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1.
Proc Natl Acad Sci U S A ; 117(7): 3551-3559, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32015121

RESUMO

Cryptococcus neoformans is an opportunistic fungal pathogen that infects ∼280,000 people every year, causing >180,000 deaths. The human immune system recognizes chitin as one of the major cell-wall components of invading fungi, but C. neoformans can circumvent this immunosurveillance mechanism by instead exposing chitosan, the partly or fully deacetylated form of chitin. The natural production of chitosans involves the sequential action of chitin synthases (CHSs) and chitin deacetylases (CDAs). C. neoformans expresses four putative CDAs, three of which have been confirmed as functional enzymes that act on chitin in the cell wall. The fourth (CnCda4/Fpd1) is a secreted enzyme with exceptional specificity for d-glucosamine at its -1 subsite, thus preferring chitosan over chitin as a substrate. We used site-specific mutagenesis to reduce the subsite specificity of CnCda4 by converting an atypical isoleucine residue in a flexible loop region to the bulkier or charged residues tyrosine, histidine, and glutamic acid. We also investigated the effect of CnCda4 deacetylation products on human peripheral blood-derived macrophages, leading to a model explaining the function of CnCda4 during infection. We propose that CnCda4 is used for the further deacetylation of chitosans already exposed on the C. neoformans cell wall (originally produced by CnChs3 and CnCda1 to 3) or released from the cell wall as elicitors by human chitinases, thus making the fungus less susceptible to host immunosurveillance. The absence of CnCda4 during infection could therefore promote the faster recognition and elimination of this pathogen.


Assuntos
Amidoidrolases/metabolismo , Quitosana/metabolismo , Cryptococcus neoformans/enzimologia , Proteínas Fúngicas/metabolismo , Amidoidrolases/genética , Parede Celular/enzimologia , Parede Celular/genética , Quitina/química , Quitina/metabolismo , Quitosana/química , Criptococose/microbiologia , Cryptococcus neoformans/química , Cryptococcus neoformans/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Humanos , Especificidade por Substrato
2.
BMC Infect Dis ; 20(1): 19, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31910878

RESUMO

BACKGROUND: Pyrazinamide still may be a useful drug for treatment of rifampin-resistant (RR-TB) or multidrug-resistant tuberculosis (MDR-TB) in China while awaiting scale up of new drugs and regimens including bedaquiline and linezolid. The level of pyrazinamide resistance among MDR-TB patients in China is not well established. Therefore, we assessed pyrazinamide resistance in a representative sample and explored determinants and patterns of pncA mutations. METHODS: MDR-TB isolates from the 2007 national drug resistance survey of China were sub-cultured and examined for pyrazinamide susceptibility by BACTEC MGIT 960 method. pncA mutations were identified by sequencing. Characteristics associated with pyrazinamide resistance were analyzed using univariable and multivariable log-binominal regression. RESULTS: Of 401 MDR-TB isolates, 324 were successfully sub-cultured and underwent drug susceptibility testing. Pyrazinamide resistance was prevalent in 40.7% of samples, similarly among new and previously treated MDR-TB patients. Pyrazinamide resistance in MDR-TB patients was associated with lower age (adjusted OR 0.54; 95% CI, 0.34-0.87 for those aged ≧60 years compared to < 40 years). Pyrazinamide resistance was not associated with gender, residential area, previous treatment history and Beijing genotype. Of 132 patients with pyrazinamide resistant MDR-TB, 97 (73.5%) had a mutation in the pncA gene; with 61 different point mutations causing amino acid change, and 11 frameshifts in the pncA gene. The mutations were scattered throughout the whole pncA gene and no hot spot region was identified. CONCLUSIONS: Pyrazinamide resistance among MDR-TB patients in China is common, although less so in elderly patients. Therefore, pyrazinamide should only be used for treatment of RR/MDR-TB in China if susceptibility is confirmed. Molecular testing for detection of pyrazinamide resistance only based on pncA mutations has certain value for the rapid detection of pyrazinamide resistance in MDR-TB strains but other gene mutations conferring to pyrazinamide resistance still need to be explored to increase its predictive ability .


Assuntos
Antituberculosos/uso terapêutico , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Pirazinamida/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Adulto , Fatores Etários , Amidoidrolases/genética , Antituberculosos/efeitos adversos , Sequência de Bases/genética , China/epidemiologia , Diarilquinolinas/uso terapêutico , Genes Bacterianos/genética , Genótipo , Humanos , Linezolida/uso terapêutico , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Mutação Puntual/genética , Polimorfismo de Nucleotídeo Único/genética , Prevalência , Pirazinamida/efeitos adversos , Rifampina/efeitos adversos , Rifampina/uso terapêutico , Fatores de Risco , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico
3.
Microb Cell Fact ; 19(1): 4, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31910844

RESUMO

BACKGROUND: Swep is an excellent carbamate herbicide that kills weeds by interfering with metabolic processes and inhibiting cell division at the growth point. Due to the large amount of use, swep residues in soil and water not only cause environmental pollution but also accumulate through the food chain, ultimately pose a threat to human health. This herbicide is degraded in soil mainly by microbial activity, but no studies on the biotransformation of swep have been reported. RESULTS: In this study, a consortium consisting of two bacterial strains, Comamonas sp. SWP-3 and Alicycliphilus sp. PH-34, was enriched from a contaminated soil sample and shown to be capable of mineralizing swep. Swep was first transformed by Comamonas sp. SWP-3 to the intermediate 3,4-dichloroaniline (3,4-DCA), after which 3,4-DCA was mineralized by Alicycliphilus sp. PH-34. An amidase gene, designated as ppa, responsible for the transformation of swep into 3,4-DCA was cloned from strain SWP-3. The expressed Ppa protein efficiently hydrolyzed swep and a number of other structural analogues, such as propanil, chlorpropham and propham. Ppa shared less than 50% identity with previously reported arylamidases and displayed maximal activity at 30 °C and pH 8.6. Gly449 and Val266 were confirmed by sequential error prone PCR to be the key catalytic sites for Ppa in the conversion of swep. CONCLUSIONS: These results provide additional microbial resources for the potential remediation of swep-contaminated sites and add new insights into the catalytic mechanism of amidase in the hydrolysis of swep.


Assuntos
Amidoidrolases/metabolismo , Bactérias/metabolismo , Biodegradação Ambiental , Herbicidas/metabolismo , Amidoidrolases/genética , Clorprofam/metabolismo , Clonagem Molecular , Comamonadaceae/metabolismo , Comamonas/metabolismo , Poluentes Ambientais/metabolismo , Hidrólise , Consórcios Microbianos , Fenilcarbamatos/metabolismo , Propanil/metabolismo
4.
Am J Physiol Renal Physiol ; 318(2): F509-F517, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31904280

RESUMO

Endothelial dysfunction, characterized by reduced bioavailability of nitric oxide and increased oxidative stress, is a hallmark characteristic in diabetes and diabetic nephropathy (DN). High levels of asymmetric dimethylarginine (ADMA) are observed in several diseases including DN and are a strong prognostic marker for cardiovascular events in patients with diabetes and end-stage renal disease. ADMA, an endogenous endothelial nitric oxide synthase (NOS3) inhibitor, is selectively metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Low DDAH levels have been associated with cardiac and renal dysfunction, but its effects on DN are unknown. We hypothesized that enhanced renal DDAH-1 expression would improve DN by reducing ADMA and restoring NOS3 levels. DBA/2J mice injected with multiple low doses of vehicle or streptozotocin were subsequently injected intrarenally with adenovirus expressing DDAH-1 (Ad-h-DDAH-1) or vector control [Ad-green fluorescent protein (GFP)], and mice were followed for 6 wk. Diabetes was associated with increased kidney ADMA and reduced kidney DDAH activity and DDAH-1 expression but had no effect on kidney DDAH-2 expression. Ad-GFP-treated diabetic mice showed significant increases in albuminuria, histological changes, glomerular macrophage recruitment, inflammatory cytokine and fibrotic markers, kidney ADMA levels, and urinary thiobarbituric acid reactive substances excretion as an indicator of oxidative stress, along with a significant reduction in kidney DDAH activity and kidney NOS3 mRNA compared with normal mice. In contrast, Ad-h-DDAH-1 treatment of diabetic mice reversed these effects. These data indicate, for the first time, that DDAH-1 mediates renal tissue protection in DN via the ADMA-NOS3-interaction. Enhanced renal DDAH-1 activity could be a novel therapeutic tool for treating patients with diabetes.


Assuntos
Adenoviridae/genética , Amidoidrolases/biossíntese , Arginina/análogos & derivados , Diabetes Mellitus Experimental/terapia , Nefropatias Diabéticas/prevenção & controle , Terapia Genética , Vetores Genéticos , Rim/enzimologia , Albuminúria/enzimologia , Albuminúria/genética , Albuminúria/prevenção & controle , Amidoidrolases/genética , Animais , Arginina/metabolismo , Citocinas/genética , Citocinas/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/genética , Fibrose , Mediadores da Inflamação/metabolismo , Rim/patologia , Masculino , Camundongos Endogâmicos DBA , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo , Transdução de Sinais , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo
5.
Gene ; 726: 144196, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31669648

RESUMO

Accumulating evidence has indicated the important roles of circular RNAs (circRNAs) in different tumors. However, their detailed regulatory mechanisms in glioma are not fully understood. In this study, the functional role of a novel circRNA, circ-EZH2, was investigated by cell counting kit-8 (CCK-8), colony formation, flow cytometry, and transwell experiments. The regulatory mechanism of circ-EZH2 was explored by bioinformatics analysis, quantitative real-time PCR (qRT-PCR), Western blot and dual-luciferase reporter assay. We identified that circ-EZH2 was overexpressed in glioma tissues and cell lines. Further studies revealed that ectopic expression of circ-EZH2 significantly promoted cell growth, migration and invasion but inhibited cell apoptosis. By contrast, silencing of circ-EZH2 induced the opposite effects. Additionally, we found circ-EZH2 served as a miRNA sponge for miR-1265 to release its suppression on DDAH1 and CBX3. Rescue assays further revealed that the oncogenic function of circ-EZH2 was partly dependent on its modulation of DDAH1 and CBX3. Our study unraveled a novel molecular pathway in glioma and may provide a new perspective for the treatment of glioma.


Assuntos
Amidoidrolases/genética , Proliferação de Células/genética , Proteínas Cromossômicas não Histona/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Glioma/genética , MicroRNAs/genética , Invasividade Neoplásica/genética , Apoptose/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Glioma/patologia , Humanos , Invasividade Neoplásica/patologia
6.
Appl Biochem Biotechnol ; 190(1): 293-304, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31346919

RESUMO

Cephalosporin C acylase (CCA) is the key enzyme in the production of 7-aminocephalosporanic acid (7-ACA) via a one-step enzymatic process. To improve the soluble expression level of CCA in recombinant Escherichia coli at elevated temperatures, a library of T7 promoter mutants was created by site-saturation mutagenesis, and a series of mutated promoters were subsequently screened. Green fluorescent protein (GFP) was fused to the C-terminus of CCA to facilitate library screening, and the expression of the CCA and GFP fusion proteins was investigated under the control of the T7 promoter. Twenty-four mutants were selected by detecting the fluorescence intensity of colonies on agar plates to form a library with different expression levels. The enzyme activities of the mutants were positively correlated with their fluorescence intensities. The highest enzyme activity among these mutant promoters was 1.3-fold higher than the enzyme activity resulting from the wild-type promoter when the cells were cultured at 32 °C for 16 h. In addition, the transcription and expression levels of several typical promoters were discussed, and the effects of GFP fusion on the enzyme activity of CCA were investigated.


Assuntos
Amidoidrolases/genética , Bacteriófago T7/genética , Cefalosporinas/metabolismo , Escherichia coli/genética , Ensaios de Triagem em Larga Escala , Mutação , Regiões Promotoras Genéticas , Amidoidrolases/metabolismo , Genes Virais , Proteínas de Fluorescência Verde/genética , Transcrição Genética
7.
Nucleic Acids Res ; 48(4): 1886-1904, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31853544

RESUMO

Imbalance in the level of the pyrimidine degradation products dihydrouracil and dihydrothymine is associated with cellular transformation and cancer progression. Dihydropyrimidines are degraded by dihydropyrimidinase (DHP), a zinc metalloenzyme that is upregulated in solid tumors but not in the corresponding normal tissues. How dihydropyrimidine metabolites affect cellular phenotypes remains elusive. Here we show that the accumulation of dihydropyrimidines induces the formation of DNA-protein crosslinks (DPCs) and causes DNA replication and transcriptional stress. We used Xenopus egg extracts to recapitulate DNA replication invitro. We found that dihydropyrimidines interfere directly with the replication of both plasmid and chromosomal DNA. Furthermore, we show that the plant flavonoid dihydromyricetin inhibits human DHP activity. Cellular exposure to dihydromyricetin triggered DPCs-dependent DNA replication stress in cancer cells. This study defines dihydropyrimidines as potentially cytotoxic metabolites that may offer an opportunity for therapeutic-targeting of DHP activity in solid tumors.


Assuntos
Amidoidrolases/genética , Transformação Celular Neoplásica/genética , Replicação do DNA/genética , Transcrição Genética , Animais , Antineoplásicos/uso terapêutico , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Uracila/análogos & derivados , Uracila/metabolismo , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento
8.
Int J Mol Sci ; 21(1)2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31861829

RESUMO

Chitin deacetylase (CDA) is a chitin degradation enzyme that strictly catalyzes the deacetylation of chitin to form chitosan, which plays an important role in regulating growth and development, as well as the immune response. In this study, a chitin deacetylase 3 gene (CDA3) was identified with a complete open reading frame (ORF) of 1362 bp from the genome database of Diaphorina citri, encoding a protein of 453 amino acids. Spatiotemporal expression analysis suggested that D. citri CDA3 (DcCDA3) had the highest expression level in the integument and third-instar nymph stage. Furthermore, DcCDA3 expression level can be induced by 20-hydroxyecdysone (20E). Injection of Escherichia coli and Staphylococcus aureus induced the upregulation of DcCDA3 in the midgut, while DcCDA3 was downregulated in the fat body. After silencing DcCDA3 by RNA interference, there was no influence on the D. citri phenotype. In addition, bactericidal tests showed that recombinant DcCDA3 inhibited gram-positive bacteria, including S. aureus and Bacillus subtilis (B. subtilis). In conclusion, our results suggest that DcCDA3 might play an important role in the immune response of D. citri.


Assuntos
Amidoidrolases/imunologia , Hemípteros/imunologia , Proteínas de Insetos/imunologia , Amidoidrolases/química , Amidoidrolases/genética , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/imunologia , Hemípteros/química , Hemípteros/genética , Imunidade , Proteínas de Insetos/química , Proteínas de Insetos/genética , Transcriptoma
9.
Proc Natl Acad Sci U S A ; 116(49): 24770-24778, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31740614

RESUMO

Fatty acid amide hydrolase (FAAH) degrades 2 major classes of bioactive fatty acid amides, the N-acylethanolamines (NAEs) and N-acyl taurines (NATs), in central and peripheral tissues. A functional polymorphism in the human FAAH gene is linked to obesity and mice lacking FAAH show altered metabolic states, but whether these phenotypes are caused by elevations in NAEs or NATs is unknown. To overcome the problem of concurrent elevation of NAEs and NATs caused by genetic or pharmacological disruption of FAAH in vivo, we developed an engineered mouse model harboring a single-amino acid substitution in FAAH (S268D) that selectively disrupts NAT, but not NAE, hydrolytic activity. The FAAH-S268D mice accordingly show substantial elevations in NATs without alterations in NAE content, a unique metabolic profile that correlates with heightened insulin sensitivity and GLP-1 secretion. We also show that N-oleoyl taurine (C18:1 NAT), the most abundant NAT in human plasma, decreases food intake, improves glucose tolerance, and stimulates GPR119-dependent GLP-1 and glucagon secretion in mice. Together, these data suggest that NATs act as a class of lipid messengers that improve postprandial glucose regulation and may have potential as investigational metabolites to modify metabolic disease.


Assuntos
Amidoidrolases/genética , Glicemia/metabolismo , Síndrome Metabólica/metabolismo , Ácidos Oleicos/metabolismo , Taurina/análogos & derivados , Amidoidrolases/metabolismo , Substituição de Aminoácidos , Animais , Glicemia/análise , Modelos Animais de Doenças , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/fisiologia , Etanolaminas/sangue , Etanolaminas/metabolismo , Feminino , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Teste de Tolerância a Glucose , Humanos , Injeções Intravenosas , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Síndrome Metabólica/sangue , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/genética , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Ácidos Oleicos/administração & dosagem , Ácidos Oleicos/sangue , Período Pós-Prandial/efeitos dos fármacos , Período Pós-Prandial/fisiologia , Receptores Acoplados a Proteínas-G/metabolismo , Taurina/administração & dosagem , Taurina/sangue , Taurina/metabolismo
10.
Genet Test Mol Biomarkers ; 23(12): 837-842, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31750736

RESUMO

Background: Hashimoto's thyroiditis (HT) is a common autoimmune disease characterized by lymphoid infiltration of the thyroid gland, including both T- and B-cells. Early studies have shown that HT is a complex disorder affected by both environmental and genetic factors. Recently, the single nucleotide polymorphism (SNP) rs2276886 associated with the CXCL9 gene was identified as associated with autoimmune thyroid disease susceptibility in Japanese populations. The aim of the present study was to validate this result for HT in a Chinese Han population. Methods: Study subjects, including 688 HT cases and 1456 healthy controls, were recruited, and 10 SNPs located within the CXCL9 gene were genotyped. Genetic association analyses were performed by fitting logistic models. Bioinformatics tools, including RegulomeDB and GTEx were utilized to investigate the functional consequences of the SNPs found to be significantly associated with HT. Results: SNP rs2276886 was identified as significantly associated with the risk of HT (odds ratio [OR] = 1.25, p = 0.0006). No significant expression quantitative trait loci (eQTL) signals could be identified for CXCL9. Significant eQTL signals were found for other genes, including ART3, CXCL10, CXCL11, NAAA, PPEF2, and SCARB2. This SNP physically maps to the CXCL9 gene region; however, further bioinformatic analyses indicated that this SNP might be associated with the gene NAAA. Conclusions: The rs2276886 SNP was found to be significantly associated with HT susceptibility. However, our findings suggest that this SNP which maps to the chromosomal region 4q21.1 likely effects the NAAA gene (as opposed to the CXCL9 gene), but still contributes to the susceptibility to HT in Han Chinese populations.


Assuntos
Amidoidrolases/genética , Quimiocina CXCL9/genética , Doença de Hashimoto/genética , Adulto , Alelos , Amidoidrolases/metabolismo , Grupo com Ancestrais do Continente Asiático/genética , Doenças Autoimunes/genética , Estudos de Casos e Controles , Quimiocina CXCL9/metabolismo , China , Cromossomos Humanos Par 4/genética , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença , Genótipo , Doença de Graves/genética , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco
11.
Bull Exp Biol Med ; 168(2): 264-269, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31782002

RESUMO

We developed a protocol for detection of mutations in the pncA gene associated with M. tuberculosis resistance to pyrazinamide by analyzing melting curves of 7 overlapping amplicons with artificial heteroduplex formation (H-HRM) formed by co-amplification of wild-type DNA and test DNA and compared its efficiency and robustness with those of classical HRM analysis. Using HRM and H-HRM, we analyzed 35 PZAR DNA isolates carrying mutations in the pncA gene, 3 PZAR isolates without mutations in the pncA gene, and 20 PZAS isolates without mutations in the pncA gene were analyzed. The sensitivity and specificity of HRM for detection of mutations in the pncA gene were moderate: 88.57% (CI 73.26%-96.80%) and 82.61% (CI 61.22%-95.05%), respectively. The sensitivity of the H-HRM test was 97.14% (CI 85.08%-99.93%) and specificity was 95.65% (CI 78.05%-99.89%), with a significant improvement in accuracy - 96.55% vs. 93.85% for HRM. In general, despite addition stage of equalizing the concentrations of the test and control mycobacterial DNA, H-HRM showed greater stability and reproducibility at standard settings of the melting curve analysis software.


Assuntos
Amidoidrolases/genética , Antituberculosos/farmacologia , Farmacorresistência Bacteriana/genética , Mycobacterium tuberculosis/genética , Pirazinamida/farmacologia , Sequência de Bases , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Desnaturação de Ácido Nucleico/genética , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNA , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologia
12.
mSphere ; 4(5)2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31597720

RESUMO

Cryptococcus gattii R265 is a hypervirulent fungal strain responsible for the recent outbreak of cryptococcosis in Vancouver Island of British Columbia in Canada. It differs significantly from Cryptococcus neoformans in its natural environment, its preferred site in the mammalian host, and its pathogenesis. Our previous studies of C. neoformans have shown that the presence of chitosan, the deacetylated form of chitin, in the cell wall attenuates inflammatory responses in the host, while its absence induces robust immune responses, which in turn facilitate clearance of the fungus and induces a protective response. The results of the present investigation reveal that the cell wall of C. gattii R265 contains a two- to threefold larger amount of chitosan than that of C. neoformans The genes responsible for the biosynthesis of chitosan are highly conserved in the R265 genome; the roles of the three chitin deacetylases (CDAs) have, however, been modified. To deduce their roles, single and double CDA deletion strains and a triple CDA deletion strain were constructed in a R265 background and were subjected to mammalian infection studies. Unlike C. neoformans where Cda1 has a discernible role in fungal pathogenesis, in strain R265, Cda3 is critical for virulence. Deletion of either CDA3 alone or in combination with another CDA (cda1Δ3Δ or cda2Δ3Δ) or both (cda1Δ2Δ3Δ) rendered the fungus avirulent and cleared from the infected host. Moreover, the cda1Δ2Δ3Δ strain of R265 induced a protective response to a subsequent infection with R265. These studies begin to illuminate the regulation of chitosan biosynthesis of C. gattii and its subsequent effect on fungal virulence.IMPORTANCE The fungal cell wall is an essential organelle whose components provide the first line of defense against host-induced antifungal activity. Chitosan is one of the carbohydrate polymers in the cell wall that significantly affects the outcome of host-pathogen interaction. Chitosan-deficient strains are avirulent, implicating chitosan as a critical virulence factor. C. gattii R265 is an important fungal pathogen of concern due to its ability to cause infections in individuals with no apparent immune dysfunction and an increasing geographical distribution. Characterization of the fungal cell wall and understanding the contribution of individual molecules of the cell wall matrix to fungal pathogenesis offer new therapeutic avenues for intervention. In this report, we show that the C. gattii R265 strain has evolved alternate regulation of chitosan biosynthesis under both laboratory growth conditions and during mammalian infection compared to that of C. neoformans.


Assuntos
Amidoidrolases/genética , Quitosana/metabolismo , Cryptococcus gattii/metabolismo , Cryptococcus gattii/patogenicidade , Proteínas Fúngicas/genética , Amidoidrolases/metabolismo , Animais , Parede Celular/química , Parede Celular/imunologia , Criptococose/microbiologia , Cryptococcus gattii/genética , Feminino , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
13.
Proc Natl Acad Sci U S A ; 116(46): 23232-23242, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31659023

RESUMO

PM20D1 is a candidate thermogenic enzyme in mouse fat, with its expression cold-induced and enriched in brown versus white adipocytes. Thiazolidinedione (TZD) antidiabetic drugs, which activate the peroxisome proliferator-activated receptor-γ (PPARγ) nuclear receptor, are potent stimuli for adipocyte browning yet fail to induce Pm20d1 expression in mouse adipocytes. In contrast, PM20D1 is one of the most strongly TZD-induced transcripts in human adipocytes, although not in cells from all individuals. Two putative PPARγ binding sites exist near the gene's transcription start site (TSS) in human but not mouse adipocytes. The -4 kb upstream site falls in a segmental duplication of a nearly identical intronic region +2.5 kb downstream of the TSS, and this duplication occurred in the primate lineage and not in other mammals, like mice. PPARγ binding and gene activation occur via this upstream duplicated site, thus explaining the species difference. Furthermore, this functional upstream PPARγ site exhibits genetic variation among people, with 1 SNP allele disrupting a PPAR response element and giving less activation by PPARγ and TZDs. In addition to this upstream variant that determines PPARγ regulation of PM20D1 in adipocytes, distinct variants downstream of the TSS have strong effects on PM20D1 expression in human fat as well as other tissues. A haplotype of 7 tightly linked downstream SNP alleles is associated with very low PMD201 expression and correspondingly high DNA methylation at the TSS. These PM20D1 low-expression variants may account for human genetic associations in this region with obesity as well as neurodegenerative diseases.


Assuntos
Adipócitos/metabolismo , Amidoidrolases/metabolismo , PPAR gama/metabolismo , Tecido Adiposo/metabolismo , Amidoidrolases/genética , Animais , Expressão Gênica , Regulação da Expressão Gênica , Variação Genética , Humanos , Masculino , Camundongos , Obesidade/genética , Fenótipo , Tiazolidinedionas
14.
Nat Plants ; 5(11): 1167-1176, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31636399

RESUMO

Soil-borne fungal pathogens that cause crop disease are major threats to agriculture worldwide. Here, we identified a secretory polysaccharide deacetylase (PDA1) from the soil-borne fungus Verticillium dahliae, the most notorious plant pathogen of the Verticillium genus, that facilitates virulence through direct deacetylation of chitin oligomers whose N-acetyl group contributes to host lysine motif (LysM)-containing receptor perception for ligand-triggered immunity. Polysaccharide deacetylases are widely present in fungi, bacteria, insects and marine invertebrates and have been reported to possess diverse functions in developmental processes rather than virulence. A phylogenetics analysis of more than 5,000 fungal proteins with conserved polysaccharide deacetylase domains showed that the V. dahliae PDA1-containing subtree includes a large number of proteins from the Verticillium genus as well as the Fusarium genus, another group of characterized soil-borne fungal pathogens, suggesting that soil-borne fungal pathogens have adopted chitin deacetylation as a major virulence strategy. We showed that a Fusarium PDA1 is required for virulence in cotton plants. This study reveals a substantial virulence function role of polysaccharide deacetylases in pathogenic fungi and demonstrates a subtle mechanism whereby deacetylation of chitin oligomers converts them to ligand-inactive chitosan, representing a common strategy of preventing chitin-triggered host immunity by soil-borne fungal pathogens.


Assuntos
Amidoidrolases/metabolismo , Quitina/metabolismo , Gossypium/microbiologia , Doenças das Plantas/microbiologia , Microbiologia do Solo , Verticillium/patogenicidade , Acetilação , Amidoidrolases/genética , Fusarium/enzimologia , Fusarium/patogenicidade , Gossypium/metabolismo , Lycopersicon esculentum/metabolismo , Verticillium/enzimologia , Virulência
15.
Appl Microbiol Biotechnol ; 103(21-22): 8839-8851, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31642949

RESUMO

D-p-hydroxyphenylglycine (D-HPG) functions as an intermediate and has important value in antibiotic industries. The high pollution and costs from chemical processes make biotechnological route for D-HPG highly desirable. Here, a whole-cell transformation process by D-hydantoinase(Hase) and D-carbamoylase(Case) was developed to produce D-HPG from DL-hydroxyphenylhydantoin(DL-HPH) in Escherichia coli. The artificially designed ribosome binding site with strong intensity significantly facilitated the protein expression of limiting step enzyme Case. Next, the cell wall permeability was improved by disturbing the peptidoglycan structure by overproduction of D,D-carboxypeptidases without obviously affecting cell growth, to increase the bioavailability of low soluble hydantoin substrate. By fine-tuning regulation of expression level of D,D-carboxypeptidase DacB, the final production yield of D-HPG increased to 100% with 140 mM DL-HPH substrate under the optimized transformation conditions. This is the first example to enhance bio-productivity of chemicals by cell wall engineering and creates a new vision on biotransformation of sparingly soluble substrates. Additionally, the newly demonstrated 'hydroxyl occupancy' phenomenon when Case reacts with hydroxyl substrates provides a referential information for the enzyme engineering in future.


Assuntos
Reatores Biológicos/microbiologia , Engenharia Celular/métodos , Parede Celular/genética , Escherichia coli/metabolismo , Glicina/análogos & derivados , Biossíntese de Proteínas/genética , Amidoidrolases/genética , Amidoidrolases/metabolismo , Carboxipeptidases/metabolismo , Parede Celular/metabolismo , Escherichia coli/genética , Engenharia Genética , Glicina/biossíntese , Permeabilidade , Biossíntese de Proteínas/fisiologia
16.
Eur J Pharmacol ; 863: 172708, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31568785

RESUMO

Obesity is a serious public health problem characterized by abnormal or excessive fat accumulation, which is caused by an energy imbalance between calories consumed and calories expended. MiRNAs have been involved in the regulation of occurrence and progression of obesity. This study aims to investigate the role of miR-324-5p in regulating the adipose tissue mass and preliminarily probe into its effect on progression of obesity. MiR-324-5p was upregulated in the epididymal white adipose tissues (eWAT), inguinal white adipose tissues (iWAT) and brown adipose tissues (BAT) of the mice fed with high fat diet (HFD). Under room temperature (RT) or thermoneutrality (TN) condition, when tail intravenously injected with miR-324-5p antagomir (anta-miR-324-5p), the fat mass and total weight of mice were both significantly suppressed. The suppressive effect was more distinct under TN than RT. The weight of iWAT and BAT were both inhibited by anta-miR-324-5p under TN. Moreover, PM20D1 was a direct target gene of miR-324-5p. In primary iWAT cells, the expression of PM20D1 was significantly increased by anta-miR-324-5p, whereas decreased by the miR-324-5p mimic. Furthermore, anta-miR-324-5p noticeably increased the cellular oxygen consumption in primary BAT and iWAT cells. Our findings indicated that inhibition of miR-324-5p increased PM20D1-mediated fat consumption and reduced body weight in mice, suggesting that miR-324-5p may be a novel therapeutic target against obesity.


Assuntos
Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/metabolismo , Amidoidrolases/metabolismo , Peso Corporal/genética , Tecido Adiposo Marrom/patologia , Tecido Adiposo Branco/patologia , Amidoidrolases/genética , Animais , Antagomirs/genética , Progressão da Doença , Camundongos , Camundongos Endogâmicos BALB C , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , Consumo de Oxigênio/genética , Termogênese/genética , Regulação para Cima/genética
17.
Int J Mycobacteriol ; 8(3): 211-217, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31512595

RESUMO

Background: This study explored the genetic diversity of Mycobacterium tuberculosis isolates in Egypt by spoligotyping in combination with pncA gene sequencing, spoNC. Methods: First, isolates were selected from 400 isolates positive for M. tuberculosis that referred to Central Labs Ministry of Health and then were subjected to the study analyses. Results: Twenty one isolates were found to be multidrug resistant (MDR) and 29 isolates were sensitive for isonizide (INH) and rifampicine (RIF) after testing by phenotypic drug susceptibility testing (DST) and Mycobacteria Growth Indicator Tube (MGIT). Spoligotyping yielded 45 patterns belonging to seven families that previously reported in neighboring countries such as Iraq, Syria, Iran, and Turkey. While four isolates were orphans. Conclusion: Application of spoNC on obtained spoligotype patterns enhances to reduce the clustering rate. Bejing family the predominant (34%) were subdivided by pncA sequence into three sensitive DST pncA wild type, three MDR-DST isolates showing cys14Arg mutation in pncA, two sensitive DST isolates with pncA Gly97Asp mutation, and three sensitive DST pncAVal128Gly mutation. The next most common CASI_DELHI family (16%) were subdivided by pncA sequencing into CASI_DELHI (st 381, MDR) including two pncA silent mutation ser65ser (tcc > tct) and CASI_DELHI (st26, sensitive) which included six pncA (wild-type) results, and Latin-American-Mediterranean 6 family (6%) all had PncA Gly97Asp mutation. We concluded that spoNC provides good snap shot for MDR surveillance and its country origin and performs early identification of outbreaks in Egypt.


Assuntos
Amidoidrolases/genética , Mutação , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Adolescente , Adulto , Idoso , Antituberculosos/farmacologia , Técnicas de Tipagem Bacteriana , Egito , Monitoramento Epidemiológico , Feminino , Variação Genética , Genótipo , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium tuberculosis/efeitos dos fármacos , Estudos Prospectivos , Pirazinamida/farmacologia , Rifampina/farmacologia , Análise de Sequência de DNA , Escarro/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Adulto Jovem
18.
Int J Antimicrob Agents ; 54(5): 587-591, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31400469

RESUMO

The increasing use of polymyxins as last-resort drugs for managing infections by Acinetobacter baumannii has led to the emergence of resistance. This study aimed to determine the resistance mechanisms in Acinetobacter baumannii isolates with colistin MIC ≥ 4 mg/L and to relate the mechanisms of resistance with the difficulties in detecting them. Absolute agreement among the different methodologies (Phoenix automatized system, broth and agar dilution, and a rapid colorimetric test) in the 140 colistin-susceptible isolates was observed; whereas in the 25 resistant isolates, the performance varied according to the colistin MIC value. Most of the discrepancies (irrespective of the methodology that was used) were observed in isolates with an MIC value close to the breakpoint. The number of errors in each method in the resistant isolates was as follows: rapid test, four of 25 (16%); agar dilution, eight of 25 (32%); Phoenix system, 13 of 25 (52%) and its manual reading at 24 h, eight of 25 (32%). Categorical errors were detected in 13 isolates: slow growth was the main reason in five isolates, whereas in the remaining eight isolates, slow growth was detected together with a low proportion of colistin-resistant subpopulations and the colistin MIC value was close to the breakpoint value. To understand the probable reason for the observed MIC values, sequencing of genes associated with colistin resistance was performed. Mutations at lpxA, lpxC, and pmrB genes were detected and it was observed that isolates carrying mutations in lpxC presented slow growth at killing curves.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/isolamento & purificação , Aciltransferases/genética , Amidoidrolases/genética , Humanos , Testes de Sensibilidade Microbiana
19.
Food Microbiol ; 84: 103259, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31421778

RESUMO

Bacillus licheniformis is frequently associated with food spoilage due to its ability to form highly resistant endospores. The present study reveals that B. licheniformis spore peptidoglycan shares a similar structure to spores of other species of Bacillus. Two enzymatic activities associated with depolymerisation of the cortical peptidoglycan, which represents a crucial step in spore germination, were detected by muropeptide analysis. These include lytic transglycosylase and N-acetylglucosaminidase activity, with non-lytic epimerase activity also being detected. The role of various putative cortex-lytic enzymes that account for the aforementioned activity was investigated by mutational analysis. These analyses indicate that SleB is the major lysin involved in cortex depolymerisation in B. licheniformis spores, with CwlJ and SleL having lesser roles. Collectively, the results of this work indicate that B. licheniformis spores employ a similar approach for cortical depolymerisation during germination as spores of other Bacillus species.


Assuntos
Bacillus licheniformis/enzimologia , Bacillus licheniformis/genética , Mutação , Esporos Bacterianos/enzimologia , Amidoidrolases/genética , Proteínas de Bactérias/genética , Parede Celular , Microbiologia de Alimentos/métodos , Viabilidade Microbiana , Peptidoglicano/química , Esporos Bacterianos/crescimento & desenvolvimento
20.
Artigo em Inglês | MEDLINE | ID: mdl-31454682

RESUMO

Heortia vitessoides Moore is a notorious defoliator of Aquilaria sinensis (Lour.) Gilg trees. Chitin deacetylases (CDAs) catalyze the N-deacetylation of chitin, which is a crucial process for chitin modification. Here, we identified and characterized HvCDA1 and HvCDA2 from H. vitessoides. HvCDA1 and HvCDA2 possess typical domain structures of CDAs and belong to the Group I CDAs. HvCDA1 and HvCDA2 were highly expressed before and after the larval-larval molt. In addition, both exhibited relatively high mRNA expression levels during the larval-pupal molt, the pupal stage, and the pupal-adult molt. HvCDA1 and HvCDA2 transcript expression levels were highest in the body wall and relatively high in the larval head. Significant increases in the HvCDA1 and HvCDA2 transcript expression levels were observed in the larvae upon exposure to 20-hydroxyecdysone. RNA interference-mediated HvCDA1 and HvCDA2 silencing significantly inhibited HvCDA1 and HvCDA2 expression, with abnormal or nonviable phenotypes being observed. Post injection survival rates of the larvae injected with dsHvCDA1 and dsHvCDA2 were 66.7% and 46.7% (larval-pupal) during development and 23.0% and 6.7% (pupal-adult), respectively. These rates were significantly lower than those of the control group insects. Our results suggest that HvCDA1 and HvCDA2 play important roles in the larval-pupal and pupal-adult transitions and represent potential targets for the management of H. vitessoides.


Assuntos
Amidoidrolases/metabolismo , Pupa/enzimologia , Amidoidrolases/genética , Animais , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/enzimologia , Muda/genética , Muda/fisiologia , Mariposas/enzimologia , Pupa/crescimento & desenvolvimento , Interferência de RNA
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