 days), we observed a striking impairment in immune signaling in COVID-19 patients who developed VAP. Longitudinal metatranscriptomic analysis revealed disruption of lung microbiome community composition in patients with VAP, providing a connection between dysregulated immune signaling and outgrowth of opportunistic pathogens. These findings suggest that COVID-19 patients who develop VAP have impaired antibacterial immune defense detectable weeks before secondary infection onset.
RESUMEN
Caloric restriction (CR) is the leading non-pharmaceutical dietary intervention to improve health- and lifespan in most model organisms. A wide array of cellular pathways is induced in response to CR and CR-mimetics, including the transcriptional activator Nuclear factor erythroid-2-related factor 2 (Nrf2), which is essential in the upregulation of multiple stress-responsive and mitochondrial enzymes. Nrf2 is necessary in tumor protection but is not essential for the lifespan extending properties of CR in outbred mice. Here, we sought to study Nrf2-knockout (KO) mice and littermate controls in male C57BL6/J, an inbred mouse strain. Deletion of Nrf2 resulted in shortened lifespan compared to littermate controls only under ad libitum conditions. CR-mediated lifespan extension and physical performance improvements did not require Nrf2. Metabolic and protein homeostasis and activation of tissue-specific cytoprotective proteins were dependent on Nrf2 expression. These results highlight an important contribution of Nrf2 for normal lifespan and stress response.
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Restricción Calórica , Longevidad , Animales , Longevidad/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/genéticaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Neonates at risk of childhood atopy and asthma exhibit perturbation of the gut microbiome, metabolic dysfunction and increased concentrations of 12,13-diHOME in their faeces. However, the mechanism, source and contribution of this lipid to allergic inflammation remain unknown. Here, we show that intra-abdominal treatment of mice with 12,13-diHOME increased pulmonary inflammation and decreased the number of regulatory T (Treg) cells in the lungs. Treatment of human dendritic cells with 12,13-diHOME altered expression of PPARγ-regulated genes and reduced anti-inflammatory cytokine secretion and the number of Treg cells in vitro. Shotgun metagenomic sequencing of neonatal faeces indicated that bacterial epoxide hydrolase (EH) genes are more abundant in the gut microbiome of neonates who develop atopy and/or asthma during childhood. Three of these bacterial EH genes (3EH) specifically produce 12,13-diHOME, and treatment of mice with bacterial strains expressing 3EH caused a decrease in the number of lung Treg cells in an allergen challenge model. In two small birth cohorts, an increase in the copy number of 3EH or the concentration of 12,13-diHOME in the faeces of neonates was found to be associated with an increased probability of developing atopy, eczema or asthma during childhood. Our data indicate that elevated 12,13-diHOME concentrations impede immune tolerance and may be produced by bacterial EHs in the neonatal gut, offering a mechanistic link between perturbation of the gut microbiome during early life and atopy and asthma during childhood.
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Asma/inmunología , Bacterias/clasificación , Epóxido Hidrolasas/genética , Heces/química , Ácidos Linoleicos/análisis , Animales , Bacterias/enzimología , Bacterias/genética , Fenómenos Fisiológicos Bacterianos , Proteínas Bacterianas/genética , Modelos Animales de Enfermedad , Femenino , Microbioma Gastrointestinal , Humanos , Tolerancia Inmunológica , Recién Nacido , Masculino , Ratones , Linfocitos T Reguladores/metabolismoRESUMEN
Caloric restriction (CR) is the most potent nonpharmacological intervention known to both protect against carcinogenesis and delay aging in laboratory animals. There is a growing number of anticarcinogens and CR mimetics that activate NAD(P)H:quinone oxidoreductase 1 (NQO1). We have previously shown that NQO1, an antioxidant enzyme that acts as an energy sensor through modulation of intracellular redox and metabolic state, is upregulated by CR. Here, we used NQO1-knockout (KO) mice to investigate the role of NQO1 in both the aging process and tumor susceptibility, specifically in the context of CR. We found that NQO1 is not essential for the beneficial effects of CR on glucose homeostasis, physical performance, metabolic flexibility, life-span extension, and (unlike our previously observation with Nrf2) chemical-induced tumorigenesis.
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Composición Corporal , Restricción Calórica/efectos adversos , Longevidad , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Neoplasias Experimentales/prevención & control , Estrés Oxidativo , Animales , Biomarcadores de Tumor/metabolismo , Carcinogénesis , Immunoblotting , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Experimentales/etiología , Neoplasias Experimentales/metabolismoRESUMEN
Gut microbiota bacterial depletions and altered metabolic activity at 3 months are implicated in childhood atopy and asthma. We hypothesized that compositionally distinct human neonatal gut microbiota (NGM) exist, and are differentially related to relative risk (RR) of childhood atopy and asthma. Using stool samples (n = 298; aged 1-11 months) from a US birth cohort and 16S rRNA sequencing, neonates (median age, 35 d) were divisible into three microbiota composition states (NGM1-3). Each incurred a substantially different RR for multisensitized atopy at age 2 years and doctor-diagnosed asthma at age 4 years. The highest risk group, labeled NGM3, showed lower relative abundance of certain bacteria (for example, Bifidobacterium, Akkermansia and Faecalibacterium), higher relative abundance of particular fungi (Candida and Rhodotorula) and a distinct fecal metabolome enriched for pro-inflammatory metabolites. Ex vivo culture of human adult peripheral T cells with sterile fecal water from NGM3 subjects increased the proportion of CD4+ cells producing interleukin (IL)-4 and reduced the relative abundance of CD4+CD25+FOXP3+ cells. 12,13-DiHOME, enriched in NGM3 versus lower-risk NGM states, recapitulated the effect of NGM3 fecal water on relative CD4+CD25+FOXP3+ cell abundance. These findings suggest that neonatal gut microbiome dysbiosis might promote CD4+ T cell dysfunction associated with childhood atopy.
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Asma/epidemiología , Linfocitos T CD4-Positivos/inmunología , Microbioma Gastrointestinal/genética , Hipersensibilidad/epidemiología , ARN Ribosómico 16S/genética , Asma/inmunología , Bifidobacterium/genética , Linfocitos T CD4-Positivos/metabolismo , Candida/genética , Diferenciación Celular/inmunología , Preescolar , Faecalibacterium/genética , Heces/química , Femenino , Factores de Transcripción Forkhead/metabolismo , Microbioma Gastrointestinal/inmunología , Humanos , Hipersensibilidad/inmunología , Lactante , Recién Nacido , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Interleucina-4/inmunología , Masculino , Oportunidad Relativa , Rhodotorula/genética , Análisis de Secuencia de ARN , Linfocitos T/inmunologíaRESUMEN
UNLABELLED: Significant gut microbiota heterogeneity exists among ulcerative colitis (UC) patients, though the clinical implications of this variance are unknown. We hypothesized that ethnically distinct UC patients exhibit discrete gut microbiotas with unique metabolic programming that differentially influence immune activity and clinical status. Using parallel 16S rRNA and internal transcribed spacer 2 sequencing of fecal samples (UC, 30; healthy, 13), we corroborated previous observations of UC-associated bacterial diversity depletion and demonstrated significant Saccharomycetales expansion as characteristic of UC gut dysbiosis. Furthermore, we identified four distinct microbial community states (MCSs) within our cohort, confirmed their existence in an independent UC cohort, and demonstrated their coassociation with both patient ethnicity and disease severity. Each MCS was uniquely enriched for specific amino acid, carbohydrate, and lipid metabolism pathways and exhibited significant luminal enrichment of the metabolic products of these pathways. Using a novel ex vivo human dendritic cell and T-cell coculture assay, we showed that exposure to fecal water from UC patients caused significant Th2 skewing in CD4(+) T-cell populations compared to that of healthy participants. In addition, fecal water from patients in whom their MCS was associated with the highest level of disease severity induced the most dramatic Th2 skewing. Combined with future investigations, these observations could lead to the identification of highly resolved UC subsets based on defined microbial gradients or discrete microbial features that may be exploited for the development of novel, more effective therapies. IMPORTANCE: Despite years of research, the etiology of UC remains enigmatic. Diagnosis is difficult and the patient population heterogeneous, which represents a significant barrier to the development of more effective, tailored therapy. In this study, we demonstrate the clinical utility of the gut microbiome in stratifying UC patients by identifying the existence of four distinct interkingdom pathogenic microbiotas within the UC patient population that are compositionally and metabolically distinct, covary with clinical markers of disease severity, and drive discrete CD4(+) T-cell expansions ex vivo These findings offer new insight into the potential value of the gut microbiome as a tool for subdividing UC patients, opening avenues to the development of more personalized treatment plans and targeted therapies.
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Bacterias/clasificación , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/patología , Microbioma Gastrointestinal , Microbiota , Saccharomycetales/clasificación , Bacterias/genética , Bacterias/crecimiento & desarrollo , Productos Biológicos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Colitis Ulcerosa/microbiología , ADN Ribosómico/química , ADN Ribosómico/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Células Dendríticas/inmunología , Etnicidad , Humanos , Activación de Linfocitos , Redes y Vías Metabólicas/genética , ARN Ribosómico 16S/genética , Saccharomycetales/genética , Saccharomycetales/crecimiento & desarrollo , Análisis de Secuencia de ADNRESUMEN
Pathogens selectively target host cells using adhesion molecules and secreted virulence factors that may utilize protein, lipid, or carbohydrate ligands on the cell surface. The human intestinal pathogen Vibrio cholerae secretes a pore-forming toxin, V.cholerae cytolysin (VCC), which contains two domains that are structurally similar to known carbohydrate-binding proteins. These tandem domains are attached to the carboxy-terminus of the cytolytic domain and contain a ß-trefoil fold and a ß-prism fold. VCC has been shown to bind glycosylated proteins, and removal of the ß-prism domain leads to a large decrease in lytic activity against rabbit erythrocytes. Despite these clues, the identity of the glycan receptors of VCC and the role of glycan binding in toxin activity remain unknown. To better understand this specificity, we used a combination of structural and functional approaches to characterize the carbohydrate-binding activity of the VCC toxin. We first probed the monosaccharide-binding activity of VCC and demonstrated that the toxin exhibits millimolar affinity for aldohexoses. To understand this specificity, we solved the crystal structure of the VCC ß-prism domain bound to methyl-α-mannose. Next, we utilized a mammalian glycan screen to determine that the ß-prism domain preferentially binds complex N-glycans with a heptasaccharide GlcNAc(4)Man(3) core (NGA2). Fluorescence anisotropy and surface plasmon resonance indicated an approximately 100-nM affinity of the ß-prism domain for the heptasaccharide core. Our results suggest that carbohydrate-binding domains on the VCC toxin facilitate high-affinity targeting of mammalian cell membranes, which may contribute to the ability of VCC to lyse cells at picomolar concentrations.