RESUMO
BACKGROUND: Obesity is the main risk factor leading to the development of various respiratory diseases, such as asthma and pulmonary hypertension. Pulmonary microvascular endothelial cells (PMVECs) play a significant role in the development of lung diseases. Aconitate decarboxylase 1 (Acod1) mediates the production of itaconate, and Acod1/itaconate axis has been reported to play a protective role in multiple diseases. However, the roles of Acod1/itaconate axis in the PMVECs of obese mice are still unclear. METHODS: mRNA-seq was performed to identify the differentially expressed genes (DEGs) between high-fat diet (HFD)-induced PMVECs and chow-fed PMVECs in mice (|log2 fold change| ≥ 1, p ≤ 0.05). Free fatty acid (FFA) was used to induce cell injury, inflammation and mitochondrial oxidative stress in mouse PMVECs after transfection with the Acod1 overexpressed plasmid or 4-Octyl Itaconate (4-OI) administration. In addition, we investigated whether the nuclear factor erythroid 2-like 2 (Nrf2) pathway was involved in the effects of Acod1/itaconate in FFA-induced PMVECs. RESULTS: Down-regulated Acod1 was identified in HFD mouse PMVECs by mRNA-seq. Acod1 expression was also reduced in FFA-treated PMVECs. Acod1 overexpression inhibited cell injury, inflammation and mitochondrial oxidative stress induced by FFA in mouse PMVECs. 4-OI administration showed the consistent results in FFA-treated mouse PMVECs. Moreover, silencing Nrf2 reversed the effects of Acod1 overexpression and 4-OI administration in FFA-treated PMVECs, indicating that Nrf2 activation was required for the protective effects of Acod1/itaconate. CONCLUSION: Our results demonstrated that Acod1/Itaconate axis might protect mouse PMVECs from FFA-induced injury, inflammation and mitochondrial oxidative stress via activating Nrf2 pathway. It was meaningful for the treatment of obesity-caused pulmonary microvascular endotheliopathy.
Assuntos
Carboxiliases , Células Endoteliais , Pulmão , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2 , Obesidade , Succinatos , Animais , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Camundongos , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Carboxiliases/metabolismo , Carboxiliases/genética , Obesidade/metabolismo , Obesidade/complicações , Masculino , Succinatos/farmacologia , Pulmão/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/irrigação sanguínea , Células Cultivadas , Microvasos/metabolismo , Microvasos/efeitos dos fármacos , Microvasos/patologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Dieta Hiperlipídica/efeitos adversos , Endotélio Vascular/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , HidroliasesRESUMO
Pyrroindomycins (PYRs) represent the only spirotetramate natural products discovered in nature, and possess potent activities against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. Their unique structure and impressive biological activities make them attractive targets for synthesis and biosynthesis; however, the discovery and generation of new PYRs remains challenging. To date, only the initial components A and B have been reported. Herein, we report a mutasynthesis approach for the generation of nine new PYRs with varying acyl modifications on their deoxy-trisaccharide moieties. This was achieved by blocking the formation of the acyl group 1,8-dihydropyrrolo[2,3-b]indole (DHPI) via gene pyrK1 inactivation and supplying chemical acyl precursors. The gene pyrK1 encodes a DUF1864 family protein that probably catalyzes the oxidative transformation of L-tryptophan to DHPI, and its deletion results in the abolishment of DHPI-containing PYRs and the accumulation of three new PYRs either without acyl modification or with DHPI replaced by benzoic acid and pyrrole-2-carboxylic acid. Capitalizing on the capacity of the ΔpyrK1 mutant to produce new PYRs, we have successfully developed a mutasynthesis strategy for the generation of six novel PYR analogs with various aromatic acid modifications on their deoxy-trisaccharide moieties, showcasing the potential for generating structurally diverse PYRs. Overall, this research contributes significantly to understanding the biosynthesis of PYRs and offers valuable perspectives on their structural diversity.
Assuntos
Antibacterianos , Staphylococcus aureus Resistente à Meticilina , Antibacterianos/química , TrissacarídeosRESUMO
Streptomycin-resistant (SM-resistant) Mycobacterium tuberculosis (M. tuberculosis) is a major concern in tuberculosis (TB) treatment. However, the mechanisms underlying streptomycin resistance remain unclear. This study primarily aimed to perform preliminary screening of genes associated with streptomycin resistance through conjoint analysis of multiple genomics. Genome-wide methylation, transcriptome, and proteome analyses were used to elucidate the associations between specific genes and streptomycin resistance in M. tuberculosis H37Rv. Methylation analysis revealed that 188 genes were differentially methylated between the SM-resistant and normal groups, with 89 and 99 genes being hypermethylated and hypomethylated, respectively. Furthermore, functional analysis revealed that these 188 differentially methylated genes were enriched in 74 pathways, with most of them being enriched in metabolic pathways. Transcriptome analysis revealed that 516 genes were differentially expressed between the drug-resistant and normal groups, with 263 and 253 genes being significantly upregulated and downregulated, respectively. KEGG analysis indicated that these 516 genes were enriched in 79 pathways, with most of them being enriched in histidine metabolism. The methylation level was negatively related to mRNA abundance. Proteome analysis revealed 56 differentially expressed proteins, including 14 upregulated and 42 downregulated proteins. Moreover, three hub genes (coaE, fadE5, and mprA) were obtained using synthetic analysis. The findings of this study suggest that an integrated DNA methylation, transcriptome, and proteome analysis can provide important resources for epigenetic studies in SM-resistant M. tuberculosis H37Rv.
Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Metilação de DNA , Transcriptoma , Proteoma/metabolismo , Estreptomicina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/genéticaRESUMO
BACKGROUND: Rifampicin (RIF) and multidrug-resistant tuberculosis (TB) are major public health threats. As conventional phenotypic drug susceptibility testing requires two-eight weeks, molecular diagnostic assays are widely used to determine drug resistance. METHODS: Clinical Mycobacterium tuberculosis isolates with consistent drug susceptibility results, tested using microbroth dilution and proportion methods in Löwenstein-Jensen medium from patients with TB in Guangdong province were utilized to evaluate MeltPro TB and whole-genome sequencing (WGS) assays in detecting resistance to RIF, isoniazid (INH), ethambutol (EMB), fluoroquinolones (FQ), and streptomycin (SM). Solid phenotypic drug susceptibility testing was used as the gold standard to evaluate the detection capacity of MeltPro TB on clinical sputum samples of patients with TB. RESULTS: Similar to WGS, MeltPro TB successfully detected RIF, INH, and SM resistance with sensitivities of 86.3, 84.8, and 86.6 %, respectively. However, the resistant isolate detection rates were only 58.1 and 69.6 % for EMB and FQ-resistant strains. For clinical specimens, MeltPro TB still showed good detectable rates of RIF and INH resistance, with sensitivities of 82.4 % and 95.2 %, respectively. Detectable rates of FQ and EMB resistance were low: 77.8 % and 35.3 %, respectively. CONCLUSIONS: MeltPro TB can detect known DNA mutations associated with drug resistance in Mycobacterium tuberculosis strains with comparable efficacy to WGS. For FQ and EMB resistance testing, MeltPro TB requires optimization and is unsuitable for general use. MeltPro TB can be used for diagnosis of RIF and multidrug-resistant tuberculosis to rapidly initiate appropriate anti-TB drug therapy.
Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Humanos , Mycobacterium tuberculosis/genética , Testes de Sensibilidade Microbiana , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Isoniazida , Etambutol , Rifampina/farmacologia , Rifampina/uso terapêutico , Fluoroquinolonas/uso terapêutico , Mutação , China/epidemiologiaRESUMO
BACKGROUND: Pulmonary hypertension (PH), an infrequent disease, is characterized by excessive pulmonary vascular remodeling and proliferation of pulmonary artery smooth muscle cells (PASMCs). However, its underlying molecular mechanisms remain unclear. Uncovering its molecular mechanisms will be beneficial to the treatment of PH. METHODS: Differently expressed genes (DEGs) in the lung tissues of PH patients were analyzed with a GEO dataset GSE113439. From these DEGs, we focused on TRIM59 which was highly expressed in PH patients. Subsequently, the expression of TRIM59 in the pulmonary arteries of PH patients, lung tissues of PH rat model and PASMCs cultured in a hypoxic condition was verified by quantitative real-time PCR (qPCR), western blot and immunohistochemistry. Furthermore, the role of TRIM59 in PAMSC proliferation and pathological changes in PH rats was assessed via gain-of-function and loss-of-function experiments. In addition, the transcriptional regulation of YAP1/TEAD4 on TRIM59 was confirmed by qPCR, western blot, luciferase reporter assay, ChIP and DNA pull-down. In order to uncover the underlying mechanisms of TRIM59, a protein ubiquitomics and a CoIP- HPLC-MS/MS were companied to identify the direct targets of TRIM59. RESULTS: TRIM59 was highly expressed in the pulmonary arteries of PH patients and lung tissues of PH rats. Over-expression of TRIM59 accelerated the proliferation of PASMCs, while TRIM59 silencing resulted in the opposite results. Moreover, TRIM59 silencing mitigated the injuries in heart and lung and attenuated pulmonary vascular remodeling during PH. In addition, its transcription was positively regulated by YAP1/TEAD4. Then we further explored the underlying mechanisms of TRIM59 and found that TRIM59 overexpression resulted in an altered ubiquitylation of proteins. Accompanied with the results of CoIP- HPLC-MS/MS, 34 proteins were identified as the direct targets of TRIM59. CONCLUSION: TRIM59 was highly expressed in PH patients and promoted the proliferation of PASMCs and pulmonary vascular remodeling, thus contributing to the pathogenesis of PH. It is indicated that TRIM59 may become a potential target for PH treatment.
Assuntos
Hipertensão Pulmonar , Humanos , Ratos , Animais , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Remodelação Vascular/genética , Espectrometria de Massas em Tandem , Transdução de Sinais , Proliferação de Células/genética , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Hipóxia/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitinação , Miócitos de Músculo Liso/metabolismo , Células Cultivadas , Fatores de Transcrição de Domínio TEA , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismoRESUMO
Antibiotic resistance of Mycobacterium tuberculosis (Mtb) is a major public health concern worldwide. Therefore, it is of great significance to characterize the mutational pathways by which susceptible Mtb evolves into drug resistance. In this study, we used laboratory evolution to explore the mutational pathways of aminoglycoside resistance. The level of resistance in amikacin inducing Mtb was also associated with changes in susceptibility to other anti-tuberculosis drugs such as isoniazid, levofloxacin and capreomycin. Whole-genome sequencing (WGS) revealed that the induced resistant Mtb strains had accumulated diverse mutations. We found that rrs A1401G was the predominant mutation in aminoglycoside-resistant clinical Mtb isolates from Guangdong. In addition, this study provided global insight into the characteristics of the transcriptome in four representative induced strains and revealed that rrs mutated and unmutated aminoglycoside-resistant Mtb strains have different transcriptional profiles. WGS analysis and transcriptional profiling of Mtb strains during evolution revealed that Mtb strains harbouring rrs A1401G have an evolutionary advantage over other drug-resistant strains under the pressure of aminoglycosides because of their ultra-high resistance level and low physiological impact on the strain. The results of this study should advance our understanding of aminoglycoside resistance mechanisms.
Assuntos
Aminoglicosídeos , Mycobacterium tuberculosis , Aminoglicosídeos/farmacologia , Mycobacterium tuberculosis/genética , Transcriptoma , Antituberculosos/farmacologia , LevofloxacinoRESUMO
BACKGROUND: Obesity has been identified as a risk factor for acute lung injury/acute respiratory distress syndrome (ALI/ARDS). However, the underlying mechanisms remain elusive. This study aimed to investigate the role of fatty acid synthase (FASN) in lipopolysaccharide (LPS)-induced ALI under obesity. METHODS: A high-fat diet-induced obese (DIO) mouse model was established and lean mice fed with regular chow diet were served as controls. LPS was intratracheally instilled to reproduce ALI in mice. In vitro, primary mouse lung endothelial cells (MLECs), treated by palmitic acid (PA) or co-cultured with 3T3-L1 adipocytes, were exposed to LPS. Chemical inhibitor C75 or shRNA targeting FASN was used for in vivo and in vitro loss-of-function studies for FASN. RESULTS: After LPS instillation, the protein levels of FASN in freshly isolated lung endothelial cells from DIO mice were significantly higher than those from lean mice. MLECs undergoing metabolic stress exhibited increased levels of FASN, decreased levels of VE-cadherin with increased p38 MAPK phosphorylation and NLRP3 expression, mitochondrial dysfunction, and impaired endothelial barrier compared with the control MLECs when exposed to LPS. However, these effects were attenuated by FASN inhibition with C75 or corresponding shRNA. In vivo, LPS-induced ALI, C75 pretreatment remarkably alleviated LPS-induced overproduction of lung inflammatory cytokines TNF-α, IL-6, and IL-1ß, and lung vascular hyperpermeability in DIO mice as evidenced by increased VE-cadherin expression in lung endothelial cells and decreased lung vascular leakage. CONCLUSIONS: Taken together, FASN inhibition alleviated the exacerbation of LPS-induced lung injury under obesity via rescuing lung endothelial dysfunction. Therefore, targeting FASN may be a potential therapeutic target for ameliorating LPS-induced ALI in obese individuals.
Assuntos
Lesão Pulmonar Aguda , Ácido Graxo Sintases , Síndrome do Desconforto Respiratório , Animais , Camundongos , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/prevenção & controle , Lesão Pulmonar Aguda/tratamento farmacológico , Células Endoteliais/metabolismo , Ácido Graxo Sintases/antagonistas & inibidores , Lipopolissacarídeos , Pulmão/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/metabolismo , Síndrome do Desconforto Respiratório/metabolismoRESUMO
BACKGROUND: It is established that pulmonary disorders are comorbid with metabolic disorders such as obesity. Previous studies show that the stimulator of interferon genes (STING) signaling plays crucial roles in obesity-induced chronic inflammation via TANK-binding kinase 1 (TBK1) pathways. However, it remains unknown whether and how the STING signaling is implicated in the inflammatory processes in the lung in obesity. METHODS: Human lung tissues were obtained from obese patients (n = 3) and controls (n = 3). Mice were fed with the high-fat diet or regular control diet to establish the diet-induced obese (DIO) and lean mice, and were treated with C-176 (a specific STING inhibitor) or vehicle respectively. The lung macrophages were exposed to palmitic acid (PA) in vitro. The levels of STING singaling and metabolic inflammation factors were detected and anlyzed. RESULTS: We find that STING+/CD68+ macrophages are increased in lung tissues in patients with obesity. Our data also show that the expressions of STING and the levels of proinflammatory cytokines are increased both in lung tissues and bronchoalveolar lavage fluid (BALF) in obesity compared to controls, and inhibition of the STING blunted the obesity-induced lung inflammation. Mechanistically, our data demonstrate that the STING signaling pathway is involved in the PA-induced inflammation through the STING-TBK1-IRF3 (interferon regulatory factor 3)/NF-κB (nuclear factor kappa B) pathways in the lung macrophages. CONCLUSIONS: Our results collectively suggest that the STING signaling contributes to obesity-associated inflammation by stimulating proinflammatory processes in lung macrophages, one that may serve as a therapeutic target in ameliorating obesity-related lung dysfunctions.
Assuntos
Pneumonia , Transdução de Sinais , Animais , Humanos , Camundongos , Inflamação/metabolismo , NF-kappa B/metabolismo , Obesidade/complicaçõesRESUMO
Endothelial dysfunction is essential in pulmonary arterial hypertension (PAH) pathogenesis and is considered to be a therapeutic target of PAH. Curcumol is a bioactive sesquiterpenoid with pharmacological properties including restoring endothelial cells damage. This study aimed to evaluate the effect of curcumol on PAH rats and investigate its possible mechanisms. PAH was induced by subcutaneous injection of 60 mg/kg monocrotaline (MCT) in male Sprague Dawley rats. Curcumol (12.5, 25, and 50 mg/kg/day) were administered by intragastric administration for 3 weeks. The results demonstrated that curcumol dose-dependently alleviated MCT-induced right ventricular hypertrophy and pulmonary arterial wall thickness. In addition, endothelial-to-mesenchymal transition (EndMT) in the pulmonary arteries of MCT-challenged rats was inhibited after curcumol treatment, as evidenced by the restored expressions of endothelial and myofibroblast markers. The possible pharmacological mechanisms of curcumol were analyzed using network pharmacology. After screening the common therapeutic targets of PAH and curcumol by searching related databases and comparison, pathway enrichment was performed and AKT/GSK3ß was screened out as a possible signaling pathway which was relevant to the therapeutic mechanism of curcumol on PAH. Western blot analysis verified this in lung tissues. Moreover, combination of TNF-α, TGF-ß1 and IL-1ß-induced EndMT in primary rat pulmonary arterial endothelial cells were blocked by curcumol, and this effect was resembled by PI3K/AKT inhibitor LY294002. Above all, our study suggested that curcumol inhibited EndMT via inhibiting the AKT/GSK3ß signaling pathway, which may contribute to its alleviated effect on PAH. Curcumol may be developed as a therapeutic for PAH in the future.
Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Sesquiterpenos , Animais , Masculino , Ratos , Modelos Animais de Doenças , Células Endoteliais , Hipertensão Pulmonar Primária Familiar/patologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/tratamento farmacológico , Monocrotalina/efeitos adversos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Hipertensão Arterial Pulmonar/induzido quimicamente , Artéria Pulmonar/patologia , Ratos Sprague-Dawley , Sesquiterpenos/farmacologia , Sesquiterpenos/uso terapêutico , Sesquiterpenos/metabolismo , Transdução de Sinais , Transdiferenciação CelularRESUMO
Pulmonary hypertension (PH) is a life-threatening cardiopulmonary disease characterized by pulmonary vascular remodeling. Excessive growth and migration of pulmonary artery smooth muscle cells (PASMCs) are believed to be major contributors to pulmonary vascular remodeling. Ubiquitin-specific protease 15 (USP15) is a vital deubiquitinase that has been shown to be critically involved in many pathologies. However, the effect of USP15 on PH has not yet been explored. In this study, the upregulation of USP15 was identified in the lungs of PH patients, mice with SU5416/hypoxia (SuHx)-induced PH and rats with monocrotaline (MCT)-induced PH. Moreover, adeno-associated virus-mediated functional loss of USP15 markedly alleviated PH exacerbation in SuHx-induced mice and MCT-induced rats. In addition, the abnormal upregulation and nuclear translocation of YAP1/TAZ was validated after PH modeling. Human pulmonary artery smooth muscle cells (hPASMCs) were exposed to hypoxia to mimic PH in vitro, and USP15 knockdown significantly inhibited cell proliferation, migration, and YAP1/TAZ signaling in hypoxic hPASMCs. Rescue assays further suggested that USP15 promoted hPASMC proliferation and migration in a YAP1/TAZ-dependent manner. Coimmunoprecipitation assays indicated that USP15 could interact with YAP1, while TAZ bound to USP15 after hypoxia treatment. We further determined that USP15 stabilized YAP1 by inhibiting the K48-linked ubiquitination of YAP1. In summary, our findings reveal the regulatory role of USP15 in PH progression and provide novel insights into the pathogenesis of PH.
Assuntos
Hipertensão Pulmonar , Animais , Humanos , Camundongos , Ratos , Proliferação de Células , Hipertensão Pulmonar/genética , Hipóxia/complicações , Hipóxia/genética , Hipóxia/metabolismo , Pulmão/patologia , Miócitos de Músculo Liso/metabolismo , Transdução de Sinais , Proteases Específicas de Ubiquitina/metabolismo , Remodelação Vascular/genéticaRESUMO
Currently, there are no particularly effective biomarkers to distinguish between latent tuberculosis infection (LTBI) and active pulmonary tuberculosis (PTB) and evaluate the outcome of TB treatment. In this study, we have characterized the changes in the serum metabolic profiles caused by Mycobacterium tuberculosis (Mtb) infection and standard anti-TB treatment with isoniazid-rifampin-pyrazinamide-ethambutol (HRZE) using GC-MS and LC-MS/MS. Seven metabolites, including 3-oxopalmitic acid, akeboside ste, sulfolithocholic acid, 2-decylfuran (4,8,8-trimethyldecahydro-1,4-methanoazulen-9-yl)methanol, d-(+)-camphor, and 2-methylaminoadenosine, were identified to have significantly higher levels in LTBI and untreated PTB patients (T0) than those in uninfected healthy controls (Un). Among them, akeboside Ste and sulfolithocholic acid were significantly decreased in PTB patients with 2-month HRZE (T2) and cured PTB patients with 2-month HRZE followed by 4-month isoniazid-rifampin (HR) (T6). Receiver operator characteristic curve analysis revealed that the combined diagnostic model showed excellent performance for distinguishing LT from T0 and Un. By analyzing the biochemical and disease-related pathways, we observed that the differential metabolites in the serum of LTBI or TB patients, compared to healthy controls, were mainly involved in glutathione metabolism, ascorbate and aldarate metabolism, and porphyrin and chlorophyll metabolism. The metabolites with significant differences between the T0 group and the T6 group were mainly enriched in niacin and nicotinamide metabolism. Our study provided more detailed experimental data for developing laboratory standards for evaluating LTBI and cured PTB.
Assuntos
Infecção Latente , Tuberculose Pulmonar , Humanos , Isoniazida/uso terapêutico , Rifampina/uso terapêutico , Cromatografia Líquida , Prognóstico , Espectrometria de Massas em Tandem , Tuberculose Pulmonar/diagnósticoRESUMO
Ethambutol (EMB) is a first-line antituberculosis drug currently being used clinically to treat tuberculosis. Mutations in the embCAB operon are responsible for EMB resistance. However, the discrepancies between genotypic and phenotypic EMB resistance have attracted much attention. We induced EMB resistance in Mycobacterium tuberculosis in vitro and used an integrated genome-methylome-transcriptome-proteome approach to study the microevolutionary mechanism of EMB resistance. We identified 509 aberrantly methylated genes (313 hypermethylated genes and 196 hypomethylated genes). Moreover, some hypermethylated and hypomethylated genes were identified using RNA-seq profiling. Correlation analysis revealed that the differential methylation of genes was negatively correlated with transcription levels in EMB-resistant strains. Additionally, two hypermethylated candidate genes (mbtD and celA1) were screened by iTRAQ-based quantitative proteomics analysis, verified by qPCR, and corresponded with DNA methylation differences. This is the first report that identifies EMB resistance-related genes in laboratory-induced mono-EMB-resistant M. tuberculosis using multi-omics profiling. Understanding the epigenetic features associated with EMB resistance may provide new insights into the underlying molecular mechanisms.
Assuntos
Mycobacterium tuberculosis , Tuberculose dos Linfonodos , Antituberculosos/farmacologia , Farmacorresistência Bacteriana/genética , Etambutol/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , Proteoma , Serina ProteasesRESUMO
Linear nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) template the modular biosynthesis of numerous nonribosomal peptides, polyketides and their hybrids through assembly line chemistry. This chemistry can be complex and highly varied, and thus challenges our understanding in NRPS and PKS-programmed, diverse biosynthetic processes using amino acid and carboxylate building blocks. Here, we report that caerulomycin and collismycin peptide-polyketide hybrid antibiotics share an assembly line that involves unusual NRPS activity to engage a trans-acting flavoprotein in C-C bond formation and heterocyclization during 2,2'-bipyridine formation. Simultaneously, this assembly line provides dethiolated and thiolated 2,2'-bipyridine intermediates through differential treatment of the sulfhydryl group arising from L-cysteine incorporation. Subsequent L-leucine extension, which does not contribute any atoms to either caerulomycins or collismycins, plays a key role in sulfur fate determination by selectively advancing one of the two 2,2'-bipyridine intermediates down a path to the final products with or without sulfur decoration. These findings further the appreciation of assembly line chemistry and will facilitate the development of related molecules using synthetic biology approaches.
Assuntos
2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Flavoproteínas/química , 2,2'-Dipiridil/síntese química , 2,2'-Dipiridil/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Cisteína/química , Cisteína/metabolismo , Flavoproteínas/metabolismo , Compostos Heterocíclicos/química , Compostos Heterocíclicos/metabolismo , Modelos Químicos , Estrutura Molecular , Peptídeo Sintases/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Policetídeo Sintases/metabolismo , Policetídeos/química , Policetídeos/metabolismo , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismoRESUMO
Tuberculosis caused by the pathogen Mycobacterium tuberculosis (MTB), remains a significant threat to global health. Elucidating the mechanisms of essential MTB genes provides an important theoretical basis for drug exploitation. Gene mtsp17 is essential and is conserved in the Mycobacterium genus. Although Mtsp17 has a structure closely resembling typical steroidogenic acute regulatory protein-related lipid transfer (START) family proteins, its biological function is different. This study characterizes the transcriptomes of Mycobacterium smegmatis to explore the consequences of mtsp17 downregulation on gene expression. Suppression of the mtsp17 gene resulted in significant down-regulation of 3% and upregulation of 1% of all protein-coding genes. Expression of desA1, an essential gene involved in mycolic acid synthesis, and the anti-SigF antagonist MSMEG_0586 were down-regulated in the conditional Mtsp17 knockout mutant and up-regulated in the Mtsp17 over-expression strain. Trends in the changes of 70 of the 79 differentially expressed genes (Log2 fold change > 1.5) in the conditional Mtsp17 knockout strain were the same as in the SigF knockout strain. Our data suggest that Mtsp17 is likely an activator of desA1 and Mtsp17 regulates the SigF regulon by SigF regulatory pathways through the anti-SigF antagonist MSMEG_0586. Our findings indicate the role of Mtsp17 may be in transcriptional regulation, provide new insights into the molecular mechanisms of START family proteins, and uncover a new node in the regulatory network of mycobacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/metabolismo , Ativação Transcricional , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Mycobacterium smegmatis/genética , Domínios Proteicos , Fator sigma/genética , Fator sigma/metabolismoRESUMO
Soil microbial communities maintain multiple ecosystem functions in terrestrial ecosystems. The response of soil microbial communities to vegetation restoration in desertification environments is still poorly understood. Therefore, the purpose of our study was to evaluate the dynamic changes of the soil microbial community during the growth of Pinus sylvestris var. mongolic (P. sylvestris) plantations. We collected soil samples from five P. sylvestris plantations with different stand age. High-throughput sequencing was performed to determine the microbial community structure. The dynamic relationship between soil microbial community and edaphic factors was analyzed using the co-occurrence network, mantel test and partial least squares path modeling. The results showed that the soil microbial alpha diversity and community structure were significantly various among the plantations (P < 0.001). The number of nodes and edges in microbial co-occurrence network gradually decreased and the interrelationships between species became weak with stand age. The Available phosphorus was the most significant factor affecting the structure of bacterial community (R2 = 0.952), while the total phosphorus was the most significant factor affecting the structure of fungal community (R2 = 0.745). However, soil moisture had no significant effect on the microbial community. pH (0.73) and available nitrogen (0.91) had the largest positive total effects on bacterial and fungal community, respectively. Stand age (-0.65) was an indirect factor with the largest negative total effects on the bacterial community. Therefore, we concluded that the soil microbial community was not limited by soil moisture during the natural restoration process of P. sylvestris plantations in the desertification environment and the phosphorus utilization efficiency played a leading role in shaping the soil microbial community.
Assuntos
Microbiota , Pinus sylvestris , Pinus , China , Fósforo , Areia , Solo , Microbiologia do SoloRESUMO
Ample evidence indicates that obesity causes dysfunctions in the lung. Previous studies also show that cholinergic anti-inflammatory pathways play crucial roles in obesity-induced chronic inflammation via α7 nicotinic acetylcholine receptor (α7nAChR) signaling. However, it remains unclear whether and how obesity affects the expressions of α7nAChR in myeloid cells in the lung. To address this question, we treated regular chow diet-fed mice or high-fat diet induced obese mice with lipopolysaccharide (LPS) or vehicle via endotracheal injections. By using a multicolor flow cytometry approach to analyze and characterize differential cell subpopulations and α7nAChR expressions, we find no detectable α7nAChR in granulocytes, monocytes and alveolar macrophages, and low expression levels of α7nAChR were detected in interstitial macrophages. Interestingly, we find that a challenge with LPS treatment significantly increased expression levels of α7nAChR in monocytes, alveolar and interstitial macrophages. Meanwhile, we observed that the expression levels of α7nAChR in alveolar and interstitial macrophages in high-fat diet induced obese mice were lower than regular chow diet-fed mice challenged by the LPS. Together, our findings indicate that obesity alters the expressions of α7nAChR in differential lung myeloid cells.
Assuntos
Dieta Hiperlipídica , Pulmão/metabolismo , Células Mieloides/metabolismo , Obesidade/etiologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Imunofenotipagem , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/imunologia , Obesidade/imunologia , Obesidade/metabolismo , Obesidade/patologiaRESUMO
In recent years, the treatment of tuberculosis is once again facing a severe situation because the existing antituberculosis drugs have become weaker and weaker with the emergence of drug-resistant Mycobacterium tuberculosis (Mtb). The studies of cell division and cell cycle-related factors in Mtb are particularly important for the development of new drugs with broad-spectrum effects. Mycobacterium smegmatis (Msm) has been used as a model organism to study the molecular, physiological, and drug-resistant mechanisms of Mtb. Bioinformatics analysis has predicted that MSMEG_6171 is a MinD-like protein of the septum site-determining protein family associated with cell division in Mycobacterium smegmatis. In our study, we use ultrastructural analysis, proteomics, metabolomics, and molecular biology techniques to comprehensively investigate the function of MSMEG_6171. Overexpression of MSMEG_6171 in Msm resulted in elongated cells, suggesting an important role of MSMEG_6171 in regulating cell wall morphology. The MSMEG_6171 overexpression could enhance the bacterial resistance to vancomycin, ethionamide, meropenem, and cefamandole. The MSMEG_6171 overexpression could alter the lipid metabolism of Msm to cause the changes on cellular biofilm property and function, which enhances bacterial resistance to antibiotics targeting cell wall synthesis. MSMEG_6171 could also induce the glyceride and phospholipid alteration in vivo to exhibit the pleiotropic phenotypes and various cellular responses. The results showed that amino acid R249 in MSMEG_6171 was a key site that can affect the level of bacterial drug resistance, suggesting that ATPase activity is required for function.
RESUMO
Drug-resistant Mycobacterium tuberculosis (M. tuberculosis) has become an increasingly serious public health problem and has complicated tuberculosis (TB) treatment. Levofloxacin (LOF) is an ideal anti-tuberculosis drug in clinical applications. However, the detailed molecular mechanisms of LOF-resistant M. tuberculosis in TB treatment have not been revealed. Our study performed transcriptome and methylome sequencing to investigate the potential biological characteristics of LOF resistance in M. tuberculosis H37Rv. In the transcriptome analysis, 953 differentially expressed genes (DEGs) were identified; 514 and 439 DEGs were significantly downregulated and upregulated in the LOF-resistant group and control group, respectively. The KEGG pathway analysis revealed that 97 pathways were enriched in this study. In the methylome analysis, 239 differentially methylated genes (DMGs) were identified; 150 and 89 DMGs were hypomethylated and hypermethylated in the LOF-resistant group and control group, respectively. The KEGG pathway analysis revealed that 74 pathways were enriched in this study. The overlap study suggested that 25 genes were obtained. It was notable that nine genes expressed downregulated mRNA and upregulated methylated levels, including pgi, fadE4, php, cyp132, pckA, rpmB1, pfkB, acg, and ctpF, especially cyp132, pckA, and pfkB, which were vital in LOF-resistant M. tuberculosis H37Rv. The overlapping genes between transcriptome and methylome could be essential for studying the molecular mechanisms of LOF-resistant M. tuberculosis H37Rv. These results may provide informative evidence for TB treatment with LOF.
Assuntos
Farmacorresistência Bacteriana/genética , Epigenoma , Levofloxacino/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Transcriptoma , Antibacterianos/farmacologia , Metilação de DNA , Genes BacterianosRESUMO
The diagnosis and treatment of fever of unknown origin (FUO) are huge challenges to clinicians. Separating the etiologies of FUO into infectious and non-infectious disease is conducive to clinical physicians not only on making decisions rapidly concerning the prescription of suitable antibiotics but also on further analysis of the final diagnosis. In order to develop and validate a diagnostic tool to efficiently distinguish the etiologies of adult FUO patients as infectious or non-infectious disease, FUO patients from the departments of infectious disease and internal medicine in three Chinese tertiary hospitals were enrolled retrospectively and prospectively. By using polynomial logistic regression analysis, the diagnostic formula and the associated scoring system were developed. The variables included in this diagnostic formula were from clinical evaluations and common laboratory examinations. The proposed tool could discriminate infectious and non-infectious causes of FUO with an area under receiver operating characteristic curve (AUC) of 0.83, sensitivity of 0.80 and specificity of 0.75. This diagnosis tool could predict the infectious and non-infectious causes of FUO in the validation cohort with an AUC of 0.79, sensitivity of 0.79 and specificity of 0.70. The results suggested that this diagnostic tool could be a reliable tool to discriminate between infectious and non-infectious causes of FUO.
Assuntos
Doenças Transmissíveis/diagnóstico , Febre de Causa Desconhecida/diagnóstico , Doenças não Transmissíveis/epidemiologia , Adulto , China/epidemiologia , Doenças Transmissíveis/epidemiologia , Doenças Transmissíveis/patologia , Diagnóstico Diferencial , Febre de Causa Desconhecida/epidemiologia , Febre de Causa Desconhecida/patologia , Humanos , Modelos LogísticosRESUMO
The present study aimed to establish a list of parameters indicative of pathogen invasion and develop a predictive model to distinguish the etiologies of fever of unknown origin (FUO) into infectious and non-infectious causes. From January 2014 to September 2017, 431 patients with FUO were prospectively enrolled in the study population. This study established a list of 26 variables from the following 4 aspects: host factors, epidemiological factors, behavioral factors, and iatrogenic factors. Predefined predicted variables were included in a multivariate logistic regression analysis to develop a predictive model. The predictive model and the corresponding scoring system were developed using data from the confirmed diagnoses and 9 variables were eventually identified. These factors were incorporated into the predictive model. This model discriminated between infectious and non-infectious causes of FUO with an AUC of 0.72, sensitivity of 0.71, and specificity of 0.63. The predictive model and corresponding scoring system based on factors concerning pathogen invasion appear to be reliable screening tools to discriminate between infectious and non-infectious causes of FUO.